INTRODUCTION - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/9853/7/07_chapter 1.pdfTechnology means the application of science for ... All living cells contain different types

INTRODUCTION

Murali Krishna. CH Page 1

1.0 INTRODUCTION

Biotechnology is the use of living organisms, either in natural or

modified form, for commercial or industrial purpose. Bio means the

science of life that includes all living organisms (Getu et al, 2011).

Technology means the application of science for commercial and

industrial product production. Biotechnology is itself not a product or

range of products; rather it should be regarded as a range of enabling

technologies involving the practical application of organisms or their

cellular components to manufacturing and service industries

associated with environmental management (James et al, 2011).

Biotechnology, among all technologies, is the fast growing applied

science today. (Stephen et al, 2000).

Unlike a single scientific discipline, biotechnology is also

commonly referred to as the clever science of biology forming a link

between the biological, physical sciences and technological

achievements. In fact, it is an amalgamative field of microbiology,

biochemistry, molecular biology, cell biology, immunology, protein

engineering, enzymology, classified breeding techniques and the full

range of bioprocess technologies (Nelo et al, 2001).

Historically, biotechnology was an art rather than a science,

exemplified in the manufacture of wines, beers, cheeses, etc, where

the techniques of manufacture were well worked out and reproduced

but the molecular mechanisms were not understood ( Bhat et al,

2000) .With the major advances in microbiology and biochemistry,

these processes have been better understood and improved (Gurpreet

et al, 2011).Modern biotechnological processes now encompass a wide

range of new products including enzymes, antibiotics, vaccines and

monoclonal antibodies, the production of which has been optimized by

a host of new molecular innovations, allowing unprecedented changes

to be made to living systems (Rani et al, 2003).

INTRODUCTION

Murali Krishna .CH Page 2

1.1 ENZYMOLOGY

Enzyme technology is an off-shoot of fundamental science

related to cellular metabolism. With the development of science of

microbiology, biochemistry, a better understanding of the wide range

of enzymes present in living cells and their mode of action was

achieved (Reeta et al, 2009).Although enzymes are formed only in

living cells, many can be isolated without loss of catalytic function in

vitro (Kiran et al, 2010).This unique ability of enzymes to perform their

specific chemical transformations in isolation has led to an ever-

increasing use of enzymes in industrial processes, collectively termed

as “enzyme technology” (Ajay et al, 2010).

1.1.1 SOURCES OF ENZYMES

All living cells contain different types of enzymes and without

which none of the living body survives. Depending upon the need of

activity enzymes are produced extracellularly or intracellularily (Joel

et al, 1998). Enzymes can be obtained from plant (-amylase, papain,

bromelain, urease, ficin, polyphenol oxidase (tyrosinase),

lipoxygenase, etc.), animal (Pepsin, lipase, lysozyme, rennin, trypsin,

phosphor-mannase, chymotrypsin, etc.) and microbial (-amylase,

pencillin acylase, protease, invertase, lactase, dextranase, pectinase,

pullulanase, etc.) sources (Zheng et al, 2011).

In general, the enzymes from plant and animals are considered

to be more important than those from microbial sources, but for both

technical and economical reasons, microbial enzymes are considered

to be more important (Sumitra et al, 2004). Therefore increasing

efforts are being pursued to produce enzymes by microbial

fermentation (Arpana et al, 2011).

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1.1.2 ADVANTAGES OF MICROBIAL ENZYMES

Animal sources for enzymes are very limited.

Microorganisms are attractive because of their biochemical

diversity.

Microbes have short generation time and require smaller

area; 20 kg of rennin is produced in 12 h by B. subtilis with

100 liter fermentor whereas one calf stomach gives 10 kg

after several months.

Microbial feasibility of bulk production and ease of

extraction.

Microbes use inexpensive media for growth and production of

enzymes.

Microbes screening is easy compared to plant and animal

sources for enzyme production.

Microbial strains are vernalable for genetic engineering to

produce abnormally huge amounts.

