2012 Capstone. Final

8/13/2019 2012 Capstone. Final

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PURIFICATION AND KINETIC PARAMETERS CHARACTERIZATION OF

ALKALINE PROTEASES

THROUGH SUBMERGED FERMENTATION TECHNIQUE.

Submitted to

The Faculty of the Biotechnology

Lovely Professional University,Phagwara

In Partial Fulfillment

Of the requirements for the Degree

Bachelor Of Technology

.

BY

Siddartha Phukan(10904501),

Dapinder Pal Singh(10901023),

Ravikant Rocky(10907506),

Ayush Kaundal (10902530).

Project Supervisor

Er. Robinka Khajuria


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ACKNOWLEDGEMENT

It is our pleasure to be indebted to various people, who directly or indirectly contributed in

the development of this work and who influenced our thinking, behavior, and acts during the

course of study.

We take this opportunity to express my gratitude to our esteemed teacher Dr. Neeta Raj

Sharma Head of School of Biotechnology and Biosciences for his able guidance throughout

the period of this work.

We would also like to express our gratitude to the COD,HOD and the faculty members of the

Department.

We express our sincere gratitude to Er. Robinka Khajuria (mentor) who provided her

valuable suggestions and precious time in accomplishing our project report.

Then, we would like to thank all our team mates who worked cordially and effectively

throughout the time period and helped in completing the report.

We would like to thank the almighty and our parents for their moral support and our friends

with whom we shared day-to-day experience and received lots of suggestions that improved

our quality of work.

THANK YOU!

Siddartha Phukan

Dapinder Pal Singh

Ravikant Rocky

Ayush Kaundal


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CERTIFICATE

This is to certify that Mr. Siddartha Phukan (10904501), Mr. Dapinder Johal (10901023),

Mr. Ravikant Rocky(10907506) and Mr. Ayush Kaundal (10902530), students of B.Tech

(Biotechnology) are pursuing the project titled Purification and Kinetic Parameters

Characterization of an Alkaline Protease through Submerged Fermentation Technique

towards partial fulfillment of requirement for the award of the degree of B.Tech

(Biotechnology).

To the best of my knowledge, the present work is the result of t h e i r originalinvestigation and study. No part of the p r o j e c t r e p o r t has b e e n submitted for

any other degree or diploma.

Date: Signature of Advisor

Name:

UID:


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DECLARATION

We Siddartha Phukan (10904501), Dapinder Johal (10901023), Ravikant Rocky(10907506)

and Ayush Kaundal (10902530), are pursuing the Project titled: Purification and Kinetic

Parameters Characterization of an Alkaline Protease through Submerged Fermentation

Technique for the award of Degree of Bachelor of Technology. The information given in

this project is true to the best of our knowledge.

Date: Investigators

Mr. Siddartha Phukan (10904501)

Mr. Dapinder Johal (10901023)

Mr. Ravikant Rocky (10907506)

Mr. Ayush Kaundal (10902530),

TABLE OF CONTENTS-


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S.No. Page No.

1 Introduction 6

22.12.22.32.42.52.6

2.72.8

Review of LiteratureProteasesSourcesTypes of proteasesProduction techniquesEffectProtease Enzyme Assay:

ApplicationsFuture scope

8-1989

10111218

1819

3 Rationale of study. 19-20

4 Aim and Objective of the study. 20

55.15.25.35.4

5.55.65.75.85.8.15.8.25.9

Research methodology.Collection of soil sampleIsolation of the microorganism.Storage and maintenance of strain.Primary screening for proteases .

Growth of culture.Preparation and storage of crude exract.Screening for the localization of enzymes.Quantitative Assay.Protease Assay.Colour development.Selection of Best producer of each enzyme.

20-2520202020

2020202122232425

66.16.2

6.3

6.47

8

Characterization of protease enzyme.Effect of pH on activity and stability of enzyme.Effect of temperature on activity and stability ofenzyme.Effect of inhibitors, salts and detergents onenzyme activity.Effect of various metal ions on enzyme activity.Expected result

Reference

252526

26

2626

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ABBREVIATIONS


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SmF--------------------------Submerged Fermentation SSF---------------------------Solid State Fermentation EDTA------------------------Ethylenediaminetetraacetic acid HCL--------------------------Hydrochloric Acid SDS---------------------------Sodium Dodecyle sulphate dH2O-------------------------Distilled Water NA----------------------------Nutrient Agar NB----------------------------Nutrient broth DNA--------------------------Deoxyribonucleic Acid MgSo4-------------------------Magnesium Sulphate NaCl---------------------------Sodium chloride FeSo4--------------------------Ferrous sulphate psi-----------------------------Per square inch CaCl2---------------------------------Calcium chloride TCA---------------------------Trichloroacetic Acid BSA---------------------------Bovine Serum Albumin KH2Po4-----------------------Potassiumdihydrogen phosphate Rpm---------------------------Rotation per minute NaOH-------------------------Sodium Hydroxide DNHB Casein--------------3,5-dinitro-2-hydroxybenzyl-casein FITC casein------------------Fluorescein isothiocyanate Casein AIDS-------------------------- Acquired immunodeficiency syndrome SDM--------------------------Site-Directed Mutagenesis PMSF-------------------------Phenylmethyl Sulfonyl fluoride-

