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.

Enzymes 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 V

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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.

Usually means the cleavage of chemical bonds by the addition of water.

When a carbohydred is broken into is component sugar molecule by hydrolysis (e.g. sucrose
being broken down into glucose and fructose, this is termed saccharification.

Generally, hydrolysis or saccharification is a step in the degradation of a substance OR in the

language of chemistry The reaction of cation and anion or both with Water molecule due to
which pH is altered, cleavage of

H-O bond in hydrolysis takes place.

Hydrolysis can be the reserve of a condensation reaction In which two molecule join together
into a larger one and Eject a water molecule. Thus hydrolysis adds water to break down, whereas
condensation builds up by removing Water.

Types:-

Usually hydrolysis is chemical process in which a molecule of water is added to a substance.

Sometimes this addition causes both substance and water molecule to break down.

In such reaction, one fragment of the target molecule ( or parent molecule) gains a hydrogen ion.

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Salts:-

A common kinds of hydrolysis occurs when a salt of a weak base (or both) is dissolve is water.
Water spontaneously ionizes

Into hydroxide anions and hydronium cations.

The salts also dissociates into its constituent anions and cations.

For example, sodium acetate dissociates in water into sodium and acetate ions.

Sodium ions react very little with the hydroxide ions whereas the acetate ions combine with
hydronium ions to produce acetic Acid (H 2so4) in water is accompanied by hydrolysis to given
hydronium and bisulfate, the sulfuric acids conjugate base.

For a more technical discussion of what occurs during such a hydrolysis

INDRISTIAL IMPORTANCE OF HYDROLASE:-

INTRODUCTION

Hypersaline environments are widely distributed on the earths continent where they exist either
as natural water bodies such as permanent saline lakes, ephemeral salt pans and salt marshes, or
as artificial solar salterns which comprise a series of interconnecting evaporation and crystallizer
ponds constructed for the production of salt, potash and soda. Hypersaline environments can be
divided into two broad categories. These are the thalassohaline and athalassohaline.
Thalassohaline water bodies have similar salt composition to seawaterwith sodium and chloride
being the dominant ions; common examples include the Great Salt Lake in Utah, playas, brine
springs from underground salt deposits andsolar salterns (Litchfield and Gillevet, 2002). In
contrast, athalassohaline water bodies such as the Dead sea, Lake Magadi in Kenya, Wadi Natrun
in Egypt, the sodalakes of Antarctica and Big Soda Lake and Mono Lake inCalifornia are
dominated by potassium, magnesium, or sodium (Oren, 2002; Litchfield and Gillevet, 2002).
Hypersaline water bodies are commonly 9-10 times more concentrated than sea water, which is

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generally defined as having 3.5% (w/v) dissolved salts. Both natural and artificial hypersaline
environments harbour remarkably high and diverse microbial cell densities. Microorganisms that
thrive in these environments have been broadlyclassified into halophilic microorganism (that is,
require salt for their viability) and halotolerant microorganisms which are able to grow in the
absence as well as in the presence of NaCl. Halophiles can be further divided into slight
halophiles that grow optimally in 3% (w/v) total salt, moderate halophiles [optimal growth at 3 -
15% (w/v) salt] and extreme halophiles that grow optimally at 25% (w/v) salt (Ventosa et al.,
1998). Eubacteria are mainly represented within the halotolerant, slight halophiles and moderate
halophiles, with only a few genera been shown to be extremely halophilic. To adapt to saline
conditions, these bacteria have developed various strategies to maintain cell structure and
function. These include the accumulation of osmolytes such as ectoine and hydroxyectoine,
glycine and betaine (Vargas et al., 2008). While most research performed on hypersaline
environments has focused on the microbial diversity and ecology
of these environments, there is growing interest in the extracellular hydrolytic enzymes from
moderately halophilic bacteria. Most halophilic hydrolase producers
have been assigned to the family Halomonadaceae and were shown to produce industrially
relevant enzymes such as cellulases, amylases, xylanases, proteases and lipases (Snchez-Porro
et al., 2003a; Govender et al., 2009; Rohban et al., 2009). It is generally believed that while these
halophilic enzymes perform the same enzyme function as their non-halophilic counterparts, they
can catalyse such reactions under different conditions, such as high salt environments. In
addition, some of the enzymes derived from bacterial strains that were isolated from soda lakes
and solar salterns originating from athalassohaline environments could display
polyextremophilicity due to their adaptation to high salt and alkaline pH typical of soda lakes.
Consequently, these bacteria would be an excellent source of enzymes that exhibit optimal
activities at various ranges of salt concentrations and pH.