Synthesis of foreign enzymes is possible by genetically

engineered microorganisms.

Microbes do not show any seasonal variations unlike plants.

1.1.3 INDUSTRIAL IMPORTANCE OF MICROBIAL ENZYMES

Microbial enzymes are widely used in several industries,

notably in detergent, food processing, brewing and pharmaceuticals

(Vander et al, 2002).In fact, their use has been recorded since ancient

times without known the functional utility in oriental countries

(Samrat et al, 2011).They are also used for diagnostic, scientific and

analytical purposes (Biazus et al, 2007).In the present era, enzymes

such as proteases, glucoamylases, glucose isomerase, and pectinases

became part of our daily life and extensively used as commodities

(Maria et al, 2005). Some of the microbial enzymes used industrially

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are shown in Table 1.1. It may be noted that most of these are

hydrolases.

Most industrially important enzymes are extracellular i.e.

secreted by the cells into the ambient medium and they have to be

recovered by removal and separation from the cellular and other solid

material (Masafumi et al, 2010).

1.1.4 DETERMINATION OF ENZYME ACTIVITY

The enzyme activity is determined using substrate, cofactors,

allosteric effectors, the concentration and type of inhibitors, ionic

strength, pH, temperature and initial reaction time etc (Aliyu et al,

2011).

Many assay procedures for measurement of enzyme activity are

available. The rate of substrate conversion serves as a measure of the

activity (Aw et al, 1969).The knowledge of enzyme activity is necessary,

to follow the production and isolation of enzymes, to understand and

determine the properties of commercial preparations and to ascertain

the correct amount of enzyme to be added to a particular commercial

process (Lonsane et al, 1990).

The first step in deciding on a suitable assay is to choose the

appropriate substrate. Some of the substrates that have been used

for the assay of hydrolases are as follows (Cynthia et al, 2011).

Amylases and Amyloglucosidases: Raw or soluble starch and

modified starch of known dextrose equivalent.

Cellulases: Cellulose powder, cellular phosphate, filter paper and

ground bran.

Pectinases: Pectic acid, pectin, pectinic acid and freeze-dried fruit

puree.

Proteases: Casein, egg albumin, gelatin, hemoglobin, milk powder

and raw meat. (Pandey et al, 2000)

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TABLE 1.1: MICROBIAL SOURCES OF SOME REPRESENTATIVE

ENZYME USED INDUSTRIALLY

Enzyme Source

Amylase Aspergillus oryzae, B. licheniformis, B.cereus, B.

megaterium, B.polymyxa.

Cellulase Aspergillus niger., Trichoderma reesei.

Dextranase Penicillium sp., Trichoderma sp.

Glucoamylase A. niger, Rhizopus sp.

Glucose Isomerase Bacillus coagulans, Actinoplanes, sp.,

Arthrobacter sp., Streptomyces sp.

Invertase Saccharomyces cerevisiae

Lactase Kluyveromyces fragilis, K.lactis, A.niger

Lipase Rhizopus sp., Candida lipolytica, Geotrichum

candidum.

Pectinase Aspergillus sp.

Protease Aspergillus sp., Bacillus sp., Streptomyces griseus

Rennet Mucor pusillus, Endothia parasitica.

Once the substrate is selected, the assay is carried out under

predetermined temperature, pH and incubation period (Ashis et al,

2009). At the end of incubation period, the reaction is readily stopped

by the use of pH change or heat or by adding sufficient enzyme

inhibitor (Elizibath et al, 1998). The extent of reaction is then

determined by a suitable chemical or physical method (Maryam et al,

2010).

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1.2 FERMENTATION STUDIES FOR IMPROVED PRODUCT

PRODUCTION

The term fermentation is derived from the Latin word fervere to

boil thus describing the appearance of the action of the yeast on

extracts of fruit or malted grain. The boiling experience is due to the

production of CO2 bubbles caused by the anaerobic catabolism of the

sugars present in the extract (Sangeeta et al, 2009).