1). Introduction


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A living system controls its activity through enzymes. An enzyme is a protein molecule that

is a biological catalyst with three characteristics. First, the basic function of an enzyme is to

increase the rate of a reaction. Most cellular reactions occur about a million times faster than

they would in the absence of an enzyme. Second, most enzymes act specifically with only

one reactant (called a substrate) to produce products. The third and most remarkable

characteristic is that enzymes are regulated from a state of low activity to high activity and

vice versa. The use of enzymes in the diagnosis of disease is one of the important benefits

derived from the intensive research in biochemistry since the 1940's. Enzymes have provided

the basis for the field of clinical chemistry. It is, however, only within the recent past few

decades that interest in diagnostic enzymology has multiplied. Many methods currently on

record in the literature are not in wide use, and there are still large areas of medical research

in which the diagnostic potential of enzyme reactions has not been explored at all. [Zabin

K.et al.,2011]

Enzymes such as proteases, amylases, carboxymethylcellulases, cellulases and lipases are

extensively used in the industries for the manufacture of pharmaceuticals, foods, beverages

and confectioneries as well as in textile and leather processing, paper industry and waste

water treatment. The majority of the enzymes used in the industry are microbial in origin

because microbial enzymes are relatively more stable than the corresponding enzymes

derived from plants and animals.

Naturally-occurring microorganisms are the most productive producers of enzymes. This

knowledge has been exploited by industry for more than 50 years. Bacteria and fungi are the

microorganisms best suited to the industrial production of enzymes. They are easy to handle,

can be grown in huge tanks without light, and have a very fast growth rate.

Enzyme-based biocatalysis provides a means to carry out chemical processes efficiently and

economically. This fact is increasingly recognized as reflected by a rapidly growing enzyme

market which was valued at approximately $1.5 billion already in 2000 (Cherry and

Fidantsef, 2003) and is expected to increase by an average annual growth rate of at least

10%. The future success of enzyme technology will depend on the development of efficient

and cost effective processes for the production and downstream processing of enzymes

.


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Proteases refers to a group of enzymes whose catalytic function is to hydrolyze (breakdown)

proteins. They are also called proteolytic enzymes or proteinases. Proteolytic enzymes are

very important in digestion as they breakdown the peptide bonds in the protein foods to

liberate the amino acids needed by the body. Additionally, proteolytic enzymes have been

used for a long time in various forms of therapy. Their use in medicine is notable based on

several clinical studies indicating their benefits in oncology, inflammatory conditions, blood

rheology control, and immune regulation. Protease is able to hydrolyze almost all proteins as

long as they are not components of living cells. Normal living cells are protected against

lysis by the inhibitor mechanism. The aim of the project is to isolate protease producers from

soil sample and perform qualitative and quantitative assays for protease.


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2. Literature review

2.1 Proteases

Protease refers to a group of enzymes whose catalytic function is to hydrolyze (breakdown)

peptide bonds of proteins. They are also called proteolytic enzymes or proteinases. Proteases

differ in their ability to hydrolyze various peptide bonds. Each type of protease has a specific

kind of peptide bonds it breaks. Examples of proteases include: fungal protease, pepsin,

trypsin, chymotrypsin, papain, bromelain, and subtilisin. Proteolytic enzymes are very

important in digestion as they breakdown the protein foods to liberate the amino acids needed

by the body, proteolytic enzymes have been used for a long time in various forms of therapy.

Their use in medicine is gaining more and more attention as several clinical studies are

indicating their benefits in oncology, inflammatory conditions, blood rheology control, and

immune regulation

Alkaline protease, an enzyme used in the hydrolysis of protein was produced from Bacillus

sp. From a total number of 80 Bacillus strains, 11 were promising based on their ability to

produce clear zones on Nutrient agar plates fortified with 1-2% Casein.Proteases are one of

the most important industrial enzymes and accounting for the 60-65% of total global

industrial enzyme market . Of these, 25% is constituted by alkaline proteases, 3% by trypsin,

10% by rennin and 21% by the other proteases. [Sankar. et al, 2012]

2.2Sources

Industrial enzymes are obtained from three major sources: plant , animal and

microorganisms. The extraction of enzyme from plant or animal sources is limited and

seasonal in case of plant source. However the manufacturing of enzymes by fermentation

process is unlimited and can be produced round the year. Proteolytic enzymes are produced

by a wide range of microorganisms: bacteria, mould and yeast.

Bacillus species are the specific extracellular protease producers in bacterial kingdom.

Bacillusspecies can grow in a pH range of 7.0 11.0 and produces extracellular proteases.

The proteolytic enzymes produced by Bacillus species are used as cleansing additives in


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detergents to facilitate the release of proteinaceous materials in stains due to grime, blood,

milk. [Gul. et al, 2008]

Molds of the generaAspergillus, PenicilliumandRhizopusare especially useful for producing

proteases, as several species of these genera are generally regarded as safe (Sandhya et al.,

2005).

The use of alkaline protease as active ingredient in laundry detergent is the single largest

application of this enzyme (Nehra et al., 2002). For the production of enzymes for industrial

use, isolation and characterization of new promising strain is a continuous process (Kumar et

al., 2002). They are generally produced by using submerged fermentation due to its apparent

advantages in downstream in spite of the cost intensiveness for medium components

(Prakasam et al., 2005). Reports on bleach stable alkaline protease from fungal sources are

scanty (Mulimani et al., 2002). Therefore, a need was felt to explore native fungal isolates,

capable of producing alkaline proteases and at the same relatively stable at the operating

conditions.[Devi. et al,2008]

Table 1: Some sources of Proteases and their industrial Application

Microorganism Type of protease Industry

Bacteria

Bacillus licheniformis Alkaline Detergent

Bacillus amyloliquefaciens Alkaline Detergent

Bacillus flrmus Alkaline Detergent

Bacillus megaterium Alkaline Detergent

Bacillus pumilis Alkaline Detergent

Streptomyces griseus Alkaline, neutral Detergent, leather, food

Bacillus subtilis Neutral Leather, food

Fungi

Aspergillusjlavus Alkaline Detergent

Aspergillus sojae Alkaline , neutral Detergent, leather, food

Aspergillus oryzae Alkaline, neutral Detergent, leather, food

Pericularia oryzae Neutral Leather, food

Endothia parasitica Acid Pharmaceutical, food

Mucor miehei Acid Pharmaceutical, food

2.3) TYPES OF PROTEASES


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Proteases are currently classified into six broad groups with respect to their polarity:

Serine proteases Threonine proteases Cysteine proteases Aspartate proteases Metalloproteases Glutamic acid proteases.

