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A type I pullulanase of Bacillus cereus Nws-bc5 screening from stinky tofu brine: Functional expression in Escherichia coli and Bacillus subtilis and enzyme characterization

Paper ID Volume ID Publish Year Pages File Format Full-Text
34448 45026 2014 10 PDF Available
Title
A type I pullulanase of Bacillus cereus Nws-bc5 screening from stinky tofu brine: Functional expression in Escherichia coli and Bacillus subtilis and enzyme characterization
Abstract

•The first reported pullulanase with the producing strain screening from the stinky tofu brine.•The gene pulBC was expressed in E. coli and B. subtilis respectively.•The pullulanase gene expression coding from B. cereus with detailed PulBC enzymatic properties.•The recombinant pullulanase was expressed extracellularly in B. subtilis WB800N with the amyQ signal sequence.

Using enrichment procedures, the strain showing distinct pullulan degradation ability was isolated from the stinky tofu brine and identified as Bacillus cereus (named B. cereus Nws-bc5) by 16S rRNA sequence analysis. Meanwhile, the pullulanase gene (named pulBC) involved in pullulan degradation was obtained from Nws-bc5. The gene has an open reading frame of 2142-bp, and shows highest identity with the pullulanase from B. cereus Q1 (CP000227.1). The gene pulBC was expressed in Escherichia coli and Bacillus subtilis WB800 respectively. The expression level of recombinant PulBC expressed in E. coli and B. subtilis was 5.6 and 0.8 mg/ml respectively. The purified recombinant enzyme (molecular weight 81.4 kDa) was able to attack the α-1,6-linkages in pullulan specifically to generate maltotriose as the major product. The pH and temperature optima of the recombinant enzyme were pH 6.0 and 40 °C respectively. Moreover, PulBC showed much higher stability under alkaline conditions and was stable at pH 6.0–9.0. The pullulanase activity was enhanced significantly by Ca2+. The specific activity of purified PulBC was 44.7 U/mg (pullulan) and 29.3 U/mg (soluble starch). The Km and Vmax values of purified PulBC were 0.45 mg/ml and 45.3 μmol/min, respectively.

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Keywords
Stinky tofu brine; Strain screening; Pullulanase; Gene cloning; Enzymatic properties; Bacillus subtilis expression
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A type I pullulanase of Bacillus cereus Nws-bc5 screening from stinky tofu brine: Functional expression in Escherichia coli and Bacillus subtilis and enzyme characterization
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 49, Issue 11, November 2014, Pages 1893–1902
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