Sun Qian, Wang Xi-Ping, Yan Qiao-Juan, Chen Wei, Jiang Zheng-Qiang
College of Food Science and Nutritional Engineering, Beijing Key Laboratory of Functional Food from Plant Resources, China Agricultural University, PO Box 294, No.17 Qinghua Donglu, Haidian District, Beijing, 100083, China.
Appl Biochem Biotechnol. 2014 Sep;174(1):174-85. doi: 10.1007/s12010-014-1044-6. Epub 2014 Jul 18.
Purification and characterization of a chymosin from Rhizopus microsporus var. rhizopodiformis were investigated in the present study. A newly isolated R. microsporus var. rhizopodiformis F518 produced a high level of milk-clotting activity (1,001 SU/mL). A chymosin from the fungus was purified 3.66-fold with a recovery yield of 33.2 %. The enzyme appeared as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 37.0 kDa. It was optimally active at 60 °C and was stable up to 40 °C. The purified enzyme was an acid protease with an optimum pH of 5.2 and retained 80 % of residual activity within pH 2.0-8.0. The inhibition of 96 and 100 % by pepstatin A at 0.01 and 0.02 mM, respectively, revealed that the enzyme is an aspartic protease. Thus, high milk-clotting activity of the chymosin with good stability will strengthen the potential use of the chymosin as a substitute for calf rennet in cheese manufacturing.
本研究对微小毛霉根状变种凝乳酶的纯化及特性进行了研究。新分离的微小毛霉根状变种F518产生了高水平的凝乳活性（1,001 SU/mL）。从该真菌中纯化得到的凝乳酶纯化倍数为3.66倍，回收率为33.2%。该酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上呈现为单一蛋白条带，分子量为37.0 kDa。其最适活性温度为60℃，在40℃以下稳定。纯化后的酶是一种酸性蛋白酶，最适pH为5.2，在pH 2.0 - 8.0范围内保留80%的残余活性。胃蛋白酶抑制剂A在0.01和0.02 mM时分别抑制96%和100%，表明该酶是一种天冬氨酸蛋白酶。因此，具有良好稳定性的凝乳酶的高凝乳活性将增强其作为奶酪制造中小牛凝乳酶替代品的潜在用途。