Serine proteases

Serine proteases (or serine endopeptidases) areenzymes that cleavepeptide bonds in

proteins,in whichserine serves as thenucleophilicamino acid at the (enzyme's)active site.

They are found ubiquitously in botheukaryotes andprokaryotes.Serine proteases fall into

two broad categories based on their structure:chymotrypsin-like (trypsin-like) orsubtilisin-

like. In humans, they are responsible for co-ordinating various physiological functions,

including digestion, immune response, blood coagulation and reproduction.

Threonine proteases

Threonine proteases are a family ofproteolyticenzymes harbouring a threonine(Thr) residue

within the active site. The prototype members of this class of enzymes are the catalytic

subunits of theproteasome.

Cysteine proteases

Cysteine proteases also known as thiol proteases are enzymes that degrade polypeptides.These proteases share a common catalytic mechanism that involves a nucleophilic cysteine

thiol in a catalytic dyad.

Cysteine proteases are commonly encountered infruits includingpapaya,pineapple,fig and

kiwifruit. The proportion of protease tends to be higher when the fruit is unripe. In fact,

dozens oflatices of different plantfamilies are known to contain cysteine proteases. Cysteine

proteases are used as an ingredient inmeat tenderizers.
http://en.wikipedia.org/wiki/Serine_proteasehttp://en.wikipedia.org/wiki/Threonine_proteasehttp://en.wikipedia.org/wiki/Cysteine_proteasehttp://en.wikipedia.org/wiki/Aspartate_proteasehttp://en.wikipedia.org/wiki/Metalloproteasehttp://en.wikipedia.org/w/index.php?title=Glutamic_acid_protease&action=edit&redlink=1http://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Peptide_bondhttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Serinehttp://en.wikipedia.org/wiki/Nucleophilichttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Active_sitehttp://en.wikipedia.org/wiki/Eukaryoteshttp://en.wikipedia.org/wiki/Prokaryoteshttp://en.wikipedia.org/wiki/Chymotrypsinhttp://en.wikipedia.org/wiki/Subtilisinhttp://en.wikipedia.org/wiki/Threonine_proteasehttp://en.wikipedia.org/wiki/Proteolytichttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Threoninehttp://en.wikipedia.org/wiki/Catalysishttp://en.wikipedia.org/wiki/Proteasomehttp://en.wikipedia.org/wiki/Cysteine_proteasehttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Catalysishttp://en.wikipedia.org/wiki/Nucleophilehttp://en.wikipedia.org/wiki/Cysteinehttp://en.wikipedia.org/wiki/Thiolhttp://en.wikipedia.org/wiki/Fruitshttp://en.wikipedia.org/wiki/Papayahttp://en.wikipedia.org/wiki/Pineapplehttp://en.wikipedia.org/wiki/Common_fighttp://en.wikipedia.org/wiki/Kiwifruithttp://en.wikipedia.org/wiki/Fruithttp://en.wikipedia.org/wiki/Latexhttp://en.wikipedia.org/wiki/Family_%28biology%29http://en.wikipedia.org/wiki/Meat_tenderizerhttp://en.wikipedia.org/wiki/Meat_tenderizerhttp://en.wikipedia.org/wiki/Family_%28biology%29http://en.wikipedia.org/wiki/Latexhttp://en.wikipedia.org/wiki/Fruithttp://en.wikipedia.org/wiki/Kiwifruithttp://en.wikipedia.org/wiki/Common_fighttp://en.wikipedia.org/wiki/Pineapplehttp://en.wikipedia.org/wiki/Papayahttp://en.wikipedia.org/wiki/Fruitshttp://en.wikipedia.org/wiki/Thiolhttp://en.wikipedia.org/wiki/Cysteinehttp://en.wikipedia.org/wiki/Nucleophilehttp://en.wikipedia.org/wiki/Catalysishttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Cysteine_proteasehttp://en.wikipedia.org/wiki/Proteasomehttp://en.wikipedia.org/wiki/Catalysishttp://en.wikipedia.org/wiki/Threoninehttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Proteolytichttp://en.wikipedia.org/wiki/Threonine_proteasehttp://en.wikipedia.org/wiki/Subtilisinhttp://en.wikipedia.org/wiki/Chymotrypsinhttp://en.wikipedia.org/wiki/Prokaryoteshttp://en.wikipedia.org/wiki/Eukaryoteshttp://en.wikipedia.org/wiki/Active_sitehttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Nucleophilichttp://en.wikipedia.org/wiki/Serinehttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Peptide_bondhttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/w/index.php?title=Glutamic_acid_protease&action=edit&redlink=1http://en.wikipedia.org/wiki/Metalloproteasehttp://en.wikipedia.org/wiki/Aspartate_proteasehttp://en.wikipedia.org/wiki/Cysteine_proteasehttp://en.wikipedia.org/wiki/Threonine_proteasehttp://en.wikipedia.org/wiki/Serine_protease


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Aspartic proteases are a family of protease enzymes that use an aspartate residue for

catalysis of their peptide substrates. In general, they have two highly-conserved aspartates in

the active site and are optimally active at acidic pH. Nearly all known aspartyl proteases are

inhibited by pepstatin.