Pasteur applied the term “fermentation” to those anaerobic

reactions through which microorganisms obtained energy for growth

in the absence of oxygen. Today fermentation has much broader

meaning (Balasubramaniam et al, 2011). It applies to both aerobic

and anaerobic metabolic activities of the microorganisms wherein

specific chemical changes are brought about by an organic in

substrate (Nabuo et al, 2011).

A variety of substances such as alcohols, organic acids, amino

acids, vitamins, antibiotics, enzymes, single cell proteins, hormones

etc, are produced through fermentations by employing different

microorganisms (Maryam et al, 2011). The success of fermentation

greatly depends on the use of right type of organism that can produce

the desired product at minimum cost and in large quantities (Maria et

al, 2011). There are different types of fermentations in usage for the

production of various industrial products. They include solid state,

submerged, dual or multiple fermentations (Ya-Lie Tang et al, 2011).

1.2.1 SUBMERGED FERMENTATION

In this type of fermentation organisms grew in a vigorously

aerated and agitated liquid nutrient medium in fermenters (Abdul et

al, 2010).These fermenters are usually made of non-corrosive type of

steel may be either open tank or closed tank and the type of

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fermentation may be either batch or continuous type (Shigetoshi et al,

1993).

1.2.1.1 BATCH FERMENTATION

In this type, organism is grown in a known amount of

culture medium for a defined period of time. The growth &

biochemical synthesis are allowed to proceed until maximum yields

have been obtained (Sangeeta et al, 2009). At this point fermentation

is stopped for recovery of the product. Then the fermenters are

cleaned resterilized and another batch of fermentation is started (Haq

et al, 1998).

During the process of fermentation neither inoculum nor

nutrient solution is added (Speight et al, 2009). However oxygen in the

form of air, an antifoaming agent and acid or base to control pH is

added (Vidyalakshmi et al, 2009). Due to metabolism of the cells the

composition of the culture medium, cell concentration will change

constantly from time to time ( Hashemi et al, 2011).

In a closed type of fermentation the multiplying micro

organisms exhibit 4 typical characteristic growth phases, lag phase,

log phase, stationary phase and death phase.

A modified and enhanced mode of conventional closed batch

fermentation is called fed batch fermentation (Ya-Jie Tang et al, 2009).

This is characterized by the addition of substrate in increments as the

fermentation is progressed (Gunjan et al, 2011).

1.2.1.2 CONTINUOUS FERMENTATION

Continuous fermentations are those in which fresh nutrient

medium is added continuously or intermittently to the fermentation

vessel, accompanied by a corresponding continuous or intermittent

withdrawal of portion of the medium for recovery of cells or

fermentation products (Gulay et al, 2004).

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1.2.2 IMMOBILIZATION

Immobilization of cells is the attachment of cells or their

inclusion in distinct solid phase that permits exchange of substrates

and products, inhibitors, but at the same time it separates the

catalytic cell biomass from the bulk phase containing substrates and

products, therefore it is expected that the microenvironment

surrounding the immobilized cells do not necessarily be the same as

their free-cell counter parts experience (Zoe et al, 2006).

Immobilization commonly is accomplished using high molecular

hydrophilic polymeric gels such as alginate, carrageenan, agarose, etc.

In these cases, the cells are immobilized by entrapment in the

pertinent gels (Arpana et al, 2011).

1.2.2.1 ADVANTAGES OF IMMOBILIZED CELLS OVER FREE CELLS

The use of immobilized whole microbial cells and/or

organelles eliminates the often tedious, time consuming, and

expensive steps involved in isolation and purification of extra cellular

enzymes (Noda et al, 2001). It also tends to enhance the stability of

the enzyme by retaining its natural catalytic surroundings during

immobilization and subsequent continuous operation (Dhanya et al,

2009). The ease of conversion of batch processes into a continuous

mode and maintenance of high cell density without washout

conditions even at very high dilution rates, are a few of the many

advantages of immobilized cell systems (Gulay et al, 2006). Recent

reports on higher retention of plasmid-bearing cells have further

extended the scope of whole-cell immobilization to recombinant

product formation. Another important advantage of immobilization,

particularly in the case of plant cells, is the stimulation of secondary

metabolite formation and elevated excretion of intracellular

metabolites (Mosafumi et al, 2010).

