The
pulA1 gene, encoding a novel thermostable type I pullulanase PulA1 from
Bacillus sp. CICIM 263, was identified from genomic DNA. The open reading frame of the
pulA1 gene was 2655 base pairs long and encoded a polypeptide (PulA1) of 885 amino acids with a calculated molecular mass of 100,887 Da. The
pulA1 gene was expressed in
Escherichia coli and
Bacillus subtilis. Recombinant PuLA1 showed optimal activity at pH 6.5 and 70 掳C. The enzyme demonstrated moderate thermostabi
lity as PuLA1 maintained more than 88% of its acitivity when incubated at 70 掳C for 1 h. The enzyme could completely hydrolyze pullulan to maltotriose, and hydrolytic activity was also detected with glycogen, starch and amylopection, but not with amylose, which is consistent with the property of type I pullulanase. PulA1 may be suitable for industrial app
lications to improve the yields of fermentable sugars for bioethanol production.
Keywords:
Bacillus sp.; thermostable type I pullulanase; gene cloning and expression