Metalloproteases

A metalloproteinase, or metalloprotease, is anyproteaseenzyme whosecatalytic mechanism

involves ametal.Most metalloproteases requirezinc,but some usecobalt.The metalion is

coordinated to theprotein via threeligands.The ligands co-ordinating the metal ion can vary

withhistidine,glutamate,aspartate,lysine,andarginine.The fourth coordination position istaken up by alabile water molecule.

Treatment withchelating agents such asEDTA leads to complete inactivation. EDTA is a

metal chelator that removes zinc, which is essential for activity. They are also inhibited by

the chelator .

The threonine and glutamic-acid proteases were not described until 1995 and 2004,

respectively. The mechanism used to cleave a peptide bond involves making an amino acid

residue that has the cysteine and threonine (proteases) or a water molecule (aspartic acid,

metallo- and glutamic acid proteases) nucleophilic so that it can attack the peptide carboxyl

group. One way to make a nucleophile is by acatalytic triad,where ahistidine residue is used

to activateserine,cysteine,orthreonine as a nucleophile.

2.4 PRODUCTION TECHNIQUES

The two important fermentation methodologies include submerged fermentation (SmF) and

solid state fermentation (SSF). Submerged fermentation is more extensively used for

production of enzymes on commercial scale but SSF is also used in certain cases. Economic

feasibility of a production process depends primarily on the strain. Hence high yielding and

stable strains should be chosen by proper screening strategy. Further optimization of process

parameters like media, pH, temperature, aeration, agitation etc by statistical methods will

enhance the cost-effectiveness of a process.
http://en.wikipedia.org/wiki/Metalloproteasehttp://en.wikipedia.org/wiki/Proteasehttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Catalytichttp://en.wikipedia.org/wiki/Metalhttp://en.wikipedia.org/wiki/Zinchttp://en.wikipedia.org/wiki/Cobalthttp://en.wikipedia.org/wiki/Ionhttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Ligandhttp://en.wikipedia.org/wiki/Histidinehttp://en.wikipedia.org/wiki/Glutamatehttp://en.wikipedia.org/wiki/Aspartatehttp://en.wikipedia.org/wiki/Lysinehttp://en.wikipedia.org/wiki/Argininehttp://en.wikipedia.org/wiki/Labilehttp://en.wikipedia.org/wiki/Chelating_agenthttp://en.wikipedia.org/wiki/EDTAhttp://en.wikipedia.org/wiki/Threoninehttp://en.wikipedia.org/wiki/Glutamic_acidhttp://en.wikipedia.org/wiki/Peptide_bondhttp://en.wikipedia.org/wiki/Cysteinehttp://en.wikipedia.org/wiki/Aspartic_acidhttp://en.wikipedia.org/wiki/Carboxylhttp://en.wikipedia.org/wiki/Catalytic_triadhttp://en.wikipedia.org/wiki/Histidinehttp://en.wikipedia.org/wiki/Serinehttp://en.wikipedia.org/wiki/Cysteinehttp://en.wikipedia.org/wiki/Threoninehttp://en.wikipedia.org/wiki/Threoninehttp://en.wikipedia.org/wiki/Cysteinehttp://en.wikipedia.org/wiki/Serinehttp://en.wikipedia.org/wiki/Histidinehttp://en.wikipedia.org/wiki/Catalytic_triadhttp://en.wikipedia.org/wiki/Carboxylhttp://en.wikipedia.org/wiki/Aspartic_acidhttp://en.wikipedia.org/wiki/Cysteinehttp://en.wikipedia.org/wiki/Peptide_bondhttp://en.wikipedia.org/wiki/Glutamic_acidhttp://en.wikipedia.org/wiki/Threoninehttp://en.wikipedia.org/wiki/EDTAhttp://en.wikipedia.org/wiki/Chelating_agenthttp://en.wikipedia.org/wiki/Labilehttp://en.wikipedia.org/wiki/Argininehttp://en.wikipedia.org/wiki/Lysinehttp://en.wikipedia.org/wiki/Aspartatehttp://en.wikipedia.org/wiki/Glutamatehttp://en.wikipedia.org/wiki/Histidinehttp://en.wikipedia.org/wiki/Ligandhttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Ionhttp://en.wikipedia.org/wiki/Cobalthttp://en.wikipedia.org/wiki/Zinchttp://en.wikipedia.org/wiki/Metalhttp://en.wikipedia.org/wiki/Catalytichttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Proteasehttp://en.wikipedia.org/wiki/Metalloprotease


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In general, production of proteases is either a constitutive or partially inducible property. The

culture conditions that promote protease production are found to be significantly different

from that promoting cell growth. The alkaline protease comprises 15.6% nitrogen (Kole et al,

1988). Usually in production of alkaline proteases at an industrial scale technical media are

used (Aunstrup, 1980). There is no defined medium established for production of alkaline

proteases, since the constituents of a medium and their concentrations vary with organism

and fermentation conditions.

Production of Alkaline Protease:

An alkaline protease from Bacillus subtilis was produced under some pre- optimizes

fermentation conditions (4mL/100mL of inoculum size, 7% substrate concentration at pH 11

for 48 hrs fermentation time period and 2% molasses was also used as additional supplement

for substrate to get better production of alkaline protease from Bacillus subtilis. After

stipulated time period, the fermented cultures were harvested by centrifugation at 10000g

for 10 min. at 4C to get clear supernatant containing enzyme solution. The clear supernatant

was used as crude enzyme extract for protease assay and also for purification purposes.

