两种新型L-苏氨酸醛缩酶的鉴定及活性检测方法

doi:10.13560/j.cnki.biotech.bull.1985.2020-0695

摘要/Abstract

摘要：

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。

关键词: L-苏氨酸醛缩酶, β-羟基-α-氨基酸, 生物合成, 活性检测, 链霉菌

Abstract:

The aim of this study is to conduct the bio-enzymatic synthesis of β-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 synthesize β-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 for β-hydroxy-α-amino acids,but also provide the research basis for molecular modification to improve its catalytic activity and selectivity in the future.

Key words: L-threonine aldolase, β-hydroxy-α-amino acid, biosynthesis, activity detection, streptomyces

任思羽, 程新宽, 张宇辉, 庄建文, 马龙. 两种新型L-苏氨酸醛缩酶的鉴定及活性检测方法[J]. 生物技术通报, 2021, 37(3): 233-240.

REN Si-yu, CHENG Xin-kuan, ZHANG Yu-hui, ZHUANG Jian-wen, MA Long. Identification of Two New Types of L-threonine Aldolases and Development of Its Activity Detection Method[J]. Biotechnology Bulletin, 2021, 37(3): 233-240.

图/表 9

图1 含有β-羟基-α-氨基酸药物的结构 β-羟基-α-氨基酸;2：甲砜霉素;3：氟苯尼考;4：屈昔多巴

图2 l-苏氨酸醛缩酶催化的醇醛缩合反应

图3 醛缩酶的SDS-聚丙烯酰胺凝胶电泳分析结果 M：蛋白Marker;（-）：未加入IPTG诱导的样品;T：菌体破碎后混合液;P：破碎后的沉淀;S：破碎后的上清液;UB：与镍柱结合后的上清液;1：20 mmol/L咪唑洗脱液;2：25 mmol/L咪唑洗脱液;3：50 mmol/L咪唑洗脱液;4：100 mmol/L咪唑洗脱液;5：200 mmol/L咪唑洗脱液;6：250 mmol/L咪唑洗脱液;7：500 mmol/L咪唑洗脱液;8：1 mol/L咪唑洗洗脱液

图4 薄层层析色谱法检测反应产物 1：甘氨酸标准品;2：D/L-threo-苯基丝氨酸标准品;3：SCO1844实验组;4：SFR7A实验组;5：只含有苯甲醛与苷氨酸阴性对照组;6：只含有苯甲醛阴性对照组;7：只含SCO1844醛缩酶阴性对照组;8：只含SFR7A醛缩酶阴性对照组;9：D/L-threo-苯基丝氨酸标准品

图5 氨基酸的衍生化过程

图6 醛缩酶催化醇醛缩合反应的高效液相色谱分析 A：L-PsTA阳性对照组;B：SCO1844实验组;C：SFR7A实验组;D：三组液相峰叠加结果

图7 苯甲醛与Purpald发生显色反应

图8 Purpald显色测定结果及标准曲线测定

图9 Purpald显色法检测醛缩酶的活性 B：苯甲醛;B-G：苯甲醛-甘氨酸;SCO-B-G：SCO1844-苯甲醛-甘氨酸实验组;SFR-B-G：SFR7A-苯甲醛-甘氨酸实验组;G：甘氨酸;SCO：SCO1844;SFR：SFR7A

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