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The use of immobilized system offers many advantages over

conventional free cell fermentation including (Arpana et al, 2011).

Prolonged activity and stability of the biocatalyst.

Higher cell densities per unit bioreactor volume, which

leads to high volumetric productivity, shorter

fermentation times and elimination of non-productive cell

growth phases.

Increased substrate uptake and yield improvement.

Feasibility of continuous processing.

Increased tolerance to high substrate concentration and

reduced end product inhibition.

Feasibility of low-temperature fermentation leading to

improved product quality.

Easier product recovery through reduction of separation

and filtration requirements, thus reducing cost for

equipment and energy demands.

Regeneration and reuse of the biocatalyst for extended

periods in batch operations, without removing it from the

bioreactor.

Reduction of risk of microbial contamination due to high

cell densities and fermentation activity.

Ability to use smaller bioreactors with simplified process

designs and therefore lower capital costs.

Reduction of maturation times for some products.

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1.2.3 SOLID STATE FERMENTATION

Solid-state fermentation (SSF) may be defined as the

fermentation involving solids in absence or near absence of free water

(Audinarayana et al, 2005). But the substrate, which is used for

growth and metabolism of the microorganisms, must have enough

moisture (Pandey et al., 2000). SSF holds potential for the production

of enzymes and credited as the beginning of the fermentation

technique in ancient time (Reeta et al, 2009). Therefore it is not

surprising that all the fermentation processes used in ancient time

were based on the principles of SSF (Ashis et al, 2009).

SSF can be the best being employed for the processing of the

agric- industrial residues (Gurpreet et al, 2011). Because solid-state

processes have lower energy requirements, produce lesser wastewater,

and are environment friendly to resolve the problem of solid waste

disposal (Ajay et al, 2010). Further utilization the agro industrial

residues in the SSF offer a unique process development for value

addition of these low cost residues (Pushpa et al, 2009). At present

SSF processes are used at commercial scale for the production of

microbial products such as feed, fuel, food, industrial chemicals and

pharma products (Sarc et al, 2002). Its application in bioprocess such

as bioleaching, biobeneficiation, bioremediation, biopulping etc, has

offered several advantages (Pandey et al, 2000).

The key aspect of the SSF is the selection of proper substrate,

which should be in-soluble and should acts both as a physical

support and source of nutrients (Reeta et al, 2009). The substrate

should be a solid material, which can be naturally occurring such as

agric crops materials, agro industrial residues or inert supports (Lin

Hui et al, 2010). There are two major considerations for the selection

of substrate; one is a specific substrate, which requires suitable value

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addition or disposal (Maryam et al, 2010). The second could be related

with the goal of producing a specific product from suitable substrate

(Frank et al, 2011).

Agricultural industrial residues are generally considered the

best substrates for SSF processes (Cynthia et al, 2011). Some of the

substrates that have been used in SSF process includes Cane

bagasse, Wheat bran, Maize bran, Gram bran, Wheat bran, Rice

straws, Rice husk, Soy hull, grape vine, Trimmings, Saw dust, Banana

waste, Tea waste, Palm oil waste, Sugar beet pulp, Sweet sorghum

pulp, Apple pomace, peanut meat, coconut & Mustard oil cake, wheat

& Corn flours, Steamed rice and Starch etc (Pushpa et al, 2009).

However wheat bran holds the key and the most commonly

used in various processes (Lin Hui et al, 2010). The selection of

substrates for enzymes depends upon several factors, mainly the cost

of substrate (Frank et al, 2011). The substrate may provide the needed

nutrients to the microorganisms growing in it, but some of the

nutrients may be present in sub optimal concentrations or even

absent in the substrate. In such cases this can be over come by

supplying nutrients externally (Ya-Jie Tang et al, 2011).

