INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY

GOVERNMENT COLLEGE UNIVERSITY LAHORE

Production and Characterization of Pectinases by

Aspergillus niger for Industrial use

Nauman Jamil Khan

Reg. No. 7 5 - G C U - P h D - B I O - 1 0

SESSION: 2010-2016

INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY

GOVERNMENT COLLEGE UNIVERSITY LAHORE

Production and Characterization of Pectinases by

Aspergillus niger for Industrial use

Submitted to Government College University Lahore

In partial fulfillment of the requirement

for the award of degree

of

Doctor of Philosophy

In

Biotechnology

By

Nauman Jamil Khan

Reg. No. 7 5 - G C U - P h D - B I O - 1 0

SESSION: 2010-2016

“IN THE NAME OF ALLAH,

THE MOST BENEFICENT AND THE MERCIFUL”

Read: in the name of your lord who created man from a clot.

Read: and your lord is the most bounteous who taught by the pen.

Taught man that which he did not know.

Al Quran

___________________

DECLARATION

I, Nauman Jamil Khan with Registration # 075-GCU-PHD-BIO-10, as student

of Doctor of Philosophy in the subject of Biotechnology session 2010-2016, hereby

declare that the matter printed in this thesis titled “Production and Characterization of

pectinases by Aspergillus niger for industrial use” is my own work and has not been

printed, published and submitted as search work, thesis or publication in any form in any

University, Research Institution etc in Pakistan and abroad.

Dated:

Signature of deponent

___________________ ________________

PLAGIARISM UNDERTAKING

I, Mr. Nauman Jamil Khan, Registration No. 075-GCU-PhD-BIO-10, solemnly declare

that the research work presented in the thesis titled “Production and Characterization

of pectinases by Aspergillus niger for Industrial Use” is solely my research work, with

no significant contribution from any other person. Small contribution / help whenever

taken have been acknowledged and that complete thesis has been written by me.

I understand the zero tolerance policy of HEC and Government College

University Lahore, towards plagiarism. Therefore, I as an author of the above titled thesis

declare that no portion of my thesis has been plagiarized and any material used as

reference has been properly referred/cited.

I understand that if I am found guilty of any formal plagiarism in the above titled

thesis, even after the award of PhD degree, the University reserve the right to withdraw

my PhD degree and that HEC/University has the right to publish my name on

HEC/University website, in the list of culprits of plagiarism.

Dated:

Signature of Deponent

RESEARCH COMPLETION CERTIFICATE

It is certified that the research work contained in this thesis entitled “Production and

Characterization of pectinases by Aspergillus niger for industrial use.” has been

carried out and completed by Nauman Jamil Khan with Registration # 075-GCU-PHD-

BIO-10 under my supervision during his PhD studies in the subject of Biotechnology.

ACKNOWLEDGEMENTS

I am especially thankful to Allah S.W.T. who gave me the strength and potential to carry

out this research. We invoke peace of Holy Prophet HAZRAT MUHAMMAD

(P.B.U.H), who is forever a torch of guidance for humanity as a whole.

I am highly grateful to Prof. Dr. Ikram-ul-Haq (SI), Distinguished National

Professor/Professor Emeritus Biotechnology and my supervisor for his support,

supervision and unending guidance. His enthusiasm for research was pivotal in

energizing me to do this research. His encouraging attitude and the provision of all

facilities helped me tremendously especially at times when making up for any of my

shortcomings.

I extend my gratitude to Director Institute of Industrial Biotechnolog and all the staff

members of Institute of Industrial Biotechnology especially Dr. Ali Nawaz.

I am also thankful to my parents whose prayers and support have imbibed in me the spirit

to work progressively and have undoubtedly been crucial to the success of my research

work.

Nauman Jamil Khan

DEDICATED

TO MY

ALMA MATER

GOVERNMENT COLLEGE UNIVERSITY

LAHORE

LIST OF FIGURES

Figure

No.

Title Page

No.

1. Microscopic image of Aspergillus niger (40X) 40

2. Pectinolytic activities of variants of Aspergillus niger after UV

rays treatment.

41

3. Pectinolytic ability of EMS treated variants Aspergillus niger

strain IIB-13 after EMS (1%) treatment for 10 mins

42

4. Pectinase production of nitrous acid (0.1M) treated mutants for 5

min of Aspergillus niger strain IIB-13.

43

5. Rate of pectinase synthesis using wild IIB-13 and mutant H-97

strain of Aspergillus niger by submerged fermentation.

44

6. Effect of Incubation temperature on pectinase activity using

wild IIB-13and mutant H-97strains of Aspergillus niger by

submerged fermentation.

45

7. Effect of pH on the production of pectinase using the wild IIB-

13 and mutant H-97 strains of Aspergillus niger by submerged

fermentation.

46

8. Effect of fermentation medium for pectinase production using

the wild IIB-13 and H-97 mutant strain of Aspergillus niger by

submerged fermentation.

47

9. Effect of Carbon source for pectinase production using the wild

IIB-13 and mutant H-97 strain of Aspergillus niger by

submerged fermentation.

48

10. Effect of Pectin Concentration on the production of Pectinase

using the wild IIB-13 and mutant H-97 strains of Aspergillus

niger by submerged fermentation.

48

11. Effect of Nitrogen source for best pectinolytic activity using wild

IIB-13 and mutant H-97 strains of Aspergillus niger by

submerged fermentation

50

12. Effect of yeast extract concentration on the production of

pectinase using the wild IIB-13 and mutant H-97 strains of

Aspergillus niger by submerged fermentation

50

13. Effect of Incubation time on the production of pectinase using

mutant strain H-97 of Aspergillus niger in stirred fermenter

51

14. Kinetic studies of incubation time effect on the production of

pectinase using mutant strain H-97 of Aspergillus niger in

stirred fermenter

52

15. Effect of aeration rate on the production of pectinase using

mutant strain of Aspergillus niger in stirred fermenter

53

16. Kinetic evaluation of aeration effect on the production of

Pectinase using mutant strain of Aspergillus niger in stirred

fermenter.

53

17. Effect of agitation rate on the production of pectinase using

mutant strain of Aspergillus niger in stirred fermenter

54

18. Kinetic studies of agitation rate for the production of pectinase

using mutant strain of Aspergillus niger in stirred fermenter

55

19. Investigation of inoculum size on the production of pectinase

using mutant strain of Aspergillus niger in stirred fermenter

56

20. Kinetic studies of agitation rate for the production of pectinase

using mutant strain of Aspergillus niger in stirred fermenter.

56

21. Purification of pectinase using LPC using Bio-Rad

UnosphereTM

Q (BioscaleTM

mini) column.

59

22. SDS-PAGE analysis of purified Pectinase. 60

23. Line weaver-Burke Double Reciprocal Plot to calculate the Km

and Vmax for Pectinase.

61

24. Effect of temperature on enzyme activity of pectinase. 62

25. Arrhenius plot to calculate the activation energy and enthalpy of

Activation for pectianse catalyzed enzyme reaction

62

26. Temperature stability profile of pectinase. 63

27. Effect of different pH on Pectinase activity. 64

28. pH stability profile of pectinase. 65

29. Effect of metal ions on Pectinase activity. 66

30. Stability profile of pectinase in the presence of metal ions. 67

31. Effect of Surfactants on Pectinase activity 68

LIST OF TABLES

Table

No.

Title Page No.

1 Ammonium Sulphate Precipitation 31

2 Secondary Screening of Pectinolytic strains of Aspergillus

niger

35

3 Overall summary of Pectinase purification 58

LIST OF ABBREVIATIONS

Symbols Abbreviations

% (Percentage)

µl (micro liter)

ml (milliliter)

Conc. (Concentration)

dH2O (distilled water)

GOD (Glucose oxidase)

CaCO3 (Calcium Carbonate)

MgSO4 (Magnesium Sulphate)

NaNO3 (Sodium Nitrate)

Na2HPO4 (Di sodium hydrogen phosphate)

(NH4)2HPO4 (Di ammounim hydrogen phosphate)

DNS (Dinitrosalycyclic acid)

BSA (Bovine serum albumin)

°C (degree celcius)

min (Minute)

pH (Power of Hydrogen Ion Concentration)

rpm (revolution per minute)

Sec (Seconds)

LIST OF EQUIPMENTS

Serial # Equipments

1 Shaking Incubator at 37oC/22

oC (Innova

R 43 Incubator shaker series)

2 Incubator at 37oC (ECOCELL)

3 Cold Cabinet (WB10 Memmert, Germany).

4 Centrifuge machine (SIGMA laboratory centrifuges 3K30) at 40C

5 Centrifuge machine ((Model: EBA 20, Hettich, UK)

6 pH meter (INOLAMTM

)

7 Electric balance (Types AUW 2200 No D 450013067, SHIMODU

Corporation Japan)

8 Fermentor ( New Brunswick Scientific, BIOFLO 110/ Bioreactor,

Germany)

9 Laminar flow cabinet (Technico Scientific Supply)

10 Spectrophotometer (Model: 7200 Aquarius, Cecil CE, England).

11 Ion exchange chromatography column (Bio-Rad Bio-scale Mini Macro-

Prep DEAE cartridge)

12 Casting system (Mini-PROTEAN Tetra cell, Bio-Rad)

TABLE OF CONTENTS

Declaration

Certificate

Dedication

Acknowledgements

List of Figures

List of Tables

List of

Abbreviations List

of Equipment

Abstract

Chapter #1: Introduction 1-7

Chapter #2: Literature Review 8-24

Chapter #3: Materials and Methods 25-34

Chapter#4: Result 35-68

Chapter #5: Discussion 69-75

References 76-86

Annexure 87-88

1

Abstract

Two hundred and sixteen fungal strains were isolated from two hundred fifty soil samples

collected from different localities of Punjab and subjected to screening for their pectinolytic

ability using submerged fermentation. One hundred and fifty one pectinolytic strains were

subjected to secondary screening and isolate IIB-13 gave the maximum pectinase yield

(10.78±0.45U/ml/min) and identified as Aspergillus niger using morphological

characteristics. Random mutagenesis (physical and chemical) was used to improve the

pectinolytic activity of strain IIB-13. Nitrous acid mutant H-97 resulted in three fold increase

(32.16 + 0.05U/ml/min) in pectinase production. This H-97strain was further subjected to

optimization of media, carbon and nitrogen sources, temperature and pH. Maximum

pectinase (37.01±0.11 U/ml/min) was produced using medium containing K2HPO4 (4.0 g),

yeast extract (0.6 g), KH2PO4 (1.28 g), (NH4)2SO4 (2.0 g), MgSO4 (1.1 g) and pectin (10 g)

per 1.0 L of distilled water at pH and temperature (7.0 and 30°C) respectively after 60 hours

of fermentation. Fermenter studies (7.5 L) resulted in increased pectinase activity

(40.31±0.07 U/ml/min) with decreased incubation time of 48 h using 1% inoculums with

optimized reaction conditions i.e. temperature (30°C), pH (07), agitation (200 rpm) and

aeration (1vvm). Fermentation kinetics was applied to validate the results. The crude enzyme

was subjected to purification by applying ammonium sulfate precipitation and Ion exchange

chromatography which resulted in 44.20 % yield with increased activity i.e. 75.18±0.04

U/mg. Km value of 2.30 mg/ml using pectin as substrate showed its maximum specificity

for the enzyme. Activation energy (Ea) i.e. -28.95 KJ/mol and enthalpy of activation (ΔH)

i.e. -26.73 KJ/mol were calculated employing thermodynamic studies. Characterization of

enzyme revealed catalytic activity optimum temperature and pH as 25°C and pH 7. The

enzymes catalytic activity was observed to be reduced by some heavy metals such as Hg2+,

Pb2+, Cu+ and Cd2+.

2

1. Introduction

Pectinases are the group of enzymes that are used for the degradation of pectin. Pectin

consists of α-1, 4 galacturonic acid, carboxyl groups and is esterified with methanol &

rhamnogalacturonan backbone. Side chains are linked via glycosidic linkages to 3 & 4

carbonic of rhamnose units and are composed of neutral sugars (De-Gregorio et al. 2002).

De-polymerization of Pectin substances is conducted by hydrolysis and trans elimination

reactions. Pectinases are categorized on the basis of their breakdown activity of the

galacturonan part of the pectin molecule. The three main pectinase groups are:

Polygalacturonases, pectin methyl esterases and pectin lyases. Actually, the

Polygalacturonases are de-polymerizing enzymes catalyzing α-1,4 glycosidic linkages into

pectin chain. Pectin methyl esterase hydrolyzes the methyl ester of galacturonide chain and

release methanol while pectin lyases breakdown α-1,4 glycosidic linkages by trans

elimination (Voragen and Visser, 2004).

Structure of pectin

Pectin is found in large amount in the cell wall of higher plants besides cellulose, hemi-

cellulose and soluble proteins. These are main components found in middle lamella in

between the walls of smaller cells and are prominent in parenchyma tissue of primary cell

wall. It also acts as a thin adhesive layer (Whitaker, 1990). It is also found in large amount in

the peels of fruits i.e. mango, orange, apple, pineapple, papaya, tomato, potato, and lemon

(Pilnik and Voragen, 1993). Pectin plays a vital role for the cohesion of plant tissues, its

structural integrity and in binding and cementing the cells. (Rombouts and Pilnik, 1980).

3

Microorganisms are usually widely used for industrial and commercial production of

pectinase as they are not affected by seasonal and climatic factors (Patil and Dayan, 2006).

The production of enzyme can be enhanced easily by changing the environmental modifiers

like temperature, pH, medium, addition of surfactants, stage of growth, rate of growth and

genetic manipulations (Busto et al. 1995). Moreover, microbial enzymes are produced in

greater quantity by using established fermentation techniques (Ray et al. 2007). Aspergillus

niger, Rhizopus and Penicillium are some of the common fungi which are used for the

production of pectinase. Aspergillus niger is considered to be most effective producer of

pectinase enzyme (Khairnar et al. 2009). It is also used as a model organism for the analysis

and study of eukaryotic protein secretion and recognized as GRAS (Generally regarded as

safe) (Baker, 2006). Aspergillus niger is a soil saprobe belonging to the phylum Ascomycota,

which produce large conidia, radiated from a vesicle or spherical conidiophores found in a

globose head. It contains a wide range of oxidative and hydrolytic enzymes which degrade

plant polysaccharides (Rapper et al. 1965).

Pectinase is produced both by submerged and solid state fermentation but generally pectinase

production is carried out by submerged fermentation system using regulatory aspects i.e.

molds and the production of enzyme is induced by using pectin (Maldonado et al. 1989).

Aspergillus niger was isolated from citrus fruit peels and local environment and screening

was done to select the efficient isolate i.e. the one producing the substantial amount of

extracellular pectinase (Khairnar et al. 2009). The efficiency of enzyme can be further

enhanced by optimizing biochemical and physical parameters. The production of pectinase

by Aspergillus niger can be influenced by many parameters like pH, temperature, aeration,

incubation time, agitation and composition of growth medium (Thakur et al. 2012).

Carbon sources are very important and play a significant role for the production of pectinase.

As they act as energy source for the growth of microorganisms producing the enzyme. The

simple sources which are used for pectinase’ production contain sucrose, maltose, fructose,

lactose, glucose and citrus pectin, while complex sources include dry peel powder of many

fruits like orange, potato, pineapple, apple and bagasse of orange (Bali, 2003). Moreover,

nitrogen sources also influence the pectinase production as they are responsible for the

formation of protein and amino acid content of the microb. Both complex and simple nitrogen

sources e.g. yeast extract, peptone, urea, (NH4)2SO4, corn steep liquor etc can be used for the

production of pectinase. pH of the growth medium has a great effect on enzyme production

4

and growth of organism, which in turn effects the pectinase production. Optimum range of

pH for production of pectinase is 4.0-7.0 pH (Niture and Pant, 2004). Initial Incubation

temperature is also a highly valuable controlling factor for production of pectinase, as if the

temperature is too low the organism’s metabolism will slow down while if it’s too high then

proteins get denatured (Kitpreechavnichet al. 1984). Its value usually varies from 25-35ºC for

maximum production of pectinase (Rapper et al. 1965). Maximum activity of enzyme is also

linked with incubation period. Optimal incubation period is required as if the time is very

short desired metabolite will not be produced sufficiently. Maximum production of pectinase

is attained within 72-120 hours of incubation (Venugopalet al. 2007).

Aeration influence the fungal grown and play a positive role for the production of enzymes

(Dekker and Barbosa, 2001). As we provide more aeration to the medium we observe more

enzyme activity. The size of inoculum also influences pectinases production. Less amount of

inoculum will result in low enzyme production as the cells are insufficient cells to produce

enzyme while in large inoculum, the microorganisms will deplete the nutrients quickly.

Strain improvement can be done to improve the yield of enzyme. Genetic variability is

caused due to mutagenesis by using the various energy sources like X-rays, UV, gamma rays

and chemical treatment. They produce mutants for greater and enhanced production of

required enzyme. UV radiation and chemical mutagenesis (Ethidium bromide and nitrous

acid) are the commonly used mutagens for strain improvement. UV rays in the range 200-300

nm are absorbed by the nucleic acids and cause alteration of DNA by breaking dimer of

nitrogenous bases, altering the base pairs of DNA molecule and disrupting DNA molecule

(Conn and Stumph, 1994).

5

Ion exchange chromatography technique is one such purification technique which is based on

the principle that proteins carry either negative or positive charge and are insoluble and

separation of protein can be achieved. Cation and anion exchangers i.e. two types of ion

exchangers are generally used in this process (Wilson and Walker, 2007). Isoelectric point of

pectinase is observed in pH range 4-5 (Zia et al. 2007). On this basis, anion exchange

chromatography is used for the purification of pectinase. Elution is done by using NaCl as

salt gradient. However, salt gradients and mixed pH have been examined previously, many

techniques involving the phenomena of precipitation have been used before and after ion

exchange chromatography for good and purified yield of pectinase including: ammonium

sulphate precipitation (Zia et. al., 2007).