[Ahmed et al, 2011]

2.5 EFFECT

Effect of carbon.

Various sources of carbon such as glucose, maltose, lactose, sucrose, fructose, galactose and

xylose were used to replace lactose which was the original carbon source in growth medium.

Results showed that glucose was found to be the best carbon source that induced the

production of protease by B. subtilis, when compared to other carbon sources. This

observation is in agreement with the production of alkaline protease by Bacilluscereus strain

146.[ Mrudula et al,2012] .


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Effect of nitrogen

Among the nitrogen sources tested, maximum protease production was recorded with urea.

Similar findings have been reported for protease production by Bacillus licheniformis .

Shafee et al.(year)reported that repression of protease with the addition of inorganic nitrogen

sources. Peptone have been reported as best for production of protease by Bacillus

licheniformis, whereas beef extract resulted in the highest level of protease activity.[ Mrudula

et al,2012] .

Effect of temperature.

The effect of temperature on protease activity can be studied by incubating the supernatant at

different temperatures (30, 40, 50, 60, 70 and 80C. The tyrosine equivalent released aremeasured spectrophotometrically at 280nm and the activity can determined using the

standard curve of known concentration for tyrosine as reported by[Lawal et al, 2011.]

Effect of pH.

The effect of different pH values of different buffers on protease activities was studied using

different crude enzymes. The crude protease was incubated at different pH values of different

appropriate buffers ; citrate buffer (pH 48.0), borate buffer (pH 9.0 ) and borate buffer (pH10-12). pH measurements were made by a standard pH meter. [Lawal et al, 2011]

Effect of metal ions

The effect of metal ions on the purified enzyme were determined by treating with different

metals ions including Al2+, Ca2+, Co2+, Cu2+, Fe2+, Hg2+, Mg2+, Mn2+ and Zn2+ at

concentration of 5 mM for 30 min at room temperature. [Sankar et al,2012]

Effect of inhibitors

The protease inhibitors, namely, ethylene diamine tetra acetic acid (EDTA), diisopropyl

fluorophosphates (DFP), dithiothreitol (DTT), and phenyl methyl sulfonyl fluoride (PMSF)

were also tested against the enzyme under optimum reaction conditions. Aliquots of the

protease were pre-incubated with different protease inhibitors at concentration of 5 mM for

30 min at room temperature and the residual activity of the enzyme was assayed. [Sankar et

al, 2012]


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2.6 Protease Enzyme Assay:

Various assays have been reported by different workers to estimate the quantity of enzyme

produced. These assays can be quantitative or qualitative. In both cases proteolytic activity ismeasured either by estimating the products of proteolysis or the residual protein substrate.

Numerous assay and detection methods for proteolytic activity are available with varying

levels of simplicity rapidity and sensitivity of detection limits and ranges. Most often these

methods are tailor made to suit the experimental conditions. [S. M. A. Habib et al. 2011]

Qualitative analysis

Proteolytic activities of Vibro sp., Lactobacillus brevis, Zymomonas sp., Athrobacter sp.,

Corynebacterium sp. andBacillus subtilis were detected on the basis of appearance of clear

zones around the bacterial colonies. Luria casein agar (1 %) plates were used. [Femi-ola et

al,2012]

Quantitative assays

The culture conditions and media for growth of the alkaline protease by the microorganisms

were optimized to give maximum production. In the method described by Mc Donald and

Chen (1965) 2ml of 1% casein was prepared in glycineNaOH buffer (pH 10) was incubated

with 1ml enzyme at 600C for 15 min. The reaction was stopped by adding 3ml 10% TCA

followed by centrifugation. The supernatant was then titrated in 5ml alkaline copper reagent.

After 15 minute 0.5 ml follin reagent was added and absorbance read at 700nm. [McDonald

and Chen,1965]

One unit of enzyme activity was defined as the amount of enzyme which releases a

micromole tyrosine under standard assay condition of 45 C, pH 8.5 and reaction time one

hour. Lowry method was used for protein determination. Similar method has also been

reported in the works of [,Mukhtar and Haq 2008, Femi-ola et al, 2012.]


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An overview of protease assay methods Assay

Assay Substrates Reaction involved

Qualitative assaysProtein agar plate assay Skim milk, casein,

gelatin, BSA, keratin

Enzymatic hydrolysis of

substrate creating a zone

of clearance in culture

Radial diffusion assay Skim milk, casein,

gelatin, BSA, keratin



of clearance in culture

supernatantThin layer enzyme assay Skim milk, casein,

gelatin, BSA, keratin,

fibrinogen, egg-albumin,

mucin, Immunoglobulin

G



of clearance in broth

Quantitative Assays

Spectrophotometric assays

Substrate Wavelength

Casein 700 nm

660nm

750nm

Hammerstein Casein 600nm275nm

DNHB casein 366 nm

Immobilized ostazin

blue S-2G dyed-casein

620 nm

Thermally modified

casein complexed with

black drawing ink

400 nm


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Azocasein 440 nm

340 nm

420 nm

480 nm

Thermally modified

azocasein

366400 nm

Azoalbumin 440 nm

bl-Crystalline aggregate 405 nm

Thermally modified

gelatin complexed with

congo red or nigrosin

490, 570 nm

Chemically modified

(formaldehyde/

gluteraldehyde

mediated) gelatin

complexed with black

drawing ink

800900 nm

Tripeptide substrate 400 nm

Fluorescent oligopeptide energy transfer assay

Dansylated hexapeptide 310410 nm

ELISA-based protease assay

Biotinylated BSA 405 nm

Magnet-based protease assay

Magnet dye stained

gelatin

605 nm

Fluorescence-based protease assay

FITC casein, FTC

hemoglobin 575 nm.

