a project report studying the effect of salt concentration on the production of alkaline protease...

8/12/2019 A PROJECT REPORT STUDYING THE EFFECT OF SALT CONCENTRATION ON THE PRODUCTION OF ALKALINE PROTEA

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A

PROJECT REPORT

ON

TO STUDY THE EFFECT OF SALT CONCENTRATION ON

THE PRODUCTION

OF ALKALINE PROTEASE BY BACILLUS SUBTILIS AND

IMMOBILIZATION OF

WHOLE CELL

SUBMITTED BY GUIDED BY

Himanshu Ramteke Bidyut Mazumdar

Reecha Sahu Sharmistha Banerjee

Somnath Mukhopadhyay


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CHAPTER 1

Introduction

Protease, a hydrolytic enzyme shares 60% of total worldwide sale of industrial enzymes.

Proteases possess some characteristics of biotechnological interest due to which these have

become the most important industrial enzymes (Barindra, et. al., 2006). Almost all proteases are

heat resistant (Mehler, 1957), vary widely in their specific activities, optimum pH, pH stability

range (2-13), heat sensitivity, substrate specificity, active site, catalytic mechanism and stability

profiles (Raymonde & Othmer, 1941; Ward, 1985). On the basis of their acid-base behavior,

proteases have been classified in to three categories i.e., acid, neutral and alkaline proteases. The

acid proteases are those which have pH optima in the range of 2.0-5.0 and are mainly fungal in

origin (Haq & Mukhtar, 2007). Proteases having pH optima in the range of 7.0 or around arecalled neutral proteases. They are mainly of plant origin however; some bacteria and fungi also

produce neutral proteases. While proteases that have pH optima in the range of 8.0-11.0 are

grouped under the category of alkaline proteases. Some of the important alkaline proteases are

solanain, hurain and proteolytic enzymes of Bacillus and Streptomycesspecies (Hameed et al.,

1996; Lee et al., 2002; Tang et al., 2004).

B. subtilis can be isolated from many environments terrestrial and aquatic making it

seem that this species is ubiquitous and broadly adapted to grow in diverse settings within the

biosphere. However, like all members of the genus Bacillus, B. subtilis can form highly resistant

dormant endospores in response to nutrient deprivation and other environmental stresses [1,2].

these spores are easily made airborne and dispersed by wind [3,4]. Thus, spores might migrate

long distances, land in a given environment but never germinate there. Considering that the

traditional methods for isolating B. subtilis require that the organism be in its spore form, there is

no guarantee that when a strain is isolated from a particular environment it was actually growing

at that location. Thus, the question of where B. subtilis grows has not been so simple to answer

[5,6]. B. subtilis is often referred to as a soil dweller. The organism was most often in its

vegetative form when associated with decaying organic material [8]. Although this early study is

the only one to date that has directly examined growth in natural soils, further support for the

idea that B. subtilis can lead a saprophytic lifestyle comes from recent experiments in which


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spores were inoculated into artificial soil microcosms saturated with filter-sterilized soluble

organic matter extracted from soil [9]. B. subtilis has also been isolated repeatedly from aquatic

environments [2325]. However, there is no published account that directly demonstrates the

growth of B. subtilis in natural waters. Although growth in marine water might occur, the

abundance of B. subtilis in these environments might also be explained by its observed

association with the GI tract of marine organisms [26] and other biotic surfaces [25]. In

summary, current data indicate that the apparent ubiquity of B. subtilis is not solely a

consequence of spore persistence in these environments. Instead, B. subtilis seems to grow in

diverse environments including soils, on plant roots, and within the GI tract of animals.

