A Secreted Expression Vector Construction of β-Glucosidase Gene and Its Expression in Picchia pastoris

摘要

About 2500 bp gene without signal peptide from Aspergillus niger encoding sequence bgl1 was connected to the Pichia pastoris expression vector pPICZ α-A, resulting in the recombinant expression plasimid pPICZα-A-bgl1. It was transformed into P. pastoris GS115 competent cells after Pme I digestion. With the induction of methanol, bgl1 gene in P. pastoris has got a higher level secretory expression. Shake flask experiments showed that there was a corresponding increase in extracellular protein secretion in fermentation broth as the methanol induction time increased. After 8d induced fermentation, the content of extracellular protein reached 1.36 mg /ml and the β-glucosidase activity was up to 7.75IU/ml which was 2.8 times compared to original strain. The relative molecular weight of recombinant β-glucosidase was about 125kD by SDS-PAGE analysis. The optimal enzyme reaction temperature and pH were 60 and 4.0 °C respectively; it maintained good stability below 50 and at pH 3 ～7.