575 nm with excitation at

490 nm


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2.7Applications

Proteases are commercially important industrial enzymes accounting 60% of the total enzyme

sales with two thirds of the proteases produced are from microorganisms. Microbial enzymes

are replacing chemical catalysts in manufacturing chemicals, food, leather goods,

pharmaceuticals, and textiles. Among proteases, alkaline proteases are employed mainly as

detergent additives because of their distinctive abilities to assimilate proteinaceous stains

such as blood, chocolate, and milk. Currently, alkaline protease-based detergents are

preferred over the conventional synthetic detergents, as they have better cleaning properties,

higher efficiency at lower washing temperatures, and safer dirt removal conditions.

Preferably, proteases used in detergent formulation must have a high activity level and

stability over a wide range of pH and temperature. One of the major drawbacks affecting the

stability of enzymes recovered from thermophiles at alkaline pH is that enzymes from

alkalophiles confer stability over wide pH range but are generally thermolabile. So, there is

always a need for proteases with all desirable properties to become accustomed with

application conditions, and also, it is necessary to check the stability of the enzyme at

elevated temperatures and pH. Applications, such as protease for detergent industries need

concentrated and cleaned enzyme to amend with detergent to get good performance during

storage and application as well. The enzyme is cleaner when the medium is simple and

defined, where, as in case of sludge medium, fermented enzyme contains many other sludge

particles and other impurities, so enzyme has to be clarified and concentrated to get higher

activity. [Bezawada et al, 2011]

2.8 Future scope

Proteases are a unique class of enzymes, since they are of immense physiological as well as

commercial importance. They possess both degradative and synthetic properties. Sinceproteases are physiologically necessary, they occur ubiquitously in animals, plants, and

microbes. However, microbes are a goldmine of proteases and represent the preferred source

of enzymes in view of their rapid growth, limited space required for cultivation, and ready

accessibility to genetic manipulation. Microbial proteases have been extensively used in the

food, dairy and detergent industries since ancient times. There is a renewed interest in

proteases as targets for developing therapeutic agents against relentlessly spreading fatal

diseases such as cancer, malaria, and AIDS. Advances in genetic manipulation ofmicroorganisms by SDM of the cloned gene opens new possibilities for the introduction of


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predesigned changes, resulting in the production of tailor-made proteases with novel and

desirable properties. The advent of techniques for rapid sequencing of cloned DNA has

yielded an explosive increase in protease sequence information. Analysis of sequences for

acidic, alkaline, and neutral proteases has provided new insights into the evolutionary

relationships of proteases. Despite the systematic application of recombinant technology and

protein engineering to alter the properties of proteases, it has not been possible to obtain

microbial proteases that are ideal for their biotechnological applications. Industrial

applications of proteases have posed several problems and challenges for their further

improvements. A recent trend has involved conducting industrial reactions with enzymes

reaped from exotic microorganisms that inhabit hot waters, freezing Arctic waters, saline

waters, or extremely acidic or alkaline habitats. The proteases isolated from extremophilic

organisms are likely to mimic some of the unnatural properties of the enzymes that are

desirable for their commercial applications. Exploitation of biodiversity to provide

microorganisms that produce proteases well suited for their diverse applications is considered

to be one of the most promising future alternatives. Introduction of extremophilic proteases

into industrial processes is hampered by the difficulties encountered in growing the

extremophiles as laboratory cultures. Revolutionary robotic approaches such as DNA

shuffling are being developed to rationalize the use of enzymes from extremophiles. The

existing knowledge about the structure-function relationship of proteases, coupled with gene-

shuffling techniques, promises a fair chance of success, in the near future, in evolving

proteases that were never made in nature and that would meet the requirements of the

multitude of protease application.

A century after the pioneering work of Louis Pasteur, the science of microbiology has

reached its pinnacle. In a relatively short time, modern biotechnology has grown dramatically

from a laboratory curiosity to a commercial activity. Advances in microbiology and

biotechnology have created a favorable niche for the development of protease and will

continue to facilitate their applications to provide a sustainable environment for mankind and

to improve the quality of human life.


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3)RATIONALE OF STUDY

The proteases available today in the market are derived from microbial sources. This is due to

their high productivity, limited cultivation space requirement, easy genetic manipulation,

broad biochemical diversity and desirable characteristics that make them suitable for

biotechnological applications.

Production of enzymes by microorganism is a wide field of application of enzyme

technology in which cheap raw materials or the byproducts can be used as substrate for the

production of useful products. The aim of our work is to isolate high-yielding strains of

protease producers and optimize their production method.


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4) AIMS AND OBJECTIVES

For the proposed investigation, soils samples from the areas around Tanneries, Milk

Processing industries will be investigated for the Isolation of Strains producing novel alkaline

proteases. The following studies were undertaken to achieve the same:

1. Isolation of protease producers from soil sample.2. Qualitative and Quantitative assays for Proteases.3. Characterization of the isolates.4. Estimation optimum temperature for enzyme activity.5. Estimation of optimum pH for maximum enzyme activity.6. Effect of different carbon sources of enzyme production.7. Effect of metal ions on enzyme activity.8. Effect of inhibitors on enzyme activity.


