Secretory Expression of Saccharomycopsisfibuligera R64 α-Amylase with Native Signal Peptide in Pichiapastoris

Saccharomycopsisfibuligera R64 α-amylase (Sfamy) can degrade raw starch making it attractive for industrial application. The enzyme has been widely used in the food and textile industries, and recently in the generation of renewable energy. Here we report the secreted expression of Sfamy with native signal peptide in Pichiapastoris using the methanol controlled alcohol oxidase (AOX1) promoter. The gene for Sfamy with native signal sequence (WTSfamy) was amplified from S. fibuligera genomic DNA employing PCR method and was cloned using pPICZA expression vector for Pichiapastoris. Expression of Sfamy in P. pastoris was induced by addition of 0.75% methanol every 24 h. The result showed that Sfamy was secreted by P. pastoris to the culture supernatant. The highest activity of Sfamy in the culture supernatant was found at 72 h induction time, that was 32.29 U/mL. The result showed that Sfamy with native signal sequence was recognize by P.pastoris secretion machine, which was confirmed by SDS-PAGE analysis with the molecular weight 54 kDa.