The particle size of the substrate is critical usually smaller

substrate particles provide larger surface for microbial attack (Chen et

al, 2011). The other important factor is the moisture content; the

water activity (aw) of the medium has been attributed as a

fundamental parameter for mass transfer of the water and solute

transfer across the microbial cells (Umberto et al, 2011). This

parameter could be used to modify the metabolic production or

excretion of microbial product so that water has profound impact on

the physicochemical properties of the solids and this in turn effect the

overall process productivity (Solange et al, 2010). Other parameters

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that influence the product production under SSF include incubation

temperature, medium pH and available surface area (Parveen et al,

2011, Maryam et al, 2010).

1.3 STRATEGIES TO IMPROVE THE ENZYME PRODUCTIVITY

BY STATISTICAL METHODS

The traditional pattern of performing experiments to optimize a

production process using an experimental design by identifying

various independent factors and levels, and later conducting the

experiments by altering “one variable at a time” (OVAT), while keeping

all others at a predetermined level is very inefficient in many cases,

since it involves carrying out many experiments which are time-

consuming and laborious (Aliyu et al, 2011). Moreover, these OVAT

designs often overlook the interactions among the variables.

Statistically designed experiments consist of several well-planned

individual experiments conducted together (Priya et al, 2011).

Normally in designing of a statistical based experiment, it involves

several steps such as

Selection of responses (performance characteristics of interest)

that will be observed.

Identification of the factors (the independent or influencing

factors) to be studied.

The different treatments (or levels) at which these factors will be

set in different individual experiments.

Consideration of blocks (the observable noise factors that may

influence the experiments as a source of error of variability).

The major drawback of the statistical approach is that there are

no precise guidelines for the sequence of experiments to be conducted

and the level combinations of different independent variables for each

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experiment (Radhouane et al, 2008). The system of laying out the

conditions of experiments involving multiple factors was first proposed

by Sir R.A. Fisher in 1920s, popularly termed as “fractional design of

experiments” (Aliyu et al, 2011). A full fractional design identifies all

the possible combinations for a given set of factors (Kiranbabu et al,

2010).Since most industrial experiments usually demand a significant

number of factors, a full factorial design results in performing a large

number of experiments (Chi-Zhang et al, 2010). To reduce the number

of experiments to a practical level, only a small set from all the

possibilities is selected (Jiangya et al, 2011). The method of selecting a

limited number of experiments which generates the most information

is known as a partial fractional design (Hashemi et al, 2011).

1.4 AMYLASES

Amylases are amongst the most studied enzymes (Noomen et al,

2009). There is renewed interest in the study of proteolytic enzymes,

mainly due to the recognition that these enzymes not only play an

important role in the cellular metabolic processes but have also

gained considerable attention in the industrial community (Priya et al,

2011). Their enormous diversity of function makes them one of the

most fascinating groups of enzymes for application at different sectors

of life in both physiological and commercial fields (Marc et al, 2002).

This vast diversity of amylases, in contrast to the specificity of their

action, attracted worldwide attention in attempts to exploit their

physiological and biotechnological applications (San-Lang Wang et al,

2011).

Amylases are starch degrading enzymes that catalyze the

hydrolysis of internal alpha 1-4 glycosidic bonds in polysaccharides

with the retention of alpha anomeric configuration in the products

(Takata et al, 1992). They are found in all forms of organisms

regardless of kingdom. Alpha amylases are ubiquitous enzymes

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produced by plants, animals and microbes where they play a

dominant role in carbohydrate metabolism (Damien et al, 2010).

Amylases from plants and microbe sources have been employed for

centuries in brewing industry (Madson et al, 2011). Fungal amylases

are widely used for the preparation of oriental foods (Mabel et al,

2006). Amylases of bacteria, fungi and viruses are increasingly

studied due to the relative ease of large scale production (low

downstream cost as they are extracellular in nature) as compared to

amylases from plants and animals and their importance in

subsequent application at industry (Ashis et al, 2009).