Ammonium sulphate precipitation is based on the hydrophobic groups of proteins. When the

proteins are completely mixed, hydrophobic groups of proteins get into forced contact with

water and during this process, they are arranged in surroundings of the protein. Removal of

water molecules takes place by the addition of high concentration of salts and as a result the

proteins precipitate due to accumulation of hydrophobic portions of proteins. This

methodology is applied to separate larger proteins from the smaller protein. Proteins which

have large amount of hydrophobic portions will precipitate out earlier than those having

smaller portions of hydrophobic group.

Ammonium sulphate is utilized in this technique as it is sufficiently soluble and

comparatively more economical (Bankaret. al., 2009).

SDS-PAGE is used in finding the protein’s comparative molecular weight and to purify the

enzyme as separation of proteins is due to their size. SDS is an anionic detergent and causes

denaturation of the protein. Linear structure of proteins molecules having negative charge is

obtained after denaturation of proteins by SDS. The separation of SDS-protein complexes is

observed when they pass through the gel. Smaller the size of protein, the lesser it is trapped

from relocation and resultantly proteins are parted depending on their comparative molecular

masses. The proteins are seen as blue bands after PAGE. Protein markers are run in parallel to

samples to analyze the molecular weight of protein (Wilson and Walker, 2007).

6

Kinetic factors like Vmax and Km are very important from indudtrial point of view. A suitable

way of determining the required optimum amount of substrate for efficient working of

enzyme is Km. From industrial point of view Km can be explained in the way that if a certain

substrate has to be used by an industry, then the enzyme with lower value of Km will be

utilized by the industry for achieving faster rate of reaction. This will prove beneficial for the

industry from economical point of view and as it will require less time. Higher the specificity

of the enzyme for the substrate; lower is the value of Km of the enzyme for particular

substrate (Zia et. al., 2007).

The greater catalysis rate of an enzyme for a substrate with the change in temperature is

known as thermodynamic aspect of the enzyme. Activation energy (Ea) causes disorder in the

system and is called entropy i.e. (ΔS) and the total heat content of a system or a substance is

called as enthalpy and is represented with as (ΔH). It is considered that disorder or entropy of

the system is cumulative in a non-reversible reaction. Changes with reference to entropy and

enthalpy are two most commonly studied parameters of thermodynamics.

Enzymes are very sensitive in their performance and slight change in temperature can cause

changes in enzyme rate of activity / reaction. It has been observed that rate of enzyme

reaction doubles for every 10◦C rise in temperature. It has further been observed that

maximum enzymes lose their activity and become almost denatured at the temperature ranges

of 40°C to 70°C. Every enzyme has a different temperature at which enzyme shows

maximum activity. Various temperature ranges have been observed for maximum

productivity of different enzymes. The temperature at which maximum activity of a particular

enzyme is observed is known as its optimum temperature (Wilson and Walker, 2007). The

enzyme activity is also linked with pH as it is dependent on the ionization state of active site

amino acids. Generally, proteins are highly active within a range of pH 5-9 (Voet and Voet,

2011).

6

Metal ions show various behaviors as inhibitor or effectors. It is learnt that activity of many

enzymes is inhibited in the presence of Ag+, Cu2+, Pb2+, Hg2+, Ni2+ ,Cd2+, , Fe2+, Mn2+ and

Zn2+. The characteristic properties of metal ions have studied on fungi and yeast (Zia et. al.,

2010). The studies of enzyme activity with reference to the presence of various metal ions

provide valuable information to the industries particularly for enhancing the working efficacy

of enzymes in industry.

Pectinases are utilized in many commercial processes. The estimated industrial scale

utilization of the enzymes is about 75% (Satyanarayana and Panda, 2002). The most common

application of pectinases in industries is the extraction of fruit juice and its clarification.

Pectin causes turbidity and viscosity of fruit juice. Pectinases along with other enzymes

(amylases) are used for clarification process (Blancoaet al. 1999). Pectinases are also used in

maceration, by which pulpy juices and baby foods are manufactured by transforming

organized tissues into the intact cells (Pilnik and Voragen, 1993).

Bio-scouring of cotton fibers is carried out with pectinases, in this process non-cellulosic

impurities are eliminated from fibers. In textile processing pectinases are used along with

lipases, cellulases and hemi-cellulases to remove sizing agents from cotton (Hoondal et al.

2002). Pectinase is also successfully used in flax retting to remove pectin and isolate the

fibers and in biotechnological degumming of plant based fibers (Kapoor et al. 2000). In

vegetable processing industry, pectinases are used for pretreatment of waste water to help the

removal of pectic material, which can further be decomposed by activated sludge treatment

(Hoondal et al. 2002). Pectinases are also used in fermentation of tea and coffee as they speed

up the fermentation process of tea by destroying pectin, they also reduce the foam forming

properties of instant tea powders and coat of coffee beans can be isolated by using this

enzyme (Carr et al. 1985). In paper and pulp industries pectinase is widely used for the

manufacturing of paper. It can also be used in the production of animal feed as it reduces the

feed viscosity and increase absorption of nutrients (Hoondal et al. 2001). The purification of

plant viruses can be achieved by using pectinase and cellulases, which provides pure

preparation of viruses by releasing the virus from tissues (Salazar and Jayasinghe, 1999). The

stability and chromaticity of red wines is enhanced by using pectinase. It is mixed before

adding yeast to macerated fruits which improve color, turbidity (visual characteristics) and

stability of wines (Revilla and Ganzalez, 2003).

7

Objectives:

This work is carried out for fungal strain improvement and process optimization of

pectinase production using agricultural waste material. The specific objectives of the

present work are:

1. Isolation, screening and optimization of potent Aspergillus niger for pectinase

production.

2. Strain improvement using random mutagenesis.

3. Cultural conditions optimization for improved enzyme activity.

4. Scaling up of pectinase production in laboratory scale stirred fermentor.

5. Down streaming and characterization of pectinase.

6. Industrial application.

8

2. Literature Review

Maldonado et al. (1986) exuded pectinase from Asperigillus. Instead of pectin, the carbon

source was pretreated lemon peels. Asperigillus sp. strains obtained from decaying lemons

was experimented for extracellular pectinase production. Extracellular polygalacturonase

production was approximately the same when unwashed fresh lemon peel was used rather

than pectin. Acuna-Arguelles et al. (1995) applied Aspergillus niger for production of

extracellular Pectinases through solid state and submerged fermentation. The optimal

productivity of exo and endo pectinase and pectin lyase was then achieved through solid state

fermentation. In solid state fermentation, it has been observed that the pectinase activities

were stable at the extreme pH and temperature but Km value of endo-pectinase and pectin-

lyase were greater as compare to their activities in submerged culture techniques.

Electrophoresis using sodium dodecyl sulphate or polyacrylamide gel alongwith the

enzymatic extracts achieved for these two culture methods, have equal number of protein

bands but it was observed that some differences thus found in their electrophoretic position. It

indicates that the culture method technique: submerged or solid-state can be responsible to

induce changes in various pectinolytic enzymes produced by A. niger.

Cavalitto et al. (1995) isolated pectinase producing fungus, the production of pectinases by

Aspergillus foetidus NRRL 341 was done using solid state fermentation. Increase in the

pectinases activity was then observed with the increase in acidity of the culture medium.

Optimum growth was observed, when varying concentrations of 0.2, 0.3, 0.4 and 0.5 M HCl

were added to the culture medium and then inoculated with the diluted spore suspension of

Aspergillus foetidus. Maximum activity was recorded after 36 hours (2535 U/g) in the

medium comprises 0.4 M HCl. In the medium containing 0.2 and 0.3 M HCl, optimal activity

of pectinases (1860 U/g) was observed after 30 hours. It was indicated that acidity of culture

medium was inversely proportional to the pectin esterase activity and was directly

proportional to the activity of polygalacturonase.

Maldonado et al. (1998) checked production of pectin-esterase and polygalacturonase from

Aspergillus niger by submerged and solid state fermentation. Medium constitutes pectin thus

produced greater enzyme in fermentation in the 48 h of fermentation time. Glucose mixed as

the carbon source to medium because it enhanced the production of polygalacturonase(3.16

U/L) and pectin esterase production (1.14 U/L) by solid state fermentation. However,

9

pectinase production prohibited in submerged fermentation. Thus, the production of enzyme

is affected in each of the fermentation system by the addition of glucose.

Ezike et al. (2000) stated that Pectinases are class of enzymes that catalyze the hydrolysis of

pectins. Value addition to wastes generated from processing and consumption of orange fruits

was achieved by the extraction of pectin from waste peels and frequently used in production

of pectinase by submerged fermentation using Aspergillus niger. Results show that at pH of

2.2, a pectin yield of 15.5% was thus obtained from orange peels at 70°C, and of three

pectinolytic fungi isolated from the natural environment and thus induced with pectin from

orange peels as sole carbon source for production of pectinase; A.niger produced more

pectinase than that of others. On partial purification, 2-fold increase in pectinase activity

whose pH and temperature optima were 5.0 and 40°C were thus obtained. Value addition

could be obtained on orange wastes from extraction of pectin from it and its successive use in

the pectinase production. The results of study could be used as source of environmental clean-

up where waste is converted to wealth as the pectinase could further be used on industrial

scale in orange juice clarification.

Teixeira et al. (2000) studied activity of pectin esterase, exopolygalacturonase and

endopolygalacturonase obtained from the Aspergillus japonicus586 was taken from different

carbon sources in the liquid media with various carbons substrates concentrations. The

medium was inoculated with 5.106spores/ml, maintained under agitation (140 rpm), at 30◦C

for 122 h. The activity of enzyme was determined after 24 h of the filtration. It was indicated

that the best enzymes activity was bound in presence of 0.5% pectin: pectin esterase, 0.2%

glycerol (endopolygalacturonase) and 0.2% pectin and 0.5% pectin alongwith 0.5% glucose

(exopolygalacturonase). The presence of different carbon sources greatly influence the

activity of pectin esterase, and exo-and endolygalacturonase. However, when we add pectin,

glucose and saccharose, in the medium of the high concentrations, repression effect on the

activity of analyzed enzymes is thus observed.

Sunnotel et al. (2002) examined degradation of citrus pectin from the selected seven various

strains. It was thus observed that 05 bacterial soil isolates produced the alkaline pectinases

and 02 fungal strains produced pectinases in the acidic media. The aforementioned isolates

produced significant amount of pectin lyase and polygalacturonase with optimum activities

(30.1 and 29.1 U/ml). Fungal strains obtained from Aspergillus sp produced maximum

10

quantity of highly stable pectinases as compared to PN 1 bacterial sp. The Aspergillus niger

produced the highly stable pectinases which is very important factor in the industrial

applications.

Dhillon et al. (2004) stated that in the food processing industry, produced large waste

materials including pulp, peels and seeds which eventually causes disposal problems and thus

leads to pollution. It has been indicated that dried citrus peels contained maximum quantity of

proteins, carbohydrates and Pectin. The pectin is the inducer for production of pectinolytic

enzymes by microbial system. Dried citrus peels were used as substrates for the production of

pectinase (polygalacturonase) by fungus Aspergillus niger. Maximum enzymes was procured

with 15 % substrate when incubated at 30ºC for 120 h.

Martin et al. (2004) worked on fungal strains Moniliella SB9 and Penicillium EGC5 were

produced by solid state fermentation. These strains consumed the bagasse of wheat bran,

orange and sugar cane as substrate and exuded pectin lyase and polygalacturonase. Pectin

lyase produced by Moniliella showed its optimal activity at 450C temperature and 10 pH,

whereas the same enzyme produced by Penicillium gave its maximal activity at pH 9 and

400C. Polygalacturonase originated from Moniliella showed its activity maximally at pH 4.5

and 550C, while the same enzyme isolated from Penicillium gave its activity optimally at pH

4.5-5 and 400C.

Palaniyappan et al. (2005) indicated that group of hydrolytic enzymes, Pectinases, play an

imperative role in industry of alcoholic beverages and food processing. Comparison of

synthetic pectin with natural substrate for the production of pectinase using Aspergillus niger

was studied. The natural substrate comprise of corn flour and wheat flour. This study was

done in order to suggest a plausible commercially suitable substrate as compared to the

standards. Several factors like pH, fermentation period, substrate concentration, rpm,

temperature and the effect of carbon sources were considered. The experimental work

divulged the maximum production of Pectinases from niger in the presence of wheat along

with the starch as a supplementary carbon source. The optimal conditions were noticed as

wheat (1%), pH 5.5, temperature 300C, time 72 hours, rpm 170 and carbon source (starch)

0.025 %.

11

Phutela et al. (2005) identified 120 various isolates for the production of pectin esterase and

polygalacturonase by the use of thermophilic fungi. Fungus was known and identified as

Aspergillus fumigatus type Fres. MTCC 4163. Optimization was carried out through solid-

state fermentation for the production of enzymes pectinase and polygalacturonase. Highest

production of enzyme was attained in a medium having yeast extract, (NH4)2SO4, carbon

sucrose and wheat bran when cultivated at 50ºC for 2-3 days. Pectinase produced highest

enzyme activity of 1116 U/g and polygalacturonase produced 1270 U/g at pH: 4.0 and 5.0,

respectively.

Phutela et al. (2005). Thermophilic fungus screened primarily from 120 different isolates

produced Pectinases and Polygalacturonase. The isolated fungus was recognized as

Aspergillus fumigates Fres. MTCC 4163. Maximum production of enzymes was obtained by

growing the culture in sucrose, yeast extract, wheat bran, and (NH4)2 SO4. The static

incubation at 500C for 2-3 days was accomplished. Maximum enzyme activities were

obtained i.e. 1270 Ug-1 for polygalacturonase and 1116 Ug-1 for pectinase at pH 5.0 and 4.0,

correspondingly.

Silva et al. (2005) used Penicillium viridicatum RFC3 which was fermented with 1:1 orange

bagasse and wheat bran. Its solid state fermentation produced pectin lyase (PL), endo-

polygalacturonase (endo-PG) and exo-polygalacturonase (exo-PG). The substrate with 70%

and 80% moisture content was prepared and each was cultivated in Polypropylene pack and

Erlenmeyer flask. In contrast to exo-PG and endo-PG, PL production was more in medium of

80 % moisture in Erlenmeyer flask. Moisture had no effect on exo-PG and endo-PG. 41.30 U

mL−1, 8.90 U mL−1 and 0.70 U mL−1 were the highest activities shown by the enzymes PL,

exo-PG and endo-PG, respectively. All the three enzymes were produced more when

polypropylene packs were used instead of Erlenmeyer flask. 70 % moisture was provided in

this case. The maximum production 100 U mL−1 pectin lyase was achieved. Exo-PG and

endo-PG showed 8.33 U mL−1 and 0.7 U mL−1 production, correspondingly. Fall in reducing

sugar content and rise in pH of the medium was observed. PenicilliumviridicatumRFC3 was

capable of producing pectin esterase and depolymerizing enzymes: amylase, CMCase,

protease and xylanase.

Debing et al. (2006) studied the production of pectinase by solid state fermentation using

Aspergillus niger. Initially, the substrate was optimized and then two different temperature

12

treatments were applied in fermentation. First: 30ºC for 30 hours and secondly 23ºC for 42

hours. Maximization was conducted with the conditions that: pH 6.5 and 60% of humidity.

Wheat bran was taken as substrate with dextrose (8%) as carbon source and (NH4)2SO4 as

nitrogen source which gave maximum production of pectinase (36.3 U/g).

Patil et al. (2006) assessed the local available agro-wastes rich in pectin like lemon peel,

sunflower head and sorghum stem as substrates for pectinase production by Aspergillus niger

DMF 27 and Aspergillus niger DMF 45 by using solid state and submerged fermentation. The

greater quantity of exopectinase (17.2 U/g) was obtained from sunflower head followed by

lemon peel: exopectinase activity 10.2 U/g through solid-state fermentation. When additional

carbon and nitrogen sources were added to agro-wastes, production level of pectinase was

enhanced. Glucose proved less effective source in solid state fermentation. Ammonium

sulphate which is used as nitrogen source for pectinase production increased the amount of

enzyme in solid state and submerged fermentation.

Giese et al. (2008) worked on the production of laccase and pectinase by Botryosphaeria

rhodina MAMB-05 in solid state (SSF) and submerged fermentation (SmF). Orange bagasse

contained pulp tissues, seeds and rind. The industrial food waste comprises of orange bagasse

oozed during the processing of orange juice. Botryosphaeria rhodina MAMAB-05 was

grown on essential oils extracted orange bagasse in SSF and SmF. In SSF, maximum enzyme

titre (laccase, 46 U mL-1 and pectinase 32 U mL-1) was acquired without added nutrients. In

SmF, the obtained or extricated orange essential oil was supplemented to nutrient medium

comprising of 1% glucose which resulted in fungal growth inhibition. It led to lower the

enzyme activities. The research revealed the need of removal of essential oils fraction from

citrus waste. After elimination, the citrus waste can be prolifically and successfully used as a

substrate for pectinase production.

Viswanathan et al. (2008) applied six identified strains of the Aspergillus awamorii NCIM

885, filamentous fungi: Aspergillus niger NCIM 548, Aspergillus niger NCIM

616,Aspergillus foetidus NCIM 505,Aspergillus foetidus NCIM 1027, and Aspergillus

foetidus NCIM 510 for comparison for their exopectinase activity by using tamarind kernel

powder as a substrate in the submerged fermentation. Effect of polygalacturonic acid and of

pure pectin was examined on the pectinase production. The carbon sources in the combined

form and as individual forms were used to indicate the effect of carbon sources. The best

13

activity was observed of Aspergillus foetidus NCIM 505 after 72 hours of incubation.

Khairnar et al. (2009) said that fermentation technology has significant importance in the

biotechnology. Biorector is a vessel which is seed of fermentation technology used for the

production of many enzymes, food products, single cell proteins, antibiotic etc. In this

experiment the production of pectinase by submerged fermentation by the application of

different parameters such as various strains, substrates, fermentation duration were studied to

attain the maximum pectinase production by using Aspergillus niger species.

Pedrolli et al. (2009) indicated that pectinases are large number of class of enzymes that

perform catalysis to hydrolyze pectic polysaccharides of tissues of plant into small molecules

such as galacturonic acids. It has been used to enhance the clarity and yields of fruit juices.

