Study Of The Production And Propetries Of The Microbial Milk-clotting Enzyme

Cheese derives from milk and forms by coagulation of the milk protein casein withrennet or some other milk-clotting enzymes. Cheese is valueable for human health due to itsnutritions, including almost all proteins and minerals of the milk, rich in various fat-solublevitamins and other trace elements. Only few cheeses are produced by adding acids or lemonjuice to curdle milk, while most cheeses are coagulated by adding the milk-clotting enzyme.As a key factor, the milk-clotting enzyme has a significant influence on the flavor and textureof the cheese during cheese production. The demand of milk-clotting enzyme was increasedsteadily, however, calf rennet cannot meet the requiremen of the market. Therefore, newsource and substitutes of milk-clotting enzyme are studied by many researchers since1950s.In this study we screened a strain which displayed a milk-clotting activity(MCA). Thestrain was identified as Bacillus amyloliquefaciens by16S rDNA BLAST in the GenBank andmorphological and physiological characteristics analysis.In order to improve the yield of milk-clotting enzyme (MCE), a two-stage oxygen supplycontrol strategy was proposed and successfully applied in the MCE fermentation. During thefirst16h KLa was controlled at72.2h-1to obtain high cell growth rate (v) and MCE activity(MCA) productivity (rMCA). Subsequently, KLa was controlled at33.9h-1to maintain highspecific MCA productivity (qMCA). MCA peaked at36h with the MCA of6590.41SU·mL-1with this strategy, which was18h earlier than other investigated processes. The concept andresults described represent the basis of an industrial scale-up process to achieve high MCEyield, MCA productivity and MCA/proteolytic activity (PA).The milk-clotting enzyme was purified by ammonium sulfate precipitation, DEAEanion-exchange chromatography and Sephacryl S-100gel filtration chromatography. Thepurified enzyme after these steps showed a protein band which corresponded to27.5kDa onSDS-PAGE. The enzyme rendered the highest MCA at70℃and pH5.5respectively, whileproteolytic activity (PA) was highest at50℃and not affected much with pH variation. Thepurified enzyme was stable at pH4-6, retaining88%MCA and92%PA. The purified enzymepossessed a property of low thermal stability. Several mineral elements had little effect onboth the activities. In contrast, Al3+, Co2+, Ni2+, Cu2+, Cd2+and La3+inhibited the MCA andPA.The purified enzyme was separated by SDS-PAGE, then the target protein was digestedby trypsin. The peptides were analyzed by MALDI-TOF-MS/MS and the enzyme wasidentified as subtilisin BPN.The degenerate primer DNAs based on subtilisin BPN sequence was designed and thegene was inserted to the vector pET-28a expressed in E. coli BL21(DE3). The overexpressionsubtilisin BPN proteins were found in the insoluble cytoplasmic fraction as inclusion bodies.The inclusion bodies were dissolved in PBS buffer with8mol·L-1urea followed withpurification by SP cation-exchange chromatography. The obtained protein in inclusion bodiesexhibited the milk-clotting ability and proved it expressed sucssessfully in E. coliBL21(DE3).