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ECOLOGICAL DISTRIBUTION OF HYDROLASEPRODUCING

BACTERIA

Hypersaline environments maintain remarkably high microbial cell densities and are biologically
very productive ecosystems. Various culture-dependent and nutriational analyses carried in
tandem with molecular cultureindependent techniques have been used to characterize
the microbial communities in hypersaline environments. Halophilic and extremely halotolerant
microorganisms are present in each of the three domains of life: archaea, bacteria and eukarya
(Oren, 2002). The domain bacteria typically contains many types of halophilic and halotolerant
microorganisms that spread over a large number of phylogenetic subgroups. Most of these fall in
the family Halomonadaceae (class Gammaproteobacteria, order Oceanospirillales) and they are
moderate rather than extreme halophiles (Oren, 2002). Research on hydrolytic enzymes from
halophilic organisms was pioneered by Nordberg and Hofsten at the end of the 1960s (Nordberg
and Hofsten, 1969). Since then, a considerable amount of effort has been dedicated towards the
evaluation of extracellular salt-tolerantenzymes of the moderately halophilic bacteria and the
use of such enzymes in biotechnological processes (Ventosa et al., 1998). Several researchers
have screened halophilic bacteria from different hypersaline environments through direct plating
on agar media amended with substrates specific for enzymes of interest. A wide variety
of bacteria that secrete extracellular hydrolytic enzymes such as amylases, proteases, lipases,
DNases, pullulanases and xylanases have been isolated and characterized (Snchez-Porro et al.,
2003b; Rohban et al., 2009; Govender et al., 2009). Greater hydrolytic activity is commonly
observed amongst Gram-positive moderately halophilic bacteria than Gram-negative bacteria.
Most of the Gram-positive bacteria belong to the Bacillus group including Salibacillus,
Halobacillus, Oceanobacillus, Gracilibacillus, Virgibacillus, Thalassobacillus and Piscibacillus
(Snchez-Porro et al., 2003b; Rohban et al., 2009). Hydrolase-producing Gram-negative bacteria
commonly comprise species of Salinivibrio, Chromohalobacter and Halomonas (Snchez-Porro
et al., 2003b;Rohban et al., 2009). Amylases, lipases, proteases, xylanases xylanases and
cellulases are widely distributed amongst halophilic bacteria. While this could be unexpected, it
is understandable since most natural and artificial hypersaline environments are open system
with an influx and presence of plant and animal matter at any given time. Consequently, the

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microbial population in these environments can be expected to harbour the machinery to exploit
the nutrient resources present in their surrounddings.

OSMOADAPTATION IN HALOPHILIC BACTERIA:-

Hypersaline environments are characterized by high but variable osmotic strength and
microorganisms present in these environments must be able to adapt to the changes in
osmolarity. Most halophilic and halotolerant bacteria maintain viability in these environments by
accumulating low-molecular weight water-soluble organic compounds commonly referred to as
compatible solutes to counteract the deleterious effect of high salinity on cell physiology and loss
of cell water (Louis and Galinski, 1997; CnovasB et al., 1998; Bursy et al., 2008). Both Gram-
positive (e.g. Marinococcus halophilus, Streptomyces coelicolor, Nesterenkonia halobia) and
Gram-negative bacteria (e.g. Halomonas elongata, Chromohalobacter salexigenes) are known to
accumulate ectoines as the predominant class of osmolytes while other compounds such as
glycine and betaine are only accumulated in small amounts (Louis and Galinski, 1997). These
compounds are synthesized de novo or may be taken up from the external environment and they
can be amassed by the cell in very high concentrations to provide osmotic balance without
affecting essential cellular functions (Vargas et al., 2008; Bursy et al., 2008). Several studies
have shown that osmolytes such as ectoines may serve as general stress protectants as they are
produced both in response to salt and heat stresses (Bursy et al., 2008; Vargas et al., 2008). The
ectoine synthesis pathway has been extensively studied and the ectoine synthesis gene
cluster (ectABC) was found to be highly conserved among the ectoine-producing bacteria
(Caldern et al., 2004; Vargas et al., 2008). The genes ectA, ectB and ectC encode the enzymes
diaminobutrytic acid acetyl transferase, diaminobutrytic acid transaminase and ectoine synthase,
respectively, which, altogether constitute the ectoine biosynthetic pathway (Caldern et
al., 2004; Kuhlmann and Bermer, 2002; Vargas et al., 2008). While ectoine is generally thought
to serve as an osmoprotectant, it has also been reported to play a critical role in stabilizing
proteins and supporting correct folding of polypeptides under denaturing conditions
(Bursy et al., 2008).

