Microbial diversity

Microbial diversity encompasses the spectrum of variability among all type of Microorganism in
the natural world and is altered by human intervention.

Microorganism includes Microorganisms distribution in nature, their relationship with each

other and other living organisms, their effects on human beings and animal and plants.

The diversity of a microbial consortium can vary and change with environmental factors for
example- Temp, ammonium concentration, CO2 concentration.

Microbes are everywhere. They live in the steaming hot springs coloring stones.

Thermophillic bacteria are found in the warnest places of the earth.

Some microbes are enimies and some are good.

Nitrogen fixing bacteria are able to live in the root of legumes by forming nodules.

Micro organisms are very diverse in colour, shape, function and metabolic activity.

Microbes are occur in the universe. Microbes are widely distributed in air, water, soil, sea,
mountain, hot springs and also in the body of living plants and animals including the human.

As evolution preceded new kinds of micro organisms appeared. Therefore the diversity of
microbial world incread.

Microbiology is the study of living organism of microscopic size which includes bacteria, fungi,
algae, protozoa and viruses.

Microbial diversity is one of the most reasons why the world is finely turned prefect.

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Industrial importance of micro organisms

Microscopic yeast and bacteria are used to produce a variety of products, such as bread, beer, and
carry out processes such as biogas production.

There are various types of micro organisms that are used for large scale production of industrial
items:-

1- The ability of specific micro organisms to specific enzyme and proteins has been
exploited for many purposes in industry.

2- Industrial micro organisms are used to produces many things including food, cosmetics,
pharmaceuticals and construction materials.

3- Micro organisms can be genetically modified to aid in large scale production.

4- Genetically engineered microbes produce insulin in a pure form.

5- Aspergillus is used in the production of alcoholic beverages and pharmaceutical

development.

6- Microorganisms are used in biotransformation of several chemicals to facilitate the

reduction of environmental pollution.

7- Different strains of bacteria and fungus are used for fermentation dairy production of
wide variety of cultured milk products.

8- Lactic acid bacteria are used for coagulation of milk that can be processed to yield a wide
variety of cheeses.

9- Microorganisms such as lactobacillus is used in food and health industry.

10- Molds are used rotting of grapes for production of varieties of wines.

11- Mushrooms ( agaricusbisporus) are one of the most important fungi used as a food
source.

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 12- Alcoholic beverages as beer are produce by fermentation of cerears and grains using
different strains of yeast.

Microbial enzyme

Microbial enzyme have wide spread uses in industries and medicine.

Microbial enzymes are also more active and stable than plant and animals enzyme.

In addition, microbial enzymes are the preferred source of industrial enzymes as they can be
produce in large quanties in a short period of times and genetic manipulation can be performed
more easily on bacterial cells to increase the enzyme production.

For the production of industrial enzymes, microbial cells are selected from the group of bacteria,
fungi or yeast.

As per WHO recommendation, only enantiopure drugs are applicable for the treatment of
patients.

Most of the medicines are salt of racemic mixture which contains both enantiomers in 50:50
ratio. Only one form is affective for curing to a given disease.

Chemically it is a tedious process while one the other hand biocatalyst are very much capable to
distinguish both the form and select one form the transformation.

Enzyme are macro molecular biological catalyst.

Enzymes catalyses the chemical reaction, without being used up in the reaction.

Like all catalyst enzyme increase the reaction rate by lowering its activation energy.

Some enzyme are used commercially for example:- in the synthesis of antibiotics, some
household product use enzyme to speed up chemical reaction

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HISTORY OF ENZYME

In 1897 Edward Buchner discovered that yeast extract could ferment sugar to alcohol proving
that fermentation was promoted by molecules that continued to function when removed from
cell.

W. kuhne 1878 called these molecules enzymes .

CLASSIFICATION OF ENZYME:-

In 1961 I.U.B. (INTERNATION UNION OF BIOCHEMISTRY)

Was given the classification of enzyme.

There are six classes of enzymes that were created so that enzymes could easily be named.

These classes are

ENZYME E.C. NUMBER

Oxidoreductase I

Transferase II

Hydrolase III

Lyase IV

Isomerase V

Ligase VI

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HYDROLASE

An enzyme that catalyses the hydrolytic cleavage of C-O,

C-N, C-C is known as hydrolase.

Hydrolytic enzyme an enzyme that catalyses the hydrolysis of a chemical bond.

A-B+ H2O A-OH+B-H

Hydrolase secreated by lactobacillus yensenli in the human gut stimulates the liner to secreate
bile salts that aids in the digestion of food.

Hydrolase break the compound in the present of water.

SUB CLASSES

Hydrolase are classified as EC3 in the EC Number classification of enzyme hydrolase can be
further

Classified in to several subclasses based up on the bonds they act upon.