Microbial enzymes reveal broad biochemical diversity and

susceptible to genetic manipulation (Ahmad et al, 2010). In addition,

bacterial amylases have longer shelf life and can be stored for weeks

without significant loss of activity (Jos et al, 2011). In addition,

microorganisms elaborate a large array of amylases, which are

intracellular, and/or extracellular (Mohsen et al, 2005). Intracellular

amylases are important for various cellular and metabolic processes,

such as sporulation and differentiation, protein turnover, maturation

of enzymes and hormones and maintenance of the cellular protein

pool (Yakup et al, 2010). Extracellular amylases are important for the

hydrolysis of Starch and Cellulose in cell-free environments and

enable the cell to absorb and utilize hydrolytic products (Chi Wen Li et

al, 2011). At the same time, these extra cellular amylases have also

been commercially exploited to assist starch degradation in various

industrial processes (Encarnacion et al, 2011). Of the various types of

amylases, alpha amylases (E.C.3.2.1.1.) which catalyze the hydrolysis

of internal alpha 1-4-O-glycosidic bonds in polysaccharides with the

retention of alpha anomeric configuration in the products are widely

studied (Baharen et al, 2011). Amylases are one of the most important

industrial enzymes that have a wide variety of applications ranging

from conversion of starch into sugar syrups, to the production of

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cyclodextrins for the pharmaceutical industry (Marc et al, 2011).

These enzymes accounts for about 30% of the world’s enzyme

production.

In the present biotechnological era, microbial amylases have

been investigated for their role in the manufacturing of maltose and

also in the manufacturing of High Fructose corn syrup (Archana et al,

2011). Amylases are also used in Manufacture of Oligosaccharide

mixture, Manufacture of Maltotetrose Manufacturing of high

molecular weight branched dextrins, removal of starch sizer from

textiles etc (Damien et al, 2010, Arpana et al, 2011).

1.5 CLASSIFICATION OF AMYLASES

A number of microorganisms produce one or more of types of

amylase enzymes with different pH optima for activity (Takahiro et al,

2011). Amylases are broadly classified into four groups like endo

amylases, exo-amylases, debranching enzymes and transferases

(Parveen et al, 2011). Endo amylases cleave internal alpha 1-4 bonds

resulting in an alpha anomeric product (Ahmad et al, 2010). Exo

amylases cleave alpha 1-4 or alpha 1-6 bonds of the external glucose

residues resulting in alpha or beta anomeric products. Debranching

enzymes hydrolyze alpha 1-6 bonds exclusively leaving long linear

polysaccharides and Transferases cleave alpha 1-4 glycosidic bond of

the donor molecule and transfer part of the donor to a glycosidic

acceptor forming a new glycosidic bond (Ashis et al, 2009). Glycosidic

hydrolases are able to metabolize a large variety of saccharides that

have been divided into classes based on their mode of reaction and

families based on their well defined amino acid sequence similarities

(Norman et al, 2008).Most of the starch converting enzymes belong to

GH-13 family. GH-13 family can be further classified based on a larger

unit called clan, which is a three dimensional structure of catalytic

domain but not limited sequence similarity indicating that protein

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structure is better preserved by evolution than amino acid sequence.

Among the fourteen clans (A-N) defined by glycosidases and

transglycosidases, alpha amylase family (GH-13) belongs to the eighth

clan GH-H (Henrissat et al, 1991).

The concept of this group of enzymes as the alpha amylases was

proposed in 1992 .According to that definition the members of this

family must satisfy the following requirements (Bahareh et al, 2011,

Birch et al, 1973).

They must act on the alpha glycosidic linkages and hydrolyze

them to produce alpha anomeric monosaccharides and

oligosaccharides.

Should have four highly conserved sequences regions in their

primary structures consisting of catalytic and important

substrate binding sites.