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5) RESEARCH METHODOLOGY

Collection of Soil samples

Isolation of the Microorganism

Storage and Maintenance of Strains

Primary Screening for Protease producing organisms

Growth of Culture

Preparation and Storage of Crude Extract

Characterization of Cultures

Screening for the localization of Enzymes (extracellular and intracellular) of the

Isolated strains

Quantitative Assay

Selection of Best producer of each enzyme

Optimization of Conditions (pH, Temperature, Carbon sources, Metal ions) for optimum enzyme

product


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5.1 Collection of Soil samples:

We will collect different soil samples from different region of the city, generally from areas

where tannery or detergent industries are situated.

5.2 Isolation of the Microorganism:

0.5g of each soil sample will be dissolved in sterile distill water. Serial dilutions upto 10-3for

each sample will be carried out. 200l of these dilutions will be spread on nutrient agar

plates and incubated at 370Cfor 12 hours.

Also, 200l of undiluted samples will be spread plated on nutrient agar respectively and

incubated at the similar conditions. The morphologically different colonies obtained will be

further streaked on NA plates.

5.3 Storage and Maintenance of Strains:

The strains isolated will be stored at 4oC and maintained in the active stage by transferring

aseptically on fresh plates of NA from time to time.

5.4 Primary Screening for Protease producing organisms:

NA plates containing casein were prepared. The various cultures obtained above will be

streaked and incubated for 24 hours at 37 0celsius. A strong halo around the colony will

indicated the presence of Protease activity.

5.5 Growth of Culture:

Depending upon the size of the zone of clearance the best producers of each enzyme will be

selected. And will be grown in alkaline broth medium at 35C with gentle shaking

5.6 Preparation and Storage of Crude Extract:

The cells will be then centrifuged at 8000Xg for 7-8 minutes. The culture media.i.e,

supernatant will be saved for further testing.. The cell suspension will be kept at -20oC .The

pellet will be subjected to cell lysis by the following procedure:

Lysis Buffer will diluted with d.H2O in the ratio of 1:1.The pellet will be dissolved in 200l

of lysis buffer and incubated at 37oC for 15 minutes. The cell lysate will centrifuged at 18000

rpm for 10 minutes. The supernatant obtained i.e cell free extract will be transferred to fresheppendroffs and stored at -200C for further testing.


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.

5.7 Screening for the localization of Enzymes (extracellular and intracellular) of the

Isolated strains.

0.5g of casein powder will be dissolved in 5ml of sterile distill water and added to 95ml of

hot solution of 1.2% (w/v) agar in water. 20 ml of this emulsion will be poured in flat bottom

plastic petri dishes and left to solidify. Holes of 5 mm will be bored with a borer and 200l

supernatant and 100l of cell free extract of each sample will be loaded in respective wells.

The plates will be incubated at 30C till the zone of clearance appeared.

5.8 Quantitative Assay:

5.8.1 Protease Assay:

The following two Assay Protocols will be used for calculating the protease activity.

The following concentrations of given solutions were pipette into suitable vials for each test

sample:

Solutions Test samples Blank

0.65% casein in 50mM

phosphate buffer(pH 7.4)

5ml 5ml

Enzyme Supernatant 200l; Cell Free

Extract 100 l

----

Mixed by swirling and incubated at 370C for exactly 10 minutes. Then following solution

will be added

110mM Tricholoro

Acetic Acid

5ml 5ml

Enzyme ---- Supernatant 200l; Cell Free

Extract 100 l

Mixed by swirling and incubated at 37 C for about 30 minutes. Centrifugation will be

carried out at 15000xg for 3 mins.


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5.8.2 COLOR DEVELOPMENT:

Test Filterate 2ml ---

Blank Filterate ---- 2ml

500mM Sodium Bicarbonate 5ml 5ml

Folin & Ciocalteu's Phenol

Reagent(Diluted 4 folds)

1ml 1ml

Mixed by swirling and incubated at 37 C for 30 minutes. Centrifugation will be carried out

at 15000xg for 3 mins. The absorbance will be taken at 660nm.Standards using 1.1mM stock

solution of tyrosine will be made and subjected to same treatment. One unit will be defined as

the amont that hydrolyzed casein to produce color equivalent to 1.0 M mole (181 mg) of

tyrosine per minute at pH 7.5 at 37 C

5.9 Selection of Best producer of each enzyme:

On the basis of units calculated from the above mentioned assays highest enzyme producing

strain for each enzyme will be selected and subject to further analysis

6) CHARACTERIZATION OF PROTEASE ENZYME

6.1 Effect of pH on activity and stability of enzyme

The protein stability at different pH values will be determined by incubating purified

enzymes using different buffers: 0.1 M sodium citrate (pH 56); 0.1 M potassium phosphate

(pH 68); 0.1 M Tris-HCl (pH 79) and 0.1 M glycine-NaOH (pH 910) for 24 h at 30C.

The residual protease activities will be then determined using casein as substrate.

The dependence of reaction rate on pH will be measured at different pH values in the range

of 6.010. The reaction mixture will be that of standard assay with purified enzyme and the

pH will be adjusted to the different values by addition of either 1M HCl or 2M NaOH.

6.2 Effect of temperature on activity and stability of enzyme

The effect of temperature on the stability of enzyme will be determined by incubating

aliquots of purified enzyme for 30 min in 50 mM Tris-HCl buffer, pH 8 at different

temperature (1060C).


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Residual enzyme activity will be measured spectrophotometrically using casein as substrate.

The optimum temperature for enzyme activity will be determined with casein as the substrate

by incubation of reaction mixtures at different temperatures in the range of 15-75oC.