As pectic substances are the complex macromolecule therefore, various pectinolytic enzymes

are essentially required for their complete breakdown. These enzymes exhibit differences in

their cleavage mode and are divided into two main groups that act on pectin “smooth” regions

or on pectin “hairy” regions. Pectinases are widely distributed enzymes in bacteria, fungi and

plants. This examination reveals pectinolytic enzymes, microbial pectinase production, their

substrates, characterization and the industrial application of these enzymes.

Ali et al. (2010) experimented on crude pectinases production through submerged

fermentation by using Aspergillus niger. Orange peels rich in proteins, carbohydrates and

pectin were used to induce production of pectinolytic enzymes. Optimization of different

parameters were examined like inoculum size: 9%, substrate quantity and time period: 120 h,

temperature: 30ºC and pH: 5.0. Aspergillus niger produced optimal production after 7 days of

incubation with the1.27 M/ml activity. .

Nitinkumar et al. (2010) isolated microorganisms obtained from the industrial waste for

production of pectinase by using the selective isolation method. Potential culture was then

isolated as Penicillium sp on the basis of morphology. Extracellular pectinase was obtained in

the higher quantity using submerged fermentation. Polygalacturonase was produced at

maximum rate when cultivated in the medium having1.5% pectin, pH: 6.0 at 35˚C after 72

hours incubation. In order to produce enzymes at lesser rate, solid state fermentation was

carried out for substrate optimization. Highest activity of polygalacturonase 64.50 U/g was

achieved when used orange bagasse.

14

Thakur et al. (2010) conducted study for the production of extracellular enzyme

polygalacturonase. Different parameters were used to optimize the production of enzymes.

Highest activity of polygalacturonase was achieved at 30ºC, pH: 4.0 after 48 hours. Pectin

used as carbon source and nitrogen source such as combination of yeast extract and casein

hydrolysate was used. The highest activity of polygalacturonase was attained by using pure

pectin, followed by wheat bran. However, when Glucose was added to the culture medium it

decreased the activity of polygalacturonase. Optimum enzyme activity was observed at 42 ºC

and pH: 5.5. Enzyme showed stability at pH range of 4.5-6.5.

Akhter et al. (2011) obtained seven fungal strains from various sources. Solid state

fermentation was executed. The strain which exhibited its proficiency of producing pectinase

effectively was Aspergillus niger IM-6. Optimum activity of enzyme was found in the

presence of (NH4)2 SO4 (nitrogen source) at 400C. Incubation period was 7 days. Good results

were seen when wheat bran & potato starch were used as a substrate in solid state culture.

Enzyme production was enhanced to 116.57 U/gm by adding 9.68% pectin. Aeration

reflected good results in 750mL flask. Provided 60% moisture showed maximal production of

Pectinases.

Janani et al. (2011) carried out research for the isolation and screening of Pectinases from

agricultural waste dump soils in Tamil Nadu South India. 10 strains from the relevant soil

were taken. Out of them, 3 strains were found positive in Pectinase de-polymerization assay

plates. Extracellular Pectinase was purified by (NH4)2SO4 precipitation and dialysis. 3 strains

produced good Pectinase by submerged and semi-solid fermentation. Maximum production

was achieved using wheat bran substrate. Temperature study revealed its optimum production

at 300C (Asian Journal of Bio-chemical).

Joshi et al. (2011) reported the purification of pectinase originated from apple pomace. They

also determined the enzyme efficacy in fruit juice clarification and extraction. Solid state

fermentation was carried out containing Aspergillus niger as culture and apple pomace as

substrate for pectinase (pectin methyl esterase) production. Enzyme was purified by

(NH4)2SO4 fraction (20-80% concentration). Purified enzyme was stable for 60 days and up

to 50◦C. It completely loses its activity at 90◦C. Optimum activity was observed at pH 3.5.

Moreover, it was analyzed for juice extraction and clarification of apricot, pear, peach and

15

plum juices. Pulps were enzymatically treated and enhanced the juice recover. Apricot juice

retrieval increased from 50 to 80%. Juice recovery of pear, peach and plum increased from 60

to 72%, 38 to 63% and 52 to 78%, respectively. Pectinases increased the titratable acidity,

colour, total sugars and total soluble solids (TSS) in juices extracted enzymatically. It does

not affect the flavor on addition of more enzymes at different concentrations. Desirable

activity was shown by the enzyme and enhanced the quality of assessed fruit juices.

Maciel et al. (2011) carried research work to study the activity of polygalacturonase, pectin

esterase and pectin lyase by Aspergillus niger URM 4645 using forage palm as substrate in

solid state fermentation. The effect of various parameters for the production of pectinases i.e.

temperature, inoculum and substrate amount were also examined. The highest activity of

66.19 U/g for endopolygalacturonase and 40.615 U/g for exopolygalacturonase after

incubation time of 96 and 72 hours respectively were observed but no activity was given for

pectin esterase. Maximum enzyme production was attained by using 10 g of substrate, at

28°C with 5.0 pH. However, Polygalacturonases were found stable at 50-80°C and 3.5-11

pH.

Vasanthi et al. (2011) stated that tons of waste is produced by the citrus fruit processing

farms which causes major disposal issue and eventually causes pollution. In citrus fruit

processing’s, enough portion goes waste such as pulp, peel and seeds. Dried citrus peel

contains the rich quantity of pectin, proteins and carbohydrates; pectin producepectinolytic

enzymes by microbial systems. The sour oranges peel produced crude pectinase using

Aspergillus niger during the solid state fermentation. By finding the optimized conditions i.e.

substrate concentration: 4%, temperature: 30ºC, time/duration: 48 Hrs pH 5,nitrogen sources:

0.3% Ammonium sulfate and moisture holding capacity: 50%.Moreover,polygalacturnose

produced from the isolation of Aspergillus niger was then purified with acetone purification,

dialysis and ammonium sulphate precipitation. The purified enzyme’s protein molecular

weight was determined in the range of 35-60KDa by SDS-PAGE.

Amande et al. (2012) isolated 32 fungi from fungal contaminated fruits from genera of

Mucor, Aspergillus and Fusarium. 9 pectinolytic fungi were isolated and screened using

pectin as a carbon source on the solid agar. The selected isolates which used for the

production of Polygalacturonase by submerged fermentation. The identified separated

isolates were then utilized for the production of polygalacturonase in the submerged

16

fermentation using different substrates. The polygalacturonase production from 0.0212-

5.8850 U/ml in which Aspergillus tamari produced the optimal level of enzymes. It can be

confirmed that selected fungal species isolated from the contaminated fruits can be further

utilized for the production of polygalacturonase.

Bhardwaj et al. (2012) carried out study on 109 bacterial isolates from soil of orchard to

obtain optimized production of pectinase at 45oC and acidic pH. The isolate was identified

culturally, morphologically and biochemically as Bacillus sp. MBRL576 on the basis of

phylogenetic analysis and nucleotide homology. Production of pectinase process conditions

under submerged fermentation for Bacillus sp. MBRL576 was thus optimized. By applying

process of ion exchange chromatography, the enzyme was purified on SDS-PAGE. Crude

enzyme i.e. pectinase was thus utilized for clarification and extraction of juice from vegetable

and fruits.

Heerd et al. (2012) used Aspergillus sojae to maximize the Polygalacturonase production by

solid state fermentation. The effect of different parameters i.e. media composition, inoculums

size, incubation time, temperature were then examined in optimization and screening studies.

Agriculture and agro-industrial waste produced enzymes which is cost-efficient and

sustainable. The production of pectinase of A.sojae ATCC 20235 and A.sojae CBS 100928

was then compared under maximized conditions, it was learned that A.sojae ATCC 20235

produces the pectinase gave approximately 609 times greater polygalacturonase activity.

Furthermore, it produces highest enzyme yield: 909.5 ±2.7 U/g after 8 days at 30°C using the

30% of sugar beet pulp as inducer alongwith the wheat bran as medium wetted at 160% with

0.2 M HCI.

Hitha et al.(2012) carried out a study for screening and isolation of bacterial isolate for the

activity of pectinase. Initially 20 samples were taken from the decay of vegetables and food

and then further screened for activity of pectinase by using solid medium based on the

diameter of clear zone thus produced. Out of 20 isolates, it was then observed that P 1 and P2

showed greater pectinase activity and thus identified by 16S rDNA sequencing as

Streptomyces rochi and Bacillus subtilis. Thereafter, P1 and P2 isolate were cultured at

optimum temperature and pH. Furthermore the enzyme assay was conducted by using the

DNS method. The P1 and P2 isolates containing enzymes were applied on Banana juice and

its microbial cultures were used. Extent of clarification was calculated by measuring

17

absorbance at 660nm using UV is spectrophotometer.

Kumar et al. (2012) obtained a bacterial strain MFW7 isolated from the waste of fruit market.

This strain was investigated for pectinase production. The best pectinolytic strain identified

through preliminary screening was characterized by 16S rDNA sequencing. The recognized

strain was Bacillus sp. MFW7. Optimization parameters were applied by alternating the

physico chemical environment of the producing medium. Optimal pectinase production was

found at 35°C and 6.5 pH. Incubation period was of 72 hours. Effects of several nutrients on

the production of extracellular pectinase were monitored. Substrate taken was cassava waste.

Amalgam of lactose and peptone supported the optimum pectinase production. Molecular

weight of purified pectinase was 37 kDa. Molecular weight was observed via SDS-PAGE.

Latif et al.(2012) isolated 61 bacterial strains from different sources. Initial isolation and

screening for the pectinase production, bacterial strains was conducted by well plate method.

16 bacteria giving zones, 8-22 selected. Further screening was conducted quantitatively

through DNS method for the polygalacturonase production. Z-AT 23,Z-AT 35 and Z-AT 33

strains were considered as Bacillus sp. having maximum amount of enzymes from 1.04 to 2

u/ml. the selected strains were optimized at different temperatures and pH. Bacillus break

down pectin maximum at the pH of 7.0 - 8.0 but Z-AT 35 and Z-AT 33strains produced

maximum enzyme at 42 °C and the Z-AT 23 at 32 °C.

Oyeleke et al. (2012) designed a work for the production of pectinase by Aspergillus niger

from corn cob. Isolation and screening of enzymatic activity was performed by consuming

pectin and carboxylic methyl cellulose as a substrate. Microorganism used subjected to

various maximum conditions such as pH, enzymatic activity, temperature and biomass yield.

Highest activity of pectinase was attained on 4th and the 5th day: 1.5×10-4μg per ml per sec.

Optimum 6.0 pH for the activity was found with the enzyme yield of 1.5×10-4μg per ml per

sec. However, optimum temperature 60ºC was observed having activity of 1.6x10-4μg per ml

per sec.

Patil et al. (2012) said that large quantity of vegetable wastes is produced on the daily basis

and its disposal is also of a great challenge. The degraded vegetable waste can be used for

biosynthetic production of pectinase at cost effective rate, which have diverse industrial

applications. The isolation and screening of vegetable waste was directed to get pectinase

18

producing microorganisms from the carrot. The best isolate was then recognized as a unique

strain of Bacillus sp. It was identified on the basis of its biochemical tests and morphology

and by using 16S rRNA sequencing. This isolate was produced in the bulk quantity using

optimal conditions of pH: 9 and at 50oC temperature to obtain optimal quantity of production

i.e. 49.58% from carrot waste.

Qureshi et al. (2012) conducted experimental studies directed to produce pectinases using the

batch fermentations by using isolated Bacillus subtilis EFRL 01 on an inexpensive medium

like waste date syrup by submerged fermentation. Carbon source was then added to increase

the activity and yeast extract as a nitrogen source which was found to be very effective.

Fermentation duration batch of 48 h having pH of 8.0 gave the pectinase activity of 27000 u

per ml at 45°C. The optimum temperature, nitrogen source, pH and carbon source with their

quantities were calculated after using multiple nutrient sources and conditions for

fermentation reactions.

Tariq et al. (2012) conducted the research on isolation of bacteria producing

Polygalacturonases. The bio-chemical characterization of isolated bacteria was also carried

out. 61 strains of bacteria were isolated from numerous sources like water, soil, rotten

vegetables and fruits. Well plate method was used for initial screening. 16 bacteria formed

zones of 08 to 22 mm. These 16 bacteria were elected. Bio-chemical, cellular and

morphological characterization of chosen bacteria was also done. Selected strains were

further screened quantitatively by DNS (3,5dinitrosalicylic acid) method for polygalcturonase

production. Enzymes were produced more ranging from 1.04 to 2 U mL-1 by the following

strains: Z-AT23, Z-AT33 and Z-AT35 of Bacillus. Bacillus strains were examined at different

pH and temperatures for enzyme production. pH 7 to 8 for all Bacilli proved to be suitable for

hydrolyzing more pectin. Strain Z-AT23 produced greater quantity of enzyme at 320C.

Strains Z-AT33 and Z-AT35 had shown maximum amount of enzyme production at 420C.

19

Pasha et al. (2013) indicated that pectinases is enzymes used in industries specifically in

fruits and textile industries. Pectinases catalyzes complex polysaccharides of the plant tissues

into the smaller molecules such as galacturonic acids. The Pectinases are the mostly used

enzymes successfully in bringing down of cloudiness and bitterness of the fruit juices.

Whereas, the alkaline Pectinases have shown good results for the degumming and retting of

fiber crops, fermentation of coffee and tea, production of good quality paper, treatment of

pectic waste water and oil extractions.

Raju et al. (2013) carried out a work for the screening and isolation of pectinase producing

bacterial strains from ten samples which were obtained from dump yards of vegetable wastes

from different areas of Banglore. PSAM (Pectinase screening agar medium) helped in sorting

out or separating pectinase producing bacterial strains. Several regions of Banglore were

considered to find out the efficient bacteria for pectinase production. Screening of numerous

isolates was undertaken for owning the capability of pectinase production. 6 isolates were

found proficient. Biochemical tests were performed for the recognition of nominated isolates.

The organisms were identified as Staphyloccus aureus, Bacillus licheniformis and

Bacilluscerus.

Sandhya et al. (2013) separated fungal strains from spoiled fruits, vegetables and soil.

Initially 13 strains were separated from which Penicilluim citrinum attained from rotten

tomatoes proved to be an effective and potent producer of Pectinase. Pectinases were

cultivated using two fermentation techniques i.e. solid state and submerged. Effects of the

following: temperature, pH, nitrogen source, carbon source and salt were also analyzed. The

maximum production of pectinolytic enzymes was observed through solid state fermentation

at 300C and these enzymes were having constitutive nature.

Varghese et al. (2013) said that Pectinase are the group of enzymes that hydrolyze pectin

substances found in plant material and are observed cosmopolitan in distribution. This study

was conducted to isolate, characterize and screen pectinolytic microorganisms taken from

different soil samples of Indian city Chhattisgarh, Raipur. After initial isolation and

screening, sixteen strains then examined for pectin hydrolysis. Out of which, 13 were found

worthy in the production of pectinase enzymes. It was observed that out of them,

Streptococcus sp (SB6) produced maximum zone of clearance with greater activity of

20

enzymes pectin.

Akhtar et al. (2014) carried a research to differentiate the strains of Aspergillus niger

isolated from vegetables and fruits. Physiological, morphological and molecular approaches

played the role in distinguishing the strains. Macro and micro-morphological characters of A.

niger isolates were found to be the same. This research also revealed the closeness of strains

on the basis of rDNA spacer sequence. It was discovered that the production and activity of

different enzymes including pectinase, urease, catalase and lipase varied greatly even when

the different isolates were morphologically and genetically similar.

Khayati et al. (2014) conducted the research to study the effect of citrus pulp on pectinase

production. It has numerous applications in food processing industries. An appealing and

fascinating technology for enzyme production is solid state fermentation (SSF). Five factors

influencing the production of pectinase using Aspergillus niger in SSF were analyzed. The

factors (C/N ratio, initial pH of the medium, concentration of citrus pulp, type of solid

substrate and citrus pulp) were evaluated by Taguchi orthogonal array (OA). Type of solid

substrate, concentration and type of citrus pulp were found to be the extremely effective

factors for raising the enzyme production. In contrast to basal medium, the medium

supplemented with citrus pulp increased the pectinase production by 23%. The most

appropriate and suitable method for optimizing the experiments involved in pectinase

production (R 2= 0.946) is Taguchi's method.

Rao et al. (2014) pointed out horticulture wastes and agriculture residues could be taken as

substrates in SSF to produce pectinase enzyme using Aspergillus niger NCIM 548. For this

purpose, 16 substrates were screened. Out of these 16 substrates, Jack fruit waste proved to be

the best. The highest yield of pectinase 39.836U/gds was achieved using 10 g of Jack fruit

waste as a substrate. The conditions for optimum yield were the following: moisture content

70% v/w, (NH4)2 SO4 1.0% w/w, glucose 3.5% w/w, particle size 152-354 µm, temperature

30oC, pH 5.0 and fermentation time 72 h.

Thangratham et al. (2014) studied the pectinase production and optimization through fungal

species which were isolated and identified from agro waste dumped soil. Isolated fungal

strains namely R.oryzae, A. flavus and A. oryzae were screened for pectinase production

21

through submerged and solid state Fermentation. Out of three selected fungal species A.

flavus produced maximum amount of pectinase and it showed this activity in Pine apple (0.79

IU/ml) by using Solid State Fermentation (15.55 U/ml). Optimal production of pectinase then

observed with 72 hrs of incubation, 5.5pH at 35C. It is thus concluded that by utilizing

agricultural waste provides cost effective method for production of pectinase at large scale.

Tripathi et al. (2014) conducting the experiment with intention to segregate the Pectinase

producing Bacillus subtilis from soil. Chief purpose was to produce and purify the enzymes

pectinases. Pectinases play significant role in many industrial processes. This group of

enzymes is not only industrially important, but also has commercial significance. Several

screening processes were undertaken for isolated bacteria. Bacteria showed positive result for

Pectinases screening test. The positive result discloses the Pectinases production from

bacteria. The produced enzyme was purified by ammonium sulphate precipitation. It was then

followed by dialysis and ion exchange chromatography.