EC 3.1: Ester bond (ESTERASE)

EC 3.2: Sugars ( DNA glylasylases)

EC 3.3: Ether bonds

EC 3.4: peptide bond ( peptidases)

EC 3.5: carbon- nitrogen bonds other then

Peptide bond

EC 3.6: acid anhydride

EC 3.7: carbon-carbon bonds

EC 3.8: halids bonds

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EC 3.9: phosphorus- Nitrogen bonds

EC 3.10: sulphur- Nitrogen bonds

EC 3.11: carbon- phosphorus bonds

EC 3.12: sulphur- sulphur bonds

EC 3.13: carbon- sulphur bonds

LIPASE ENZYMES:- EC-3.1

An Lipase is a hydrolase enzyme that splits esters into an acid and an alcohol in a chemical
reaction with water called hydrolysis.

Or

An enzyme that catalyses the hydrolysis of an ester called lipase.

Lipase have been isolated from plant, animal and microorganism.

Introduction

Lipases have emerged as one of the leading biocatalysts with proven potential for contributing to
the multibillion dollar underexploited lipid technology bio-industry and have been used in in situ
lipid metabolism and ex situ multifaceted industrial applications1. Lipases are triaclyglycerol
acylhydrolases (EC 3.1.1.3) that catalyze the hydrolysis of triaclyglycerol to glycerol and fatty
acids.
They often express other activities such as phospholipase, isophospholipase, cholesterol esterase,
cutinase, amidase and other esterase type of activities2. Microbial lipases have gained special
industrial attention due to their ability towards extremes of temperature, pH, and organic
solvents, and chemo-, region-, and enantioselectivity. Lipases are ubiquitous in nature and are
produced by several plants, animals and microorganisms3. Some important lipase-producing

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bacterial genera are Bacillus, Pseudomonas and Burkholderia4 and fungal genera include5
Aspergillus,Penicillium, Rhizopus, Candida. Different species of yeasts belonging to seven
different genera include Zygosaccharomyces, Pichia, Lachancea, Kluyveromyces,
Saccharomyces, Candida, and Torulaspora6. Knowledge of the three-dimensional structure of
lipase plays an important role in designing and engineering lipases for specific purposes. More
than 12 lipases from various sources have been crystallized and extensive information on lipase
engineering has been documented. All lipases are members of the _/_ hydrolase fold
super-family. Also, lipases share a conserved active site signature, the Gly-Xaa-Ser-Xaa-Gly
motif7. Although, considerable progress has been made over the recent years towards the
developing cost-effective systems for lipases but the high cost of production of this enzyme
remains the major challenge associated with large-scale industrial applications.
Given the heterogeneity of natural environments and the enormous potential of microorganisms
to provide novel pharmaceuticals, fine chemicals and new technologies, the
biotechnology industry has a vast, largely untapped resource for the discovery of new chemicals
and novel processes.

Properties of Lipases

The number of available lipases has increased since the 1980s and used as industrial biocatalysts
because of their properties like bio-degradability, high specificity and high catalytic efficiency.
Some unique properties of lipase such as their specificity, temperature, pH dependency, activity
in organic solvents and nontoxic nature leads to their major contribution in
the food processing industries. Ethyl, isobutyl, amyl and isoamyl acetates are widely used flavor
esters9. Lipases from different sources have investigated for their hydrolytic as well
as synthetic activity. The most desired characteristics of the lipase are its ability to utilize all
mono-, di-, and tri-glycerides as well as the free fatty acids in transesterification, low product
inhibition, high activity/yield in non-aqueous media, low reaction time, resistance to altered
temperature, pH, alcohol and reusability of immobilized enzyme10. Additionally, lipases can
carry out reactions under mild conditions of pH and temperature and this reduces energy needs to
direct reactions at unusual temperatures and pressures. As a result, unstable reactants and

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products are protected from destruction. Other reasons for biotechnological potential of
microbial lipases are their stability in organic solvents and being active without the aid of
cofactors.

pH and Temperature Kinetics:

Bacterial lipases have a neutral or alkaline optimum pH with the exception of lipase from P.
fluorescens SIK W1 that had an acidic optimum pH 4.8. However, lipases from Bacillus
stearothermophilus SB-1, B. atrophaeus SB-2 and B. licheniformis SB-3 are active over a
broad12 pH range 3-12. Bacterial lipases generally have temperature optima in the range13 30-
60C. For B. licheniformis MTCC-10498 maximum lipase production was observed at pH
7.5 (~0.4 U/ml)14. However, bacterial lipases with optima in both lower and higher ranges have
been reported. Thermal stability data are available only for species
of Bacillus, Chromobacterium,Pseudomonas and Staphylococcu s. The thermostability of the
enzyme from Bacillus sp. was enhanced by the addition of stabilizers such as ethylene glycol,
sorbitol, glycerol, with the enzyme retaining activity at even after 150 min of incubation at
70C15.

