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.