摘要
1,5-戊二胺(戊二胺)具有良好的生物活性,广泛应用在农业、医药以及工业等领域。赖氨酸脱羧酶可以催化L-赖氨酸生产戊二胺,为了提高赖氨酸脱羧酶催化合成戊二胺的效率,首先在大肠杆菌中克隆表达了粘质沙雷氏菌来源的赖氨酸脱羧酶(SmcadA)。生化特征表明,SmcadA最适催化pH为6.0,最适催化温度约为40℃。随后对SmcadA的第348位氨基酸进行了突变研究,筛选获得了催化效率显著提高的突变体Gly348Ala,主要原因是该突变导致蛋白中氨基酸残基和底物之间相互作用的氢键数增加,从而影响其催化效率。最后,对重组菌株细胞进行了戊二胺合成研究,催化反应25 h,含有突变体Gly348Ala的重组菌细胞可以催化合成218.2 g/L戊二胺,野生型SmcadA重组菌仅能催化合成159.2 g/L戊二胺。该研究结果为工业化酶催化合成戊二胺提供了借鉴。
1,5-Pentanediamine(pentanediamine)has good biological activity and is widely used in agriculture,medicine and other industries.Lysine decarboxylase can catalyze L-lysine to produce pentanediamine.In order to improve the efficiency of lysine decarboxylase to catalyze the synthesis of pentanediamine,we first screened,expressed and characterized lysine decarboxylases derived from Pseudomonas putida KT2440,Gluconobacter oxydans DSM3504,Bacillus licheniformis DSM13 and Serratia marcescens(SmcadA),in Escherichia coli.The biochemical characteristics showed that the optimal catalytic pH of SmcadA was 6.0 and the optimal catalytic temperature was about 40℃.Subsequently,we conducted a mutation study on the 348th amino acid of SmcadA,screened and obtained a mutant Gly348 Ala with significantly improved catalytic efficiency.The main reason was that the mutation caused an increase in the number of hydrogen bond interactions between the amino acid residues of the protein and the substrate lysine,thereby influencing the proteins catalytic efficiency.Finally,the recombinant strain cells were studied for the synthesis of pentanediamine.The recombinant bacteria cells containing the mutant Gly348 Ala could synthesize 218.2 g/L pentanediamine,while the wild-type SmcadA recombinant bacteria could only catalyze the synthesis of 159.2 g/L pentanediamine.The results provide a reference for the synthesis of pentanediamine catalyzed by industrialized enzymes.
作者
OSIRE Tolbert
杨套伟
乔郅钠
孙杨
徐美娟
张显
邵明龙
饶志明
OSIRE Tolbert;YANG Taowei;QIAO Zhina;SUN Yang;XU Meijuan;ZHANG Xian;SHAO Minglong;RAO Zhiming(School of Biotechnology,Jiangnan University,Wuxi 214122,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2022年第1期8-14,共7页
Food and Fermentation Industries
基金
国家自然科学基金项目(31770058)
江苏省自然科学基金项目(BK20181205)。
关键词
赖氨酸脱羧酶
分子改造
氢键
酶催化
戊二胺
lysine decarboxylase
molecular modification
hydrogen bond
enzyme catalysis
pentanediamine
作者简介
第一作者:OSIRE Tolbert,硕士研究生;通信作者:饶志明,教授,E-mail:raozhm@jiangnan.edu.cn。
