重组微小毛霉凝乳酶的发酵条件及其酶学性质

Fermentation Conditions for the Production of Recombinant Mucor pusillus Rennet and Its Enzymological Characterization

通过对重组微小毛霉凝乳酶(recombinant Mucor pusillus rennet,RMPR)发酵条件的研究,确定甲醇诱导体积分数1%、初始OD600nm0.5、装瓶量100mL/500mL摇瓶、诱导时间96h为发酵最适条件。培养液离心取上清用硫酸铵分级沉淀获得粗酶液,并对其酶学性质进行研究,经测定该酶的最适反应温度为60℃,30～45℃酶较稳定,保温90min,剩余凝乳酶活力达86.5%;酶在pH5.5～7.0范围内,随pH值升高,凝乳酶活力逐渐降低,pH5.5时凝乳酶活力最高。当Ca2+浓度为0.03mol/L时凝乳酶活力最高,Na+浓度对凝乳酶活力没有明显影响,Ni2+和Fe2+对酶有抑制作用,Mn2+、K+、Mg2+对酶有一定的促进作用。

The aims of this study were to investigate the fermentation conditions for the production of recombinant Mucor pusillus rennet （RMPR） and to enzymologically characterize the enzyme obtained. An optimum enzyme activity was obtained after 96 h of induction with 1% methanol as the inducer under the following conditions：initial OD600 nm,0.5; and volume of medium contained in 500 mL flask,100 mL.The incubation both obtained was centrifugated,and the supernatant was then salted out with ammonium sulfate to prepare crude enzyme solution,and enzymological characterization was carried out for the crude enzyme solution. The optimal reaction temperature of the enzyme was 60 ℃,and it kept stable in the temperature range from 30 to 45 ℃ and the residual enzyme activity was still 86.5% after 90 min of incubation at 45 ℃. As pH increased from 5.5 to 7.0,the activity of the enzyme presented a gradual decrease and the highest enzyme activity was observed at pH 5.5. At a Ca2＋ concentration of 0.03 mol/L,the enzyme had the highest activity to clot milk,and Na＋ had no notable effect on the enzyme,and Ni＋ and Fe2＋ were both an enzyme inhibitor,while Mn2＋,K＋ and Mg2＋ all had promoting effect on the enzyme.