Expression,Purification And The Enzymatic Properties Of The Recombinant Chymosin

Milk clotting coagulants are enzymes which cut the kappa casein specifically.When kappa caseins are cut,the stable state of casein particles in the milk has been destroyed,then the casein coagulated and precipitated while the whey was separated.Use this property,milk clotting coagulants are widely used in the process of cheese to curd the milk.The milk clotting coagulants that are suitable for cheese-making requires high clotting milk activity and low nonspecific proteolytic activity,their ratio,the c/p value is a measurement which assess the quality of the cheese that made with different milk clotting coagulants.The milk clotting coagulants are added into the milk in the process of cheese-making,then the curd form and the whey is discharged,but there are still a small amount of milk clotting coagulants remaining in the curd,they participate in hydrolytic protein in the riping of the cheese,so they can effect on the texture,the flavor and the yield of cheese.According to the different sources,the milk clotting coagulants can be divided into animal rennet(AR),plant-derived coagulant(PC),microbial coagulant(MC)and fermentation produced chymosin or recombinant chymosin(FPC or RC).The calf rennets widely was used in traditional cheese-making for its low nonspecific proteolytic activity,the cheese made with it has good quality and the hige yield.Recombinant chymosin is produced by the process that the prochymosin's cDNA is cloned into the expression vector,then transformed into a host to express the prochymosin,after the subsequent processing and purification,the recombinant chymosin can obtain.The biochemical characteristics of the recombinant chymosin is almost the same compare with that of the natural chymosin,and the purity of the recombinant chymosin is high,the cheese made with the recombinant calf chymosin is comparable to the cheese made with the calf rennet.In this paper we choose two recombinant chymosins to study,the bovine chymosin and the camel chymosin,which are studied extendedly and the two recombinant chymosin have commercial produced.We synthesized the two prochymosin by the chemical method,cloned the two sequences into the expression vector pET30a successfully,named pET30a-b-proCHY and pET30a-c-proCHY respectively,then transform the two vectors into Escherichia coli BL21(DE3),the target protein is expressed with high level when added the IPTG,and the prochymosin accounted for about 66.3%and 61.4%of the total bacteria protein respectively.The recombinant prochymosin original exists in the form of inclusion body in E.coli,the inclusions are dissolved by 6 mol/L urea first,then renatured by dialysis with TE(pH8.0)to get rid of the urea gradually,we can get the folded recombinant prochymosin and the yield is about 200 mg/L,eventually.We treat the bovine recombinant chymosin with acidification/neutralization,namely activating the bovine prochymosin in vitro.We adjust the pH to 2.0 for 2h,then adjust the pH to 6.0.We found that a lot of white precipitates appeared,placed a period of time,clear and transparent at the top,bottom is white precipitate,and we can separate the solution by centrifugal treament quikly.Sampled the supernatant and precipitation and detected them by SDS-PAGE.The supernatant mainly contain the mature chymosin and the his tag-propeptide fusion peptide,the precipitation besides cantain a certain amount of chymosin,also contains almost all of the impurity protein.If the supernatant is transfered for storage,precipitation is resuspended with sterile distilled water times,can recover a certain amount of chymosin from precipitation.So,we centrifuge the activated prochymosin solution that treated by acidification/neutralization,transfer the supernatant for storage,resuspended the precipitation with sterile distilled water and centrifuge,transfer the supernatant,repeated 2 times,mix the supernatants,this is the preliminary purification of the bovine chymosin.We add the saturated ammonium sulfate into the mix supernatants to different final concentration(5%-50%),when the final concentration of the saturated ammonium sulfate obtain 35%-50%,the mature chymosin precipitate while the his tag-propeptide fusion peptide retain in the supernatants.So we chose the final concentration of the saturated ammonium sulfate 40%and 50%to precipitate the recombinant chymosin and we get the purified chymosin finally.Detecting the concentration of protein and the milk clotting activity every step of the purification,we found the recovery rate of the recombinant chymosin is 1.82%,after centrifuge and saturated ammonium sulfate precipitation treaments.This probably because a large number of proteins were removed by the process of acidification/neutralization and the removed proteins are the prochymosins that do not folded corectly.But the total milk clotting activity increased by 73.3%and the specific activity increased 94 times compare with the activated prochymosin solution.This may be relate to the remove of the propeptide and impurity proteins.The freeze-dried recombinant prochymosin and recombinant chymosin retain their activity completely when rehydration.When we add NaCl to the chymosin before freeze-drying,we can get the dispersed particles that contain the chymosin 33?50 mg/g,this form are convenient to access and preservation.We analyzed the enzymic properities of the recombinant bovine chymosin and use the commercial recombinant chymosin as a control.The results show that the optimal temperature range of milk-clotting activity of recombinant chymosin was 57?62 ?,and the chymosin was relatively stable below 40? and in pH 2?7.Metal ions such as Al3+,Fe3+ and Cu2+ significantly increased the chymosin activity,however,pepsin inhibitor pepstatin A had an obvious inhibitory effect on the chymosin activity.These enzymic properities are similar with the commercial recombinant chymosin,so the expressed recombinant chymosin has the commercial potential.Activating the camel prochymosin with different pH,only when the pH is lower than 4.5 can obtain the mature camel chymosin,and the camel chymosin can clot the fresh cow's milk and the bactrian camel milk.The activation rate of the recombinant camel prochymosin has significant difference from that of the recombinant bovine prochymosin,the activation rate of the camel prochymosin is far lower than that of the bovine prochymosin.Under the same conditions,the bovine prochymosin was activated fully within 2h and the camel prochymosin need above 3d.We found that adding NaCl to the activation system of the camel prochymosin can significantly improved the activation rate.We replaced the partly sequences of the camel prochymosin with the corresponding sequences of the bovine prochymosin.One replacement sequence is near the cutting site(propeptide41-42 and chymosinl-6)and the other replacement sequence is contain the whole propeptide and several amino acid of the chymosin(propeptidel-42 and chymosinl-6).Cloning the two replacement camel prochymosin into pET30a,expressing the proteins in Escherichia coli and the fusion proteins were renatured by the same method as mentioned above.Named the fusion proteins as MUT-1 and MUT-2,respectively.The fusion protein MUT-1 is not activated in pH2.0-8.5 and the fusion protein MUT-2 has the similar behaves with the camel prochymosin when under the activation conditions.It suggests that the activation of prochymosin not only involves electrostatic interactions between the propeptide and the enzyme active site,may also be associated with the structure or enzyme activity of chymosin.This study aims provide high expression strains for the industrial production of recombinant bovine chymosin and recombinant camel chymosin,and our results can provide the some experimental basis for the future industrial production of the two recombinant chymosins.