Abstract: In order to improve expression of glucoamylase in Pichia pastori, based on the gene sequences encoding as registered in GenBank (serial number is AY652617), using the partiality condon of P.pastoris, the gene was designed and synthesized. The modified gene was cloned into the pGAPZαA vector to construct the recombinant expression vector pGAPZαA-EC. Then the pGAPZαA-EC which was linearized by Bln Ⅰ was transformed into P.pastoris X33 by electroporation. The transformants were screened with Zeocin and multiply-copy colonies were harvested, in which glucoamylase gene was verified to be inserted into yeast chromosome stably. SDS-PAGE result showed that, a 80 ku secreted protein was produced, consistent with the expected one, concentration of which was 180 mg/L in supernatant. Expression product showed enzymatic activity by Starch-PAGE.

Key words: Aspergillus niger; glucoamylase; Pichia pastoris; secretion expression; activity identification