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HALOPHILIC HYDROLASES

There has been growing interest in scientific research on salt tolerant enzymes derived from
halophilic bacteria due to the potential industrial application of these enzymes. It is generally
believed and has been proven that many halophilic enzymes are polyextremophilic. These
enzymes not only remain active and stable in high salt environments but are often also
thermotolerant and alkaliphilic (Moreno et al., 2009). These properties made halophilic enzymes
attractive for various biotechnological applications as they would be able to catalyze reactions
under harsh conditions typical of many industrial processes. a- distinct clade away from other
extremophilic amylases (Figure 1). The enzyme was reported to display 55 and 53% identity to
the amylases from Pseudoalteromonas haloplanktis and Aeromonas hydrophila, respectively
(Coronado et al., 2000b). In contrast, the amylase from the thermophilic, moderately halophilic
anaerobic bacterium H. orenii display high homology with thermophilic amylases from
Dictyoglomus thermophilum and Bacillus species, although it has narrow substrate specificity as
it does not hydrolyse substrates such as pullulan and cyclodextrins (Mitjs and Patel, 2002). The
H. orenii amylase also lacks transferase activity which means that it can perform the same
catalytic reactions as the thermophilic amylases but will most probably generate adifferent range
of products (Tan et al., 2008

Proteases

Microbial proteases are one of the most extensively studied enzymes and they are widely applied
in industrial processes. They are commonly used as additives in laundry detergents, food
processing, pharmaceuticals, leather and diagnostic reagents, waste management as well as silver
recovery (Amoozegar et al., 2007; Karbalaei-Heidari et al., 2009). Halophilic proteases have
been isolated and characterized from several bacterial species including Bacillus sp. (Kamekura
and Onishi, 1974; Kumar et al., 2004; Setyorini et al., 2006; Shivanand and Jayaraman, 2009),
Pseudoaltermonas sp. (Sanchez-Porro et al., 2003b), Salinivobrio sp. (Amoozegar et al., 2007),
Salicola sp. (Moreno et al. 2009), Halobacillus spp. (Namwong et al., 2006; Karbalaei-Heidari et
al., 2009), Filobacillus sp. (Hiraga et al., 2005), Chromohalobacter sp. (Vidyasagar et al., 2009),
Nesterenkonia sp. (Bakhtiar et al., 2005) and Virgibacillus sp. (Sinsuwan et al., 2009). These
enzymes display optimal activity in the presence of NaCl and maintain stability over a wide pH

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range (pH 5-10). In addition, the enzymes were active at temperatures of 40 75C. While some
of the enzymes display an absolute requirement of NaCl for activation, the protease from
Chromohalobacter was reported to retain 100% stability in the absence of NaCl (Vidyasagar et
al., 2009). In addition, some of the enzymes may display polyextremophilicity.
For instance, the enzymes may be haloalkaliphilic (Gupta et al., 2005) or halothermophilic
(Vidyasagar et al., 2009). Consequently, halophilic and halotolerant bacteria harbour a pool of
proteases that will be more suitable for application in food production processes that are
performed under saline conditions but can also be applied in saline free systems. For instance,
saline fermentation processes involved in the production of various protein rich foods including
processing of fish and meat-based products and the production of soy sauce (Setyorini et al.,
2006). Moreover, the enzymes derived from halophiles make excellent additives for
1558 Afr. J. Biotechnol.

CONCLUSION AND FUTURE PROSPECTS

Halophilic and halotolerant bacteria secrete a wide range of hydrolytic enzymes into their
surrounding environment. Several of these enzymes which include amylases, proteases,
xylanases and cellulases display polyextremophilic properties. They are generally
haloalkaliphilic and thermotolerant which renders them amenable to an array of industrial
processes, normally performed at extreme conditions of temperature and pH. However, only a
limited number of these enzymes have been well characterized and only a few of them are
exploited commer-cially, mainly because research has largelyfocused on microbial diversity in
hypersaline environments rather than the industrial potential of halophiles. In order to fully reap
the benefits of newly described bacterial species, it is necessary to understand their metabolic
and physiological properties. This will allow the generation of valuable information and the
definition of therepertoire of extreme enzymes that has the potential toopen new biotechnological
applications. Therefore, it is necessary to expedite research on the sequence analyses, expression
and characterization of halophilic enzymes so that the potential of these enzymes for industrial
applications can be explored.