Stability in Organic Solvents:

Stability in organic solvents is desirable in synthesis reactions. From the available literature, it
can be inferred that lipases are generally stable in organic solvents, with few exceptions of
stimulation or inhibition. Ethanol and methanol enhanced the lipase activity of B.
thermocatenulatus and AG-8 lipase16.
Effect of Metal Ions:

Metal ions can either stimulate or inhibit microbial enzyme production. Metal cations,
particularly Ca2+, play important roles in the structure and function of enzymes,
and some of the lipases are strictly calcium dependent17. Ca2+ ions activated the enzyme, while
Zn2+, Fe2+, Fe3+ strongly inhibited its activity. Salt ions like Ca2+, Cd2+, and Fe2+ enhanced
the activity of immobilized biocatalyst while a few ions like Co2+, Zn2+, Mg2+, Mn2+, Al3+,
and Na+ had mild inhibitory effect18.

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Lipase Inhibitors:

Lipase inhibitors have been used in the study of structural and mechanistic properties of lipases.
Further, the search for lipase inhibitors is also of pharmacological interest. Lipase inhibitors are
used for designing drugs for the treatment of obesity and the problem of acne. Following is an
account of general inhibitors. Broadly, inhibitors of enzymes are classified as reversible or
irreversible. The reversible inhibitors can be further classified as nonspecific
and specific reversible inhibitors.

Non-Specific Reversible Inhibitors:

Compounds that do not act directly at the active site but inhibit lipase activity by changing the

conformation of lipase or interfacial properties are defined as non-specific inhibitors.

Surfactants, bile salts4 and proteins belong to this group of inhibitors. However, surfactants and

bile salts activate the enzyme in some cases.

Applications of Lipases

Lipases form an integral part of the industries ranging from food, dairy, pharmaceuticals,

agrochemical and detergents to oleo-chemicals, tea industries, cosmetics, leather and in several

bioremediation processes19. Because of the vast applications, newer microbes are to be screened
for production of lipases having desirable properties. The understanding of structurefunction
relationships will enable researchers to tailor new lipases for biotechnological applications20.

Lipases in Fat and Oil Processing:

Fats and oil modification is one of the prime areas in food processing industry that demand novel
economic and green technologies21. Fats and oils are important constituents of foods. Lipases
allow us to modify the properties of lipids by altering the location of fatty acid chains
in the glyceride and replacing one or more of these with new ones. In this way, a relatively
inexpensive and less desirable lipid can be modified to a higher value fat. The removal of

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phospholipids in vegetable oils (de-gumming) using highly selective microbial phospholipases is
also a recently developed environmental friendly process22. There are many studies on the
hydrolysis of fats and oil by lipases used either in the pure form, in the immobilized form or in
the cell bound form.
Lipases in Food Industry:

In the field of biotechnology there are many industrial applications that result in biotech products
that we use every-day at home. Some of these are food science applications that utilize enzymes
to produce or make improvements in the quality of different foods. Lipases have
immense application in food industry such as in cheese ripening, flavor development and EMC
technology24. Lipases are used ex situ to produce flavors, and to modify the structure by inter- or
transesterification, in order to obtain products of increased nutritional value, or suitable for
parental feeding25. Lipases have also been used for addition in food to modify flavor by
synthesis of esters of short chain fatty acids and a1cohols, which are known flavor and fragrance
compounds.
LIPASES IN DETERGENTS:

The use of enzymes in detergent formulations is now common in developed countries, with over
half of all detergents presently available containing enzymes. Laundry detergents are becoming
more and more popular because of their increasing use in washing machine, where it
impart softness, resiliency to fabrics, antistaticness, dispersible in water and mild to eyes and
skins. There are many different brands or types of laundry detergents, and mostly they claim
some special qualities27. China's demand for detergent has grown in the past decade and both
production and demand will continue to grow in the next five years. There has been a
tremendous increase in the significance of the biotechnological applications of lipases since the
last two decades where they display amazing versatility in catalytic behavior. The latest
trend in detergent industry is towards lower wash temperatures which not only save energy, but
also help to maintain the texture and quality of fabrics28. Detergent industries are the primary
consumers of enzymes, in terms of both volume and value. The use of enzymes in detergents
formulations enhances the detergents ability to remove tough stains and making the
detergent environmentally safe. Nowadays, many laundrydetergent products contain cocktails of
enzymes including proteases, amylases, cellulases and lipases.

