Journal of Medicinal Chemistry and Drug Discovery ISSN: 2347-9027 International peer reviewed Journal Special Issue Analytical Chemistry Teachers And Researchers Association National Convention/Seminar Issue 2, Vol. 1, pp 699-711, 28 February 2016, Available online at http://www.jmcdd.com
Production of alpha-amylase alpha from Bacillus subtilis using different agro waste (Rice bran) by solid state fermentation Asmita. P. Salvi Department of Biotechnology, Milind college of science, Aurangabad (Maharashtra) 431001
Abstract: Enzyme production by Fermentation carried out by solid state fermentation. SSF have been established as a superior technique for the production of enzymes. The project targeted at the production of amylase by B. subtilis using rice bran as substrate with the use of SSF techniques and tested for its activity. Effect of physical parameters such as enzyme concentration, substrate concentration, temperature and incubation time and PH were also tested. Substrate tested found most suitable for enzyme production & their potential could be effectively exploited in the future for production of various commercial products.
Keywords :- alpha-amylase, amylase, B. subtilis, agro waste, fermentation, rice bran.
Introduction: Life is intricate meshwork involving a perfect coordination of a vast majority of chemicals reactions. Some of these reactions result in synthesizing large molecules, others in cleaving large molecules and all of them either utilize energy oorr liberate energy. All these reaction occurs very slowly at the low temperature and the atmospheric pressure the condition under which living cell carry on their life processes, yet in the living cells these reactions proceeds at extremely high rate. This is due to the presence of some catalysts produced and synthesized inside the body of the organism. Enzymes are biocatalysts protein in nature; they catalyze the biochemical reaction taking place in the living cell without any overall change.
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Journal of Medicinal Chemistry and Drug Discovery ISSN: 2347-9027 International peer reviewed Journal Special Issue Analytical Chemistry Teachers And Researchers Association National Convention/Seminar Issue 2, Vol. 1, pp 699-711, 28 February 2016, Available online at http://www.jmcdd.com
Isolated ted enzymes were first used in detergents in the 1914, their protein nature proven in 1926 and their large scale microbial production stared in 1960s. A large number of industrial processes in the areas of industrial environmental and food technology utilize utili enzymes at some stage or the other. Current developments in biotechnology are yielding new application for enzymes. Now a day, most enzymes are produced in industry for various purposes, lipase, protease, amylase, cellulose and other enzymes are also produced produced in industries. Many organisms are capable to produce various enzymes. Bacteria and fungi are mostly used organism for production of enzymes. The most widely used enzyme in the industry for starch hydrolysis is alpha amylase (EC.3.2.1.1). These enzymes enzymes account 65% of enzyme market in world. wo This enzyme catalyses the endo-cleavage cleavage of the alpha1, 1, 4 glycoside linkages and the release of short oligosaccharide and limit dextrin. This enzyme is used commercially for the production of sugar syrups from starch which consist of glucose, maltose and higher oligosaccharide. It is also extensively used in starch liquefaction and paper, food, pharmaceutical and sugar induction. To meet the demands of these industries low cost medium is required for the production produc of alpha amylase. Microbial alphaamylase A large number of microorganism including Bacteria, Yeast and Fungi produce different groups of enzymes. Selection of a particular strain however, remains a tedious task, especially when commercially compete competent nt enzymes, yield are to be achieved. For example, it has been reported that while a strain of produce 19 types of enzymes. Alpha amylase was being produced by as many as 28 microbial cultures. Amylolytic enzymes too are commonly produced by filamentous fungi and the preferred strains belong to the species of as well as bacteria such as Although commercial production of amylases is carried out using both fungal and bacterial cultures. Bacterial alpha amylase is generally preferred for starch liquefaction du due to it high temperature stability. In order to achieve high temperature stability productivity with less production cost, apparently, genetically modified strain hold the key to enzyme production.
