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Production, purification and Characterization of thermostable bacterial α– amylase by solid state fermentation of agro-byproducts

KrishiKosh

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Title Production, purification and Characterization of thermostable bacterial α– amylase by solid state fermentation of agro-byproducts
 
Creator Singh, Ajay Kumar
 
Contributor Ramteke, P. W.
Masih, Harison
 
Subject oils, oilseeds, marketing, markets, sales, productivity, plant oils, retail marketing, groundnuts, accounts
Bacillus amyloliquefaciens, Bacillus licheniformis, SSF, Enzyme activity
 
Description A Thesis Entitled “Production, Purification and Characterization of Thermostable Bacterial Α–Amylase by Solid State Fermentation of Agro-Byproducts” was submitted in partial fulfillment of the requirement for the award of the degree of Doctor of Philosophy in Biotechnology by Ajay Kumar Singh.
The production of extracellular α-amylase by thermotolerant Bacillus
amyloliquefaciens and Bacillus licheniformis was studied under solid state fermentation
(SSF). Various agro- byproducts namely Wheat flour, Barley flour, Corn flour, Gram
flour, Moong husk, Arhar husk, Mustard oil cake, coconut oil cake, Banana peel, Potato
peel, Sweet Potato peel, Soybean hull, Wheat bran, Rice bran, and Sugarcane baggase
were examined for α- amylase production. Among all the substrates wheat flour was
found to be best substrate for α-amylase production (145.56 IU/ml) in phosphate buffer
as extracting medium for Bacillus amyloliquefaciens, but in case of Bacillus
licheniformis, wheat bran supported maximum growth and produced maximum α-
amylase (154.17 IU/ml) with Triton –X as extraction medium. Process optimization was
conducted using wheat flour and wheat bran in a single parameter mode showing
enhanced enzyme titre. Further, the appropriate incubation period, moisture level,
incubation temperature and inoculum concentration were determined. Maximum yields
of 149.62 IU/ml, 144.64 IU/ml, 173.28 IU/ml, 164.48 IU/ml were achieved by employing
wheat flour as substrates with Bacillus amyloliquefaciens at temperature 37°C, pH 7,
moisture content 80% and incubation period 72 h whereas Bacillus licheniformis was
found to produce optimum α- amylase at temperature 40°C (168.78 IU/ml), pH 6
(170.34 IU/ml), moisture content 80% (171.89 IU/ml) and incubation period 48 h (155.06
IU/ml). The phosphate concentration was also found to enhance α- amylase yield.
Media supplementation with carbon source as (1%) maltose and (0.15 M) inorganic
source (ammonium chloride) in SSF medium increased amylase enzyme yield (167.44
IU/ml, 167.11 IU/ml) for Bacillus amyloliquefaciens and (178.46 IU/ml and 172.36 IU/ml)
for Bacillus licheniformis, respectively. The effect of addition of external organic
nitrogenous compounds further showed a positive impact on enzyme synthesis by both
the culture. Increase in the enzyme activity was obtained when tryptone and soy
peptone at 1% concentration was added to the fermentation medium. The enzyme was
partially purified to 2.40 and 2.18 fold by ammonium sulphate precipitation and ion
exchange chromatography. The stability profile of the partially purified enzyme from
both strains exhibited maximum activity at 65°C and pH 6-7. The enzyme exhibited
marked increase in activity in presence of metal ions (Ca2+, Fe+++, Mn2+). On the basis
of comparative production optimization parameters the molecular weight of purified
alpha amylase enzyme from Bacillus licheniformis was determined and estimated as 71
kDa on SDS Polyacrylamide gel electrophoresis. The apparent Km and Vmax of alpha
amylase enzyme from Bacillus amyloliquefaciens for soluble starch were estimated to
be 0.085 mg/ml and 212.76 IU/ml respectively whereas from Bacillus licheniformis were
found to be 0.074 mg/ml and185.87 IU/ml, respectively.
 
Date 2016-12-16T10:37:13Z
2016-12-16T10:37:13Z
2016
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/90459
 
Language en
 
Format application/pdf
 
Publisher Sam Higginbottom Institute of Agriculture, Technology & Sciences (SHIATS)