Abstract: The gene of acid-stable α-amylase was amplified by PCR using Aspergillus niger genomic DNA as template, the amplified gene was cloned into the vector of pPIC9K, and the recombinant pPIC9K-asAA was then transformed into Pichia pastoris SMD1168. High copy transformants were screened in G418 plates. Regulated by α-factor, AOX1 gene promoter and termination signal of yeasts, the recombinant amylase was expressed and secreted from the cells. The expression of recombinant amylase was strictly induced by methanol under shaking culture conditions. The amylase reached its maximal activity of 2838 U/mL after induction with 2% methanol for 168 h. SDS-PAGE analysis showed that the molecular weight of recombinant amylase was approximately 58 kD. The recombinant amylase revealed the highest activity at pH 4.0 and 70 ℃. Meanwhile, the amylase was basically stable at pH 3.0－6.0 and could keep its stability for a long time and high activity at 50 ℃. Therefore, recombinant Pichia pastoris has good genetic stability.

Key words: Aspergillus niger, acid-stable α-amylase, pPIC9K, Pichia pastoris SMD1168