The screening of the strains of B. subtilis can be done by culturing the microbial sample

in selective media. One of the commonly used methods is by culturing the serially diluted sample

in casein agar media. The microorganism can be isolated on the basis of formation of a clear

zone of casein hydrolysis on the test plate and transferred to nutrient agar slant for growth and

maintenance. Another commonly used laboratory method of screening B. subtilis is the Starch

Hydrolysis Test

Extracellular protease production in microorganisms is highly influenced by media

Variation in components such as C/N ratio, the presence of easily metabolisable sugars like

glucose (Gupta et al., 2002a; Ferrero et al., 1996) and the presence of metal ions (Varela et al.,

1996). Besides this, several other factors, such as aeration, inoculums density, pH, temperature

and incubation time, also affect the amount of protease produced (Hameed et al., 1999; Gupta et

al., 2002b). In commercial practice, the optimization of media composition is done to maintain a

balance between the various media component, thus minimizing the amount of unutilized media

component at the end of the fermentation. Research efforts have been directed mainly toward

evaluating the effect of various carbon and nitrogen nutrient effective substrates on the yield of

the enzymes, requirement of the divalent metal ions in the fermentation medium and

optimization of environmental and fermentation parameters like pH, temperature, agitation,

aeration. Growth under conditions of salt stress has important effects on thesynthesis of

degradative enzymes in Bacillus subtilis. Salt stress stronglystimulates the expression of sacB,

encoding levansucrase (about ninefold),and downregulates the expression of aprE, encoding


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alkaline protease (aboutsixfold). It is suggested that the DegS-DegU two-component system is

involved in sensing salt stress.

The common methods for determining enzyme (protein) depend on using a standard

protein. There is no absolute method for determining the enzyme concentration when you have a

mixture of proteins in a solution. So in general, a specific protein which is readily available in

quite pure form and not too expensive; for example many biochemical labs use bovine serum

albumin (BSA), gelatin and casein is used.

Protein concentration can be determined according to the method described by Bradford.

One ml of Bradford reagent was added to 50l of sample and the extinction was measured after 5

min at 595 nm. Different concentrations of bovine serum albumin (BSA) were used as a protein

standard: 10, 20, 40, 60, 80, and 100 g/ ml distilled water. One ml of Bradford reagent was

added to 50 l BSA standard and the extinction was measured after 5 min at 595nm (Ibrahim

et.al. 2007).Another common method for the determination of protein content is by Folin-

Lowrys Method. The phenolic group of tyrosine and trytophan residues (amino acid) in a

protein will produce a blue purple color complex, with maximum absorption in the region of 700

nm wavelength, with Folin reagent which consists of sodium tungstate molybdate and phosphate.

Thus the intensity of color depends on the amount of these aromatic amino acids present and will

thus vary for different proteins. Most proteins estimation techniques use Bovin Serum Albumin

(BSA) universally as a standard protein, because of its low cost, high purity and ready

availability. The method is sensitive down to about 10 g/ml and is probably the most widely

used protein assay despite its being only a relative method , subject to interference from Tris

buffer, EDTA, nonionic and cationic detergents, carbohydrate, lipids and some salts. The

incubation time is very critical for a reproducible assay. The reaction is also dependent on pH

and a working range of pH 9 to10.5 is essential. Overall, about 10 to 50 times more sensitive

than the Biuret method. The Folin Standard Curve is usually not perfectly linear, so the assay

should be carried out carefully.

Modification of biotechnology and processes, using immobilized biocatalysts, has

recently gained the attention of many biotechnologists. Application of immobilized enzymes or


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whole cells is advantageous, because such biocatalysts display better operational stability10,11

and

higher efficiency of catalysis,12,13

and they are reusable. Microbial products are usually produced

either by free or immobilized cells. The use of immobilized cells as industrial catalysts can be

advantageous compared to batch fermentation process.14,15

Whole cell immobilization has been a

better choice over enzyme immobilization.16,17

Whole cell immobilization by entrapment is a

widely used and simple technique. Romo and Perezmartinez18

reported the viability of microbial

cells over a period of 18 months under entrapped conditions and it was considered as one of the

potential applications. The success achieved with the entrapment technique prompted us to study

the production of alkaline protease with immobilized cells using this technique.

