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SECRETORY EXPRESSION OF SACCHAROMYCOPSISFIBULIGERA R64 Α-AMYLASE WITH NATIVE SIGNAL PEPTIDE IN PICHIAPASTORIS
Shabarni Gaffar, Dani Permana, Dessy Natalia, Toto Subroto, Soetijoso Soemitro
Universitas Padjadjaran, 3rd Procedia Chemistry 17 ( 2015 ) 177 – 183 International Seminar on Chemistry 2014 Available online at www.sciencedirect.com
Bahasa Inggris
Universitas Padjadjaran, 3rd Procedia Chemistry 17 ( 2015 ) 177 – 183 International Seminar on Chemistry 2014 Available online at www.sciencedirect.com
native signal peptide, Pichia Pastoris, Sfamy
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 fromS.fibuligera genomic DNA employing PCR method and was cloned using pPICZA expression vector forPichiapastoris. Expression of Sfamy inP .pastoriswas induced by addition of 0.75% methanol every 24 h. The result showed that Sfamy was secreted byP.pastoristo the culture supernatant. The highest activity of Sfamy in the culture supernatant was found at 72 h inductiontime, that was32.29 U/mL. The result showed that Sfamy with native signal sequence was recognize byP. pastorissecretion machine, which was confirmed by SDS-PAGE analysis with the molecular weight 54kDa.