2012•Journal of Food Science and BiotechnologyRequires access
Cloning and Secreting Expression of the .BETA.-Glucosidase Gene from Aspergillus niger in Pichia pastoris GS115
Zhu Longbao, Bin Tang, Yugui Tao, Fei Ge, Shenghua Wei, Tao Chen, Wanzhen Li
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Abstract
Based on the β-glucosidase coding sequences from GeneBank,a β-glucosidase gene was amplified from Aspergillus niger by using RT-PCR and vector pMD-18T-bgl was constructed.The expression vector pPIC9K-bgl was constructed by subcloning the gene into plasmid pPIC9K,and then transformed into P.pastoris GS115 through electroporation after linearized by BglⅡdigestion.The recombinant P.pastoris G115 were screened in MD and YPD/G418 plate.The activity of the engineered strain reached to 38 U/mL after induction with the final concentration of 1% methanol.SDS-PAGE analysis showed that the recombinant P.pastoris GS115 had an additional protein band of approximately 90 kD,which was not present in the control,and consistent with the theoretical value of the gene product.The crude enzyme catalysis results indicated that the optimum temperature and pH for the activity were about 50 ℃ and 5.5,respectively.