Proteases execute a large variety of functions and have important biotechnological

applications. Proteases represent one of the three largest groups of industrial enzymes and find

application in detergents, leather industry, food industry, pharmaceutical industry and

bioremediation processes (Anwar and Saleemuddin, 1998; Gupta et al. 2002). Probably the

largest application of proteases is in laundry detergents, where they help removing protein based

stains from clothing (Banerjee et al. 1999). For an enzyme to be used as an detergent additive it

should be stable and active in the presence of typical detergent ingredients, such as surfactants,

builders, bleaching agents, bleach activators, fillers, fabric softeners and various other

formulation aids. In textile industry, proteases may also be used to remove the stiff and dull gum

layer of sericine from the raw silk fibre to achieve improved luster and softness. Protease

treatments can modify the surface of wool and silk fibres to provide new and unique finishes.

Alkaline proteases are envisaged to have extensive applications in leather industry. In a

tannery, a rawhide is subjected to a series of chemical treatments prior to tanning and finally

converted to finished leather. Proteases may play a vital role in these treatments by replacing

these hazardous chemicals especially involved in soaking, dehairing and bating (Puvankrishnan

& Dhar, 1986). Increased usage of enzymes for dehairing and bating not only prevents pollution

problems, but also is effective in saving energy. The advantage of using proteases for dehairing

of skins are the reduction of the sulfide contents in the effluent, recovery of the hair/wool which

is of good quality, an increased yield of leather area, easy handling of the pelts by workmen,
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simplification of the pretreatment, the elimination of the bate in the deliming stage and finally

the production of a good quality pelts/leather (Dhar, 1974; Mitra & Chakraverty, 1998).

Another interesting application of alkaline protease was developed by Fujiwara and

coworkers (Ishikawa et al. 1993). They reported the use of an alkaline protease to decompose the

gelatinous coating of X-ray films, from which silver was recovered.The proteolytic enzymes also

offer a gentle and selective debridement, supporting the natural healing process in the successful

local management of skin ulcerations by the efficient removal of the necrotic material (Sjodahl et

al. 2002). Alkaline protease has also been investigated to determine its usefulness in the clean up

of DNA at high temperature due to its stability against t SDS and temperature.


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CHAPTER 2

Review of Literature

Microorganisms excrete a wide variety of proteolytic enzyme, which are also found in

mammalian systems. They are molecules of relatively small size and are compact, spherical

structures that catalyses the peptide bond cleavage in protein (Polgar, 1989). They hydrolyse the

peptide bonds and therefore lead to disassembly of proteins. Commercially, they are very

important as 60% of the total enzyme market relies on proteases, isolated from plants, animals,

bacteria and fungi. Proteases are (physiologically) necessary for living organisms. They are

ubiquitous, found in diversity of sources.

The review of literature was summarized under the following subheadings.

2.1Microorganisms Used

2.2Isolation and screening ofBacillus subtilis2.3Effect of different carbon, nitrogen sources and varying salt concentration on cell

biomass and enzymatic activity

2.4Whole cell immobilization ofBacillus subtilisin various matrices

2.5Enzymatic assays

2.6Estimation of crude protein content

2.1Micro-organisms used

For the production of protease various microorganisms have been used as source. For the

production of serine alkaline protease, Bacillus sp. (SBP-29) was used in works performed at

Delhi University (Saurabh, S. et.al., 2007). Srinubabu, Lokeswari and Jayaraju used Aspergillus

oryzae 637for the production of protease. In work conducted by the University of Oregon Health

Sciences Center for the production of alkaline production Pseudomonas aeruginosawas used.

The media utilized for the purpose consisted of a dialysate of Trypticase soy broth (TSBD)

treated with chelating agent, Chelex 100 (Bio-Rad Laboratories, Richmond, Calif.) (Stanley J.

Cryz and Barbara H.Iglewski, 1980). Usama Beshay used Teredinobacter turnirae for production

of alkaline protease immobilized in Ca-alginate beads. The alkaline protease of Teredinobacter

turnirae possesses unique properties in terms of a high salt tolerance. (Usama Beshay, 2003).


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Protease can be produced by all microorganisms, however, only microbes that produce a

substantial amount of extra cellular protease have been exploited commercially. To date, the

major proportions of the commercial alkaline protease are derived from Bacillus sp.(Joo et al.,

2002; Manachini et al, 1998; Yang et al., 2000; Ito et al., 1998). The reason for this is their wide

temperature and pH tolerance and stability (Genckel and Tari, 2006).

