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Molecular cloning and expression of α-amylase gene of bacillus sp. to E. coli

KrishiKosh

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Title Molecular cloning and expression of α-amylase gene of bacillus sp. to E. coli
 
Creator Kaushik, Naveen Kumar
 
Contributor Chaudhary, Kamla
 
Subject Bacillus, α-amylase, Cloning
 
Description An alpha-amylase producing Bacillus sp. Ns5, was isolated
from soil. Growth and amylase production by Bacillus sp. Ns5 was
studied under different cultivation conditions. Maximum growth was
observed at 30°C. Among defined carbohydrates tested starch and
glucose, starch supported good growth and amylase production, with
the highest productivity recorded in the presence of starch 1%. The
enzyme starts releasing reducing sugar after 2 min of incubation with
substrate. The enzyme characterized for different parameters including
temperature, pH, Ca++ requirement and its Km. Enzyme was found
active under range of temperature 30-70°C with temperature optima at
45° C. pH optima of enzyme found to 5.5 with active under acidic range
of pH having Km 0.67 mg/ml. Presence of Ca++ increase activity 2 fold.
partial Sau3A fragment of Bacillus Sp. Ns5 DNA cloned in an
Escherichia coli YEp FLAG-1 yeast E. coli shuttle vector system was
shown to direct the synthesis of a alpha-amylase whose activity could
be detected in situ on petri plates using the iodine staining method. A
2kb fragment containing plasmid Kn3, the active gene was shown to
express in the periplasmic space and then to excreted out to medium.
The restriction map of the plasmid was determined for three restriction
enzymes. The enzyme production in E. coli transformant K3 was found
seven fold less than parent bacterial strain. The optimum temperature
and pH for enzyme activity were found to be 45°C and pH 5.5,
respectively. Furthermore enzyme was found to be affected by Ca++
Which increase its activity two fold.
 
Date 2016-11-28T10:59:33Z
2016-11-28T10:59:33Z
2006
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/87787
 
Language en
 
Format application/pdf
 
Publisher CCSHAU