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








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 are
called 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 Streptomyces species (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
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 the

synthesis of
degradative enzymes in Bacillus subtilis. Salt stress strongly

stimulates the expression of sacB,
encoding levansucrase (about ninefold),

and downregulates the expression of aprE, encoding
alkaline protease (about

sixfold). 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
whole cells is advantageous, because such biocatalysts display better operational stability
10,11
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 Perezmartinez
18
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,
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.

















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.
2.1 Microorganisms Used
2.2 Isolation and screening of Bacillus subtilis
2.3 Effect of different carbon, nitrogen sources and varying salt concentration on cell
biomass and enzymatic activity
2.4 Whole cell immobilization of Bacillus subtilis in various matrices
2.5 Enzymatic assays
2.6 Estimation of crude protein content

2.1 Micro-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 637 for the production of protease. In work conducted by the University of Oregon Health
Sciences Center for the production of alkaline production Pseudomonas aeruginosa was 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).
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).

2.2 Isolation of Bacillus subtilis
The bacterium can be isolated from a varied range of sources including air, water and soil.
Bacillus subtilis was 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); NH
4
Cl (3); KH
2
PO4 (1.0); K
2
HPO
4
(3.0); Na
2
SO
4
(2.0); and MgSO
4
.7H
2
O (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 subtilis IH-72 was 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 CM3 from 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).

2.3 Effect of different Carbon, Nitrogen sources and varying salt concentration on cell
biomass 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
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 that Aspergillus Oryzae utilized 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.

2.4 Enzymatic assays
Works 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 4
mL 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).

2.5 Estimation of crude protein content
Estimation 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).
2.6 Whole cell immobilization of Bacillus subtilis in various matrices
Kunamneni Adinarayana, Bezawada Jyothi and Poluri Ellaiah studied the effect of Bacillus
subtilis PE-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 of B subtilis PE-11 in
calcium alginate are more efficient for the production of alkaline protease with repeated batch
fermentation. The alginate immobilized cells of B subtilis PE-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.








CHAPTER 3
Objectives

The present study was conducted with the following main objectives-
Comparative study of Bacillus subtilis 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 Bacillus subtilis.
To perform whole cell immobilization of Bacillus subtilis for maintaining reusability
of the organism.
To determine the total protein content of the enzyme showing maximum activity.














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 Bacillus subtilis
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 of B. 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.



3.3 Morphological study of B. subtilis (Gram staining)

Solution A was prepared by dissolving 1 gram crystal violet in 20 ml of 90% ethanol
solution. 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.

3.3 Preparation 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.


3.4 Submerged 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.

3.5 Preparation of standard graph
Alkaline Reagent or Lowry Reagent was prepared.
25 mL of 0.1 N NaOH and 25 mL of 4 % Na
2
CO
3
solution was mixed to prepare reagent
2.
3 mL of 0.5 % CuSO
4
and 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.

3.6 Enzyme 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.

3.7 Determination 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 =




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 mm
3
.
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.


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 assay
R 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 nm
S1 0.177
S2 0.222
S3 0.275
S4 0.300
S5 0.402
Immobilization in calcium alginate( OD at 700 nm)
Salt concentration Enzyme assay Protein assay
Control 0.395 0.845
10 mL 0.329 0.702
20 mL 0.378 0.676
30 mL 0.311 0.118
40 mL 0.302 0.102

Immobilization in agar-agar( OD at 700 nm)
Salt concentration Enzyme assay Protein assay
Control 0.355 0.790
10 mL 0.289 0.675
20 mL 0.319 0.622
30 mL 0.309 0.079
40 mL 0.285 0.056
















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 subtilis 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 subtilis
The effect of salt concentration was studied on growth of Bacillus subtilis against a
control setup.The salt solution consists of K
2
HPO
4
,FeSO
4
.7H
2
O, MgSO
4
.7H
2
O 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
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
e
i
g
h
t

(
I
n

G
r
a
m
s
)

Salt Concentration(ml)
Cell Biomas - Damp Soil
Replicate 1
Replicate 2


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
W
e
i
g
h
t

(
i
n

g
r
a
m
s
)

Salt Concentration(ml)
Cell Biomass - Garden Soil
Replicate 1
Replicate 2



0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
Control 10ml 20ml 30ml 40ml
E
n
z
y
m
a
t
i
c

a
c
t
i
v
i
t
y
(
U
\
m
l
)

Salt Concentration(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
Control 10ml 20ml 30ml 40ml
E
n
z
y
m
a
t
i
c

A
c
t
i
v
i
t
y
(
U
n
i
t
s
\
m
l
)

Salt Concentration(ml)
Enzymatic Activity- Garden Soil
Replicate 1
Replicate 2


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
E
n
z
y
m
a
t
i
c

A
c
t
i
v
i
t
y
(
U
\
m
L
)

Salt concentration(mL)
Comparitive representation of Enzymatic
Activity
Garden 2
DAMP 2
GARDEN 1
DAMP 1






0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
Control 10ml 20ml 30ml 40ml
E
n
z
y
m
a
t
i
c

A
c
t
i
v
i
t
y

(
U
n
i
t
\
m
l
)

Salt Concentration(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
Control 10ml 20ml 30ml 40ml
S
p
e
c
i
f
i
c

a
c
t
i
v
i
t
y

(
X

1
0
-
2

U
n
i
t
s
\
m
g
)

Salt Concentration(ml)
Comparitive Representation of Specific
Activity
Calcium alginate
Agar Agar
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
Control 10ml 20ml 30ml 40ml
S
p
e
c
i
f
i
c

A
c
t
i
v
i
t
y

(
X

1
0
-
2

U
n
i
t
s
\
m
g
)

Salt Concentration(ml)
Specific Activity - Damp Soil
Replicate 1
Replicate 2
0
10
20
30
40
50
60
Control 10ml 20ml 30ml 40ml
S
p
e
c
i
f
i
c

A
c
t
i
v
i
t
y

(
X

1
0
-
2

U
n
i
t
s
\
m
g
)

Salt Concentration(ml)
Specific Activity - Garden Soil
Replicate 1
Replicate 2







0
10
20
30
40
50
60
Control 10ml 20ml 30ml 40ml
S
p
e
c
i
f
i
c

A
c
t
i
v
i
t
y

(
X

1
0
-
2

U
n
i
t
s
\
m
g
)

Salt Concentration(ml)
Comparitive Representation of Specific Activity
DAMP1
GARDEN1
DAMP2
GARDEN2
4.6 Digestion of Egg White
When egg white was digested with the help of enzyme obtained from B.subtilis isolated
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.




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