Yang et al. (2014) took orange peels directly into 7 liter fermentor to produce L-lactic acid in

the mixed culture system. The production of pectinase and cellulose was subsequently

increased by using Aspergillus niger GH-06 by inoculation of Lactobacillus casei G-02 at 16

h of culture by adding phytic acid at 3 g/l, which activities calculation as 108.6 and 17.8 U/ml

in 72 h, respectively, more than 2-folds higher as compare to original culture with single

strain. Subsequently, fermentation and saccharification, the L-lactic acid concentration of

116.3 g/l was achieved from orange peels with the time frame of 36 h with the high

conversion efficiency of 90.3%.

Arifa et al. (2015) stated that due to bio-degradability, polysaccharide pectin (methylated

ester of polygalacturonic acid) has great significance in commercial and scientific world.

Wide group of pectinases break down pectin polysaccharides of fruits and plants, thus used

for clarifying the fruit juices in industry. In industrial sector, pectinases are also used for

increasing yield of fruit juices. Dilutions (10-4 and 10-6) of rotten oranges were used to isolate

bacterial strains. Two strains were isolated. The separated organisms were recognized by

biochemical and staining tests. Pectin containing minimal essential medium was used to

determine the pectinolytic activity. Kirby Bauer agar well diffusion method was directed at

35 ± 20C. Two isolated strains L and M of bacteria were Staphylococcus aureus and Bacillus

22

cereus, respectively. Strains were identified on the basis of spore staining, gram staining and

biochemical tests. Both strains showed different pectinolytic zones depending on the

concentration of inoculum. The largest zone formed by both the strains was of 25 mm.

Pectinases produced by Staphylococcus aureus and Bacillus cereus can be commercially used

to enhance the quality of fruit juices.

Dhembare et al. (2015) checked usefulness of influence of temperature, pH and kinetics of

pectinase enzyme in foods Aspergillus niger along with the substrate soil was cultivated by

SSF. Aspergillus niger was subjected to varied conditions (temperature, pH and kinetics of

pectinase enzyme). At pH 4, maximal activity of pectinase 340.56 µg /mL/sec was observed.

Incubation time was 96 hours. Moreover, the highest pectinase activity found at 400C and the

same incubation time was 169.31 µg /mL/sec. Km and Vmax calculated were 2.5 µm and 0.020

µm/min/mg, correspondingly. Aspergillus niger extricated from soil can be a source of

industrial pectinase.

Hachemi et al. (2015) analyze the effect of cultivation factors i.e. ammonium sulfate, glucose

and water in the culture medium and the of size of dry orange waste for their bioconversion

for the production of pectinases. Polygalacturonase (PG) separated by using ion exchange

chromatography with the gradient elution 0-0,5 m/l NaCl, thereafter by sephadex-G75

column chromatography was then applied and the 51,28 KDa molecular weight was achieved.

Purified PG enzyme with pH: 5 and at35°C showed the maximum activity. Apple juice was

then treated with purified enzyme which produced clear juice, which was competitive with

the juice produced by Sigma Aldrich Aspergillus niger enzyme.

Lian et al. (2015) said that polygalacturonases are type or kind of pectinases which are used

to breakdown Alpha-1,4 glycocidics which is found between galactronic acid residues.

Polygalacturonase are widely used in the food bio-fuel and other industries related to textile

wherein thermo stable polygalacturonase are demanded at high temperature of 50-60 °C.

During the experimental studies a thermo stable polygalacturonase was separated from the

pile fermentation of Pu’er tea in China from Aspergillus fumigates.

Meena and Pawar, (2015) stated that food grade pectinases produced by the Aspergillus niger

are richly used in industrial applications. The enzyme kinetics was detected in order to

23

maximize the concentration of substrate and processing parameters with the reference to the

activity of polygalacturonic acid. Km is then measured by knowing the reaction velocities on

standardized temperature and pH range on basis of polygalacturonase activity. At 2.43mg per

mL, Km the enzyme activity found optimal at 40°C and beyond this, the activity was greatly

affected negatively coupled with the gradual reaction. The maximum activity of

polygalacturonase found at 5.78 μmol per ml per min at pH 4.5 having 9 mg per ml substrate

concentration of the activity of enzyme.

Sabah et al. (2015) recorded that experiments conducted for Pectinases production and their

use in home and commercial applications for the production of juices and wines in the early

1930s.The scientists worked hard in the1960s to observe the chemical nature of plant tissues

and frequently identified the extraction and purification of large number of enzymes and their

applications in various industrial applications in a more bifacial and efficient manners. As a

result of endless efforts, Pectinases enzymes as today are the up-coming enzymes in

commercial sector. The study was conducted to check pectinase activity of the pectinolytic

fungi and bacteria by using fruit peel waste as a substrate for Pectinase production by

different organisms in sub merged state fermentation as it is the cost effective method.

Phanerochete chrysosporium and Aspergillus niger produce Pectinases which induce

conversion of pectin into various oligosaccharides. It is important to select a potent microbial

strain for the production of Pectinases to identify the waste to enhance the production of

Pectinases. Mostly, EMS methods and random mutagenesis are used to obtain enhanced

production of enzyme. The Aspergillus niger which is selected from Citrus peel showed

highest Pectinase activity through mutagenesis study. The culture was then exposed to

200mM EMS at different time intervals i.e. 5 min, 10 min and 15 min. It has been indicated

that the plate exposed for 5 min showed the highest Pectinase activity as compared to the

non-mutated that grow on citrus peel. It has further been observed that PC mutated for 10 min

shown increased enzyme yields and activity. Similarly, Aspergillus niger exposed for 5 min

had shown an increase in pectinolytic enzyme yield and activity. The genes mutated in both

the fungi required different times of exposure to 200 milli-molar EMS to obtain maximum

production of enzyme. Pectins are high molecular weight heterogeneous and acidic structural

polysaccharides which are the major cell wall constituent’s of cereals, vegetables, fruits and

fibers. The principal component of pectin backbone constitute neutral sugars and acids i.e.

galactose, xylose and arabinose present in side chains, whereas rhamnose comprised a minor

24

component of pectin.

Ali et al. (2016) indicated the various types of pectinases and their industrial applications on

commercial basis. Pectinase breakdowns bigger plant’s pectin polysaccharides into small

molecules like galacturonic acids. These are categorized as esterases, protopectinases and

depolymerases due to their catalytic activity on pectin and its derivatives aroused in industrial

applications. Commercially, they are used in purification of fruit juices. It is used for

clarification and extraction of juices by removing bitterness and cloudiness. Pectinases have

wide range of applications, especially in textile retting, tea & coffee fermentation fiber crops

degumming, extraction of oil, production of good quality paper, pectic waste water treatment.

25

3. Materials and Methods

The chemicals used in the experimental work were purchased from scientific companies like

Sigma, Fisher, Merck, and Acros.

3.1. Soil sampling and isolation of fungi

In order to isolate desired fungi i.e. Aspergillus niger, different soil samples were collected

from various locations of Lahore and transported to the laboratory in sterile polythene bags.

The soil samples were serially diluted up to 10-9 and grown on potato dextrose agar. The

inoculated plates were incubated at 30ºC for 96 h. The cultures obtained were maintained on

potato dextrose agar slants at 4ºC in cold cabinet (Model: MPR-1410, Sanyo, Japan) and were

sub-cultured after 15 day intervals regularly.

3.2. Screening

The isolated fungal cultures were screened in a medium containing K2HPO4 (4.0 g),

(NH4)2SO4 (2.0 g), yeast extract (0.6 g), KH2PO4 (1.28 g), MgSO4 (1.1 g) per liter,

supplemented with 1% citrus pectin to obtain the production of pectinases. Crude enzyme

was used to estimate the pectinase activity (Okafor et al. 2010).

3.3. Morphological Identification

Preliminary identification of isolates was carried out on the basis of hyphal and spore

morphology (Onions et al. 1986).

3.4. Mutagenesis

3.4.1. UV Radiations

Hundred µl spore suspension (107spores ml-1) of A. niger was transferred aseptically to malt

agar plates placed under UV lamp of 20 W (Gromada and Fiedurek, 1996). UV light of

1.2×106 J/m2/s intensity was exposed to the spore suspension. Different time duration

exposures were provided to the suspension solution during 30, 60, 90, 120, 150 and 180

minutes (Azin and Noroozi, 2001).

26

3.4.2. EMS Mutation

Forty eight hours old A. niger culture was obtained and spore suspension was developed in 10

ml sterilized distilled water. 250 µL diluted suspension (10-4) was shifted to three different

eppendorf tubes along with 50 µL of 1% EMS. Samples were withdrawn at an interval of 5,

10 and 15 minutes and100 µL of each sample was transferred to malt agar plate and spread

uniformly (Lotfy et al. 2006).

3.4.3. Nitrous Acid Mutation

One ml spore suspension of A. niger of 48 hours old culture was transferred to eppendorf

tubes and centrifuged at 10,000 rpm using refrigerated bench top centrifuge (Model: 18/80

MSE, Harrier, UK) to get pellets of spores. Pellets were mixed with 1 mL of 0.1 M sodium

acetate buffer and treated with NaNO2 having 0.1 molar concentration for 5, 10 and 15 min.

Vigorous shaking of the mixture was carried out and treatment was stopped by the addition of

0.1 M phosphate buffer. Treated spore suspensions were used to inoculate 0.1 ml on the PDA

plates (Mala et al. 2001).

3.5. Submerged Fermentation

3.5.1. Vegetative inoculum

Fungal slants which are 48 h old are used to form spore suspension by adding 10 ml of

distilled water and subsequent scratching aseptically with inoculating needle. Vegetative

inoculums was produced by inoculating 25 ml of nutrient broth with 1 ml spore suspension

and incubated for 24 h at 30ºC and shaking speed of 200 rpm.

3.5.2. Fermentation batch

Submerged fermentation was used to obtain production of pectinase in 250 ml Erlenmeyer

flasks having 25 ml fermentation medium. The medium was sterilized at 121ºC, 15 psi for 15

min. 1 ml vegetative inoculum was added aseptically in fermentation medium. Flasks were

incubated in shaking incubator (Model: 10X400.XX2.C, SANYO Gallenkamp PLC, UK) at

200 rpm for 96 hours at 30ºC.

3.5.3. Extraction

Enzyme was separated by centrifugation of fermentation medium for 10 mins using

27

centrifuge speed of 6000 rpm (Model: D-37520, Osterodeam-Harz, Germany). Supernatant

was further used for estimation of enzyme activity.

3.6. Analytical Analysis

3.6.1 Pectinase Assay

3.6.1.1. Preparation of 0.1 M Sodium acetate buffer

a) One liter sodium acetate solution was prepared by dissolving 27.22 g in 300 ml

distilled water and thereafter the volume was raised up to 1.0 L.

b) One liter solution of acetic acid was prepared by dissolving 12.05 ml of glacial acetic

acid initially in 400 ml of distilled water and then the volume was finally raised up to 1.0 L.

48 ml solution (a) is mixed with 2 ml solution (b) to obtain 50 ml sodium acetate buffer

having pH 5.5.

3.6.1.2. D-Galacturonic acid Standard curve

A standard curve of D-galacturonic acid was prepared for estimation of galcturonic acid

released by action of pectinase on pectin. Stock solution was prepared by dissolving 0.1 g of

D- Galacturonic acid in 20 ml of distilled water and then the volume was raised up to 100 ml.

Ten dilutions (0.0-1.0 mg/ml) of D-galacturonic acid in buffer (0.1M sodium acetate, pH: 5.5)

were prepared from stock solution. Then 3 ml DNS reagent was mixed with one ml sample

from each dilution test tube and put in boiling water for 5 minutes. Thereafter, the test tubes

were allowed to cool and 7 ml distilled water was added and absorbance at 550 nm on

spectrophotometer was observed after setting zero with the blank. A graph was prepared

between absorbance and D-galacturonic acid concentration (Annexure I). Slope of curve was

noted and used to estimate the activity of pectinase.

3.6.1.3. Preparation of Bradford reagent

100 mg coomassie brilliant blue (G250) was dissolved in 100 ml ethanol (90% v/v).

Subsequently, the volume of solution was raised to one liter (1000 ml) after addition of 100

ml of H3PO4 (85% v/v). The solution was filtered by using Whattman filter paper No. 1 and

stored in amber colored bottle. The standard curve of BSA was plotted to determine the

28

concentration of proteins (Annexure II).

3.6.1.4. Pectinase Bioassay:

Enzyme activity of Pectinase was estimated using the method devised by Okafor et al. 2010.

Enzyme assay was carried out using 500 µl substrate (1% pectin) and 500 µl crude enzyme

for a reaction period of 10 min at 50°C. Product formation was assessed indirectly by using

DNS and color change was analyzed using spectrophotometer (CECIL CE-7200 Series, UK).

One unit of Pectinase is defined as action of 1 ml of enzyme to liberate one microgram (1 μg)

galactouronic acid per minute.

3.6.1.5. Protein Assay

The protein assay was conducted employing Bradford Reagent after the method put forward

by Bradford, 1976.

3.7. Cultural parameters optimization

Different physical parameters such as temperature, pH, incubation time and nutritional

parameters i.e. type of medium, carbon and nitrogen sources were optimized to get maximum

production of pectinase.

3.7.1. Optimization of Incubation time

Submerged fermentation was carried out for a time period of 96 hours with an equal interval

of 12 hrs i.e. 12, 24, 36, 48, 60, 72, 84 and 96 hours. Pectinase activity was analyzed after

each interval to determine the best incubation time for pectinase production using both wild

and mutant strains.

3.7.2. Optimization of Incubation temperature

Different temperatures (25, 30, 35 and 40°C) were used to perform the submerged

fermentation using both the wild and mutant strains for pectinase production.

3.7.3. Optimization of Incubation pH

pH range of 6-8 with an interval of 0.5 were used to find out the best pH for pectinase

production.

29

3.7.4. Optimization of fermentation medium

For the fermentation of pectinases, the following media were used for the purpose

3.7.4.1 Medium M-I

KH2PO4 (2.0), K2SO4 (2.0 g), (NH4)2SO4 (2.0 g) and citrus pectin (10.0 g) were mixed in 500

ml of distilled water and then the volume was raised to one liter (1000 ml). The pH was

adjusted to 5.5 with 0.1 N NaOH (Vishwanathan and Babu 2008).

3.7.4.2. Medium M II

K2HPO4 (6.0 g), KH2PO4 (6.0 g), MgSO4.7H20 (1.0 g), (NH4)2SO4 (6.0 g) and pectin (10.0 g)

were mixed in 250 ml distilled water and the volume was then raised to 1.0 L. The pH of the

solution was adjusted to 5.5 with 0.1 N NaOH (Okafor et al. 2010).

3.7.4.3. Medium M-III

K2HPO4 (0.5 g), MgSO4.7H2O (0.5 g), yeast extract (20.0 g), and pectin (10.0 g) were mixed

in 500 ml distilled water and then the volume was raised to 1.0 L. The pH of the solution was

adjusted to 5.5 with 0.1 N NaOH (Joshi et al. 2011).

3.7.4.4. Medium M-IV

K2HPO4 (4.0 g), yeast extract (0.6 g), KH2PO4 (1.28 g), (NH4)2SO4 (2.0 g), MgSO4 (1.1 g)

and pectin (10 g) were mixed in 500 ml of distilled water and then the volume was raised 500

µl to 1.0 L. The pH of the solution was adjusted to 5.5 with 0.1 N NaOH (Mrudulla and

Anithraj, 2011).

3.7.4.5. Medium M-V

FeSO4.7H20 (0.0005 g), (NH4)2SO4 (0.1 g), MgSO4.7H2O (0.5 g), KH2PO4 (0.5 g), and pectin

(10.0 g) were mixed in 500 ml of distilled water and then the volume was raised to 1.0 L. The

pH of the solution was adjusted to 5.5 with 0.1 N NaOH (Patil and Dayanand, 2006).

3.8. Optimization of Carbon Source and concentration

3.8.1. Fruit Peels Collection & Preparation

Orange and pineapple fruit peels were taken from various local fruit markets of Lahore. The

30

fruit peels were washed thoroughly and peeled into clean trays with sterilized and clean sharp

knife. Peels were dried in hot air oven at 50ºC until constant weight was achieved (Rangrajan

et al. 2010). Thereafter, the dried pieces were grinded into powder and were sealed in

polyethylene bags for further use. Different carbon sources such as maltose, pineapple,

orange, pectin from citrus, sucrose and glucose were also tested for enhanced production of

pectinase.

3.8.2. Optimization of Nitrogen Source and concentration

Nitrogen sources such as ammonium chloride, corn steep liquor, urea, yeast extract and

peptone were used to determine the best pectinase production.

3.9. Upscale processing

Up scaling of fermentation process was carried out in a stirred fermenter of 7.5 liters (Model:

BF-110 BioFlo/ CelliGen by New Brunswick). The parameters such as incubation time under

controlled conditions, agitation rate, and aeration rate and inoculums size were studied.

Fermentation kinetics was also applied to analyze the validity of results. The following

parameters of kinetics were calculated as described by Pirt (1975) and Lawford and Rousseau

(1993):

1. Maximum specific growth rate (μ max) per hour, which was calculated from plot

of natural log of cell mass (ln x) vs. fermentation time.

2. Product yield coefficient (Y p/x) in U/mL/mg, which was determined by the

equation:

Y p/x = dp/dx (1)

3. Specific product yield coefficient (qp) U/mL/h, which was determined by the

equation:

qp = Y p/x .μ max (2)

3.10. Ammonium sulphate precipitation

The amount of the ammonium sulphate required for precipitation was calculated (Table 1).

Ammonium sulphate precipitation was done after Saxena et. al., (2003) in a range of 10-90%

salt concentration. Desired fraction was further purified to remove excessive amount of slats

by dialysis using membrane having 12000-14000 molecular cut off value in a 0.1 M

phosphate buffer.

31

Table 1: Chart of Ammonium Sulphate Precipitation

32

3.11. Ion Exchange Chromatography

3.11.1. Reagents Used

a) Binding Buffer (A) (0.1 M Phosphate buffer, pH 6.0)

Solution A: KH2PO4 solution (1 M) was prepared by dissolving 13.6 g KH2PO4 in distilled

water to attain the final volume of 100 mL.