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Lipases in Pulp and Paper Industry:

The pulp and paper industry processes huge quantities of lignocellulosic biomass

every year. The technology for pulp manufacture is highly diverse, and numerous opportunities
exist for the application of microbial enzymes. Historically, enzymes have found some uses in
the paper industry, but these have been mainly confined to areas such as modifications of raw
starch. The enzymatic pitch control method using lipases have been in use in a large-scale
paper-making process as a routine operation since early 1990s30. Pitch or the hydrophobic
components of wood (mainly triglycerides and waxes), causes severe problems in pulp and
paper manufacture. Lipases are used to remove the pitch from the pulp produced for paper
making11.
Nippon Paper Industries, Japan, have developed a pitch control method that uses the
Candida rugosa fungal lipase to hydrolyze up to 90% of the wood triglycerides.

Lipases in Oleochemical Industry:

The current trend in the oleochemical industry involves the use of immobilized lipases to initiate
various reactions (hydrolysis, alcoholysis, and glycerolysis) using mixed substrates. Thus, the
use of immobilized enzyme ensures high productivity as well as
continuous running of the processes. This offers a greatest hope for successful fat
splitting/modification without substantial investment in expensive equipment as well as in
expenditure of large amounts of thermal energy24. The scope for the application
of lipases in the oleochemical industry is enormous as it saves energy and minimizes thermal
degradation during hydrolysis, glycerolysis, and alcoholysis.

Lipases in Environmental Management:

Employment of lipases in bioremediation processes is a new aspect in lipase biotechnology. The

wastes of lipid- processing factories and restaurants can be cleaned by the help of lipases from

different origins. In this sector, lipases could be used by either ex situ or in situ31. Due to the

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rapid development observed in industries, environmental pollution became more and more

critical. Lipaseproducing strains played a key role in the enzymological remediation of polluted

soils32. Cold-adapted lipases have great potential in the field of wastewater treatment,

bioremediation in

fat contaminated cold environment and active compounds synthesis in cold condition33 while in
temperate regions, large seasonal variations in temperature reduce the efficiency of
microorganisms in degrading pollutants such as oil and lipids. The enzymes active at low and

moderate temperature may also be ideal for bioremediation process.

LIPASES IN TEA PROCESSING:

The quality of black tea is dependent largely on the dehydration, mechanical breaking, and

enzymatic fermentation to which tea shoots are subjected. During manufacture of black tea,

enzymatic breakdown of membrane lipids initiate the formation of volatile products with

characteristic flavor properties, emphasize the importance of lipid in flavor development. Lipase
produced by Rhizomucor miehei enhanced the level of polyunsaturated fatty acid observed by
reduction in total lipid content.

Lipases as Biosensors:

A promising new field is the use of microbial lipase as biosensors. Biosensors can be chemical or
electronic in nature. An important analytical use of lipases is determination of lipids for clinical
purpose. The basic concept is to utilize a lipase to generate glycerol from triacylglycerol and
quantify the released glycerol or alternatively the non-esterified fatty acid by chemical and
enzymatic method. This principal enables physicians precisely to diagnose patients with
cardiovascular complaints. Non-specific lipase, especially of C. rugosa with high specific
activity has been selected to allow rapid liberation of glycerol. C. rugosa lipase biosensor, which

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optically conjugates to bio-recognition group in DNA, has been developed as probe.

Lipases as Diagnostic Tools:

Lipases are also important drug targets or marker enzymes in the medical sector. They can be
used as diagnostic tools and their presence or increasing levels can indicate certain infection or
disease. Lipases are used in the enzymatic determination of serum triglycerides to generate
glycerol which is subsequently determined by enzyme linked International Research Journal of
Biological Sciences.

International Science Congress Association:

colorimetric reactions. The level of lipases in blood serum can be used as a diagnostic tool for
detecting conditions such as acute pancreatitis and pancreatic injury36. Acute pancreatitis
usually occurs as a result of alcohol abuse or bile duct obstruction. Although serum trypsin level
ultrasonography, computed tomography and endoscopic retrograde cholangiopancreatography
are the most accurate laboratory indicators for pancreatitis but serum amylase and lipase levels
are still used to confirm the diagnosis of acute pancreatitis37. Diagnosis of chronic pancreatitis
and revealing the presence of exocrine pancreatic insufficiency has also been determined by
measuring serum amylase, pancreatic isoamylase, lipase, trypsinogen and elastase38. Lipase of
pathogenic bacteria such as P. acnes39, Corynebacterium acnes and Staphylococcus
aureus40 has also been found to have the influence on skin rash in acne patients.
Lipases in Cosmetics and perfumery:

Lipases have potential application in cosmetics and perfumeries because it shows activities in

surfactants and in aroma41 production. Retinoids (Vitamin A and derivatives) are of great

commercial potential in cosmetics and pharmaceuticals such as skin care products.

Water-soluble retinol derivatives were prepared by catalytic reaction of immobilized lipase42.

