栗酒裂殖酵母来源黄素单加氧酶的性质及催化合成S-甲基-l-半胱氨酸亚砜的应用

A flavin-containing monooxygenase from Schizosaccharomyces pombe: characterization and application in the synthesis of S-methyl-l-cysteine sulfoxide

摘要:

S-甲基-l-半胱氨酸亚砜(S-methyl-l-cysteine sulfoxide,SMCO)是一种具有多种功能特性的非蛋白质含硫氨基酸。目前关于催化S-甲基-l-半胱氨酸(S-methyl-l-cysteine,SMC)生物合成SMCO的酶鲜有报道。本研究将栗酒裂殖酵母(Schizosaccharomyces pombe)来源的黄素单加氧酶基因(spfmo)通过大肠杆菌(Escherichia coli) BL21(DE3)进行异源表达，并对其酶学性质进行了分析。重组SpFMO的最适催化条件为30℃、pH 8.0，该条件下比酶活为72.77 U/g；此外，适当的Mg²⁺可以提高SpFMO的酶活；经酶动力学分析，该酶对底物SMC的酶促反应动力学参数K_m值为23.89 μmol/L，催化效率k_cat/K_m为61.71 L/(min·mmol)。在最适条件下，SpFMO催化SMC在9 h内生成SMCO的产率为12.31%。本研究为酶法合成SMCO提供了一定参考。

Abstract:

S-methyl-l-cysteine sulfoxide (SMCO) is a non-protein sulfur-containing amino acid with a variety of functions. There are few reports on the enzymes catalyzing the biosynthesis of SMCO from S-methyl-l-cysteine (SMC). In this study, the flavin-containing monooxygenase gene derived from Schizosaccharomyces pombe(spfmo) was heterologously expressed in Escherichia coli BL21(DE3) and the enzymatic properties of the expressed protein were analyzed. The optimum catalytic conditions of the recombinant SpFMO were 30 ℃ and pH 8.0, under which the enzyme activity reached 72.77 U/g. An appropriate amount of Mg²⁺ improved the enzyme activity. The enzyme kinetic analysis showed that the K_m and k_cat/K_m of SpFMO on the substrate SMC were 23.89 μmol/L and 61.71 L/(min·mmol), respectively. Under the optimal reaction conditions, the yield of SMCO synthesized from SMC catalyzed by SpFMO was 12.31% within 9 h. This study provides reference for the enzymatic synthesis of SMCO.