Solution B: 0.1 M solution of K2HPO4 was prepared by dissolving 17.4 g of the salt in

distilled water by raising the final volume to 100 mL.

68.8 ml of solution A was mixed with 32.2 mL of solution B and the final volume of the

buffer was raised to one liter (1000 mL).

b) Elution Buffer (B) (0.1 M NaCl in buffer A)

5.85 g of NaCl was dissolved in 1000 ml of the buffer A and was mixed homogeneously.

Then buffer was filtered through Whatman filter paper no. 1 before the use.

3.12. Chromatography Method

Pectinase was purified by using ion exchange chromatography. Bound and unbound fractions

obtained after chromatography were assessed for enzyme activity. Pectinase containing

fraction was further analyzed for protein purity using SDS PAGE after Saxena et. al. (2003).

33

3.13. Characterization of Enzyme activity

3.13.1. Effect of temperature on pectinase activity and stability

Activity assay of the purified enzyme was conducted at various temperatures (15-35°C).

Stability of enzyme was examined at various temperatures (20, 30, 40, 50 and 60°C) for

various incubation duration (30-120 mins).

3.13.2. Effect of pH on pectinase activity and stability

Phosphate buffer of various pH (5.5-8) was used to analyze the activity of enzyme. Phosphate

buffers of different pH (5.5, 6, 6.5 and 7) were used to analyze the effect on stability of

pectinase for 24-72 hrs.

3.13.3. Effect of metal ions on pectinase activity and stability

1% solutions of metal ions i.e. mercury sulfate, cobalt chloride, manganese chloride, Calcium

Chloride, Lead Nitrate, Magnesium Chloride, Cadmium Nitrate, Copper Sulfate and Nickel

Chloride were prepared and mixed with enzyme to analyze the effect on activity. Incubation

of metal ions with enzyme was also carried out for 72 hours to check the stability profile of

pectiase with metal ions.

3.13.4. Effect of surfactants on enzyme activity

EDTA, Tween 80 and SDS in a concentration of 1% were incubated with enzyme to check

the effect of surfactants on enzyme activity.

3.14. Kinetic Analysis

Kinetic parameters i.e. Km and Vmax were calculated by using the Line weaver-Burk double

reciprocal plot after analyzing the enzyme activity for substrate concentration ranging from

10-80 mM (Line weaver and Burk, 1934).

3.15. Thermodynamic Studies

The thermodynamic parameters, such as activation energy (Ea), change in entropy (ΔS) and

change in enthalpy (ΔH), were examined as by Siddiqui et. al. (1997)

3.16. Statistical Analysis

The Computer statistical software Costat, cs6204W.exe was used for statistical analysis

34

(Snedecor and Cochrane, 1980). Substantial difference among the replicates has been

presented as Duncan’s multiple range tests in the form of probability (p) values.

35

4. Results

Isolation and screening of pectinolytic fungi

Two hundred and nine different fungal strains were isolated from two hundred and

fifty soil samples that were collected from different areas of Lahore. Primary screening

of fungal isolates was based upon ability to form pectin lysis zones. The results showed

that 151 fungal isolates were pectinolytic. The screened pectinolytic strains were

subjected to secondary screening by submerged fermentation. It was observed that

strain IIB-13 produced maximum pectinase activity (10.78±0.45U/ml/min) as shown in

Table 1. Therefore, this strain (IIB-13) was selected for further studies.

Table 2: Secondary screening of pectnolytic strains of Aspergillus niger.

Sr. No. Strain Activity (U/ml/min)

1 IIB-1 1.28±0.15

2 IIB-3 7.78±0.05

3 IIB-7 9.78±0.01

4 IIB-8 4.34±0.21

5 IIB-9 2.28±0.25

6 IIB-11 1.74±0.10

7 IIB-12 6.70±0.08

8 IIB-13 10.78±0.45

9 IIB-14 6.44±0.14

10 IIB-17 9.21±0.16

11 IIB-18 5.09±0.24

12 IIB-19 1.25±0.02

13 IIB-20 1.28±0.45

14 IIB-21 4.48±0.11

15 IIB-22 3.22±0.02

16 IIB-23 7.19±0.16

17 IIB-24 2.38±0.02

18 IIB-25 7.98±0.05

19 IIB-27 8.49±0.09

20 IIB-29 2.64±0.04

21 IIB-30 4.51±0.19

22 IIB-31 1.29±0.10

36

23 IIB-32 5.03±0.15

24 IIB-33 6.11±0.02

25 IIB-37 9.37±0.08

26 IIB-38 8.44±0.17

27 IIB-39 0.89±0.04

28 IIB-40 1.55±0.31

29 IIB-42 6.79±0.28

30 IIB-43 6.34±0.11

31 IIB-44 7.28±0.08

32 IIB-45 1.08±0.01

33 IIB-46 2.52±0.11

34 IIB-47 3.64±0.15

35 IIB-48 4.23±0.21

36 IIB-49 7.29±0.24

37 IIB-50 8.09±0.25

38 IIB-51 9.26±0.19

39 IIB-52 9.51±0.17

40 IIB-53 3.97±0.14

41 IIB-55 7.28±0.17

42 IIB-56 2.57±0.24

43 IIB-57 1.28±0.07

44 IIB-58 2.59±0.02

45 IIB-59 4.56±0.12

46 IIB-60 3.89±0.28

47 IIB-61 6.27±0.31

48 IIB-62 7.89±0.16

49 IIB-63 5.76±0.20

50 IIB-64 2.09±0.15

51 IIB-65 8.46±0.08

52 IIB-66 2.59±0.10

53 IIB-67 4.29±0.05

54 IIB-68 7.19±0.11

55 IIB-71 8.09±0.02

56 IIB-75 4.57±0.41

37

57 IIB-76 6.46±0.21

58 IIB-77 5.12±0.04

59 IIB-78 7.12±0.0.38

60 IIB-79 4.40±0.15

61 IIB-80 2.36±0.10

62 IIB-81 5.98±0.08

63 IIB-82 1.26±0.31

64 IIB-83 7.92±0.39

65 IIB-84 8.01±0.43

66 IIB-85 4.29±0.26

67 IIB-86 1.03±0.17

68 IIB-87 6.79±0.19

69 IIB-88 9.98±0.11

70 IIB-89 4.09±0.09

71 IIB-92 2.36±0.35

72 IIB-97 1.78±0.15

73 IIB-98 3.77±0.10

74 IIB-99 3.08±0.25

75 IIB-100 2.99±0.31

76 IIB-101 4.19±0.28

77 IIB-102 5.01±0.29

78 IIB-103 7.49±0.06

79 IIB-104 3.81±0.02

80 IIB-105 1.19±0.10

81 IIB-106 0.71±0.11

82 IIB-107 2.70±0.30

83 IIB-108 3.56±0.36

84 IIB-109 2.12±0.08

85 IIB-110 4.17±0.31

86 IIB-113 7.09±0.45

87 IIB-114 8.15±0.29

88 IIB-115 6.49±0.17

89 IIB-116 7.08±0.07

90 IIB-117 1.07±0.36

38

91 IIB-118 4.12±0.13

92 IIB-119 0.25±0.07

93 IIB-120 4.08±0.19

94 IIB-121 8.36±0.14

95 IIB-122 6.09±0.26

96 IIB-123 7.29±0.31

97 IIB-125 4.11±0.28

98 IIB-126 3.97±0.29

99 IIB-127 5.78±0.34

100 IIB-129 4.89±0.11

101 IIB-131 0.78±0.08

102 IIB-132 5.49±0.01

103 IIB-133 6.79±0.23

104 IIB-134 7.19±0.26

105 IIB-136 5.19±0.38

106 IIB-137 6.03±0.41

107 IIB-138 7.08±0.02

108 IIB-139 4.57±0.44

109 IIB-140 6.19±0.09

110 IIB-141 1.59±0.16

111 IIB-142 3.15±0.27

112 IIB-143 7.85±0.20

113 IIB-144 4.72±0.15

114 IIB-145 0.77±0.10

115 IIB-146 1.43±0.05

116 IIB-147 1.05±0.09

117 IIB-148 1.76±0.01

118 IIB-149 2.07±0.08

119 IIB-150 6.12±0.06

120 IIB-152 9.10±0.16

121 IIB-153 7.49±0.17

122 IIB-154 5.78±0.12

123 IIB-155 6.18±0.39

124 IIB-156 4.28±0.12

39

125 IIB-157 5.16±0.01

126 IIB-159 3.79±0.14

127 IIB-161 7.49±0.18

128 IIB-165 7.12±0.09

129 IIB-166 0.64±0.21

130 IIB-167 9.05±0.11

131 IIB-168 2.12±0.08

132 IIB-169 6.42±0.41

133 IIB-170 1.09±0.34

134 IIB-172 5.24±0.21

135 IIB-175 8.67±0.28

136 IIB-176 3.01±0.21

137 IIB-177 3.78±0.39

138 IIB-178 5.12±0.44

139 IIB-179 6.10±0.02

140 IIB-180 5.28±0.05

141 IIB-186 5.86±0.02

142 IIB-187 4.31±0.21

143 IIB-188 1.15±0.10

144 IIB-189 9.03±0.15

145 IIB-190 7.64±0.11

146 IIB-196 3.71±0.08

147 IIB-201 5.39±0.01

148 IIB-205 1.13±0.04

149 IIB-211 8.51±0.02

150 IIB-212 9.03±0.15

151 IIB-215 7.53±0.10

40

Morphological Identification

The strain IIB-13 was subjected to morphological identification and was identified as

Aspergillus niger (Onions et. al., 1986). The strain showed white colonies initially

which later turned into black spores with septate and hyaline hyphae. Conidiophores

were found to be 949-1060 μm in length by micrometry. They were hyaline, finely

roughened, smooth-walled and had conical terminal vesicles (42 μm in diameter),

which supported a single row of phialides with 14-16 μm in length.

Figure 1: Microscopic image of Aspergillus niger (40X)

Mutagenesis UV

irradiations

UV irradiation was used in an attempt to obtain mutant strains with improved

pectinase yield. For this purpose, IIB-13 wild strain which showed maximum activity

during secondary screening was subjected to UV light treatment. After treatment, 27

mutant variants were obtained. Pectinolytic activity was measured for all strains. It

was observed that out of these variants, mutant strain U-21 exhibited maximum

41

pectinase activity (12.29±0.05 U/ml/min) among mutant strains as shown in figure 2.

Figure 2: Pectinolytic activity of Aspergillus niger strain IIB-13 variants after UV

rays (1.2×106

J/m2/s) treatment for 90 mins

EMS Treatment

Like physical treatment (UV light treatment), chemical treatment that includes 1 %

EMS treatment was also applied to wild strain IIB-13. A total of 36 mutant strains

were obtained but none of them yielded high pectinase activity as compared to wild

strain IIB-13 (Figure 3).

42

Figure 3: Pectinolytic ability of variants of Aspergillus niger strain IIB-13 after EMS

(1%) treatment for 10 mins

Nitrous Acid Treatment

Similar to EMS, the wild strain IIB-13 was also treated with 0.1 M concentration of

nitrous acid. After nitrous acid treatment, 39 mutant variants were obtained. Out of

these 39 isolates, H-97 gave the maximum yield (32.16 + 0.15 U/ml/min). The

pectinase yield produced by H-97 was twofold higher than the wild strain IIB-13

(Figure 4). Therefore, this strain was selected for further study.

12

10

8

6

4

2

0

WildE-29 E-31 E-33 E-35 E-37 E-39 E-41 E-43 E-45 E-47 E-49 E-51 E-53 E-54 E-56 E-58 E-60 E-62

EMS Variants

Enzym

e A

ctiv

ity (U

/ml)

43

Figure 4: Pectinase production of nitrous acid (0.1M) treated mutants for 5 min of

Aspergillus niger strain IIB-13.

Optimization of fermentation parameters

Effect of Incubation Time

Submerged fermentation was carried out at 25˚C and pH 6for a Incubation period of

48 hours. Both the wild IIB-13 and mutant strain H-97 were cultures by this technique

for the comparison in their activities and cultural parameters requirements. The

pectinase activity was increased gradually with the passage of time and reached

maximum value (11.56±0.15 U/ml/min) after 60 hours of incubation of the wild strain

IIB-13. Further, increase in incubation time showed decrease in the pectinolytic

activity. Like wild strain, similar trend was observed for the mutant strain H-97 with

maximum activity (32.19±0.20 U/ml/min) was obtained after 60 hours of incubation

as depicted in figure 5.

40

35

30

25

20

15

10

5

0

Nitrous Acid Mutants

En

zym

e A

ctiv

ity

(U/m

l)

44

Figure 5: Rate of pectinase synthesis using wild IIB-13 and mutant H-97 strains of

Aspergillus niger by submerged fermentation.

Fermentation Conditions: Temperature 25˚C and pH 6

Effect of Incubation Temperature

Pectinase production was carried out in 250 ml erlenmeyer flask at different

temperatures (25-40˚C) using wild IIB-13 and mutant H-97 strains. Maximum

pectinase activity was observed at 30˚C (Figure 6) for both wild strain IIB-13

(12.48±0.02 U/ml/min) and mutant strain H-97 (33.01±0.10 U/ml/min). The increase

and decrease from 30˚C resulted in the gradual decrease in the enzyme activity. The

activity was decreased drastically at 40˚C for both wild (6.01±0.05 U/ml/min) and

mutant (4.29±0.17 U/ml/min) strains.

Mutant Wild

40

35

30

25

20

15

10

20

18

16

14

12

10

8

6

4

5

0

2

0

12 24 36 48 60 72 84 96

Incubation Time (hours)

En

zym

e

Act

ivit

y (

U/m

l/m

in)

En

zym

e

Act

ivit

y (U

/ml/

min

)

45

Figure 6: Effect of incubation temperature on pectinase activity using wild IIB-13 and

mutant H-97 strains of Aspergillus niger by submerged fermentation.

Fermentation Conditions: pH 6 and 60 hours incubation period

Effect of Incubation pH

Effect of pH (6-8) was analyzed culturing both wild IIB-13 and mutant H-97 strains

of Aspergillus niger by submerged fermentation at 30 ˚C for 60 hrs. The pectinolytic

activity was increased gradually from pH 6 to pH 6.5 and then reached to maximum

activity (13.21±0.11 U/ml/min) at pH 7 by using wild strain IIB-13 (Figure 7).

Similarly, mutant strain also exhibited identical trend as showed by wild strain and

maximum activity (34.59±0.25 U/ml/min) was observed at pH 7. Further increase in

pH value resulted in the significant decrease in the pectinolytic activity.

Mutant Wild

40

35

30

25

20

15

10

5

0

20

18

16

14

12

10

8

6

4

2

0

25 30 35 40

Temperature (˚C )

En

zym

e A

ctiv

ity (U

/ml/

min

)

Enzym

e A

ctiv

ity

(U/m

l/m

in)

46

Figure 7: Effect of pH on the production of pectinase using the wild IIB-13 and

mutant H-97 strains of Aspergillus niger by submerged fermentation.

Fermentation Conditions: Temperature 30˚C and 60 hours of incubation

Fermentation Medium Selection

Pectinase activity was analyzed using five different types (M-I, M-II, M-III, M-IV and

M-V) of fermentation mediums (Figure 9). Maximum pectinase activity by wild strain

was achieved by using M-III medium and yielded 13.57±0.21 U/ml/min enzyme

activity. However, some variations were observed in case of mutant strain as it

produced highest pectinase activity (35.08±0.30 U/ml/min) in medium M-IV. For

each fermentation medium, variations in pectinase activity was observed for wild and

mutant strain (Figure 8).

Mutant Wild

40 16

35 14

30 12

25 10

20

15

8

6

10 4

5 2

0 0

6 6.5 7

pH

7.5 8

En

zym

e A

ctiv

ity

(U/m

l/m

in)

47

Figure 8: Effect of fermentation medium on pectinase production using the wild IIB-

13 and H-97 mutant strain of Aspergillus niger by submerged fermentation.

Fermentation Conditions: Temperature 30˚C, pH 7 and 60 hours of incubation.

Effect of carbon source

Effect of different carbon source in a fermentation medium was recorded. The wild

and mutant strains showed similar behavior when grown in medium containing

maltose, sucrose, glucose and pectin as carbon source. However, change in activity

was observed when pineapple extract and orange peel extract were used as a carbon

source as shown in figure 09. Maximum enzymatic activity by wild strain (13.84±0.14

U/ml/min) and mutant strain (35.77±0.17 U/ml/min) was achieved by using pectin as

a substrate (Figure 10). Concentration of pectinase was further investigated ranging

from 0.5-2.5 % in the medium with one percent was found as best as shown in figure

10.

Mutant Wild

40 16

35 14

30 12

25 10

20

15

8

6

10 4

5 2

0 0

M-I M-II M-III M-IV M-V

Fermentation Medium

Act

ivit

y (U

/ml/

min

) E

nzy

me

En

zym

e A

ctiv

ity

(U/m

l/m

in)

48

Figure 09: Effect of carbon source for pectinase production using the wild IIB-13 and

mutant H-97 strain of Aspergillus niger by submerged fermentation.

Fermentation Conditions: Temperature 30˚C, pH 7 and 60 hours of incubation

Figure 10: Effect of Pectin Concentration on the production of pectinase using the

wild IIB-13 and mutant H-97 strains of Aspergillus niger by submerged fermentation.

Fermentation Conditions: Temperature 30˚C, pH 7 and Incubation time 60 hours

Mutant Wild

40 16

35 14

30

25

12

10

20

15

8

6

10 4

5

0

2

0

Maltose Pineapple Orange Pectin Sucrose Glucose

Carbon Source

En

zym

e A

ctiv

ity

(U/m

l/m

in)

En

zym

e A

ctiv

ity

(U/m

l/m

in)

49

Effect of Nitrogen source

Effect of nitrogen source was investigated for the production of pectinase in a

fermentation medium. Five different nitrogen sources were used (Figure 11) and it

was found that wild and mutant strains showed different pectinase production pattern

when grown in different nitrogen sources like ammonium chloride, corn steep liquor,

urea and peptone. However, both wild IIB-13 and mutant H-97 strains yielded

maximum pectinase production of 14.78±0.24 U/ml/min and 36.96±0.11 U/ml/min

respectively when yeast extract was used as nitrogen source. While investigating the

effect of concentration of yeast extract, best result for wild strain was calculated to be

15.13±0.24 U/ml/min with 2% yeast extract. In contrary, mutant strain gave

maximum activity of 37.01±0.11 U/ml/min using 1% yeast extract as a nitrogen

source (Figure 12).

