Expression Of Bovine Chymosin In Pichia Pastoris And The Study Of The Enzyme Properties

Bovine chymosin is extracted from bovine stomach which belongs to aspartic proteinase family. It can specifically hydrolyze the peptide bond between Phe105-Met106in the K-casein causing milk-clotting, thus it plays an important role in the cheese-production. It’s principal function is condensing milk and improving the cheese’flavor. According to the different sources, the chymosin can be divided into four categories:animal chymosin, plant chymosin, microbe chymosin and genetic engineering chymosin. Compared to the chymosin from other sources, the genetic engineering chymosin displays the obvious advantage in the aspect of the cost of production and stability of sources.In this study, the gene fragments of prochymosin were obtained by PCR, then it was cloned into the expression vector pPIC9K. The recombinant plasmid pPIC9K-prochy was constructed successfully, which was confirmed by enzyme digestion and sequence reaction. Then it was linearized by Sal I and transformed into pichia pastoris GS115and KM71by electroporation. Transformants were obtained by MD selective medium plates and incubated at28℃for48-72hours. At last, two strains were screened by induced culture in160coloies of GS115and KM71respectively, which displayed the high enzyme activity. The two strains were named GS115-pPIC9K-prochy-32and KM71-pPIC9K-prochy-21. In this experiment, prochymosin was expressed in P.pastoris successfully. SDS-PAGE analysis revealed that the recombinant bovine prochymosin was about44KDa,which was slightly larger than the expected protein molecular size because of the glycosylation. The enzyme activity was tested according to the method of Arima K. The enzyme activity expressing by GS115-pPIC9K-prochy-32and KM71-pPIC9K-prochy-11reached25.0SU/mL and11.5SU/mL respectively. We compared the enzyme properities from the different pichia pastoris. Results showed that the enzyme activity expressing by GS115-pPIC9K-prochy-32was over twice than the KM71-pPIC9K-prochy-21. Furthermore, the former’s stability was superior to the latter. When treated at50℃for50min, the residual activity of the former was66.7%contrasted with the0%of the latter. The optimal temperature and pH was50℃and5.7respectively of the two recombination strains. When treated at60℃for20min, both of them lost their activity. Furthermore, the induced culture conditions of recombinant strain were optimized, then taking samples after induced228h. Under the condition of the optimum temperature50℃and pH5.7, The enzyme activity expressed by GS115-pPIC9K-prochy-32and KM71-pPIC9K-prochy-11reached317SU/mL and180SU/mL respectively.