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 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

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.

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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:-

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.

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 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 :-

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.

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 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.

METERIAL REQUIREMENT

A-ChemicalS
Beef extract:- control 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

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 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. funnel
IX. glass rod
X. inoculation loop

METHOD

1-Sample Collection
Sample 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

lighter the list of source is as fallowing:-

S.N. SOURCE AREA

1. Soil Ground soil
2. Soil Mango root

3. Soil Dairy soil

4. Water Bathing water sample
5. Soil Wheat soil

2:-Media preparation :-
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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:-

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.

C-Autoclaving

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After the preparation of NBM and NAM we placed broth media into the autoclave for
sterilization.

A basic principle of autoclave is that when the pressure of a gas increases the Temp of a gas
increases proportionally.

Under this condition steam at a pressure about 15psi attaing Temp ( 121 Oc ) will kill all
organisms in about 15 min.

After autoclaving the media we it colled.

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.

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.

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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.

HYDROLASE ASSAY

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

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.

ENZYME ASSAY

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 Enzyme assay for performed to serve two different purposes
1- To identify a special enzyme to prove its presence or absence in a distinet specimen
like an organism or a tissue.
2- To determine the amount of the enzyme in the sample.
For enzyme reaction it must be considered that enzyme Reaction depended on more

factors that Ph, Temperature, and ionic strength.

3-ESSENTIAL CONDITION FOR ENZYME ASSAYS:-

The aim of any assay is to observe the time depended formation of the product.

A sample but important condition is that substrate and product must differ in the observed
feature.

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:-

Fruit juice manufacturing :-

pectins are sulestrances in fruit lamella and cell walls which contains also hemicelluloses
and cellulose.

21 | P a g e
 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 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 :-

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:-

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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.

Making lactose free products for patients suffering from lactose intolerance lactose breaks
lactose to glucose and galactose.

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 .

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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.

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.

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CF = 1000,000/1,000

CF = 1,000 cells per ml.

REVIEW

1. Hammock BD, Moody DE, Sevanian A (1985) Epoxide hydrolases in the catabolism of sterols
and isoprenoids. In: Law, J.H. and H.C. Rilling (ed) Methods in Enzymology, Volume III,
Steroids and Isoprenoids, Part B. Academic Press, Orlando, Florida, pp 303311

2. Wixtrom RN, Hammock BD (1985) Membrane-bound and soluble-fraction epoxide

hydrolases: Methodological aspects. In: Zakim D, D.A. Vessey (eds) Biochemical Pharmacology
and Toxicology, Vol. 1: Methodological Aspects of Drug Metabolizing Enzymes. John Wiley &
Sons, Inc., New York, pp 193

3. Morisseau C, Hammock BD (2002) Epoxide hydrolases. In: Creighton TE (ed) Wiley

Encyclopedia of Molecular Medicine. John Wiley & Sons, Inc., New York, NY, pp 11941197

4. Newman JW, Morisseau C, Hammock BD (2005) Epoxide hydrolases: their roles and
interactions with lipid metabolism. Prog. Lipid Res. 44(1):151

5. Morisseau C, Hammock BD (2005) Epoxide hydrolases: mechanisms, inhibitor designs, and

biological roles. Annu. Rev. Pharmacol. Toxicol. 45:311333

6. Inceoglu B, Schmelzer KR, Morisseau C, Jinks SL, Hammock BD (2007) Soluble epoxide
hydrolase inhibition reveals novel biological functions of epoxyeicosatrienoic acids (EETs).
Prostaglandins Other Lipid Mediat. 82(1-4):4249

7. Morisseau C, B.D. Hammock (2007) Measurements of soluble epoxide hydrolase (sEH)

activity. In: J.S. Bus, L.G. Costa, E. Hodgson, D.A. Lawrence, D.J. Reed (eds) Techniques for

25 | P a g e
analysis of chemical biotransformation. Current Protocols in Toxicology. August, Supplement
33. John Wiley & Sons, Inc., New Jersey, pp 4.23.1-4.23.18