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Lipases in Medical Applications:

Lipases isolated from the wax moth (Galleria mellonella) were found to have a bacteriocidal
action on Mycobacterium tuberculosis (MBT) H37Rv. This preliminary study may be regarded
as part of global unselected screening of biological and other materials for
detecting new promising sources of drugs43. Lipase from Candida rugosa has been used to
synthesize Iovastatin, a drug that lower serum cholesterol level. The asymmetric hydrolysis
of 3-phenylglycidic acid ester which is a key intermediate in the synthesis of diltiazem
hydrochloride, a widely used coronary vasodilator, was carried out with S. mmcescens lipase.

Conclusion

Although lipases have several interesting immense applications in the food, detergent,
pharmaceutical, leather, cosmetic and paper industries, their industrial uses still remain limited
by their high production costs, commercialization in small amounts, and low performance of
some lipase-mediated processes. The applications of lipases are broadening rapidly and new
applications are still to be explored in these industries.

1 - ENZYME APPLICATION :-

Bacterial proteinase are still the most important detergent Enzymes. Some product have
been genetically engineered to be more stable in the hostile environment of washing
machines with several different chemical present.
Lipid degrading enzymes lipase were used in power and lipid detergents to
decomposed fats.
Amylases are used in detergent to remove starch based strains.
cellulose have been part of detergents since early 90s.

And Enzyme Complex capable of degrading crystalline to cellulose to glucose.

2-ENZYME APPLICATION: DRINK INDUSTRY:-

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 Fruit juice manufacturing :-
pectins are sulestrances in fruit lamella and cell walls which contains also hemicelluloses
and cellulose.
Pectinase xylamase and cellulose improve the literation of the juice from the pulp.
Pectinase and amylase are used in juice clarification.

WINE PRODUCTION :-
Enzymes are widely used to optain a better extraction of the necessing component and
that improving the yield.

ENZYME APPLICATION TEXTILES :-

The use of enzyme in textile industry is on of the most rapidly growing fields in
industrial enzymology.
Starch use for a long time been used as a protective of filters in weaving of
falerice. this is celled sizing.
Enzyme are used to remove the starch in a process desizing.
Amylase are used in this process since they do not human the textile fibeers.
laccase- A polyphenol oxidase from fungi in used to degrade lignin the aromatic
polymer found in all plant materials.
cellulases- Remove cellulose micro filter which are formed during washing.

iv- ENZYME APPLICATION : ANIMAL FEED: -

The net effect of enzyme usage in has been increased animal weight. The first commercial
success was addition of betaglucanase into barlay based feed diest. Barley contains Beta- glucon,
which causes high viscosity in the chicken gut. In Addition to poulty, enzymes are use in pig
feeds and turkey feeds.

v- ENZYME APPLICATION : BACKING :-

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 Alpha- amylases have been most widely studied in connection with improved bread
quality and increased shelf life.
Both fungi and bacterial amylase are use in bread making and excess may lead to sticky
to dough.

VI- ENZYME APPLICATION :PULP AND PAPER:-

The major application is the use of xylanases in pulp bleaching for paper.

Vii-ENZYME APPLICATION : LEATHER:-

Leather industry uses ptoteolytic and lipolytic enzymer in leather processing.

Enzyme are used to remove animal skin,hair,and any unwanted parts.
lipase are used in this phase or in bating phase to specifically remove grease.

VIII- ENZYME APPLICATION :BIOETHENAL , BIOFULE:-

Bioethenal is a biofued in a care.

it can be produced from starchy palnt materials.
Enzyme are use to convert starch to bioethenal.
At present, corm is a widely used source of starch.
Other Plants including wheat bamboo or other grasses can be used we

sources for boiethenal production.

IX- ENZYME APPLICATION : MEDICINE:-

As drugs for treatment of disease.
In diagnosis.
In treatment of medicine.
To remove toxic substance.

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 LIPASE REACTION:-

194 A.R. macrar and R.C. hammond:-

Triacylglyceral+free fatty acid

+_ H2o

194 A.R. macrar and R.C. hammond

Triacylglyceral

+_ H2o

Diacylglyceral +free fatty acid

+_ H2o

Monoacylglyceral + free fatty acid

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+_ H2o

Gylcerol + Free fatty acid.

To start have needed 10 ml of Original functional culture.

A Seriol diluation is the step wise dilution of a subtance in solution. usually

the dilution factor of each step-3 constant, Resulting in a geometric
progressing.

The concentration in organic. A ten fold serial dilution method

1m,0.1m,0.001 m,0,001m.

Prepare serial tube with 9 ml of dilution liquid.

The undiluted sample was added to the first tube and then serial diluting into the following tube.

Perform the first dilution

1 ml of undiluted solution was taken from test tube with a pipette and
Transferred in to the test tube containing 9ml of the dilution liquid and mix
thoroughly and labeled A .