2.2Isolation of Bacill us subtil isThe bacterium can be isolated from a varied range of sources including air, water and soil.

Bacillus subtiliswas isolated from the soil by enrichment and selective screening on skim milk

agar plate. The modified basal medium used for protease production contained (g/L) Casamino

acid (5); NH4Cl (3); KH2PO4 (1.0); K2HPO4(3.0); Na2SO4(2.0); and MgSO4.7H2O (0.10) with

the pH of 7.0. The production medium (50 mL) was inoculated with 2.0% of inoculum (Saurabh,

S. et al, 2007). The bacterial strain of Bacillus subtilisIH-72was isolated from a soil sample

collected from the tannery area. One gram soil was suspended in 10 ml of sterilized saline and

was subjected to serial dilution. 0.5 ml from an appropriate dilution was speeded on a test plate

containing nutrient broth-casein-agar medium. The strain was isolated on the basis of formation

of a clear zone of casein hydrolysis on the test plate and was transferred to nutrient agar slant for

growth and maintenance (Hamid Mukhtar and Ikram-Ul-Haq, 2008). In a work conducted by Do

Thi Bich Thuy of Heu University, Vietnam, Bacillus subtilis was isolated from shrimp shell

(water source) in Hue University of Agriculture and Forestry (Do Thi Bich Thuy, 2004). Manas

R. Swain, Shaktimay Kar, Gourikutti Padmaja and Ramesh C. Ray isolated Bacillus subtilis

strain CM3from culturable cow dung microflora. The culture was maintained on Nutrient agar

plates at 4C. The microorganism was used for the production of extracellular -amylase (Manas

R. Swain et al, 2006).

2.3Effect of different Carbon, Nitrogen sources and varying salt concentration on cellbiomass and enzymatic activity

To screen out the most favorable carbon source, different sources were studied by Afia

Ghafoor and Shahida Hasnain at variable concentrations ranging from 0.5-4%. It includes

Glucose, Fructose, Sucrose, Maltose and Lactose, while different nitrogen sources were also

employed involving Casein, Peptone, Tryptone, Beef Extract and Yeast Extract to achieve the


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maximum enzyme production (Afia Ghafoor and Shahida Hasnain, 2009). Magdi, Hezayen et.

al. reported that gelatin enhanced protease production when added to at a concentration 0.1%

(w/v). The results revealed that the maximum protease production was 30 I.U. at 0.1% (w/v).The

importance of the inorganic nitrogen source for the production purpose of bacterial protease by

Bacillus subtilis KO strain was evaluated by introducing different inorganic nitrogen sources

ammonium phosphate, ammonium oxalate, ammonium acetate, ammonium chloride, ammonium

sulphate and sodium nitrate into the production media. It was also found that ammonium acetate

and ammonium oxalate has an inhibitory effect on the production media (Magdi A.M.Y., Francis

F. H., et al, 2009).

In the works conducted by G. Srinubabu, N. Lokeswari And K. Jayaraju in 2006, It was

found thatAspergillus Oryzaeutilized carbon sources glucose, fructose, sucrose, lactose, dextrin

and starch among them glucose was found to be the best carbon source, for nitrogen sources

various inorganic and organic media components were investigated among them peptone is

found to be the best nitrogen source. 1% cottonseed followed by 2% Soya bean meal was found

to be the best inducer. With optimized media two-fold increase in the protease production. The

fungus growth depends on the concentration of carbon, nitrogen and salt solution, where as the

enzyme production was also influenced by the culture time, pH and interaction between these

two variables.

2.4Enzymatic assaysWorks conducted in the Department of Microbiology in Delhi University aimed at studying

the productivity of serine alkaline protease by Bacillus species, the protease assay involved the

following method. 0.5 mL of Glycine-NaOH buffer (pH 10, 0.2 M) was added to 0.5 mL of

appropriately diluted enzyme and was incubated with 1 mL of 1% Casein solution (Prepared in

Glycine-NaOH buffer, pH 10) for 15 min at 60 C. The reaction was stopped by the addition of 4mL of 5% (v/v) Trichloroacetic acid (Saurabh, S. et al, 2007). Protease activity was determined

according to the method of Anson using casein as a substrate (Do Thi Bich Thuy, 2004). The

method of McDonald and Chen (1965) was used for the assay of protease (Hamid Mukhtar and

Ikram-Ul-Haq, 2008). Afia Ghafoor and Shahida Hasnain used the method of Kunitz (1947) to

monitor the protease activity (Afia Ghafoor and Shahida Hasnain, 2009).