Figure 11: Effect of nitrogen source for best pectinolytic activity using wild IIB-13

and mutant H-97 strain of Aspergillus niger by submerged fermentation.

Fermentation Conditions: Temperature 30˚C, pH 7 and Incubation time 60 hour

50

Figure 12: Effect of yeast extract concentration on the production of pectinase using

the wild IIB-13 and mutant H-97 strains of Aspergillus niger by submerged

fermentation.

Fermentation Conditions: Temperature 30˚C, pH 7 and Incubation time 60 hours

Fermenter studies

Aspergillus niger nitrous acid mutant strain H-97 was employed for analyzing the

production of Pectinase in a fermenter of 7.5 liters with working volume of 5 liters.

Incubation Time

Submerged fermentation was carried out for 72 hours, at already optimized cultural

parameters at flask level, in a stirred fermenter. It was observed that Incubation time

for the production of pectinase was reduced to 48 hours as compared to 60 hours

which was observed when production was carried in flasks. It was observed that

enzyme production was also increased to 39.57±0.11 U/ml/min at temperature 30˚C

and pH 7 as shown in figure 13. Kinetics parameters also validated that enzyme

Mutant Wild

45 20

40

35

30

25

20

15

10

5

0

18

16

14

12

10

8

6

4

2

0

0.5 1 1.5 2 2.5

Yeast Extract Concentration (%) (w/v)

En

zym

e A

ctiv

ity

(U/m

l/m

in)

En

zym

e A

ctiv

ity

(U/m

l/m

in)

51

activity was increased gradually and reached to maximum after 48 hours with product

yield coefficient (0.01956 U/ml/mg) and specific product yield coefficient (0.148898

U/ml/h) as shown in figure 14.

Figure 13: Effect of Incubation time on the production of pectinase using mutant

strain H-97 of Aspergillus niger in a stirred fermenter.

Fermentation Conditions: Temperature 30˚C, pH 7, aeration rate 0.5vvm and agitation

150 rpm.

52

Figure 14: Kinetic studies of incubation time effect on the production of pectinase

using mutant strain H-97 of Aspergillus niger in stirred fermenter.

Fermentation Conditions: Temperature 30˚C, pH 7, aeration rate 0.5vvm and agitation

150 rpm.

Effect of Aeration:

Aeration rate was varied between 0.5 to 2.5 vvm to check its effect on the

production of the pectinase. It was found that pectinase activity was increased initially

(39.86±0.15 U/ml/min) when aeration rate was enhanced from 0.5 vvm to 1 vvm.

However activity started to decrease gradually when aeration rate was further

increased up to 2.5 vvm with constant increment of 0.5 vvm as shown in figure 15.

Cell mass production also followed the similar pattern. Product yield coefficient

(0.019411±0.16 U/ml/mg) and specific product yield coefficient (0.147762±0.01

U/ml/h) were found maximum at 1 vvm for the production of pectinase (Figure 16)

53

Figure 15: Effect of aeration rate on the production of pectinase using mutant strain of

Aspergillus niger in stirred fermenter.

Figure 16: Kinetic evaluation of aeration effect on the production of pectinase

using mutant strain of Aspergillus niger in stirred fermenter

54

Agitation rate:

Production of the pectinase was analyzed by changing the agitation rate from

100 rpm to 250 rpm with an increase of 50 rpm. Maximum production of the

pectinase (40.27±0.15 U/ml/min) and cell mass (2.12 g/ml ±0.21) was observed when

agitation rate of 200 rpm was maintained (figure 17). Fermentation kinetics studies

also indicated 200rpm as best agitation rate for the production of pectinase with

maximum product yield coefficient (0.018977±0.16U/ml/mg) and specific product

yield coefficient (0.144462±0.01 U/ml/h) as indicated in figure 18.

Figure 17: Effect of agitation rate on the production of pectinase using mutant strain

of Aspergillus niger in stirred fermenter.

55

Figure 18: Kinetic studies of agitation rate for the production of pectinase using

mutant strain of Aspergillus niger in stirred fermenter.

Inoculum size:

Vegetative inoculums size was varied between 0.5 to 2.5% to evaluate its

effect on pectinase production. Pectinase production was initially increased by

increasing the inoculums size from 0.5 to 1%. Little variation in production was

observed when inoculums size was further increased to 1.5 % with best activity

(40.31±0.07 U/ml/min) and cell mass growth (2.13±0.05 g/ml) was obtained using 1

% inoculum size (Figure 19). More increase in inoculums size to 2 and 2.5 % resulted

in gradual decrease in pectinase activity. Kinetic evaluation depicted that product

yield coefficient (0.018872±0.16 U/ml/mg) and specific product yield coefficient

(0.143658±0.01 U/ml/h) was found maximum using 1 % inoculums size as can be

seen in figure 20.

56

Figure 19: Investigation of inoculum size on the production of pectinase using mutant

strain of Aspergillus niger in stirred fermenter.

Figure 20: Kinetic studies of agitation rate for the production of pectinase using

mutant strain of Aspergillus niger in stirred fermenter.

57

Purification of Pectinase

Ammonium Sulphate Precipitation

Fractionation of crude pectinase was carried out with ammonium sulphate addition

ranging from 10 % to 90 %. Pectinase (60 %) was precipitated between 50-70 %

fractions of ammonium sulphate as shown in Table 2. The enzyme collected in

precipitated fractions was subjected to dialysis for removal of excess amount of salts.

Ammonium sulphate precipitation resulted in increased specific activity from 12.12

± 0.01 U/mg to 50.31 ± 0.04 U/mg.

Anion Exchange Chromatography

Partially purified enzyme was further subjected to anion exchange chromatography.

All unbound and eluted bound fractions were collected and analyzed which showed

no pectinase activity in unbound fraction. Pectinase was purified in a bounded

fractions which were eluted with NaCl concentration gradient between 0-80%

(Figure 21). This purification of Pectinase yielded 75.18 ± 0.04 U/mg specific

activity with 6.20 fold purification. The percentage yield was calculated as 44.20 %

(Table 02). Purified enzyme was further applied to SDS-PAGE for analyzing the

purity of the enzyme. A very clear single band was observed on gel having size of 35

kDa (Figure 22).

58

Table 03: Over all summary of Pectinase purification

Sample

Total

units

Total

Protein

(mg)

Specific

Activity

(U/mg)

Yield

(%)

Purification

Fold

Crude extract

200,128

16,500

12.12

100

1

(NH4)2SO4

Precipitation

120,750

2,400

50.31

60.37

4.15

Ion Exchange

Chromatography (Anion)

88,412 1,176 75.18 44.20 6.20

59

Figure 21: Purification of pectinase using LPC using Bio-Rad UnosphereTM

Q

(BioscaleTM

mini) column.

60

1 2 3 4 5

Figure 22: SDS-PAGE analysis of purified Pectinase. Lane 1: Protein marker

(Fermentas SN 0431). Lane 2 and Lane 3: Crude Pectinase produced by mutant H-

97 strain of Aspergillus niger . Lane 4 and 5: Purified pectinase by ammonium

sulphate precipitation and anion exchange chromatography of crude pectinase (35

kDa).

Characterization of pectinase:

Kinetic parameters of pectinase

Kinetic parameters i.e. Km and Vmax for pectinase were estimated using

Lineweaver-Burk double reciprocal plot (Figure 23). Varied concentrations of pectin

as substrate (10-80mM) were applied. The Vmax for the enzyme was calculated as

76.98 ± 0.02 U/mg. The Km for the enzyme for pectin degradation was determined as

2.30 mg/ml.

70 kDa

55 kDa

45 kDa

35 kDa

25 kDa

10 kDa

61

Figure 23: Line weaver-Burke Double Reciprocal Plot to calculate the Km and Vmax

for Pectinase.

Effect of temperature on Pectinase activity

Pectinase activity was analyzed at different temperatures i.e. 15-35oC to find out

optimum temperature. Starting from 15oC enzyme activity started to increase steadily

and reached to maximum (76.98 ±0.12 U/mg) at 25oC. The activity difference at 25oC

and 30oC was not appreciable but slightly more activity was observed at 25oC as

shown in figure 24. The reduced enzyme activity was noticed at 35oC (51.02 ±0.22

U/mg). Arrhenius plot (Figure 25) revealed activation energy (Ea) and enthalpy of

activation (ΔH) as -28.95 KJ/mol and -26.73 KJ/mol respectively.

62

Figure 24: Effect of temperature on enzyme activity of pectinase.

Figure 25: Arrhenius plot to calculate the activation energy and enthalpy of Activation

for pectianse catalyzed enzyme reaction.

90

80

70

60

50

40

30

20

10

0

15 20 25 30 35

Temperature oC

Sp

ecif

ic A

ctiv

ity

(U

/mg

)

63

Effect of temperature on Pectinase stability

Pectinase stability was evaluated within a temperature range of 20o to 60oC

with time interval of 30 to 120 minutes (Figure 26). Pectinase residual activity was

found to remain stable up to 30oC for the maximum reaction time (120 minutes).

Above 30oC, residual activity started to decrease with each increment in temperature

i.e. 40oC, 50oC and 60oC as 61, 44 and 22% respectively.

Figure 26: Temperature stability profile of pectinase.

120

100

80

60

40

20

0

0 30 60 90 120

Incubation Time (min)

20 °C 30 °C 40 °C 50 °C 60 °C

Res

idu

al

Acti

vity

%

64

Effect of pH on Pectinase activity

Pectinase activity was monitored under different pH conditions i.e. 5-7.5 to find out

the optimum pH required to attain the maximum activity. Pectinase activity started to

increase gradually from pH 5 to pH 5.5 and reached to its maximum (77.01 ±0.02

U/mg) at pH 6 and then started to decrease steadily from pH 6.5 to pH 7.5 as it is

evident from figure 27.

Figure 27: Effect of different pH values on Pectinase activity.

90

80

70

60

50

40

30

20

10

0

5 5.5 6 6.5 7 7.5

pH

Speci

fic

Act

ivit

y (U

/mg)

65

Effect of pH on stability of Pectinase

Pectinase stability was analyzed using phosphate buffer of pH range 5.5-7 for

incubation periods of 24, 48 and 72 hours. Residual activity was remained unchanged

when pectinase was stored in buffer of pH 6 (Figure 28). Decrease in the residual

activities i.e. 82, 74 and 61% was observed at pH 5.5, 6.5 and 7.

Figure 28: pH stability profile of pectinase.

110

100

90

80

70

60

50

40

0 24 48 72

Incubation Time (h)

5.5 6 6.5 7

Resi

du

al

Act

ivit

y %

66

Effect of metal ions on Pectinase activity

In the presence of different metal ions such as Cobalt, Mercury, Calcium, Manganese,

Lead, Copper, Magnesium, Cadmium, Nickle and Iron pectinase activity was

analyzed (Figure 29). No change in residual activity of enzyme was recorded in the

presence of six (Co, Ca, Mn, Mg, Ni and Iron) out of the 10 metal ions used in the

study. Remaining four metal ions (Hg, Pb, Cu and Cd) have resulted in decrease of

pectinase activity. It was observed that mercury reduced the maximum activity of the

enzyme with residual activity of 56%. While Cadmium, Copper and lead resulted in

76, 71 and 63 % decrease in enzyme activity respectively.

Figure 29: Effect of Metal ions on Pectinase activity.

67

Effect of metal ions on Pectinase stability

Different metal ions (Cobalt, Mercury, Calcium, Manganese, Lead, Copper,

Magnesium, Cadmium, Nickle and Iron) were used to study the effect on the stability

of the pectinase in their presence for a time period of 24-72 hours. Pectinase activity

reduced in the presence of all the metal ions. Cobalt has least (79% residual activity)

and mercury has the maximum effect with 34 % residual activity of enzyme as shown

in figure 30.

Figure 30: Stability profile of pectinase in the presence of metal ions.

110

100

90

80

70

60

50

40

30

20

0 24 48 72

Time (h)

Co Hg Ca Mn Pb Cu Mg Cd Ni Fe

Resi

du

al

Act

ivit

y %

68

Effect of surfactants on Pectinase activity

Pectinase activity in the presence of surfactants such as EDTA, SDS and Tween 80

was checked the effect of these surfactants on enzyme activity. Enzyme activity of

pectinase decreased significantly in the presence of all the surfactants. Tween 80

emerged as having adverse effect on enzyme with residual activity of 43 %. However,

little less harshness was observed in the case of EDTA and SDS with residual

activities of 61 and 59 % respectively (Figure 31).

Figure 31: Effect of Surfactants on Pectinase activity.

69

5. Discussion

The main purpose of the study was indigenous production of efficient pectinase

enzyme by Aspergillus niger for industrial purposes. The whole thermodynamic and

kinetic insight of the enzyme was further investigated in order to determine optimum

conditions of functional pectinase. One hundred and forty one strains of Aspergillus

niger were isolated which are based upon morphological characteristics like white

colonies which later turned into black spores with hyaline and septate hyphae. These

findings correspond with the investigations of Has et al. (2014). The isolated strains

were further screened for pectinolytic potential by the process of submerged

fermentation. Pectic substrate was degraded when pectinase enzyme in crude form

was used. This enzyme degrades pectic substance into smaller subunit which includes

glacturonic acid. Presence of galactouronic acid can be estimated by the reaction with

DNS due to the presence of reducing ends (Miller, 1959).

After screening, best yielding strain IIB 13 (10.78±0.45 U/ml/min) was subjected to

the chemical and physical mutation in order to obtain higher yield. Physically,

ultraviolet rays were used to mutate the strain. These rays can cause change in the

DNA due to the formation of thymine dimers which result in a mutated strain (Singh

et al. 2006). Nitrous acid and EMS were used as chemical mutagen. EMS causes the

alkylation of DNA which leads to point mutation that causes the formation of the O-6-

ethylguanine and subsequent transition of GC pair to AT pair. Nitrous acid causes the

hypoxanthine deamination which then results in the mutation in the DNA sequence

(Greene et al. 2003). The efficient mutant (H 37) with higher production of pectinase

(32.16 + 0.05 U/ml/min) was obtained after the mutation of nitrous acid (Figure 4).

Nitrous acid mutants are more stable as compared to other two might because of the

fact that the process of deamination is not easily reversible so these chemical mutants

can be maintained for a longer time period. Our findings are in accordance with

Molina et. al. (2001).

Different incubation durations were studied for the production of pectinase in order to

find the optimum time period (60 hours) for maximum production of enzyme

(32.19±0.20 U/ml/min) (Figure 5). Further increase in the incubation time resulted in

decrease of enzyme production. It might be because of the depletion of

70

nutrients from medium. Due to the unavailability of nutrients, bacterial cells might

have entered the stationary phase in which production of secondary metabolites occur.

These metabolites usually cause inhibition of the enzyme conformation. Decrease in

the production of enzyme might be due to the drop in pH of the medium that can

change the overall chemical conformation of the media. Furthermore, insufficient

incubation time period causes the inadequate growth of the microorganisms and hence

the production of enzyme. On the other hand, increase in the incubation time period

than optimum value causes the overgrowth of cell mass and results in the cells

breakage. Similar results were reported by Ray et al. (2008) in which it was

demonstrated that longer period of incubation resulted in less activity. The results

identical to our study were also presented by El-Hadi et al. (2013). Different

temperatures of incubation, such as 25-40˚C, were observed in the present study and it

was noticed that the maximum pectinase production (33.01±0.10 U/ml/min) was

found at 30°C by employing mutant strain (Figure 6). This is due to the reason that the

organism used for production of the pectinase is mesophile which prefers to grow at

moderate range of temperature and this type of organism showed retarded or limited

growth at high or low temperature (Zubair et al. 2002). The biochemistry and

morphology of these mesophilic microorganisms is such that it permits growth at its

specific temperature. At low temperatures, organism can grow but it may possess less

metabolic activity due to inhibition of their metabolic processes at low temperature.

At high temperature, components of cell wall and proteins undergo the process of

denutrition, and may cause the death of organism. Hence, optimum enzyme

production above or below specified temperature value cannot be achieved. Similar

type of work was reported by Sandhya et al. (2013) in which it was determined that

maximum activity of enzyme was achieved at optimum temperature. The extent and

timing of production of pectinase by Aspergillus niger is much dependent on the pH

of the fermentation medoium. The production of pectinase was studied by using

different values of pH ranging from 6.0 to 8.0. Maximum activity of pectinase

(34.59±0.25 U/ml/min) was found at pH 7.0 using mutant strain (Figure 7). It might

be due to the fact that the organism used for the production of pectinase shows

maximum growth at neutral pH. Microbes are sensitive to the concentration of

hydrogen ions in environment. The change in the concentration of hydrogen ions of

the medium affects large proteins such as enzymes. This results in the change of

enzyme conformation and they further bring about changes of the ionic charges on the

71

molecule. It effects charge interactions between and within polypeptides.

Accordingly, secondary and tertiary structure of proteins, especially enzymes, is

affected by pH values which is responsible for decreased enzyme activity. At lower

pH, polarity of the cell membrane is affected due to ionization of some amino acids

which are present in bilayer of the cell membrane. This change prevents the easy

movement of substrates and nutrients which are necessary for extracellular production

of the enzyme pectinase. Hence, the activity and production of enzyme appears to

decrease when pH is fluctuated from the optimum value. A different pH from the

optimum pH may lead to the enzyme denaturation (MacDiarmid et al. 1988).

Optimum pH for the production of bacterial pectinase ranges from 5.0-7.0 (Kumar et

al. 2012) and the results were in accordance to those reported by Tariq et al. (2012).

