用硫酸铵分级沉淀分离提取重组牛凝乳酶(GS115/pPICZαA-chymosin),并经过Sephadex G-75分子筛和DEAE-52离子交换2次层析柱纯化,酶的比活由672 U/mg提高到3 865 U/mg。对纯化后重组牛凝乳酶酶学性质研究表明该酶的最适反应温度为55℃,30～45℃酶活较稳定,保温120 min,剩余活力达73.5%;最适反应pH值为5.0,并在pH 3.0～7.0范围内酶活保持稳定。Ca2+离子浓度0.02%时凝乳酶活力最高,NaCl浓度小于10%时可促进酶反应,除Ni2+和Cu2+对酶有抑制作用外,Fe2+,Fe3+,Mn2+,Mg2+对酶有促进作用。

Recombinant chymosin expressed in Pichia pastoris(GS115 / pPICZαA-chyomsin) was partially puri-fied and characterized.Recombinant chymosin was obtained by fractional precipitation with ammonia sulfate,followed by the chromatography of DEAE-52 and Sephadex G-75.After purification the specific activity of the enzyme prepara-tion increased from 672 U/mg to 3 865 U/mg.The milk clotting activity of the purified enzyme was stable between pH 3.0 ～ 7.0 with maximum activity at pH 5.0.The activity of the purified enzyme was with increasing CaCl2 concentra-tion up to 0.02 mol/L.However,a gradual increased reduction of the activity was observed by increasing NaCl con-centration between 1% ～ 20%.Ni + and Cu2 + had an inhibitory effect on the recombinant chymosin.Fe2 +,Fe3 +,Mn2 +,Mg2 + had a stimulating effect on the recombinant chymosin.