Expression Of Bovine Chymosin Gene In Bacillus Megaterium WH320

Chymosin is the most essential to the industrial production of cheese. Currently the enzyme used in industry is mainly isolated from bovine renen stomach. The investigation has showed that the chymosin is exist in a variety of animal, plants and microbial. However, producing chymosin by microbial fermentation process will significantly decrease the production cost and enlarge the resource, has much advantage comparing with isolating the enzyme from animal and plant resources. Therefore, the investigation for production of chymosin by bio-engineering protocol was performed extensively around world. But as so far, few report and investigation was conducted in China.In order to promote the production of chymosin based on microbial fermentation process, the wild strain was screened, three Aeromonas spp. strains with the ability of producing chymosin were selected. However, the selected strain all is pathopoiesia microbial, not feasibility for production of chymosin. Based on the bovine chymosin has been used for cheese production, the gene sequence of bovine chymosin, accessed from GenBank data base, was synthesized after optimized its codon and sequencing fragment according to the gene expression system and the amino acid sequence difference between the pro-chymosin and activity chymosin. By constructing express vector plasmid pHIS1525-RC and transforming the plasmid into the protoplast of host Bacillus megaterium WH320the modified gene of bovine chymosin was successfully expressed.The recombinant protein was produced under xylose induction as an active chymosin verified by PCR, double-enzyme digestion, protein SDS-PAGE electrophoresis and determination of chymosin activity. The activity features of recombinant chymosin were investigated after purified by Ni-NTA chromatography. The characters of recombinant chymosin were few different comparing with native bovine chymosin. The molecular weight was35.6kDa, the optimium active temperature was45℃. After treated for30min under20～50℃the recombinant chymosin still maintained activity more than75%. With temperature increasing, the activity was dropped sharply. The activity losed nearcompletely after treated at70℃for30min. The optimum pH was pH5.0. The protein can keep the activity more than80%between pH4.6～pH5.8. The activity was more stable at pH3.0～7.4, and losed quickly when pH below3.0or above7.4. The culture condition of the Bacillus megaterium WH320-RC for chymosin production was also investigated. The results showed that:The best suitable carbon source was fructose. Glucose hampered strongly the chymosin production. The best suitable nitrogen sources were yeast extract and beef extract. Neutral pH and37℃was benefit for culture the strain to produce chymosin.0.5%xylose was the best concentration for inducing production of chymosin. Addition of0.2%Tween-80into culture medium enhanced30%the production of recontanant chymosin.By constructing express vector plasmid pET-22b-RC and pQE30-RC, transformed them into Escherichia coli BL21(DE3) and M15, neither of them could express the gene of bovine chymosin. The possible reason was the difference of codon bias between Bacillus megaterium and Escherichia coli. Further investigation need addression.By synthesizing the modified bovine chymosin gene, active chymosin was expressed in Bacillus megaterium WH320, which provided the basis for developing bio-ergineering technology for producing chymosin in China.