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Journal of Medicinal Chemistry and Drug Discovery ISSN: 2347-9027 International peer reviewed Journal Special Issue Analytical Chemistry Teachers And Researchers Association National Convention/Seminar Issue 2, Vol. 1, pp 699-711, 28 February 2016, Available online at http://www.jmcdd.com
Solid state fermentation for alpha amylase production The cost of enzyme production in submerged fermentation is high which necessitates reduction in production cost by alternative methods. The contents of synthetic media are very expensive and these contents might be replaced with more economically available agricultural byproducts for the reduction of cost of the medium. Agro industrial residues are generally considered the best substrate for the solid state fermentation processes and use of solid state fermentation (SSF) for the production of enzymes is noo exception to that a number of such substrate have been employed for the cultivation of microorganisms to host of enzyme. The substrates that have been used included rice bran. The selection of a substrate for enzyme production in a SSF process depends upon several factors mainly related with cost and availability of the substrate, and thus may involve screening of several agro industrial waste. In a SSF process, the solid substrate not only supplies the nutrients to the microbial culture growing in it bbut ut also serves as anchorage for the cells. The substrates that provide the entire needed nutrient to the microorganism growing in it should be considered as the ideal substrate. However, some of the nutrients may be available in suboptimal concentrations orr even absent in the substrate. In such cases, it would become necessary to supplement them externally with these. It has also been a practice to pretreated, some of the substrate before using in SSF processes, thereby making them more easily accessible for fo microbial growth. Scientific classification of Bacillus subtilis Domain
-
Bacteria
Phylum
-
Firmicutes
Class
-
Bacilli
Order
-
Bacilliales
Family
-
Bacillaceae
Genus
-
Bacillus
Species
-
B.subtilis
Binomial name :
-
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Bacillus subtilis www.jmcdd.com
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Journal of Medicinal Chemistry and Drug Discovery ISSN: 2347-9027 International peer reviewed Journal Special Issue Analytical Chemistry Teachers And Researchers Association National Convention/Seminar Issue 2, Vol. 1, pp 699-711, 28 February 2016, Available online at http://www.jmcdd.com
Chemical composition of starch :: [1] Molecular formula:- (C6H10O5)n [2] Molar mass:-
Variable
[3] Appearance:-
White powder
[4] Density:-
1.5%/cm3
[5] Melting point:- Decomposes [6] Solubility in :-
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Journal of Medicinal Chemistry and Drug Discovery ISSN: 2347-9027 International peer reviewed Journal Special Issue Analytical Chemistry Teachers And Researchers Association National Convention/Seminar Issue 2, Vol. 1, pp 699-711, 28 February 2016, Available online at http://www.jmcdd.com
Materials and methods: Microbial cultures The respective culture was Bacillus subtilis and maintained on nutrient agar slants at 40C. Materials:1. Nutrient broth 2. Bacillus subtilis Culture Agro waste products for SSF 1. Rice bran 2. Chemicals for confirmations Test for α amylase 1. Biurette Reagent 2. Iodine reagent Chemicals for study of different parameters ( PH,Temperature,Substrate concentration) 1. Freshly prepared 1 % starch solution 2. α- Amylase ( Supernatant) 3. phosphate buffer ( PH 6, 6.5,7.0,7.5,8.0) 4. DNSA reagent Chemicals for Immobilization of αα Amylase 1. sodium Alginate ISSN: 2347-9027
