The Expression Of CALB In Pichia Pastoris And The Study Of Its Enzymic Properties

With the availability of gene recombination technology, the synthetic calB gene was linked with vector pGAPZαA-calB and pPIC9K-calB, then the reconbinant expression vectors were transferred into E. coli DH5α. The recombinant plasmids were extracted and transformed into Pichia pastoris GS115 via elecoporation. The constitutive expression and inductive expression of CALB in Pichia pastoris had been received.What'more, the fermentation proc- ess in shake-flask was optimized and we explored the optimizing parameters separately. The optimizing parameters of constitutive expression was pH=5.0, T=30℃and the optimum carbon resource was glycerol. And the optimizing parameters of inductive expression was pH =5.0, 2% methanol(V/V), the temperature of induction was 22℃and the optimum carbon resource was glycerol. Through optimization and purification, the concentration of protein in constitutive expression was 0.22mg/ml, while the concentration of protein in inductive expression achieved 0.45mg/ml. Then we studied the property of CALB that we got. The optimum temperature for maintaining CALB activity was 30℃~60℃, pH4.0 ~ pH6.0.and the optimum temperature and pH for CALB hydrolysis reactions was 50℃~60℃, pH8.0. And the property of CALB that we got and commercial CALB were similar.