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2.5Estimation of crude protein contentEstimation of protein content by the method of lowry et.al. was reported by Dahot with

bovine serum as standard (Dahot, 1993). The works of Jane Kestner and Ronald J. Elim provide

with a comparative analysis of four different methods for protein estimation. these methods

include Coomassie Brilliant Blue dye-binding, the method of Lowry et al., Biuret method and

immunonephelometry. The experiment was done to determine low concentrations of protein

present in human serum protein fractions (Cohn Fractions II, Ill, IV, and V) (H. Harold Nishi,

Jane Kestner and Ronald J. Elim, 1985).

2.6Whole cell immobilization of Bacillus subtilis in various matricesKunamneni Adinarayana, Bezawada Jyothi and Poluri Ellaiah studied the effect of Bacillus

subtilisPE-11 cells immobilized in various matrices, such as calcium alginate, k-Carrageenan,

polyacrylamide, agar-agar, and gelatin, for the production of alkaline protease. Calcium alginate

was found to be an effective and suitable matrix for higher alkaline protease productivity

compared to the other matrices studied. All the matrices were selected for repeated batch

fermentation. From the results, it is concluded that the immobilized cells ofB subtilisPE-11 in

calcium alginate are more efficient for the production of alkaline protease with repeated batch

fermentation. The alginate immobilized cells ofB subtilisPE-11 can be proposed as an effective

biocatalyst for repeated usage for maximum production of alkaline protease. The work was

carried out in Pharmaceutical Biotechnology Division, Department of Pharmaceutical Sciences,

Andhra University, Visakhapatnam.


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CHAPTER 3

Objectives

The present study was conducted with the following main objectives-

Comparative study of Bacill us subtil is isolated from garden soil and damp soil by

gram staining.

To study the effect of varying salt concentration on the activity of alkaline protease.

To determine the effect of salt concentration on the growth of Bacill us subtil is.

To perform whole cell immobilization of Bacillus subtil isfor maintaining reusability

of the organism.

To determine the total protein content of the enzyme showing maximum activity.


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CHAPTER 4

MATERIALS AND METHODOLOGY

3.1 Materials used

Glass wares used are petriplates, test-tubes, beakers, glass slides, conical flasks, pipettes, glass

rods, reagent bottles and measuring cylinders, reagents and instruments which include pH meter,

autoclave, incubator, water bath(thermo tech), incubator shaker, light microscope, centrifuge,

electronic weighing machine and laminar air flow hood.

3.2 Isolation and screening of Bacill us subtil is

Two soil samples were taken from different areas.

One was taken from a damp area and other from garden.

Soil samples were air dried.

1 gram of the dried soil sample was weighed and mixed in 10 mL of distilled water and

was serially diluted in 3 more test tubes having 10 mL of distilled water in each. Both the

soil samples were serially diluted.

Starch Agar medium was prepared composition of which is given in table no.3.1. It was

then poured in 2 petriplates.

1 ml of diluted soil samples was inoculated in a starch agar medium.

Inoculated petriplates was kept for incubation in an incubator at 37 C for 24 hours.

Iodine solution was prepared.

Screening of the bacterial colonies was done by examining the starch hydrolysis, iodine

solution was poured over the 24 hours old culture plate. Appearance of clear zone

indicates the presence of colonies ofB. subtilis as it hydrolyses the strarch.

Colonies were transferred in agar slants.

24 hour and 48 old agar slants were prepared containing the pure colonies.