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Journal of Medicinal Chemistry and Drug Discovery ISSN: 2347-9027 International peer reviewed Journal Special Issue Analytical Chemistry Teachers And Researchers Association National Convention/Seminar Issue 2, Vol. 1, pp 699-711, 28 February 2016, Available online at http://www.jmcdd.com
2. 0.05 M CaCl2 Solution 3. 0.02 M CaCl2 Solution 4. α- Amylase (Supernatant)
METHOD: Preparation of pre-inoculums inoculums B. subtilis was grown on nutrient agar slant for 24 hrs; some colonies were transferred with a sterile needle into the flask containing sterile nutrient broth for heavy growth of bacteria for 24 hrs at 370C in incubator. Bioconversion method Bacillus subtilis 5 gm of Rice bran is taken in a flask containing 50 ml of basal medium, sterilized at 15 lbs pressure for 15 min. at 1210C, inoculated with heavy culture of Bacillus subtilis and incubated for 48 hrs at 370C in incubator, centrifuged for 30 min. at 3000 rpm and supernatant was used as a source of enzyme, alpha amylase produced in the sample is estimated by nitrosamine salicylic acid (DNSA) method. Confirmation Test:
Confirmations Test for Iodine & Biurette reagent ISSN: 2347-9027
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Journal of Medicinal Chemistry and Drug Discovery ISSN: 2347-9027 International peer reviewed Journal Special Issue Analytical Chemistry Teachers And Researchers Association National Convention/Seminar Issue 2, Vol. 1, pp 699-711, 28 February 2016, Available online at http://www.jmcdd.com
Effect of different Parameters ( Effect of temperature & PH) Test performed by incubating temperature reaction mixture for 10 min. at temperature ranging from 20-100 oC. effect of PH determined by varying phosphate buffer, PH 6, 6.5, 7, 7.5, 8. Observation Table:Effect of PH
Sr
PH
no
1%
Buff
Enzyme
DNS
Star
er
(ml)
A
ch
(ml
at
540
nm
rice bran
(ml) Boiling
1
6.0
0.4
0.2
2
6.5
0.4
0.2
300C for 30 1
3
7.0
0.4
0.2
min
4
7.5
0.4
0.2
1
0.02
5
8.0
0.4
0.2
1
0.01
Blan
7.0
0.4
0.2
1
1.%
Incubation at
1
O.D
1
0.02
water Bath 10 min
for
0.01 0.05
k
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Journal of Medicinal Chemistry and Drug Discovery ISSN: 2347-9027 International peer reviewed Journal Special Issue Analytical Chemistry Teachers And Researchers Association National Convention/Seminar Issue 2, Vol. 1, pp 699-711, 28 February 2016, Available online at http://www.jmcdd.com
Effect of PH 0.06 0.05
0.05
0.04 0.03
Series 1
0.02
0.02
0.01
0.02 0.01
0.01
0 6 6.5 7 7.5 8
Observation Table:Effect of Temperature Sr
Temp
No (0C)
1%
phosphate buffer
PH
Enzyme
starch(ml)
7 (ml)
(ml)
DNSA
O.D 540nm
1
200C
0.2
0.4
0.2
1
0.02
2
300C
0.2
0.4
0.2
1
0.02
3
400C
0.2
0.4
0.2
1
Incubation 0.06
4
500C
0.2
0.4
0.2
5
600C
0.2
0.4
0.2
6
700C
0.2
0.4
0.2
Incubation 1 Of 1 respective Temperature 1
7
800C
0.2
0.4
0.2
1
0.01
8
900C
0.2
0.4
0.2
1
0.02
9
1000C
0.2
0.4
0.2
1
0.02
10
Blank
0.2
0.4
1
00
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at
0.03
respective
0.02
Tem
0.04
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Journal of Medicinal Chemistry and Drug Discovery ISSN: 2347-9027 International peer reviewed Journal Special Issue Analytical Chemistry Teachers And Researchers Association National Convention/Seminar Issue 2, Vol. 1, pp 699-711, 28 February 2016, Available online at http://www.jmcdd.com
Effect of Temperature 0.07
0.06
0.06
0.05
0.04
0.04
0.03
Series 1
0.03
0.02
0.02
0.02
0.02
0.02
0.01
0.02
0.01
0 20
30
40
50
60
70
80
90
100
Effect of Substrate concentration
Substrate concentration altered from 0.1 to 1.0ml followed by phosphate buffer (0.4 ml) and (2 ml) and incubated for 5min in boiling water bath. Then DNSA was added and after incubation for 5min in boiling water bath and estimation spectrophotometrically. Observation Table:
