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Improvement of thermal stability of a mutagenised α-amylase by manipulation of the calcium-binding site

Paper ID Volume ID Publish Year Pages File Format Full-Text
17071 42638 2013 8 PDF Available
Title
Improvement of thermal stability of a mutagenised α-amylase by manipulation of the calcium-binding site
Abstract

•We engineered of an atypical α-amylase by only single amino acid substitution.•The mutations have no significant effect on secondary and tertiary structure of the enzyme.•Single amino acid substitutions in the calcium binding sites, improved thermostability.•The thermostability was related to increasing the enzyme affinity for calcium.

Site-directed mutagenesis of an α-amylase isolated from Bacillus megaterium WHO has been performed to evaluate the roles of the calcium binding site residues in enzyme thermostability. The strategy used was to replace residues in the hypothetical calcium binding loops of B. megaterium WHO α-amylase (BMW-amylase) by equivalent positions at Halothermothrix orenii α-amylase (AmyA) as a thermophilic amylase by QuikChange site directed mutagenesis. Asn-75, Ser-76, and His-77 were mutated in the second calcium binding site which resulted in an increase in thermostability. All mutants retained their hydrolytic activity although their kcat parameter decreased in compare to the wild type and in the presence of calcium ions. In S76P and H77E, the Km for starch decreases while overall activity (kcat/Km) was increased. In the presence of calcium, conversion of His-77 to Glu resulted in a 4-fold enhancement in enzyme half life and a 9 °C upward shift in T50, which was observed in compare to the wild type. Further analysis suggested the H77E mutant as the most stable which increased the affinity of the enzyme for calcium ion and its optimum temperature was 5 °C higher than the wild type.

Keywords
α-Amylase; Bacillus megaterium; Calcium; Mutagenesis; Thermostability
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Improvement of thermal stability of a mutagenised α-amylase by manipulation of the calcium-binding site
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 53, Issues 6–7, 10 December 2013, Pages 406–413
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us