文献类型：期刊文献

英文题名：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

作者：Wei, Wei[1];Ma, Jing[1];Guo, Su[1];Wei, Dong-zhi[1]

机构：[1]E China Univ Sci & Technol, Newworld Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2014

卷号：49

期号：11

起止页码：1893

外文期刊名：PROCESS BIOCHEMISTRY

收录：;EI(收录号：20144500155322);WOS:【SCI-EXPANDED(收录号:WOS:000345482800015)】；

基金：The GeneBank accession numbers for the B. cereus Nws-bc5 16S rDNA gene is KF939332, and KF647846 for pulBc gene. This research was financially supported by the Fundamental Research Funds for the Central Universities of China (No. WF1214047), the National Natural Science Foundation of China (No. C050203-31200596) and the National High Technology Research and Development Program of China (No. 2013AA102109).

语种：英文

外文关键词：Stinky tofu brine; Strain screening; Pullulanase; Gene cloning; Enzymatic properties; Bacillus subtilis expression

摘要：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 pul(Bc)) 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 Pul(Bc) 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 alpha-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 degrees 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 Pul(Bc) was 44.7 U/mg (pullulan) and 29.3 U/mg (soluble starch). The Km and Vmax values of purified Pul(Bc) were 0.45 mg/ml and 45.3 mu mol/min, respectively. (C) 2014 Elsevier Ltd. All rights reserved.