[Gene expression and characterisation of three pullulanases from Bacillus cereus GXBC-3]

Exploring excellent new pullulanase genes, and enriching pullulanase theory are of great importance to realize the industrialization of pullulanase. Three genes, pulA, pulB and pulC, encoding pullulanases, were cloned from Bacillus cereus GXBC-3 by bioinformatics analyzing the open reading frame in Bacillus cereus, annotated as putative I and II pullulanases in the GenBank database. Characteristics of these recombinant enzymes were inducible intracellular expressed in Escherichia coli, the results showed PulA was typical II pullulanase. Recombinant PulA could hydrolyze alpha-1,4- and alpha-1,6-glycosidic bonds. Its specific activity was 32.89 U/mg with an optimum temperature of 40 degrees C and optimum pH 6.5 using pullulan as substrate. And for soluble starch substrate, its specific activity was 25.71 U/mg with an optimum temperature of 50 degrees C and optimum pH 7.0. PulB and PulC were I pullulanases and only hydrolyzed alpha-1,6-glycosidic bond. The specific activities, optimum temperature and optimum pH of PulB and PulC for pullulan substrate were 228.54 U/mg, 45 degrees C, 7.0 and 229.65 U/mg, 45 degrees C, 6.5, respectively.