凝乳酶能够专一性裂解κ-酪蛋白,是制造干酪的关键酶。运用大肠杆菌表达系统对牛凝乳酶原进行了原核表达和初步纯化,活化重组凝乳酶原及测定凝乳活性,对重组凝乳酶的酶学特性进行分析。结果表明:大肠杆菌表达的重组蛋白约占菌体总蛋白的66.3%,每升培养液可纯化约200 mg的重组凝乳酶原,活化后的凝乳酶活力可达600 000 SU/g。经测定凝乳酶最适作用温度为57~62℃,并在pH 2~7、低于40℃的温度范围内稳定。金属离子中Al3+,Fe3+和Cu2+能显著增强酶活;胃蛋白酶抑制剂pepstatin A对酶有明显的抑制作用。

Chymosin is the key enzyme that can specifically cleave κ-casein and manufacture cheese.In this study,we expressed the bovine prochymosin in Escherichia coli,purified and activated the recombinant prochymosin,measured its milk-clotting activity,then analyzed the enzymatic properties of chymosin.The results showed that the prokaryotic expressed prochymosin accounted for about 66.3% of the total bacterial protein,the purified prochymosin was about 200 mg / L of culture liquid,and chymosin activity was 600 000 SU / g after prochymosin activation.The optimal temperature range of milk-clotting activity of recombinant chymosin was 57 ~ 62 ℃,and the chymosin was relatively stable in pH 2 ~ 7 and below 40℃.Metal ions such as Al3 +,Fe3 +and Cu2 +significantly increased the activity of chymosin,however,pepsin inhibitor pepstatin A had an obvious inhibitory effect on the activity of chymosin.It would provide a high expression strain for the industrial production of chymosin.