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Purification and biochemical characterization of a novel α-amylase from Bacillus licheniformis NH1: Cloning, nucleotide sequence and expression of amyN gene in Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
35631 45099 2008 12 PDF Available
Title
Purification and biochemical characterization of a novel α-amylase from Bacillus licheniformis NH1: Cloning, nucleotide sequence and expression of amyN gene in Escherichia coli
Abstract

A thermostable α-amylase from a newly isolated Bacillus licheniformis NH1 was purified, characterized and the gene was isolated, sequenced and expressed in Escherichia coli BL21. The enzyme (BLA.NH1) was purified to homogeneity by 40–60% ammonium sulphate precipitation, Sephadex G-100 gel filtration and Sepharose mono Q anion exchange chromatography, with a 3.08-fold increase in specific activity and 15.9% recovery. The molecular weight of the BLA.NH1 was estimated to be 58 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and gel filtration. The enzyme was highly active over a wide range of pH from 5.0 to 10.0. The relative activities at pH 5.0, 9.0 and 10.0 were about 89, 96.6 and 90%, of that at pH 6.5, respectively. The optimum temperature of the purified enzyme was 90 °C. BLA.NH1 belonged to the EDTA-sensitive α-amylase, but its activity was not stimulated by the presence of Ca2+ ions.The purified enzyme showed extreme stability towards surfactants (SDS, Tween 20 and Triton X-100) and excellent compatibility with a wide range of commercial solid and liquid detergents at 40 °C, suggesting potential application in the detergent industry. In addition, BLA.NH1 was relatively stable towards oxidizing agents, retaining 57% of its initial activity after 1 h incubation in the presence of 1% (w/v) sodium perborate.The amyN gene, which encodes the α-amylase from B. licheniformis NH1, was isolated and its DNA sequence was determined. It showed 92% homology to the sequence encoding α-amylase from B. licheniformis NCIB 8061. The sequence of the BLA.NH1 differs from that of B. licheniformis NCIB 8061 by 21 amino acids. The region, encoding the mature α-amylase was heterologously expressed in E. coli cells using the pDEST17 expression system. The recombinant (His)6-tag enzyme was purified in a single affinity chromatography step and biochemical properties of the recombinant enzyme were determined and compared to those of the native-type enzyme. Interestingly, the recombinant α-amylase showed improved thermostability compared to the native enzyme. At 85 °C, recombinant BLA.NH1 and native BLA.NH1 showed half-lives of 60 and 8 min, respectively.

Keywords
α-Amylase; amyN gene; Bacillus licheniformis; Escherichia coli; Heterologous expression; Thermostability
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Purification and biochemical characterization of a novel α-amylase from Bacillus licheniformis NH1: Cloning, nucleotide sequence and expression of amyN gene in Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 43, Issue 5, May 2008, Pages 499–510
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