The gene of acid-stable alpha-amylase was amplified by PCR using Aspergillus nigergenomic DNA as template, then the gene was cloned into the vector of pPIC9K, the recombinant vector pPIC9K-asAA was then transformed into Pichia pastorisSMD1168, high copy transformants were screened in G418 plates. Regulated by the α-Factor, promoter of AOX1 gene and termination signal of yeast genomic, the recombinant amylase was expressed and secreted out of the cells. The expression of the recombinant amylase was strictly induced by methanol, in shaking culture condition, after 168 h induction with 2% of methanol, the amylase reached maximal activity of 2838 U/ml. SDS-PAGE analysis showed that the molecular weight of the recombinant amylase was about 58 kDa. The recombinant amylase exhibited maximal activity at pH 4.0 and 70°C, the amylase was basically stable at the pH ranging from 3.0 to 6.0, and kept stable for a long time and with a high level of activity at common industrial temperature 50°C.
Key words: Aspergillus niger, acid-stable alpha-amylase, pPIC9K, Pichia pastorisSMD1168.
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