Perform the Second dilution

1 ml of solution was taken from test tube A and was added it to the 9 ml of
Dilution liquid in the test tube B.

Trish process was repeated four times to achive the desired solution.

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Ertend procedure to perform loger serial

dilution :-
Trish process me be repeefed as many times as necessary to Aenine the distred solution.

Culture The final dilution ratio in a serial dilution:-

Dt= D1*d2*d3*dn

We did 1:10 dilution of our liquid 4 times.

Dt =10*10*10*10=10,000

The final dilution factor of the forms tube in our serial dilution is 1:10,000 the conc. Of

substance is now10,000 times less then the origined and : liquid solution.

Determine The conc. of the solution following dilution.

Cf=Cinitial/D

Cf=Final conc.
Ci = starting cons.

D = dilution ratio previously determined.

CF = 1000,000/1,000

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CF = 1,000 cells per ml.

LIPASE REVIEW:-

1. Svendsen A., Review: Lipase protein engineering, b Biochemia et Biophysica Acta, 1543, 223-
238 (2000)

2. Thakur S., Lipases, its sources, properties and applications: A review, International Journal of
Scientific and Engineering Research, 3(7), 1-29 (2012)

3. Gupta R., Gupta N. and Rathi P., Bacterial lipases: An overview of production, purification
and biochemical properties, Appl. Microbiol. Biotechnol., 64, 763781
(2004)

4. Singh A.K. and Mukhopadhyay M., Overview of fungal

lipase: A review, Appl. Biochem. Biotechnol., 166, 486-520
(2012)

5. Romo-Sanchez S., Alves-Baffi M., Arvalo-Villena M., beda-Iranzo J., Briones-Prez A.,
Yeast biodiversity fromoleic ecosystems: Study of their biotechnologicalproperties, Food
Microbiol., 27, 487-492 (2010)

6. Messaoudi A., Belguith H., Gram I. and Hamida J.B.,Classification of EC 3.1.1.3 bacterial
true lipases usingphylogenetic analysis, Afr. J. Biotechnol., 9(48), 8243-8247 (2010)
7. Bhardwaj V. and Garg N., Importance of exploration ofmicrobial biodiversity, ISCA J.
Biological Sci., 1(3), 78-83(2012)

8. Verma M.L. and Kanwar S.S., Properties and application of poly(methacrylic acid-cododecyl
methacrylate-cl-N,Nmethylene bisacrylamide) hydrogel immobilized Bacillus cereus MTCC
8372 lipase for the synthesis of geranyl acetate, J. Appl. Polymer Sci., 110, 837846 (2008)

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9. Kumar A., Sharma P. and Kanwar S.S., Lipase catalyzed esters syntheses in organic media: A
review, International Journal of Institutional Pharmacy and Life Sciences, 2(2), 91-119 (2012)

10. Jaeger K-E. and Reetz T.M., Microbial lipases from versatile tools for biotechnology, Trends
Biotechnol., 16, 396403 (1998)

11. Bradoo S., Saxena R.K. and Gupta R., Two acidothermotolerant lipases from new variants
of Bacillus sp, World J. Microbiol. Biotechnol., 15, 97-102 (1999)

12. Lithauer D., Ginster A. and Skein E., Pseudomonas luteola lipase: A New Member of the 320
Residue Pseudomonas Lipase Family, Enz. Microbial Technol., 30, 209-215 (2002)

13. Sharma C.K., Sharma P.K. and Kanwar S.S., Optimizationof production conditions of lipase
from B. licheniformis MTCC-10498, Res. J. Recent Sci., 1(7), 25-32 (2012)
14. Nawani N. and Kaur J., Purification, characterization and thermostability of lipase from a
thermophilic Bacillus sp. J33, Mol. Cell. Biochem., 206, 91-96 (2000)

15. Sharma A.K., Tiwari R.P. and Hoondal G.S., Properties of a thermostable and solvent stable
extracellular lipase from a
16. El Khattabi M., Van Gelder P., Bitter W. and J. Tommassen, Role of the calcium ion and the
disulfide bond in the Burkholderia glumae lipase, J. Mol. Catal. B., 22(5- 6), 329-338 (2003)

17. Kumar A. and Kanwar S.S., Synthesis of isopropyl ferulate using silica-immobilized lipase in
an organic medium, Enzyme Res., 2011, 1-8 (2011)

18. Patil K.J., Chopda M.Z. and Mahajan R.T., Lipase biodiversity, Indian J. Sci. Tech., 4(8),
971-982 (2011)

19. Saxena R.K., Ghosh P.K., Gupta R., Davidson W.S., Bradoo S. and Gulati R., Microbial
lipases: Potential biocatalysts for the future industry, Curr. Sci., 77(1), 101 115, (1999)

