昆虫来源L-天冬氨酸-α-脱羧酶突变体的酶学性质表征  

Characterization of enzymatic properties of an insect-derived L-aspartate-α-decarboxylase variant

作　　者：叶文琪 薛岚 王超 崔文璟[1] 周哲敏[1] 刘中美[1] YE Wenqi;XUE Lan;WANG Chao;CUI Wenjing;ZHOU Zhemin;LIU Zhongmei(School of Biothechnology, Jiangnan University, Wuxi 214122, China)

机构地区：[1]江南大学生物工程学院

出　　处：《食品与发酵工业》2019年第19期63-67,共5页Food and Fermentation Industries

基　　金：国家重点研发计划政府间创新合作重点专项(2016YFE0127400);国家自然科学基金(3140078)

摘　　要：选取突变体K49R进行分离纯化、酶学性质表征,并进行全细胞催化合成β-丙氨酸,旨在研究红粉甲虫来源的L-天冬氨酸-α-脱羧酶(L-aspartate-α-decarboxylase,ADC)的酶学性质,并为生物法制备β-丙氨酸提供理论与技术支持。突变体K49R最适pH为6.5,在pH 6.0~7.0之间保持稳定;最适温度为42℃,热稳定性较野生型提高1.7倍;底物亲和力大幅提高,催化效率是野生型的1.8倍。在培养基中添加10 g/L葡萄糖可以有效提高重组菌的生物量,以及重组酶的可溶性表达量;在全细胞催化合成β-丙氨酸体系中,添加磷酸吡哆醛(pyridoxal-5-phosphate,PLP)可以缩短反应时间,提高催化效率。该研究开发了具有工业化潜力的工程菌,建立了绿色、高效的β-丙氨酸生物合成法,为β-丙氨酸生物合成的产业化奠定了重要基础。This study aimed to characterize the enzymatic properties of L -aspartate-α-decarboxylase (ADC) derived from red flour beetles in order to provide theoretical and technical support for β-alanine bioproduction. A mutant K49R derived from red flour beetles was purified and characterized, followed by conducting a whole-cell catalysis. It was found that the optimum reaction pH and temperature of K49R was 6.5 and 42 ℃, respectively, and the thermal stability of K49R was 1.7 times higher than that of the wild type. The substrate affinity of K49R was greatly improved, and the catalytic efficiency was 1.8 times higher that of the wild type. Additionally, the biomass of the recombinant Escherichia coli and the expression level of ADC were effectively increased by adding 10 g/L glucose into the medium. The addition of pyridoxal phosphate (PLP) shorted the reaction time and improved the catalytic efficiency. In conclusion, by construction of an engineered strain this study provides a green and efficient biosynthesis method to produce β-alanine, which lays an important foundation for industrializing the biosynthesis of L -aspartate.

关 键 词：Β-丙氨酸 L -天冬氨酸-α-脱羧酶 红粉甲虫 热稳定性 全细胞催化 

分 类 号：TQ226.36[化学工程—有机化工] Q55[生物学—生物化学]