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

• 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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