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20. Jaeger K.-E., Dijkstra B.W. and Reetz M.T., Bacterial biocatalysts: Molecular biology, three
dimensional structures, and biotechnological applications of lipases, Annu. Rev. Microbiol. 53,
315-351 (1999)

21. Gupta R., Rathi P. and Bradoo S., Lipase mediated upgradation of dietary fats and oils, Crit.
Rev. Food SciNutr., 43, 635-644, (2003)

22. Clausen K., Enzymatic oil-degumming by a novel microbial phospholipase, Eur. J. Lipid
SciTechnol., 103, 333-340 (2001)

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METERIAL REQUIREMENT

A-ChemicalS
Beef extract:- Central drug house LTD india
Peptone:- ovaligens
Nacl:- CDH
Agar-Agar :- MERCK
Crystal violet:- fister scientific
Iodine:- sunhence india
Safranine- thomus baker (chemical ) PVT India
Alcohol:- SDS ( Safety Data Sheet )

B- instrument
I. Autoclave
II. Hot air oven
III. Microwave
IV. Laminor air flow
V. Microscope
VI. Incubator
VII. Eletronic compact scale
VIII. Majnring cylender
IX. Centrifuge
X. Binacular microscope

C- GLASS WERE
I. Petri plate
II. test tube
III. slide
IV. conical flask
V. measuring cylinder
VI. Beaker
VII. pippate
VIII. funal
IX. glass rod
X. inoculation loop

METHOD

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1-Sample Collection
Samples were collected from different micro flora containing soil sample and water sample. For
the collection of be selected difference sites where probability of desired micro organism was

ligher the list of source is as fallowing:-

S.N. SOURCE AREA

1. Soil vessele soil
2. Soil Gutter soil

3. Soil Field soil

4. Soil Wheat soil
5. Soil Rose soil
6. Soil Cloth soil
7. Soil Dairy soil

2:-Media preparation :-

NBM for 100 ml distil water

OR

NAM for 100 ml distil water

first of all we prepared culture media

culture media is a mixture of nutrients required for micro organism to grow these are supplied by
another by either solid or liquid culture media.

composition nutrients 1000ml distil water:-

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Beef extract 3%

Peptone 5%

Nacl 5%

Agar- Agar 15%

For the preparation of nutrient broth media we did not agar- agar.

We measured all the nutrient for NB and dissolve it into flask havening 100ml distil water.( For
NB we did not use Agar- Agar)

We mixed all the nutrients properly and then divide this 100ml media between 10 test tubes. And
then close the test tube with cotton plug.

On the other hand we also prepared NAM in the conical flask and after the autoclaving.

We will pure this media into Petridis.

4- INOCULATION OF SAMPLE INTO TEST TUBE

After the autoclaving we inoculated the samples into 7 different test tubes having nutrient broth
media.
We inoculated the sample within the chamber of laminar air flow.

Laminar air flow produced dust free air laminar air flow provides contamination on free area.
There free we can perform pouring, slanting, streaking without any kind of contamination.

5. After that we placed all the test tube into incubator to provide proper Temperature. For
growth of microbes.

Working temperature of incubator for bacteria is 37oc and for fungi 27-28oc

6. After the incubation of test tube at 28oc for 24 hour.

Bacterial growth occurred. And we inoculated this bacterial into 10 Petri plate with the help of
micropipette.

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We took 50 micro pipette sample and drop it into Petri plate and spread it with the help of
spreader.( Equal into Petri Plate)

7.- Then We placed these Petri plate into the incubators. For proper growth of bacteria.

8.- After 24 hours Bacteria colonies occurred. We identify different types of bacteria on the
basis of Colour, Shape and size.
And we took a single colony and streaked it into another Petri plate containing Nutrients Agar
Media. With the help of inoculation loop.All this we did Nutrient The laminar air flow then
placed this Petri plate into incubator.

9-After 24 hour bacterial growth occurred we did gram staining to identify these bacteria.

10. By the grams staining we got some pure Bacteria and also impure bacteria.

Then we purified these impure Bacteria.

11. Then we cultured this pure bacteria into broth media. We stick these bacteria from broth
media into Perti plate.

12:- Then we streaked these pure bacteria from Perti plate into slant for the preparation of slant
we incubated the agar media into 7 test tubes.

Then close the slant tubes after the agar had cooled and then keep this slant tube at a slant
position,

13.- Incubated the slant by transferring cells with an incubating inoculating loop from a single
colony micro organism An a Plate to the slant surface and then closed the slant tables with cotton
plug.

14:- Incubated the was incubated slant until there is evidence of growth then put the tube in a
refrigerator.

LIPASE ASSAY

To check the lipase activity. TBA plates (Tributyrin Agar Palte)

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Were prepared and all out it to solidify.