Five different fermentation media i.e. M-I to M-V were used in this study in order to

find the best medium for maximum production of pectinase by bacteria depends upon

the certain essential compounds presence in their environment. Among these media,

maximum production of enzyme was found in M-IV medium (35.08±0.30 U/ml/min)

by mutant strain (Figure 8). Pectin and yeast extract in medium allows the fungi in

order to produce higher yield and also provide growth factors and additional nutrients

required for the enhanced growth. The potassium phosphates are present for the

inhibition in drop in pH of medium during bacterial growth. Similar results were

reported in LB broth medium by Joshi et al. (2011).

Carbon source is required as a source of energy and it greatly effects the production of

pectinase enzyme. The optimization of this parameter is crucial as biosynthesis of the

pectinase in all organisms seems to be regulated by the readily available

metabolizable carbon (Jyotsana et al. 2015). The need for carbon sources changes

from organism to organism. In this study, pectin was found to be the most suitable

source of carbon. The utilization of pectin in the medium was much better as

compared to other carbon sources because of capability of pectinase producing

organisms to utilize pectin with more efficiency and ease (Akhter et al. 2011). These

results are in accordance with the study of Kapoor et al. (2000). Nitrogen source

greatly effects the production of pectinase and this could be attributed to the

nucleotide and amino acid contents of microorganisms. The effect of different

72

nitrogen sources i.e. corn steep liquor, ammonium chloride, yeast extract, peptone and

urea for the production of pectinase was studied. Yeast extract turned out to be the

better nitrogenous substrate, as mutant strain gave the yield of 37.01±0.11 U/ml/min

(Figure 11). The results are in accordance with (Rajmane & Korekar, 2012). Peptone

might being a complex organic nitrogen and rich in amino acids such as aspartic acid,

glutamic acid, alanine and glycine which are needed for the structural composition of

pectinase enzyme.

Up scaling of the product was carried out using 7.5 liter conditions controlled

fermenter. Controlled conditions of temperature (30°C), aeration (1vvm), pH (8.0.),

and agitation (200 rpm) resulted in high yield of pectinase (40.27±0.15 U/ml/min) in

reduced time period (48 h) (Figure 14). The presence of all the controlled conditions

with continuous supply of oxygen might be the reason for the reduction in time to

achieve pectinase maximum activity. Recently, it has been shown that a constant

controlled agitation guarantees the equal distribution of nutrients to the fungal cells

(Rajmane et. al., 2012). Similarly, aeration rate has a major effect on the cell mass

growth and the formation of product (Torres et al. 2006). Five different vegetative

inoculum sizes were used during studies of fermenter i.e. 0.5%, 1.0%, 1.5%, 2.0% and

2.5%. Maximum activity of pectinase (40.31±0.07 U/ml/min) was obtained at 1.0%

inoculum (Figure 19). The quantity of inoculum plays vital role in the bioprocess

outcome (Ettler, 1992). A change in inoculum size may decrease the enzyme

production yield because of the inadequate or over growth of the organism. Low

levels of inoculum may be insufficient for the production of enzyme as cells might not

be enough in number to completely utilize essential amount of nutrients. But the high

inoculum level might result in rapid depletion of nutrients in the medium, hence

reducing the yield of enzyme production. In other words, at inoculum higher

concentrations, the anaerobic condition of fermentation medium because of the

tremendous growth of microorganisms led to nutritional depletion in the medium and,

hence, resulted in less production. Also, loss of homogeneity and high cell density

would lead to the production of inhibitory metabolites that might hinder the

production of enzyme (Niladevi and Prema, 2008).

Ammonium sulphate precipitation was carried out to remove junk proteins with the

maximum pectinase recovery with precipitation range of 10-90%. Maximum desired

73

protein (14%) was recovered at 60% precipitation. High salt concentration of

ammonium sulphate might be reason for the enzyme denaturation. As the ionic

strength of solution increases because of the addition of salt the hydrophobic groups

become more available, thus results in the protein precipitation. Some of accumulated

proteins do not detach completely when re-suspended and would also be the reason

for the reduction in the activity of the pectinase enzyme. Findings of Vasanthi et al.

(2011) is in accordance with our results with ammonium sulphate concentration of 50-

60% used for the recovery of pectinase. Similar types of results were also observed by

Tripathi et al. (2014) showing 50-70% as best ammonium sulphate concentration

range for pectinase precipitation.

The ammonium sulphate precipitated enzyme was subjected to anion exchange

chromatography using DEAE-cellulose resin containing pre-packed column to obtain

the purification fold of the 6.20 along with 44.20 % yield of the enzyme (Table 02).

Recovery of the protein was 7.12 % of the total protein. Pectinase was obtained in

bound fraction of proteins eluted during the phase of concentration gradient. In spite

of this, anion exchange columns are more extensively in use for the pectinase

purification. Purity of the enzyme was assessed by SDS-PAGE and it was discovered

that it is a dimeric protein with the molecular weight of single band as 32 kDa (Figure

22). Our results are in accordance with the reports of Zia et al. (2007) who reported

pectinase size of 35 kDA but against the findings of Simpson et al. (2007) that

molecular weight of single band of dimeric Pectinase was 70 kDa. It probably would

be due to strain variability that protein of different sizes with a narrow range of

difference reported.

Purified enzyme was then lyophilized to store the enzyme for longer time period with

little loss of activity (3%) during freeze drying process. Decrease in the activity might

be due to the changes occurring in the enzyme structure during the process of freezing

and drying. To minimize the de-nutrition effect, 0.2% sucrose was used as a

stabilizing agent. Simpson et al. (2007) also reported the loss of the activity (22 %)

during the process of Lyophilization. A series of variable temperature (15-35oC) was

engaged to conclude the optimum temperature for the activity of pectinase. The

enzyme displayed maximum activity at 25oC. Further increase in temperature resulted

in the loss of activity. Nelson and Cox (2004) stated that internal energy of the

74

enzyme gets raised with the increase in temperature and results in the breakage of

week hydrogen bonds causing enzyme de- nutrition. Sherbeny et al. (2005) and

Odebunmi and Owalude (2007) reported opposed and in accordance finding to our

study with 37oC and 25oC as optimum temperature for maximum activity of

pectinase, respectively. Arrhenius plot was used to carry out the thermodynamic

studies of the ectinase enzyme. The enthalpy (ΔH) and activation energy (Ea) were

calculated as-26.73 KJ/mol and -28.95 KJ/mol, respectively. Stability of the pectinase

was analyzed in a temperature range of (20-60oC) for a time period of 30-120 min. It

was observed, enzyme was stable at 20 and 30oC for observed time period and started

to denature gradually at the temperature above than 30oC. It might be because of the

conformational changes in structure which starts to take place as the temperature

increases. Rasul et. al. (2011) reported the stability of enzyme at 30oC for a short time

period as indicated in the current study. Results of Sherbeny et al. (2005) are in

contradiction to our findings showing enzyme to be stable up to 40oC.

The kinetic parameters such as Km and Vmax were calculated as 2.30 mg/ml and

76.98± 0.02 U/mg (Figure 23). Otadi and Mobayen (2007) reported Km value of 10.5

mg/ml in their study. The effect of pH on stability and activity of the enzyme was

observed. It was found that enzyme was stable at pH 6. The reason for this

observation might be the need for change in the pH to develop some conformational

changes in the enzyme catalytic site in order to catalyze a reaction more efficiently.

Similar types of consequences were placed by Otadi and Mobayen (2007) stating that

optimum pH for activity as 6.6. Sherbeny et al, (2005) demonstrated pH 5.5 as the

best pH for the optimum activity of the pectinase.

Metal ions affect on the stability and activity of the pectinase enzyme was

investigated and it was observed that activity was reduced by four metal ions i.e.

Hg++, Pb++, Cu++ and Cd++. However, the stability was affected by all the metal ions

when incubated with them for the time period of 24-72 hours. (Figure 29 and 30) All

of these are heavy metal ions and might result in the change in catalytic site of the

enzyme by some allosteric changes in enzyme. Liu et al. (1999) also proposed similar

results in addition that enzyme was also unstable to other metal ions (Cobalt,

Mercury, Calcium, Manganese, Lead, Copper, Magnesium, Cadmium, Nickle and

Iron) against which enzyme used in our study is stable except calcium. In this way

75

enzyme used in our study is more effective.

Surfactants i.e. SDS, EDTA and tween 80 were used to check their effect on the

pectinase. All of them limited the activity of pectinase enzyme (Figure 31). All of

them are chelating agents and this could certainly be the reason for the limitation in

the activity of the enzyme. Not much data in this regard is available in literature with

which results of present study could be compared.

76

6. References

Acuña-Argüelles, M.E., Gutierrez-Rojas, M., Viniegra-González, G. and Favela-Torres, E.,

1995. Production and properties of three pectinolytic activities produced by Aspergillus

niger in submerged and solid-state fermentation. Applied microbiology and

biotechnology, 43(5): 808-814..

Akhtar, N., Shoaib, A., Munir, S., Ali, A. and Khurshid, S., 2014. Isolation, identification and

enzyme production profile of A. niger. JAPS, Journal of Animal and Plant Sciences,

24(5): 1438-1443..

Akhter, N., Morshed, M.A., Uddin, A., Begum, F., Sultan, T. and Azad, A.K., 2011.

Production of pectinase by Aspergillus niger cultured in solid state media. International

Journal of Biosciences, 1(1): 33-42..

Ali, J., Jaffery, S.A., Assad, Q., Hussain, A., Abid, H. and Gul, F., 2010. Optimization of

pectinase enzyme production using sour oranges peel (Citrus aurantium L) as substrate.

Pakistan Journal of Biochemistry and Molecular Biology, 43: 126-130..

Amande, T. and Adebayo-Tayo, B., 2012. Screening of new isolates fungal strains for

polygalacturonase production in submerged fermentation.Innovative Romanian Food

Biotechnology, 11: 15..

Aspergillus hortai. Journal of Radiation Research and Applied Sciences, 7(1): 23-28.

Azin, M. and Noroozi, E., 2001. Random mutagenesis and use of 2-deoxy-D-glucose as an

antimetabolite for selection of α-amylase-overproducing mutants of Aspergillus oryzae.

World Journal of Microbiology and Biotechnology, 17(7): 747-750.

Baker, S.E., 2006. Aspergillus niger genomics: past, present and into the future. Medical

Mycology, 44(sup1): 17-21..

Bali, R., 2007. Isolation of Extracellular Fungal Pectinolytic Enzymes and Extraction of Pectin

using Kinnow Waste as Substrate (Doctoral dissertation)..

Bankar, S.B., Bule, M.V., Singhal, R.S. and Ananthanarayan, L., 2009. Glucose oxidase—an

overview. Biotechnology advances, 27(4): 489-501.01.

Bhardwaj, V. and Garg, N., 2014. Production, purification of pectinase from Bacillus sp.

MBRL576 isolate and its application in extraction of juice. Int J Sci Res, 3: 648-652.52.

Bhatti, H. N., M. Madeeha, M. Asgher and N. Batool. 2006. Purification and thermodynamic

characterization of glucose oxidase from a newly isolated strain of Aspergillus niger,

77

Can. J. Microbiol., 12: 315-321.

Blanco, P., Sieiro, C. and Villa, T.G., 1999. Production of pectic enzymes in yeasts. FEMS

Microbiology Letters, 175(1): 1-9..

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding. Analytical

Biochemistry, 72(1-2): 248-254.

Busto, M.D., Ortega, N. and Perez-Mateos, M., 1995. Induction of β-glucosidase in fungal and

soil bacterial cultures. Soil Biology and Biochemistry, 27(7): 949-954.

Carr, J. G. 1985. Tea, coffee and cocoa In B. J. G Wood, Editor Microbiology of fermented

foods. Elsevier science ltd London. (2): 133-154.

Cavalitto, S.F., Areas, J.A. and Hours, R.A., 1996. Pectinase production profile of Aspergillus

foetidus in solid state cultures at different acidities.Biotechnology Letters, 18(3): 251-

256.

Chen, L., Wu, Z.Q., Wang, C., Ouyang, J. and Xia, X.H., 2012. Exploring the temperature-

dependent kinetics and thermodynamics of immobilized glucose oxidase in microchip.

Analytical Methods, 4(9): 2831-2837.

Conn, E.P. and P.K. Stumph. 1994. Molecular basis of gene expression and regulation Outlines

of biochemistry, John Wiley and Sons, 287-298.

De Gregorio, A., Mandalari, G., Arena, N., Nucita, F., Tripodo, M.M. and Curto, R.L., 2002.

SCP and crude pectinase production by slurry-state fermentation of lemon pulps.

Bioresource Technology, 83(2): 89-94.94.

Debing, J., Peijun, L., Stagnitti, F., Xianzhe, X. and Li, L., 2006. Pectinase production by solid

fermentation from Aspergillus niger by a new prescription experiment. Ecotoxicology

and Environmental Safety, 64(2): 244-250..

Dekker, R.F. and Barbosa, A.M., 2001. The effects of aeration and veratryl alcohol on the

production of two laccases by the ascomycete Botryosphaeria sp. Enzyme and

Microbial Technology, 28(1): 81-88..

Dhillon, S.S., Gill, R.K., Gill, S.S. and Singh, M., 2004. Studies on the utilization of citrus peel

for pectinase production using fungus Aspergillus niger. International Journal of

Environmental Studies, 61(2): 199-210..

El-Hadi, A.A., El-Nour, S.A., Hammad, A., Kamel, Z. and Anwar, M., 2014. Optimization of

78

cultural and nutritional conditions for carboxymethylcellulase production by

Aspergillus hortai. Journal of Radiation Research and Applied Sciences. Vol. 7, Issue

1, Jan. 2014: 23-28.

El-Sherbeny, G.A., Shindia, A.A. and Sheriff, Y.M.M.M., 2005. Optimization of various

factors affecting glucose oxidase activity produced by Aspergillus niger. Int J Agric

Biol, 7(6): 953-958.

Ettler, P., 1992. The determination of optimal inoculum quality for submerse fermentation

process. Collection of Czechoslovak Chemical Communications, 57(2): 303-308..

Ezike, T.C,. 2012. Production and characterization of pectinases obtained from Aspergillus

niger under submerged fermentation system using pectin extracted from orange peels as

carbon source (doctoral dissertation, university of Nigerian sukka).

Favela-Torres, E., Volke-Sepúlveda, T. and Viniegra-González, G., 2006. Production of

hydrolytic depolymerising pectinases. Food Technology and Biotechnology, 44(2):

221.

Ferri, S., Kojima, K. and Sode, K., 2011. Review of glucose oxidases and glucose

dehydrogenases: a bird's eye view of glucose sensing enzymes. Journal of diabetes

science and technology, 5(5): 1068-1076.

Ge, X.Y., Xu, Y., Chen, X. and Zhang, L.Y., 2014. Improvement Of L-Lactic Acid Production

From Orange Peels In Mixed Culture System. Journal of Global Biosciences 3(1): 354-

360..

Giese, E.C., Dekker, R.F. and Barbosa, A.M., 2008. Orange bagasse as substrate for the

production of pectinase and laccase by Botryosphaeriarhodina MAMB-05 in

submerged and solid state fermentation. Bioresources 3(2): 335-345..

Greene, E.A., Codomo, C.A., Taylor, N.E., Henikoff, J.G., Till, B.J., Reynolds, S.H., Enns,

L.C., Burtner, C., Johnson, J.E., Odden, A.R. and Comai, L., 2003. Spectrum of

chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis.

Genetics, 164(2): 731-740.

Gromada, A. and Fiedurek, J., 1995. Influence of medium components and metabolic

inhibitors on glucose oxidase production by Aspergillus niger mycelium.

Actamicrobiologica Polonica, 45(1): 37-43.

Hachemi, N., Nouani, A. and Benchabane, A., 2015. Bioconversion of Oranges Wastes for

79

Pectinase Production Using Aspergillus niger under Solid State Fermentation. World

Academy of Science, Engineering and Technology, International Journal of Biological,

Biomolecular, Agricultural, Food and Biotechnological Engineering, 9(9): 956-961.

Hankin, L. and Anagnostakis, S.L., 1975. The use of solid media for detection of enzyme

production by fungi. Mycologia, 597-607.

Hannan, A., Bajwa, R. and Latif, Z., 2009. Status of Aspergillus niger strains for pectinases

production potential. Pak. J. Phytopathol, 21(1): 77-82.

Haq, I.U., Nawaz, A., Mukhtar, H., Mansoor, Z., Riaz, M., Ahmed, M. and Ameer, S.M., 2014.

Random mutagenesis of Aspergillus niger and process optimization for enhanced

production of glucose oxidase. Pak. J. Bot, 46(3): 1109-1114.114.

Heerd, D., Diercks-Horn, S. and Fernández-Lahore, M., 2014. Efficient polygalacturonase

production from agricultural and agro-industrial residues by solid-state culture of

Aspergillus sojae under optimized conditions. Springer Plus, 3(1): 1.

Hoondal, G., Tiwari, R., Tewari, R., Dahiya, N.B.Q.K. and Beg, Q., 2002. Microbial alkaline

pectinases and their industrial applications: a review. Applied Microbiology and

Biotechnology, 59(4-5): 409-418..

Jakubikova, L., Farkaš, V., Kolarova, N. and Nemcovič, M., 2006. Conidiation of

Trichodermaatroviride isolate during submerged cultivation in a laboratory stirred-tank

fermenter. Folia microbiologica, 51(3): 209-213.

Joshi, V.K., Parmar, M. and Rana, N., 2011. Purification and characterization of pectinase

produced from Apple pomace and evaluation of its efficacy in fruit juice extraction and

clarification. Indian journal of natural products and resources, 2(2): 189-197.197.

Jyotsna, K.P., Rao, A.R. and Devaki, K., 2015. Effect of nutritional factors on cellulase

production by Streptomyces albaduncus from the gut of earthworm, Eiseniafoetida.

Pest Management In Horticultural Ecosystems,21(1): 75-80..

Kalaichelvan, P., 2012. Production and optimization of pectinase from Bacillus sp. MFW7

using cassava waste. Asian Journal of Plant Science and Research, 2(3): 369-375..

