发酵乳杆菌来源L-阿拉伯糖异构酶理性设计及在D-塔格糖生产中的应用  

Rational design of L-arabinose isomerase from Lactobacillus fermentum and its application in D-tagatose production

作　　者：李娟[1,2,3] 吴敬[1,2,3] 陈晟 夏伟[1,2,3] LI Juan;WU Jing;CHEN Sheng;XIA Wei(State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,Jiangsu,China;Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China;International Joint Laboratory on Food Safety,Ministry of Education,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学食品科学与技术国家重点实验室,江苏无锡214122 [2]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122 [3]江南大学教育部食品安全国际合作联合实验室,江苏无锡214122

出　　处：《生物工程学报》2023年第3期1107-1118,共12页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(31801472,31730067);江苏省自然科学基金(BK20180604)。

摘　　要：L-阿拉伯糖异构酶(L-arabinose isomerase,L-AI)是D-半乳糖异构化生成D-塔格糖的关键酶。为提高L-阿拉伯糖异构酶对D-半乳糖的活性及在生物转化中的转化率,本研究对发酵乳杆菌(Lactobacillus fermentum)CGMCC2921来源的L-阿拉伯糖异构酶进行重组表达和生物转化应用,并对其底物结合口袋进行理性设计以提高酶对D-半乳糖亲和力和催化活性。结果显示,突变体F279I对D-半乳糖的转化率提高至野生型酶的1.4倍,进一步叠加获得的双突变体M185A/F279I的Km和kcat分别为530.8mmol/L与19.9s-1,底物亲和力显著提高,催化效率提高至野生型酶的8.2倍。以400 g/L乳糖为底物时,突变酶M185A/F279I转化率高达22.8%。本研究在乳糖高值化生产塔格糖方面具有重要的应用价值。L-arabinose isomerase(L-AI) is the key enzyme that isomerizes D-galactose to D-tagatose. In this study, to improve the activity of L-arabinose isomerase on D-galactose and its conversion rate in biotransformation, an L-arabinose isomerase from Lactobacillus fermentum CGMCC2921 was recombinantly expressed and applied in biotransformation. Moreover, its substrate binding pocket was rationally designed to improve the affinity and catalytic activity on D-galactose. We show that the conversion of D-galactose by variant F279I was increased 1.4 times that of the wild-type enzyme. The Kmand kcatvalues of the double mutant M185A/F279I obtained by superimposed mutation were 530.8 mmol/L and 19.9 s-1, respectively, and the catalytic efficiency was increased 8.2 times that of the wild type. When 400 g/L lactose was used as the substrate, the conversion rate of M185A/F279I reached a high level of 22.8%, which shows great application potential for the enzymatic production of tagatose from lactose.

关 键 词：L-阿拉伯糖异构酶 理性设计 D-半乳糖 D-塔格糖 转化率 

分 类 号：TS201.2[轻工技术与工程—食品科学]