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Sr.No 1%
D\W
Substrate Buffer Enzyme
starch
Conn
(ml)
(mg\ml) ml)
DNSA
D\W
O.D
(ml)
1
0.1
0.9
1
0.04
2
0.2
0.8
2
0.04
3
0.3
0.7
3
Kept
Kept
0.05
4
0.4
0.6
4
in
in
0.05
5
0.5
0.5
5
boiling 0.5 ml
boiling
0.06
6
0.6
0.4
6
water
water
3.2
0.05
7
0.7
0.3
7
bath
bath
ml
0.06
8
0.8
0.2
8
for
for
9
0.9
0.1
9
5min
5min
0.2 ml
0.1 ml
0.07 0.07
0
10
1.0
00
10
11
Blank
00
00
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at40 C
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0.08
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Journal of Medicinal Chemistry and Drug Discovery ISSN: 2347-9027 International peer reviewed Journal Special Issue Analytical Chemistry Teachers And Researchers Association National Convention/Seminar Issue 2, Vol. 1, pp 699-711, 28 February 2016, Available online at http://www.jmcdd.com
Effect of Substrate conc. 0.09 0.08
0.08
0.07
0.07 0.07
0.06
0.06
0.05
0.05 0.05
0.04
0.06 0.05 Series 1
0.04 0.04
0.03 0.02 0.01 0 1
2
3
4
5
6
7
8
9
10
Enzyme immobilization
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Journal of Medicinal Chemistry and Drug Discovery ISSN: 2347-9027 International peer reviewed Journal Special Issue Analytical Chemistry Teachers And Researchers Association National Convention/Seminar Issue 2, Vol. 1, pp 699-711, 28 February 2016, Available online at http://www.jmcdd.com
Enzyme immobilization was done by using 6 % sodium alginate & beads wear formed in 0.05 M, 0.02 M CaCl2 solution.
Result & Discussion Basal media for bacillus subtilis was prepared with different agro waste(Rice bran) and production of α- Amylase was confirmed by nitrosamine salicylic acid (DNSA) method. Burette method, iodine test for enzyme obtain from B. Subtilis using rice bran as a substrate & effect of PH, Temperature and Substrate concentration was determine Effect of PH determine by varying phosphate buffer PH between 6--8 from the graph the optimum PH determined was 7 and amylase was obtained on 0.05mg/ml . Enzyme activity estimation by incubating reaction mixture for 10 min at
temperature
ranging from 20-1000C shown highest activity at 400C and amylase was obtained on 0.06mg/ml. The maximum activity attain with Rice bran substrate at its highest concentration.
References: [1]
Anto Hema & Ujjval Trivadi (2006) αα Amylase production by Bacillus cereus, food Technol, Biotechnology 44(2) : 241-245. 241
[2]
Bilal Balkan and Extan Figen (2007). Production of αα Amylase from penicillium chrysogenum under solid state fermentation by using some agriculture agriculture by product, food technology biotechnology 44(4): 439-442. 439
[3]
Jain. J.L , Sanjay Jain (2006) fundamental of Biochemistry, sixth edition 333 333-336.
[4]
Pandey A, P. Selvakumar, C.R Soccol and Poonam Nigam (1992). Solid
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Journal of Medicinal Chemistry and Drug Discovery ISSN: 2347-9027 International peer reviewed Journal Special Issue Analytical Chemistry Teachers And Researchers Association National Convention/Seminar Issue 2, Vol. 1, pp 699-711, 28 February 2016, Available online at http://www.jmcdd.com
[7]
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I-U. U. Huq, H. Ashraf, J. Iqbal, M.A. Qadeer, Bioresource Technology, Technology 2003, 87(1), 57-61.
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R.C. Ray, S. Kar, S. Nayak, M.R. Swain, Food Biotechnology,, 2008, 22(3), 234 234-246
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S. Negi, R. Banerjee, Food Technol. Biotechnol., Biotechnol 2006, 44(2), 257--261.
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