1- For preparation of TBA plates we measured 0.7gm and dissolved it to into 50ml
distilled water and sterilized it by autoclaving after autoclaving.
TBA media was poured on to petri plates and allowed it to solidify.
2- After that wells were mode on TBA plates and different samples were coded from
broth culture into these wells.
Esterase producing bacteria produce clear ions arrounded the colony after 24-48 hour of
incubator at 37Oc.
The ions size was measured after 24-28 hour.

CULTURE PRESERVATION: -

The preservation of straees as always a at mast important step in micromobsy for short duration
preservation, we prepared slant in test tube and streaked single colony.

The slant were keeps in incubator at 39oc for 24-48 hour. After cluped appearance of new colours
the slant were kept at 4oc.

For the culture preservation slant was prepared and single colony was streaked from agar plate

on to slants surface and then slants was closed with cotton plug.Slant was incubated until there is

evidence of growth.Then put these slant tubes in a refrigerator

RESULT:-

CULTURE COLLECTION:-

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The sample collected from different regions were subjected. For the isolation via serial dilution
method.
In this study serial dilution method resulted in the isolation of 7 bacterial isolate by agar Plate
streaking purification.

Method these oblaeied were characterated by grains.

The morphology and characteristics is summarized in given table.

S.N. SOURCE AREA

1. Soil vessele soil
2. Soil Gutter soil

3. Soil Field soil

4. Soil Wheat soil
5. Soil Rose soil
6. Soil Cloth soil
7. Soil Dairy soil

MICROBIAL ISOLATION-

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The sample collected from different regions were subjected for the isolation via serial dilution

method. In thish study serial dilution method result in the isolation of 10 bacterial isolate by agar

plate streaking method. These oblacaed isolareso were charecterised by grams staining colony

morphology physical appearance. The morphology and characteristics is summerised.

Photographic of isolates

S- Isolate Sources Gram Shape Colony Texture

no s Colours

1. RVM Soil Gram +ve Baccilus White Slimy

2. RVM Soil Gram - Baccilus Yellow Dry

3. RVM Soil Gram - Cocai Yellow Slimy
4. RVM Soil Gram + Baccilus Creami Slimy
5. RVM Soil Gram + Cocai White Dry
6. RVM Soil Gram - Pseudomonas Yellow Dry
7. RVM Soil Gram - Baccilus Creami Dry

8. RVM Soil Gram + Small Baccilus Yellow Slimy

9. RVM Soil Gram - Cocai Creami Dry

10. RVM Soil Mixup Cocai,Baccilus White Slimy

THE ENZYME ASSAY FOR LIPASE ACTIVITY:

The try butyrine agar TBA places were proparerd for the estimation if esterase activity.

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The holes were wade on agar place with help of starile tips 50ml preshly prepared broth culture
loded in the well in order to assay the E.A.

The place were kept in incubator for 24-48hours at 37c mast of the strains wer capable to
hydrolyze Trybutyrine and producer clear .

The zone size wax measured.

SN Isolates incubation time Zone (mm)

1 RVM I 24 hour 0.1 mm
2 RVM-II 24 hour 0.1 mm
3 RVM-III 24 hour 0.7 mm
4 RVM-IV 48 hour 0.1 mm
5 RVM-V 48 hour 0.8 mm
6 RVM-VI 24 hour 0.7 mm
7 RVM-VII 24 hour 0.7 mm
8 RVM-VIII 48 hour 0.6 mm
9 RVM-IX 24 hour 0.7 mm

CULTURE PRESERVATION: -

The preservation of straees as always a at mast important step in micromobsy for short duration

preservation, we prepared slant in test tube and streaked single colony.

The slant were keeps in incubator at 39oc for 24-48 hour. After cluped appearance of new colours
the slant were kept at 4oc.

For the culture preservation slant was prepared and single colony was streaked from agar plate
on to slants surface and then slants was closed with cotton plug.

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Slant was incubated until there is evidence of growth. Then put these slant tubes in a refrigerator

DISCUSSION

Microbial organisms are ofleuly regarded as less explored natural resource unlike other such as
minerals oil forest etc.

Microbial isolation and screening are still based upon traditional methodlogies. So this is also
considered as tedioud labour intensive,time consuming and costly process.Despite of there are
several advancement in the field of biological science.

Calclusion

Microbial diversity of a useful source to nature useful enzyme from the naluse they have been
provew estibility TERATIVE bioprocess for the canvertion of Drug into more effective from of
drugs.

Several bacterial stains Apart from pharmaceutical they can be used again several molecules
such as perfamery etc.

Food bioreadiation leathers indurstiry for the disirad value addition.

In are initial study we have isolated and screened sevraval bacrerial strength for lipase enzyam
activity.

Some of the isolates such as 3D,5C,3C,5A,5C, has been found to a poteul microorganism for the
study with commercial molecules.Well load current observation into next stage.

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