Should have Asp, Glu and Asp residues as catalytic sites

corresponding to the Asp 206, Glu 230 and Asp 297 of amylase

A and

Posses a tim barrel catalytic domain

1.6 MECHANSIM OF ACTION

Alpha amylases (α-1, 4-glucan-glucanohydrolase, EC 3.2.1.1) is

am extracellular enzyme (Cherry et al, 2004). This enzyme degrades α-

1,4-glucosidic linkage of starch and related products in an endo

fashion and produce oligosaccharides (Zubeyde et al, 2008). Mode of

action, properties and product of hydrolysis differ somewhat, depend

on the source of enzyme (Edwinoliver et al, 2010). Two types of

enzymes have been recognized, termed liquefying and saccharifying.

The main difference between then is that the saccharifying enzyme

produces a higher yield of reducing sugar than the liquefying enzyme

(Harmeet et al, 2005).

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1.7 CURRENT PROBLEMS AND POTENTIAL SOLUTIONS

The overall potential of amylases in industrial process is yet to

be exploited fully. The inherent disadvantages in the use of amylase,

in particular are related to following.

The complete cost of enzyme production and downstream

processing is the one of the major obstacle against the

successful application of any technology in the enzyme

industry (Masafumi et al, 2010).

Thermal, operational and storage problems as they are

easily prone to inactivation by self degradation (autolysis),

where as good industrial catalyst should be stable under

the toughest operating conditions and for long durations

(Ajay et al, 2010).

To overcome such limitations great attention has been devoted

for studies on amylases to tackle the problem (Biazus et al, 2007).

Recent approaches for increasing amylase yield including screening

for naturally occurring enzymes with intrinsic stability or to produce

stable enzymes by means of protein engineering and optimization of

fermentation media through a statistical approach are some of them

(Aliyu et al, 2011). The simplest approach to obtain a stable enzyme is

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to look for the desired enzyme in a readily available organism (Priya et

al, 2011). Hence great interest has been generated in the search for

new thermo, alkalophilic strains and their fermentation conditions

optimization by using statistical methods to get more economical

yields for industrial applications (Asgher et al, 2007).

Keeping the above mentioned points in view, the present

investigations have been undertaken to isolate a novel alpha amylase

producing microbial strain and subsequent development of an

ecofriendly, efficient and economically suitable fermentation process

to produce high productivity titers (Solange et al, 2010).

1.8 AIMS AND OBJECTIVES OF THE PRESENT STUDY:

The overall objective of the present research is to identify a new

and novel Aspergillus species which has got the inherent capacity to

produce alpha amylase and to develop a strategy for the fermentative

production at higher scale by optimizing all the essential fermentative

kinetics and other aspects which can result in increase in yield or

productivity of the enzyme production.

1.9 PLAN OF RESEARCH WORK:

Isolation of an efficient amylase producing strain from natural

environmental habitats of various soil samples collected from

dump yards of starch processing industries in and around

Hyderabad.

Screening of efficient amylase producing Aspergillus niger strain

and its further characterization.

Preliminary screening of various components to identify basic

requirements of the isolated strain for further studies.

Study of growth kinetic parameters of the strain.

Determination of various growth profiles like biomass, sugar

consumption rate, enzyme formation rate etc.

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Designing of medium for the efficient growth of the strain and

optimization of carbon, nitrogen, minerals, and other nutrients

for effective product formation rate.

Optimization of submerged fermentative process parameters like

pH, temperature, inoculum size, incubation period etc.

Identification and evaluation of different agro wastes for

economic production of amylase enzyme from isolated

Aspergillus species.

Optimization of various process parameters in solid state

fermentation for effective amylase production.

Determination of efficient downstream process for separation

and purification of amylase enzyme.

The above mentioned objectives are briefly explained below.

1.9.1 ISOLATION OF AN EFFICIENT AMYLASE PRODUCING

ASPERGILLUS STRAIN FROM SOIL SAMPLES.

The main objective of this experiment was to isolate an amylase

producing Aspergillus strain from different soil samples collected from

various locations and dump yards of starch processing industries in

and around Hyderabad to check its ability to produce amylase enzyme

by growing them in synthetic media like SDA, PDA, Czapeck Dox agar

etc enriched with starch, so as to identify the starch degrading

amylase producing strain based on the zone of hydrolysis on starch

containing media plates.

