Cloning and Characterization of a Novel Thermophilic Amylopullulanase with a Type I Pullulanase Structure From Anoxybacillus sp WB42

Pullulanase is an important starch-debranching enzyme that is used in the food, chemical, and pharmaceutical industries. In this study, pulWB42 is cloned from a novel pullulan-degrading thermophile (Anoxybacillus sp. WB42) by homology cloning and genome walking. PulWB42 has the signature sequences of type I pullulanases, which cleave only -1,6-glycosidic bonds in pullulan, and shows 74% identity with a type I pullulanase from A. tepidamans. Homology modeling suggests that PulWB42 has a typical type I pullulanase structure, which is distinct from that of known amylopullulanases. The recombinant PulWB42 is expressed in Escherichia coli and identified as an amylopullulanase by thin layer chromatography. rPulWB42 exhibits optimal activity toward pullulan at pH 5.8 and toward amylose at pH 5.5 over a temperature range of 55-65 degrees C, with a K-m value of 5.3 +/- 1.0mgml(-1) for pullulan (0.09-fold lower than that for amylose) and k(cat) value of 152.6 +/- 13.2s(-1) for both pullulan and amylose. Moreover, the initial activity is completely retained after a 24-h incubation at 60 degrees C and pH 5.5. Calcium ions activate the -amylase activity of rPulWB42, while weakly impacting the pullulanase activity. These catalytic properties make PulWB42 an important candidate for use in starch bioprocessing.