赤拟谷盗来源天冬氨酸α-脱羧酶分子改造及催化合成β-丙氨酸工艺的建立  

Modification of aspartate α-decarboxylase from Tribolium castaneum and its application in producing β-alanine

作　　者：王超 叶文琪 薛岚 刘中美[1] 周哲敏[1] WANG Chao;YE Wenqi;XUE Lan;LIU Zhongmei;ZHOU Zhemin(Schoool of Biotechnology,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11期7-13,共7页Food and Fermentation Industries

基　　金：国家重点研发计划政府间国际科技创新合作重点专项(2016YFE0127400);江南大学自主科研计划重点项目基金(JUSRP51713B);国家自然科学基金(31400078)

摘　　要：L-天冬氨酸α-脱羧酶活性较低,稳定性较差,使得其在工业应用中受到限制。该研究旨在提高L-天冬氨酸α-脱羧酶的催化性能,促进生物法生产β-丙氨酸的工业化进程。依据嗜热蛋白酶的氨基酸内在进化趋势,对赤拟谷盗来源L-天冬氨酸α-脱羧酶进行分子改造,以期提高稳定性。实验共构建21个突变体,获得催化性能优良的突变体K221R,该突变体的比酶活较野生型提高20.3%;野生型经50℃处理30min,残余酶活接近0,而突变体K221R的残余酶活为43%。建立了基因工程菌全细胞催化天冬氨酸生成β-丙氨酸的工艺,K221R菌株的产量达到134.72g/L,摩尔转化率为94.52%,是迄今为止的最高产量。该研究构建的基因工程菌具有工业应用潜力,同时也为生物法制备β-丙氨酸提供理论与技术基础。Low catalytic ability and poor stability limit industrial applications of L -aspartate α-decarboxylase. This study was therefore conducted to improve the catalytic activity of L -aspartate α-decarboxylase to promote biological production of β-alanine in industries. Based on evolutionary information of thermophilic bacteria, L -aspartate α-decarboxylase from Tribolium castaneum was molecularly modified to improve its enzyme stability. The mutant strain K221R was screened,as it had improved thermal stability and enzymatic activity. Compared with the wild type,the specific enzyme activity of K221R increased 20.3%. Moreover,after incubating the enzyme at 50 ℃ for 30 min,the residual activity of the wild type was 0,while K221R remained 43% activity. Furthermore,up to 134.72 g/L β-alanine was produced using K221R-expression whole cells,which was the highest production level achieved up-to-date,with 94.52% molar conversion rate. In conclusion,the engineered strain containing K221 variant has great potential for industrial production of β-alanine.

关 键 词：L-天冬氨酸α-脱羧酶 热稳定性 定点突变 Β-丙氨酸 全细胞催化 

分 类 号：Q78[生物学—分子生物学]