1.9.2 SCREENING AND CHARACTERIZATION OF THE STRAIN TO

IDENTIFY ITS SPECIES

The objective of the present experiment was to identify and

characterize the exact species by performing routine biochemical tests

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with reference to Bergey’s manual and tests based on morphological

and physiological characters by plating the cultures on some selective

starch rich medium and also by microscopic observation in addition to

enzymatic assay so as to identify the best amylase producing strain

from various soil samples collected.

1.9.3 GROWTH KINETICS STUDY

The isolated strain was grown in conical flask for 24 to 48

hours and at regular intervals samples were collected and analyzed for

growth at 600 nm by spectrophotometer and sugar consumption at

540 nm by DNS method. Later once the growth is ceased or sugar is

exhausted the experiment was stopped and all the readings were

plotted on the graph so as to identify various parameters like mean

doubling time, growth curve and for determination of various growth

profiles like OD, sugar consumption pattern and product formation

pattern.

1.9.4 OPTIMIZATION OF MEDIA COMPONENTS

Different set of experiments were conducted by inoculating the

strain in different flasks with varying sugars like glucose, xylose,

fructose, sucrose etc to identify the best carbon substrate. Similar

experiments were conducted with varying nitrogen sources also to

identify the best nitrogen source for efficient enzyme production by the

isolated Aspergillus strain. Similar experiments were conducted for

selecting various other media components for high amylase

production by the isolated Aspergillus niger strain.

1.9.5 SUBMERGED FERMENTATIVE PROCESS PARAMETERS

OPTIMIZATION

Various process parameters like incubation period, pH,

temperature, inoculum size, oxygen requirements, agitation etc were

INTRODUCTION


optimized for amylase enzyme production by isolated Aspergillus niger

strain in a 2L Fermentor. Under optimized conditions enzyme

production increased considerably.

1.9.6 EVALUATION OF DIFFERENT AGRO WASTES FOR

ECONOMIC PRODUCTION OF AMYLASE

The objective of this experiment was to screen few potentially

available agro wastes like wheat bran, rice bran and green gram husk

for the production of amylase enzyme by selected Aspergillus niger

species, so as to reduce the cost of the raw materials at production

level and also an efficient way for utilization of agro wastes for

production of important enzymes.

1.9.7 SOLID STATE FERMENTATION PROCESS PARAMETERS

OPTIMIZATION

The objective of the present experiment was to identify various

parameters like growth rate of the strain and optimization of various

growth profiles like OD, dry cell weight, sugar consumption rate and

enzyme production rate, etc in solid state fermentation by growing the

strain in solid supported matrix in various flasks and also to optimize

parameters like carbon, nitrogen source, moisture content, inoculum

percentages etc.

1.9.8 DOWNSTREAM PROCESS

The objective of the present experiment was to identify the best

suited purification techniques after enzyme production by extracting

the enzyme from conical flasks by centrifugation, dialysis and by

subjecting the enzyme to various chromatographic techniques like ion

exchange ,gel permeation etc after which SDS PAGE electrophoresis

was performed to determine the exact molecular weight of the enzyme.

INTRODUCTION


1.9.9 EFFECT OF CALCIUM CHLORIDE ON THE THERMO

STABILITY OF ALPHA AMYLASE

Varying molar concentrations of calcium chloride were added to

several flasks at varying temperatures ranging from 40 to 85oC to

monitor the enzyme production by the isolated Aspergillus niger strain

to determining the thermo stability of the alpha amylase enzyme at

various temperature ranges.

1.9.10 EVALUATION OF AMYLASE ENZYME CONCENTRATION

ON DESIZING OF COTTON CLOTH BY PARTIALLY

PURIFIED ENZYME

The effect of alpha amylase enzyme on the desizing of the cotton

cloth was studied by partially purified enzyme. The concentration of

the enzyme varied from 50 – 500 U/ml.

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