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3.3 Morphological study of B. subti l is (Gram staining)

Solution A was prepared by dissolving 1 gram crystal violet in 20 ml of 90% ethanolsolution. Solution B was prepared by dissolving 0.89 gram ammonium oxalate in 80 ml

distilled water. Both the solutions were mixed and stirred till stain dissolved. Thus

obtained solution is termed as grams crystal violet.

Another solution was prepared using 3 gram potassium iodide (KI) in 300 ml distilled

water and 2 gram of iodine was mixed to obtain so called grams iodine. 0.5% saffranin

was also used as staining agent.

A loop full of culture obtained from garden and damp soil was taken on a clean grease

free slides and smear was prepared which was air dried and heat fixed.

Smear was covered with grams crystal violet for 1 min and excess was drained off and

washed under a gentle flow of running tap water.

Then smear was covered with gram iodine for 1 min and excess was drained off. 95%

alcohol was used to decolourise smear for 45 sec.

Smear was washed and flooded with basic dye for 1min, excess was drained off and

washed under gentle flow of tap water. It was air dried and observed under the

microscope.

3.3Preparation of inoculum 200 mL of inoculum media was prepared and was distributed in 4 Erlenmeyer flasks each

containing 50 ml these were then sterilized in autoclave for 15 minutes at 121C.

Flasks were cooled, 2 flasks were inoculated aseptically with a loop full of bacteria from

48 hours old slants of garden soil and other two with damp soil.

Incubation was provided at a temperature of 37C at 140 rpm.

2 flasks, 1 inoculated with garden soil and other of damp soil were kept for 48 hours, rest

2 were withdrawn after 24 hours and kept in the refrigerator.


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3.4Submerged Fermentation 300 mL of production medium was prepared in 500-mL Erlenmeyer flask and was

autoclaved.

200 ml of salt solution was prepared. Production media was distributed in different flasks varying the salt concentration from

10 ml to 40 ml making the volume upto 50 ml. All the set up were kept in replicates.

It was inoculated with 0.5 mL of the 24 hour inoculum and incubated in the rotary

incubator at speed of 140 rpm for 24 hours at 37C.

After incubation supernatant or enzyme was obtained by centrifuging it at 10000 rpm at

4C for 15 minutes.

Pellet was separated with the help of filter paper and was kept for drying.

Dried cell mass was obtained.

3.5Preparation of standard graph Alkaline Reagent or Lowry Reagent was prepared.

25 mL of 0.1 N NaOH and 25 mL of 4 % Na2CO3 solution was mixed to prepare reagent

2.

3 mL of 0.5 % CuSO4and 2 % sodium potassium tartarate solution was mixed to prepare

reagent 1.

Lowry reagent was prepared by mixing 1 and 2 in the ratio of 1:50.

10 mL of 0.2 mg/mL BSA solution is prepared.

In 5 test tubes 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL of BSA solution was added

making up the volume to 2.0 mL with distilled water.

5 ml of alkaline reagent was added in each test tube and was incubated for 15 minutes.

After incubation 0.5 mL Folins Reagent (3:1 ratio of distilled water and FC Reagent)

was added and again was kept for incubation for 30 minutes.

Reading for OD was taken at 700 nm in UV Spectrophotometer.

OD was plotted against concentration of Standard BSA to obtain a standard graph.

Standard gelatin graph was also plotted.


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3.6Enzyme assay Substrate was prepared by dissolving 1% gelatin in Glycine NaOH buffer.

2 mL of substrate was added in all test tubes. All steps were performed in replicates.

1 ml of the crude enzyme obtained was added in each test tubes.

Test tubes were incubated for 1 hour at 30C.

Flame heated for blocking the reaction.

Mixture was centrifuged in a centrifuge tube at 5000 rpm for 10 minutes.

5 ml of alkaline reagent was added to 1 ml of supernatant and incubate it at room

temperature for 15 minutes.

Add 0.5ml of FC reagent and incubate it for 30 minutes and OD was taken at 700 nm.

With the help of standard gelatin graph the concentration of enzyme was obtained.

3.7Determination of protein content To determine the protein content of the enzyme, protein assay was performed.

1 ml enzyme was added to 5 ml of alkaline reagent and was kept for incubation at room

temperature for 15 minutes.

