期刊
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
卷 97, 期 14, 页码 6279-6292出版社
SPRINGER
DOI: 10.1007/s00253-012-4538-6
关键词
Geobacillus thermoleovorans; Amylopullulanase; Truncated amylopullulanase; Starch; Pullulan
资金
- Council of Scientific and Industrial Research (CSIR)
- Department of Science and Technology, Government of India
- University of Delhi
A gene encoding amylopullulanase (gt-apu) of the extremely thermophilic Geobacillus thermoleovorans NP33 was cloned and expressed in Escherichia coli. The gene has an open reading frame of 4,965 bp that encodes a protein of 1,655 amino acids with molecular mass of 182 kDa. The six conserved regions, characteristic of GH13 family, have been detected in gt-apu. The recombinant enzyme has only one active site for alpha-amylase and pullulanase activities based on the enzyme kinetic analyses in a system that contains starch as well as pullulan as competing substrates and response to inhibitors. The end-product analysis confirmed that this is an endoacting enzyme. The specific enzyme activities for alpha-amylase and pullulanase of the truncated amylopullulanase (gt-apuT) are higher than gt-apu. Both enzymes exhibited similar temperature (60 A degrees C) and pH (7.0) optima, although gt-apuT possessed a higher thermostability than gt-apu. The overall catalytic efficiency (K (cat)/K (m)) of gt-apuT is greater than that of gt-apu, with almost similar substrate specificities. The C-terminal region of gt-apu appeared to be non-essential, and furthermore, it negatively affects the substrate binding and stability of the enzyme.
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