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Molecular cloning of a cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. TS1-1 and characterization of the recombinant enzyme

Paper ID Volume ID Publish Year Pages File Format Full-Text
18754 42741 2006 11 PDF Available
Title
Molecular cloning of a cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. TS1-1 and characterization of the recombinant enzyme
Abstract

A cyclodextrin glucanotransferase (CGTase) gene from Bacillus sp. TS1-1 was isolated and cloned into Escherichia coli. Starting from TTG codon, there was an open reading frame composed of 2163 bp (721 amino acids). The NH2 terminal position encoded a 46-amino acid of a signal peptide and followed by the mature enzyme (675 amino acids). The deduced amino acid sequence of the mature CGTase from Bacillus sp. TS1-1 exhibited 98.7% homology with 96% identity to the CGTase sequence from alkalophilic Bacillus sp. 1-1. The recombinant CGTase of Bacillus sp. TS1-1 expressed in E. coli was successfully purified to homogeneity using ammonium sulfate precipitation, followed by α-cyclodextrin-bound-epoxy-activated Sepharose 6B affinity chromatography. The purified CGTase enzymes exhibited a single band with molecular weight of 75 kDa on SDS-PAGE. Biochemical characterization of the enzyme shows an optimum temperature of 60 °C and optimum pH of 6.0. The enzyme was stable between pH 7 and 9 and temperature up to 70 °C. The Km and Vmax values calculated were 0.52 mg/ml and 54.35 mg of β-cyclodextrin/ml/min. The yield of the products from soluble starch as the substrate were 86% for β-cyclodextrin and 14% for γ-cyclodextrin after 24 h incubation at 60 °C, without adding any selective agent. The total β-CD produced under the conditions mentioned above was 3.65 g/l.

Keywords
Cyclodextrin glucanotransferase
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Molecular cloning of a cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. TS1-1 and characterization of the recombinant enzyme
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 39, Issue 1, 1 June 2006, Pages 74–84
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