0.5 ml of FC reagent was added and was again incubated for 30 minutes.

After incubation OD was taken at 700 nm.

With the help of standard BSA graph concentration of protein in crude enzyme was

obtained.

With the help of standard graphs concentration of crude protein in enzyme and

concentration of enzyme was determined in moles/ml.

Enzyme activity and specific activity of enzyme was determined.

Enzymatic activity = ()

Specific activity =


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3.10 Immobilization of bacterial cell by Calcium Alginate Bead

1.009 gm of sodium alginate was weighed and dissolved in 30 mL of distilled water to

prepare 4% solution of sodium alginate.

0.5M solution of calcium chloride solution is prepared by dissolving 3.32 gm of calcium

chloride in 60 mL of distilled water and kept in the low temperature.

Loop full of culture is taken from 48 hour old broth and mixed properly with sodium

alginate solution.

With the help of a micro pipette homogenous mixture of sodium alginate and culture

cells is added drop wise chilled calcium chloride solution to form beads of calcium

alginate.

Beads were separated and kept in Glycine NaOH buffer at low temperature.

3.11 Immobilization in agar gel

1.079 gm of agar is weighed and dissolved in 50 mL of distilled water, solution was

homogenized by heating.

Autoclaved for 15 minutes at 115C.

Loop full of culture taken form 48 hour old broth was mixed with the agar at a

temperature of about 50C.

Poured in a petriplate to get a thickness of 4 mm and is allowed to solidify.

After that the solidified agar is cut to get cubes of 4 mm3.

The cells immobilized in agar was stored in Glycine NaOH buffer at low temperature.

3.12 Digestion of egg white

An egg was taken and the egg white or the proteinous part was separated from the yolk.

1 mL of egg white was taken test tubes as a substrate and increasing concentration of

enzyme was added and allowed to digest the protein for 15 minutes.

Similarly enzymes obtained from immobilized cells in calcium alginate as well as agar-

agar were in increasing concentration in different test tubes containing egg white.


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CHAPTER 5

OBSERVATIONS

Standard BSA

O.D. at 700 nm

S1 0.227

S2 0.272

S3 0.295

S4 0.481

S5 0.489

Standard Gelatin

Effect of salt concentration- damp soil( OD taken at 700 nm)

Salt concentration Enzyme assay Protein assay

R 1 R 2 R1 R2

Control 0.416 0.414 0.998 0.969

10 mL 0.389 0.391 0.990 0.959

20 mL 0.401 0.399 0.808 0.796

30 mL 0.362 0.359 0.625 0.600

40 mL 0.344 0.349 0.439 0.398

Effect of salt concentration- garden soil( OD taken at 700 nm); R1 and R2 are the replicas

Salt concentration Enzyme assay Protein assayR 1 R 2 R1 R2

Control 0.425 0.414 0.911 0.902

10 mL 0.389 0.391 0.750 0.741

20 mL 0.401 0.399 0.738 0.721

30 mL 0.362 0.359 0.256 0.219

40 mL 0.344 0.349 0.224 0.199

O.D. at 700 nmS1 0.177

S2 0.222

S3 0.275

S4 0.300

S5 0.402


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Immobilization in calcium alginate( OD at 700 nm)


Control 0.395 0.845

10 mL 0.329 0.702

20 mL 0.378 0.67630 mL 0.311 0.118

40 mL 0.302 0.102

Immobilization in agar-agar( OD at 700 nm)


Control 0.355 0.790

10 mL 0.289 0.675

20 mL 0.319 0.622

30 mL 0.309 0.07940 mL 0.285 0.056


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RESULTS AND DISCUSSIONS

Production of enzyme was studied along with Bacillus subtilis isolated from different soil

samples. Production was poor in damp soil as compared to garden.

The results were summarized under the following main headings:-

4.1 Comparative study of Bacillus subtil is isolated from garden and damp soil.

The isolates obtained from both the soil sources were rod shaped, gram positive

bacterium but lower density of cells were visible in damp soil confirming lower population of

organism. Similar results were obtained when Saurabh,S., Jasmine,I. in 2007 isolated a Bacillus

species from soil sample.

