两种新型L-苏氨酸醛缩酶的鉴定及活性检测方法

为了实现重要医药中间体β-羟基-α-氨基酸的生物酶法合成,挖掘验证新型的L-苏氨酸醛缩酶。以pET-28a(+)作为表达载体,通过蛋白表达纯化、薄层层析色谱(TLC)和高效液相色谱(HPLC)技术分析L-苏氨酸醛缩酶及其催化产物的性质。基于4-氨基-3-肼基-5-巯基-1,2,4-三氮唑(Purpald)显色试剂开发检测醛缩酶的新方法。Streptomyces coelicolor SCO1844(天蓝色链霉菌,S.coelicolor SCO1844)和Streptomyces xinghaiensis SFR7A(星海链霉菌,S.xinghaiensis SFR7A)来源的醛缩酶被证明能够成功地合成β-羟基-α-氨基酸,且均为L-苏氨酸醛缩酶,实现了以苯甲醛和甘氨酸为底物合成l-threo/erythron-苯基丝氨酸的醇醛缩合反应。开发的可视化活性检测方法可以实现醛缩酶的快速鉴定和高通量筛选。两种新型L-苏氨酸醛缩酶的鉴定以及活性检测方法的开发,不仅丰富了生物法合成β-羟基-α-氨基酸的酶库,也为下一步对L-苏氨酸醛缩酶进行分子改造提高其催化活性和选择性奠定了研究基础。

Identification of Two New Types of L-threonine Aldolases and Development of Its Activity Detection Method

The aim of this study is to conduct the bio-enzymatic synthesis ofβ-hydroxy-α-amino acids,the important pharmaceutical intermediates,and to explore and identify new L-threonine aldolases.Using pET-28a(+)as an expression vector,protein expression purification,thin layer chromatography(TLC),and high-performance liquid chromatography(HPLC)were performed to analyze the properties of L-threonine aldolases and their catalytic products.A new activity detection method of L-threonine aldolases was developed based on 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole(Purpald)color reagent.The results demonstrated that the aldolases from Streptomyces coelicolor SCO1844 and Streptomyces xinghaiensis SFR7A were both L-threonine aldolases and could be used to synthesizeβ-hydroxy-α-amino acids.The aldol condensation reaction of benzaldehyde and glycine was achieved using these two L-threonine aldolases,producing L-threo/erythron-phenylserine.The developed visual activity detection method allows rapid identification and high-throughput screening of aldolases to be feasible.The identification of two new L-threonine aldolases and the development of activity detection methods not only enrich the biosynthetic enzyme library forβ-hydroxy-α-amino acids,but also provide the research basis for molecular modification to improve its catalytic activity and selectivity in the future.