香菇中γ-谷氨酰转肽酶的分离纯化及其酶学性质研究

通过(NH4)2SO4沉淀、Phenyl Sepharose FF疏水层析分离纯化香菇中的γ-谷氨酰转肽酶(γ-Glutamyltranspeptidase,GGT),纯化后的GGT的比活力到达了19.59 U/mg.SDS-PAGE分析表明,GGT由分子量分别为28 kDa和60 kDa的两个亚基组成.酶学性质试验结果表明,GGT反应的最适温度为37 ℃,最适pH 值为7.6;金属离子Na+、K+和Ca2+对酶有激活作用,而Ag+、Cu2+、Zn2+和Fe3+则有抑制作用;试验范围内,GGT水解γ-glutamyl-p-nitroanilide的米氏动力学参数Km 值为2.601 μmol/mL,Vmax值为0.0287 μmol/min;组成的氨基酸中,Glu和Asp含量较高,Met和Cys含量较低.

Purification and Properties of γ-Glutamyltranspeptidase from Lentinula edodes

γ-Glutamyltranspeptidase (GGT) was extracted from Lentinula edodes by ammonium sulfate precipitation and purified by Phenyl Sepharose FF column chromatography. The specificity activity of the purified GGT was 19.59 U/mg, and SDS-PAGE revealed that the enzyme consisted of two subunits of 28 kDa and 60 kDa, respectively. The optimal temperature and pH values for enzyme activity were 37 ℃ and 7.6, respectively. Na+, K+ and Ca2+ exerted a slight activating effect on GGT, whereas Cu2+, Ag+, Zn2+ and Fe3+ inhibited enzyme activity. The rate of γ-glutamyl-p-nitroanilide hydrolysis by purified GGT followed Michaelis-Menten kinetics over the substrate concentration range 50～250 mmol/L (Km=2.601 μmol/mL,Vmax=0.0287 μmol/min). Purified GGT contained highest amounts of glutamic and aspartic acids, whereas Cys and Met were present in low amounts.