Starch Agar Media Starch Hydrolysis Test

4.2 Effect of varying salt concentration on the growth of Bacillus subtil is

The effect of salt concentration was studied on growth of Bacillus subtilis against a

control setup.The salt solution consists of K2HPO4 ,FeSO4.7H2O, MgSO4.7H2O maintained at

pH 7.The cell biomass was found to be maximum in control in case of both the live samples, i.e.

in absence of salt solution and it was found to be 0.949 gm/50 mL of fermentation media in case


20/31

of garden soil.The growth of organism decrease on increasing the salt concentration from 10 to

40 mL. In a literature by G.Shinu Babu,N.Lokeshwari and K.Jayaraju in 2006 it is cited that

there is an increase in growth of fungal cells as well as enhancement in activity of alkaline

protease on increasing the salt concentration. While it has been observed by F.Kunst and

G.Rapoport in 1995 that growth under conditions of salt stress has an important effect on the

synthesis of degradative enzymes.Salt stress strongly downregulates the expression of aprE.,

encoding alkaline protease.

0.8

0.82

0.84

0.86

0.88

0.9

0.92

0.94

Control 10ml 20ml 30ml 40ml

W

eight(InGrams)

Salt Concentration(ml)

Cell Biomas - Damp Soil

Replicate 1

Replicate 2


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4.3 Effect of salt concentration on Enzymatic activity

The enzymatic activity was found to be highest in control in case of both the soil

samples. The activity was found to be 0.034 units/mL in dam soil and 0.0353 units/mL in garden

soil.

0.7

0.75

0.8

0.85

0.9

0.95

1

0 10 20 30 40

Weight(ingrams)


Cell Biomass - Garden Soil

Replicate 1

Replicate 2


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0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04


Enzymaticactivity(U

\ml)


Enzymatic Activity - Damp Soil

Replicate 1

Replicate 2

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04


EnzymaticActivity(Units\ml)


Enzymatic Activity- Garden Soil

Replicate 1

Replicate 2


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4.4 Whole Cell immobilization of Bacillus Subtillis

Whole cell immobilization was performed by immobilizing the organism in matrices

such as Calcium Alginate and Agar-Agar. Calcium alginate was found to be an effective matrix

for higher alkaline protease productivity compared to Agar-Agar. Similar results were obtained

in the work of Kunamneni Adinarayana ,Bezawada Jyothi and Pouri Ellaiah in 2005.

Ca Alginate beads

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0 10 20 30 40

EnzymaticActivity(U\mL)

Salt concentration(mL)

Comparitive representation of Enzymatic

Activity

Garden 2

DAMP 2

GARDEN 1

DAMP 1


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0

0.005

0.01

0.015

0.02

0.025

0.03

0.035


EnzymaticActivity

(Unit\ml)


Comparitive Representation of Enzymatic

Activity

Calcium alginate

Agar Agar

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35


Specificactivity

(X10-2Units

\mg)


Comparitive Representation of Specific

Activity

Calcium alginate

Agar Agar


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4.5 Specific Activity of the enzyme

The specific activity of alkaline protease obtained from B.subtilis isolated from garden

soil was found to be more as compared to that obtained from dam soil.The specific activity in

case of garden soil was 0.526 units/mg.

0

5

10

15

20

25


SpecificActivity

(X10-2Units\mg)


Specific Activity - Damp Soil

Replicate 1

Replicate 2

0

10

20

30

40

50

60


SpecificActivity

(X10-2Units\mg)


Specific Activity - Garden Soil

Replicate 1

Replicate 2


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0

10

20

30

40

50

60


SpecificActivity

(X10-2Units\mg)


Comparitive Representation of Specific Activity

DAMP1

GARDEN1

DAMP2

GARDEN2


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4.6 Digestion of Egg White

When egg white was digested with the help of enzyme obtained from B.subtilisisolated

from garden soil; hazy appearance was observed initially. But after 15 minutes a clear zone

was obtained. Similar results were obtained in case of enzymes obtained from immobilized cells.

Cells immobilized in Calcium alginate gave better results.


28/31

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