8. Chiamvimonvat N, Ho C, Tsai H, Hammock BD (2007) The soluble epoxide hydrolase as a

pharmaceutical target for hypertension. J. Cardiovasc. Pharmacol. 50(3):225237

9. Morisseau C, Hammock BD (2008) Gerry Brooks and epoxide hydrolases: four decades to a
pharmaceutical. Pest Manag. Sci. 64(6):594609

10. Harris TR, Li N, Chiamvimonvat N, Hammock BD (2008) The potential of soluble epoxide
hydrolase inhibition in the treatment of cardiac hypertrophy. Congest Heart Fail 14(4):219224

11. Imig JD, Hammock BD (2009) Soluble epoxide hydrolase as a therapeutic target for
cardiovascular diseases. Nat Rev Drug Discov 8(10):794805

12. Wang YJ, Ulu A, Zhang L, Hammock B (2010) Soluble epoxide hydrolase in atherosclerosis.
Curr Atheroscler Rep 12(3):174183

13. Wagner K, Inceoglu B, Gill SS, Hammock BD (2011) Epoxygenated fatty acids and soluble
epoxide hydrolase inhibition: novel mediators of pain reduction. J. Agric. Food Chem.
59(7):28162824

14. Zivkovic AM, Telis N, German JB, Hammock BD (2011) Dietary omega-3 fatty acids in the
modulation of inflammation and metabolic health. Calif Agric (Berkeley) 65(3):106111

15. Wagner K, Inceoglu B, Hammock BD (2011) Soluble epoxide hydrolase inhibition,

epoxygenated fatty acids and nociception. Prostaglandins Other Lipid Mediat. 96(1-4):7683

16. Yang J, Dong H, Hammock BD (2011) EETs and oxo-ETE in airway diseases. In:
Obstructive Airway Disease: Role of Lipid Mediators. CRC Press, Boca Raton, FL, pp 125150

17. Shen HC, Hammock BD (2012) Discovery of inhibitors of soluble epoxide hydrolase: a
target with multiple potential therapeutic indications. J. Med. Chem. 55(5):17891808

18. Eiserich JP, Yang J, Morrissey BM, Hammock BD, Cross CE (2012) Omics approaches in
cystic fibrosis research: a focus on oxylipin profiling in airway secretions. Ann. N. Y. Acad. Sci.
1259:19

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19. Morisseau C, Hammock BD (2013) Impact of soluble epoxide hydrolase and
epoxyeicosanoids on human health. Annu. Rev. Pharmacol. Toxicol. 53:3758

20. Xu D, Davis BB, Wang Z, Zhao S, Wasti B, Liu Z, Li N, Morisseau C, Chiamvimonvat N,

Hammock BD (2012) A potent soluble epoxide hydrolase inhibitor, t-AUCB, acts through
PPAR to modulate the function of endothelial progenitor cells from patients with acute
myocardial infarction. Int. J. Cardiol. 167(4):12981304

21. Harris TR, Hammock BD (2013) Soluble epoxide hydrolase: Gene structure, expression and
deletion. Gene 526(2):6174

RESULT:-

CULTURE COLLECTION:-

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 Field soil
2. Soil Gutter soil

3. Soil Mango soil

4. Soil Wheat soil

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5. Soil Rose soil

MICROBIAL ISOLATION-

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. VSS Soil Gram +ve Cocai,Baccilus White Dry

2. VSS Soil Gram + Baccilus Yellow Dry

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

8. VSS Soil Gram + Baccilus White Slimy

9. VSS Soil Gram - Cocai Yellow Dry

10. VSS Soil Gram + Baccilus Creami Dry

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THE ENZYME ASSAY FOR HYDROLASE ACTIVITY:

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

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 VSS I 24 hour 0.5 mm
2 VSS -II 24 hour 0.3mm
3 VSS -III 24 hour 0.7 mm
4 VSS -IV 48 hour 0.1 mm
5 VSS -V 48 hour 0.6 mm
6 VSS -VI 24 hour 0.8 mm
7 VSS -VII 24 hour 0.7 mm
8 VSS -VIII 48 hour 0.4 mm
9 VSS -IX 24 hour 0.5 mm

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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

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.

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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 2B,3D,7A has been found to a poteul microorganism for the study
with commercial molecules.Well load current observation into next stage.

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