6.3 Effect of inhibitors, salts and detergents on enzyme activity

The inhibitory effect of selected inhibitors, detergents and salts on the activity of purified

enzyme will be investigated. Incubation of the enzyme for 30 min at 30C with divalent

metal-chelating agent EDTA at a concentration of 1 mM and 5mM will be carried out to

determine metalloprotein nature of the enzyme.

Effect of different salts on the protease activity will be also determined. Effect of PMSF at a

concentration of 1 mM and 5mM will be studied by incubating the enzyme at 30C for 30min. Effect of detergents on the enzyme activity will be analysed by incubation of the

enzyme for 30 min at 30C with 2% & 8% SDS. Effect of -Mercaptoehanol at a

concentration of 1 mM and 5mM will be studied by incubating the enzyme at 30C for 30

min. Then the normal protease assay will be carried out and absorbance will measured.

6.4)Effect of various metal ions on enzyme activity

The enzyme solution will be incubated with an equal volume of monovalent and divalentmetal ions(Ca2+,Mn2+,Mg2+,Fe2+,Cu2+,Ag+, Hg+) at 30C for 30 min in 50 mM phosphate

buffer,pH 7.5. The residual activity will be determined by the protease assay.


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7) Expected Outcome

As per the methods describe in the methodology protease producing microorganisms will be

isolated from the soil samples collected from regions surrounding Tanneries and Milk-

processing industries. These isolates will then be screened for the production of proteases.

Based on the diameter of Zone of Clearance formed good producer will be selected and assay

to check the localization of the enzyme will be performed followed by quantitative analysis to

estimate amount of enzyme produced. The strain producing highest amounts of enzyme will

then used for optimization of conditions( pH, Temperature, Effect of metal ions, inhibitors,

carbon sources) for maximum enzyme yield.


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8) References

1. Ahmed I, AZ Muhammad, MNI Hafiz. Purification and kinetic parameterscharacterization of an alkaline protease produced from Bacillus subtilis through

submerged fermentation technique. World Applied Sciences Journal . 12 (6): 751-57,

2011

2. Bezawada J, Yan S, Rojan p.j ,Tyagi R.D, Surampalli R.Y. Recovery of Bacilluslicheniformis Alkaline Protease from Supernatant of Fermented Wastewater Sludge

Using Ultrafiltration and Its Characterization , Biotechnology Research

International.10(1):11-12,2011

3. Cherry JR, Fidantsef AL. Enzyme-based biocatalysis, Novozymes Biotech, Inc.,14(4):438-441.2003

4. Devi MK, Banu AR , Gnanaprabhal GR , Pradeep BV , Palaniswamy M.Purification. characterization of alkaline protease enzyme from native isolate

Aspergillus nigerand its compatibility with commercial detergents, Indian Journal of

Science and Technology.1(7) ,2008

5. Femi- ola TO, bamidele OS. Studies on the catalytic properties of partially purifiedalkaline proteases from some selected microorganisms,Malaysian journal of

microbiology,8(3):191-196,2012

6. Gul S, Rahman MU, Achakzai AKK , Khan K. Production of extracellular proteaseby locally isolated bacillus subtilis ic-5 using agriculture byproducts,

J.Chem.Soc.Pak.30(6),2008

7. Lawal AK, Olatope SO, Majolagbe YL, Alebiosu FA, Bashar JB, Kayode OF, DikeEN, Akinola SO and Elemo GN. Microbial production of alkaline protease, Prime

Journal of Microbiology Research . 1(2):27-37,2011

8.

Mrudula S, A. Apsana Begum, K. Ashwitha and Pavan Kumar Pindi. Enhancedproduction of alkaline protease by bacillus subtilis in submerged fermentation, Int J

Pharm Bio Sci . 3(3):619-631,2012

9. Muhammad N, Qazi JI, Baig S.effect of aeration and agitation rates on alkalineprotease production by bacillus licheniformis UV-9 mutant ,Turkish Journal of

Biochemistry,34(2):89-96,2009

10.Mukhtar H,IU Haq, production of alkaline protese by bacillus subtilis and itsapplication as a depilating agent in leather processing,Pak. J. Bot.40(4):1673-1679,2008


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11.McDonald CE and LL Chen Lowry modification of the Folin reagent fordetermination of proteinase activity. Ann. Biochem., 10: 175, 1965.

12.Sankar R , Deepthi , Kumar K, Lavanya , Ravi P , Sadhna , Kumar B. Purificationand characterization of an extracellular alkaline serine protease from Bacillus subtilis

NR 18, International Journal of Current Research,4(3):98-103, 2012

13.Singhal P, NigamV.K, VidyarthiA.S. Studies on production , characterization andapplications of microbial alkaline proteases, International Journal of Advanced

Biotechnology and Research, 3(3): 653-669,2012

14.Williamson LL, Borlee BR, Schloss PD, Guan C, Allen HK, Handelsman J. Intracellularscreen to identify metagenomic clones that induce or inhibit a quorum-sensing biosensor.

Appl Environ Microbiol71:63356344,2005

15.Zabin K. Bagewadi,Swati D. Garg, Pradeep B. Deshnur, Nayana S. Shetti, Productiondynamics of extracellular alkaline protease from Neisseriasps. isolated from soil,

Research Article, Biotechnol. Bioinf. Bioeng. 2011, 1(4):483-493

16.S. M. A. Habib1, A. N. M. Fakhruddin1*, S. Begum2 and M. M. Ahmed3 Isolationand Screening of Thermostable Extracellular Alkaline Protease Producing

Bacteria from Tannery Effluents Publications journal of scientific research J. Sci. Res.

4 (2), 515-522 (2012)


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2012 Capstone. Final