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Purification and characterization of five alkaline, thermotolerant, and maltotetraose-producing α-amylases from Bacillus halodurans MS-2-5, and production of recombinant enzymes in Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
17826 42699 2008 8 PDF Available
Title
Purification and characterization of five alkaline, thermotolerant, and maltotetraose-producing α-amylases from Bacillus halodurans MS-2-5, and production of recombinant enzymes in Escherichia coli
Abstract

A newly isolated strain, MS-2-5, identified as Bacillus halodurans, produced five alkaline and thermotolerant amylases. The five amylases, named amylases A–E, were separated from each other, and purified to homogeneity. The molecular masses of these amylases were different from each other, and estimated to be 90, 85, 70, 65, and 58 kDa. These amylases showed the maximal activities at 60–65 °C and pH 10.5–11. A predominant product by each enzyme reaction was maltotetraose. These amylases were classified as an α-amylase by anomeric form analysis of the reaction products. Internal amino acid sequence analyses of the purified enzymes suggested that these enzymes were produced from a single polypeptide by proteolytic degradation. The gene, named amyA, was cloned and expressed in the T7 promoter systems of Escherichia coli. To increase yield and productivity of recombinant enzyme, cultivation conditions were examined. The maximal amount of enzyme was produced when an E. coli transformant carrying amyA was cultivated at 25 °C in Luria-Bertani medium supplemented with 1.0% d-glucose, 1.0% d-sorbitol, 0.1% MgSO4·7H2O, and 2.0% yeast extract. The yield of the transformant increased 104-fold as, compared with that of the parent strain MS-2-5.

Keywords
Ap, ampicillin; Cm, chloramphenicol; LB, Luria-Bertani; G2, maltose; G3, maltotriose; G4, maltotetraose; G5, maltopentaose; NMR, nuclear magnetic resonance; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction;
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Purification and characterization of five alkaline, thermotolerant, and maltotetraose-producing α-amylases from Bacillus halodurans MS-2-5, and production of recombinant enzymes in Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 43, Issues 4–5, 6 October 2008, Pages 321–328
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