Kapoor, M., Beg, Q.K., Bhushan, B., Dadhich, K.S. and Hoondal, G.S., 2000. Production and

partial purification and characterization of a thermo-alkali stable polygalacturonase

from Bacillus sp. MG-cp-2. Process Biochemistry, 36(5): 467-473.

Karthik, L., Kumar, G. and Rao, K.B., 2011. Screening of pectinase producing microorganisms

80

from agricultural waste dump soil by Asian Journal of Biochemical and Pharmaceutical

Research, Issue 2 (Vol.1), 2011 ISSN: 2231-2560.

Khairnar, Y., K. V. Krishna, A. Boraste, N. Gupta, S. Triverdi, P. Patil, G. Gupta, A. Jhadar,

A. Mujapara, B. Joshi and D. Mishra. 2009. Study of pectinase production in

submerged fermentation using different strains of Aspergillus niger. Int, J. Microbiol.

Res., (1): 13-17.

Kitpreechavanich, V., Hayashi, M. and Nagai, S., 1984. Production of xylan-degrading

enzymes by thermophilic fungi, Aspergillus fumigatus and Humicolalanuginosa.

Journal of Fermentation Technology, 62(1): 63-69.

Kumar, S., Sharma, H.K. and Sarkar, B.C., 2010. Effect of substrate and fermentation

conditions on pectinase and cellulase production by Aspergillus niger NCIM 548 in

submerged (SmF) and solid state fermentation (SSF). Food Science and Biotechnology,

20(5): 1289-1298.

Kurnar, N.K. and Reddy, D.S.R., 2008. Optimization of Pectinase Production from M

anihotutilissima by Aspergillus niger NCIM 548 Using Statistical Experimental Design.

Research Journal of Microbiology, 3(1): 9-16.

Lawford, H.G. and Rousseau, J.D., 1993. Production of ethanol from pulp mill hardwood and

softwood spent sulfite liquors by genetically engineered E. coli. Applied biochemistry

and biotechnology, 39(1): 667-685.

Lineweaver, H. and Burk, D., 1934. The determination of enzyme dissociation constants.

Journal of the American Chemical Society, 56(3): 658-666.

Liu, J.Z., Yang, H.Y., Weng, L.P. and Ji, L.N., 1999. Synthesis of glucose oxidase and catalase

by Aspergillus niger in resting cell culture system. Letters in applied microbiology,

29(5): 337-341.

Lotfy, W.A., Ghanem, K.M. and El-Helow, E.R., 2007. Citric acid production by a novel

Aspergillus niger isolate: II. Optimization of process parameters through statistical

experimental designs. Bioresource technology, 98(18): 3470-3477.

Maciel, M.D.H.C., Herculano, P.N., Porto, T.S., Teixeira, M.F.S., Moreira, K.A. and de Souza-

Motta, C.M., 2011. Production and partial characterization of pectinases from forage

palm by Aspergillus niger URM4645. African Journal of Biotechnology, 10(13): 2469-

2475.

81

Maldonado, M.C. and De Saad, A.S., 1998. Production of pectinesterase and polygalacturonase

by Aspergillus niger in submerged and solid state systems. Journal of Industrial

Microbiology and Biotechnology, 20(1): 34-38.

Maldonado, M.C., Navarro, A. and Callieri, D.A., 1986. Production of pectinases

byAspergillussp using differently pretreated lemon peel as the carbon source.

Biotechnology Letters, 8(7): 501-504.

Maldonado, M.C., Strasser de Saad, A.M. and Callieri, D.A., 1989. Regulatory aspects of the

synthesis of polygalacturonase and pectinesterase by Aspergillus nig sp. Sciences des

aliments, 9(1): 101-110.

Maria de Lourdes, T.M., Jorge, J.A. and Terenzi, H.F., 1991. Pectinase production by

Neurosporacrassa: purification and biochemical characterization of extracellular

polygalacturonase activity. Microbiology,137(8): 1815-1823.

Martin, N., Souza, S.R.D., Silva, R.D. and Gomes, E., 2004. Pectinase production by fungal

strains in solid-state fermentation using agro-industrial bioproduct. Brazilian Archives

of Biology and Technology, 47(5): 813-819.

Martos, M.A., Martinez Vazquez, F., Benassi, F.O. and Hours, R.A., 2009. Production of

pectinases by A. niger: influence of fermentation conditions. Brazilian Archives of

Biology and Technology, 52(3): 567-572.

Meena, K.K. and Pawar, V.N., 2015. Pilot scale production and characterization of pectinase

enzyme from Aspergillus niger. International Journal of Processing and Post Harvest

Technology, 6(1): 14-18.

Meenakshisundaram, V., 2012. Optimization of pectinase enzyme production by using sour

orange peel as substrate in solid state fermentation. Asian J Biochem Pharm Res, 2(1):

16-26.

Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar.

Analytical Chemistry, 31(3): 426-428.

Molina, S.M., Pelissari, F.A. and Vitorello, C., 2001. Screening and genetic improvement of

pectinolytic fungi for degumming of textile fibers. Brazilian Journal of Microbiology,

32(4): 320-326.

Mrudulla, S and R. Anithraj. 2011. Pectinase production in solid state fermentation by

Aspergillus niger using orange peel as substrate. Global. J.Biotechnol. Biochem., 6(2):

82

64-66.

Nelson, D. L and M. M. Cox. 2004. Lehninger: Principles of Biochemistry. 4th ed. W. H.

Freeman and Co., 206-208 ISBN: 978-0716743392.

Niladevi, K.N. and Prema, P., 2008. Effect of inducers and process parameters on laccase

production by Streptomyces psammoticus and its application in dye decolourization.

Bioresource technology, 99(11): 4583-4589.589.

Niture, S.K. and Pant, A., 2004. Purification and biochemical characterization of

polygalacturonase II produced in semi-solid medium by a strain of Fusarium

moniliforme. Microbiological Research, 159(3): 305-314.

Odebunmi, E.O. and Owalude, S.O., 2009. Kinetic and thermodynamic studies of glucose

oxidase catalysed oxidation reaction of glucose, J.Appl. Sci. Environ. Msanage.

December, 2007, Vol.11(4) 95-100.

Okafor, U.A., Okochi, V.I., Chinedu, S.N., Ebuehi, O.A.T. and Onygeme-Okerenta, B.M.,

2010. Pectinolytic activity of wild-type filamentous fungi fermented on agro-wastes.

African Journal of Microbiology Research, 4(24): 2729-2734.

Onions, A.H.S., D. Allsop and H.O.W.Eggins. 1981. Smith’s Introduction to Industrial

mycology. Edward Arnold Publishers limited.

Otadi, M. and Mobayen, S., 2011. The Survey of Kinetic Behavior of Immobilized Glucose

Oxidase on Gum Tragacanth Carrier. enzyme, 2(2): 2.

Oyeleke, S.B., Oyewole, O.A., Egwim, E.C., Dauda, B.E.N. and Ibeh, E.N., 2012. Cellulase

and pectinase production potentials of Aspergillus niger isolated from corn cob. Bayero

Journal of Pure and Applied Sciences, 5(1): 78-83.

Palaniyappan, M., Vijayagopal, V., Viswanathan, R. and Viruthagiri, T., 2009. Screening of

natural substrates and optimization of operating variables on the production of

pectinase by submerged fermentation using Aspergillus niger MTCC 281. African

journal of Biotechnology, Vol. 8 No. 4 (2009) ISSN: 1684-5315.

Pasha, K.M., Anuradha, P. and Subbarao, D., 2013. Applications of pectinases in industrial

sector. International Journal of Pure and Applied Sciences and Technology, 16(1): 89.

Patil, N.P. and Chaudhari, B.L., 2010. Production and purification of pectinase by soil isolate

Penicilliumsp and search for better agro-residue for its SSF. Recent Research in

Science and Technology 2010, 2(7): 36-42.

83

Patil, R.C., Murugkar, T.P. and Shaikh, S.A., 2012. Extraction of pectinase from pectinolytic

bacteria isolated from carrot waste. International Journal of Pharma and Bio Sciences,

3: B261-B266.

Patil, S.R. and Dayanand, A., 2006. Exploration of Regional Agrowastes for the Production of

Pectinase by Aspergillus niger. Food Technology & Biotechnology, 44(2).

Pedrolli, D.B., Monteiro, A.C., Gomes, E. and Carmona, E.C., 2009. Pectin and pectinases:

production, characterization and industrial application of microbial pectinolytic

enzymes. Open Biotechnology Journal, 9-18.

Phutela, U., Dhuna, V., Sandhu, S. and Chadha, B.S., 2005. Pectinase and polygalacturonase

production by a thermophilic Aspergillus fumigatus isolated from decomposting orange

peels. Brazilian Journal of Microbiology,36(1): 63-69.

Pilknik, W. and Voragen, G.J.A., 1993. Pectic enzymes in fruit and vegetable juice

manufacture. Enzyme and Food Processing ed. Tilak Nagodawithana, Gerald Reed,

ISBN: 9780080571454:480.

Pirt, S.J., 1975. Principles of microbe and cell cultivation. Blackwell Scientific Publications.

Poletto, P., Borsói, C., Zeni, M. and Silveira, M.M.D., 2015. Downstream processing of

pectinase produced by Aspergillus niger in solid state cultivation and its application to

fruit juices clarification. Food Science and Technology (Campinas), 35(2): 391-397.

Qureshi, A.S., Bhutto, M.A., Chisti, Y., Khushk, I., Dahot, M.U. and Bano, S., 2012.

Production of pectinase by Bacillus subtilis EFRL 01 in a date syrup medium. African

Journal of Biotechnology, 11(62): 12563-12570.

Rajmane, S.D. and Korekar, S.L., 2012. Impact of carbon and nitrogen sources on pectinase

production of post-harvest fungi. Current Botany 2012, 3(3):01-03, ISSN: 2220-4822.

Raju, E.V.N. and Divakar, G., 2013. Screening and isolation of pectinase producing bacteria

from various regions in Bangalore. Int J Res Pharma Biomed Sci, 4: 151-154.

Rangarajan, V., Rajasekharan, M., Ravichandran, R., Sriganesh, K. and Vaitheeswaran, V.,

2010. Pectinase production from orange peel extract and dried orange peel solid as

substrates using Aspergillus niger. Int J Biotechnol Biochem, 6(3): 445-453.

Raper, K.B. and Fennell, D.I., 1965. The genus Aspergillus. :1-686.

Ray, A.K., Bairagi, A. and Sen, S.K., 2007. Optimization of fermentation conditions for

cellulase production by Bacillus subtilis CY5 and Bacillus circulans TP3 isolated from

84

fish gut. Acta Ichthyologica et Piscatoria, 37(1): 47-53.

Ray, S. and Banik, A.K., 1999. Effect of ammonium and nitrate ratio on glucose oxidase

activity during gluconic acid fermentation by a mutant strain of Aspergillus niger.

Indian Journal of Experimental Biology, 37: 391-395.

Revilla, I. and José, G.S., 2003. Addition of pectolytic enzymes: enological practice which

improves the chromaticity and stability of red wines. International Journal of Food

Science & Technology, 38(1): 29-36.

Rombusts, F. M and W. Pilnik. 1980. Pectic enzymes In; Microbial enzymes and

bioconversions. Academic Press, London 5th edition: 227-72.

Rowlands, R.T., 1984. Industrial strain improvement: mutagenesis and random screening

procedures. Enzyme and Microbial Technology, 6(1): 3-10.

Salazar, L. and Jayasinghe, U., 1999. Fundamentals of purification of plant viruses. Techniques

in Plant, Virology, CIP., Training Manual, JO, Virus Purification, International Potato

Centre, Peru, 1-10.

Sandana Mala, J.G., Kamini, N.R. and Puvanakrishnan, R., 2001. Strain improvement of

Aspergillus niger for enhanced lipase production. The Journal of general and applied

microbiology, 47(4): 181-186.

Sandhya, R. and Kurup, G., 2013. Screening and isolation of pectinase from fruit and vegetable

wastes and the use of orange waste as a substrate for pectinase production. Int Res J

BiolSci 2(9): 34-39.

Satyanarayana, N. G and T. Panda. 2002. Purification and biochemical properties of microbial

pectinases: A review. Proc. Biochem., (38): 987-996.

Saxena, S., Madan, T., Muralidhar, K. and Sarma, P.U., 2003. cDNA cloning, expression and

characterization of an allergenic L3 ribosomal protein of Aspergillus fumigatus.

Clinical & Experimental Immunology, 134(1): 86-91..

Siddiqui, K.S., Rashid, M.H. and Rajoka, M.I., 1997. Kinetic analysis of the active site of an

intracellular β-glucosidase from Cellulomonas biazotea. Folia Microbiologica, 42(1):

53-58.

Silva, D., Tokuioshi, K., da Silva Martins, E., Da Silva, R. and Gomes, E., 2005. Production of

pectinase by solid-state fermentation with Penicillium viridicatum RFC3. Process

Biochemistry, 40(8): 2885-2889.

85

Simpson, C., Jordaan, J., Gardiner, N.S. and Whiteley, C., 2007. Isolation, purification and

characterization of a novel glucose oxidase from Penicillium sp. CBS 120262 optimally

active at neutral pH. Protein expression and purification, 51(2): 260-266.

Singh, O.V., Sharma, A. and Singh, R.P., 2001. Gluconic acid production by Aspergillus niger

mutant ORS-4.410 in submerged and solid state surface fermentation. Indian Journal of

Experimental Biology, 39(7): 691-696.

Snedecor, G.W. and W.G. Cochran, 1980. Statistical methods. Eddition 7. Iowa State

Unversity, USA.

Sunnotel, O. and Nigam, P., 2002. Pectinolytic activity of bacteria isolated from soil and two

fungal strains during submerged fermentation. World Journal of Microbiology and

Biotechnology 18(9): 835-839.

Suresh, B. and Viruthagiri, T., 2010. Optimization and kinectics of pectinase enzyme using

Aspergillus niger by solid-state fermentation. Indian Journal of Science and

Technology 3(8): 867-870.

Tariq, A. and Latif, Z., 2012. Isolation and biochemical characterization of bacterial isolates

producing different levels of polygalacturonases from various sources. African Journal

of Microbiology Research, 6(45): 7259-7264.

Teixeira, M.F., Lima Filho, J.L. and Durán, N., 2000. Carbon sources effect on pectinase

production from Aspergillus japonicus 586. Brazilian Journal of Microbiology, 31(4):

286-290.

Thakur, A., Pahwa, R., Singh, S. and Gupta, R., 2010. Production, purification, and

characterization of polygalacturonase from Mucor circinelloides ITCC 6025. Enzyme

Research, 1(2010):7-10.

Thangaratham, T. and Manimegalai, G., 2014. Optimization and production of pectinase using

agro waste by solid state and submerged fermentation. Int. J. Curr. Microbiol. App. Sci,

3(9): 357-365.

Tran, P.P.T. and Le, V.V.M., 2011. Effects of ultrasound on catalytic efficiency of pectinase

preparation during the treatment of pineapple mash in juice processing. Int. Food Res.

J, 18: 347-354.

Tripathi, G.D., Javed, Z. and Sushma, S.A., 2014. Pectinase production and purification from

Bacillus subtilis isolated from soil. AdvApplSci Res, 5(1): 103-105.

86

Varghese, L.K., Rizvi, A.F. and Gupta, A.K., 2013. Isolation, screening and biochemical

characterization of pectinolytic microorganism from soil sample of Raipur city. J Biol

Chem Res, 30(2): 636-643.

Venugopal, T., Jayachandra, K.A. and Appaiah, A., 2007. Effect of aeration on the production

of endopectinase from coffee pulp by a novel thermophilic fungi, mycotypha sp. strain

no. AKM1801. Biotechnology, 6(2): 245-250.

Viswanathan, R. and Jagadeesh Babu, P.E., 2008. Studies on the production of pectinase from

tamarind kernel powder by submerged fermentation using Aspergillus species, and

optimization of medium using design expert. Chemical and Biochemical Engineering

Quarterly, 22(4): 481-489.

Voet, D. and J. G. Voet. 2011. Biochemistry. 4th Ed. John Wiley & Sons, New York. ISBN:

978-1-118-09244-6.

Voragen, F. and Visser, R. 2003. Advances in Pectin and Pectinase Research. eBook 236,81,

ISBN:978-94-017-0331-4.

Wang, S., Lian, Z., Wang, L., Yang, X. and Liu, Y., 2015. Preliminary investigations on a

polygalacturonase from Aspergillus fumigatus in Chinese Pu’er tea fermentation.

Bioresources and Bioprocessing, 2(1): 1.

Whitaker, J.R., 1990. Microbial pectolytic enzymes. In Microbial enzymes and biotechnology

(133-176). Springer Netherlands.

Wilson, K. and Walker, J. eds., 2010. Principles and techniques of biochemistry and molecular

biology. Cambridge university press.

Zakhartsev, M. and Momeu, C., 2007. Purification of glucose oxidase from complex

fermentation medium using tandem chromatography. Journal of Chromatography B,

858(1): 151-158.

Zhong, W.H. and Cen, P.L., 2005. Pectinase production by Aspergillus niger P-6021 on Citrus

Changshan-huyou peel in slurry-state fermentation.Chinese Journal of Chemical

Engineering, 13(4): 510-515.

Zia, M.A., Rahman, K.U., Saeed, M.K., Andaleeb, F., Rajoka, M.I., Sheikh, M.A., Khan, I.A.

and Khan, A.I., 2007. Thermal characterization of purified glucose oxidase from a

newly isolated Aspergillus niger UAF-1. Journal of Clinical Biochemistry and

Nutrition, 41(2): 132-138.

87

Zia, M.A., Sheikh, M.A. and Khan, I.A., 2010. Chemically treated strain improvement of

Aspergillus niger for enhanced production of glucose oxidase. International Journal of

Agriculture and Biology, 12(6): 964-966.

Zubair, H., K. Rehman, M.A. Sheikh, M. Arshard and M.A. Zia. 2002. Optimization of

conditions for glucose oxidase production from Aspergillus niger. Indus. J. Plant Sci.,

2: 184–89.

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