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论文类型：期刊论文
发表时间：2012-05-01
发表刊物：WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
收录刊物：SCIE、EI、PubMed、Scopus
卷号：28
期号：5
页面范围：2087-2093
ISSN号：0959-3993
关键字：Calf chymosin; Constitutive expression; Pichia pastoris GS115; Cheese preparation
摘要：Chymosin can specifically break down the Phe105-Met106 peptide bond of milk kappa-casein to form insoluble para-kappa-casein, resulting in milk coagulation, a process that is used in making cheese. In this study, in order to obtain an alternative milk coagulant which is safe and efficient, and simultaneously can produce cheese with a good taste, bovine prochymosin B was chosen and constitutively expressed to a high level in Pichia pastoris. The recombinant chymosin was expressed mainly as a secretory form, and it exhibited milk-clotting activity. It was purified by ammonium sulfate fractionation, anion exchange, followed by cation exchange chromatography. A final yield of 24.2% was obtained for the purified enzyme, which appeared as a single band in SDS-PAGE having a molecular mass of approximate 36 kDa. Proteolysis assay showed that it specifically hydrolyzed kappa-casein. It was stable at 25-50A degrees C and had optimal activity at 37A degrees C and pH 4.0. The activity of the recombinant chymosin was activated by cations such as Mn2+, Fe3+, Mg2+ and Na+, but inhibited by K+, Co2+, Zn2+, Ni2+, and to a lesser extent by Cu2+. These results suggested that recombinant bovine chymosin is an acid milk coagulant, and it could be considered as a safe and efficient enzyme suitable for use in cheese production.