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.
Sheng Wu Gong Cheng Xue Bao. 2023 Mar 25;39(3):1107-1118. doi: 10.13345/j.cjb.220805.
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 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 and values of the double mutant M185A/F279I obtained by superimposed mutation were 530.8 mmol/L and 19.9 s, 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.
L-阿拉伯糖异构酶（L-AI）是将D-半乳糖异构化为D-塔格糖的关键酶。在本研究中，为提高L-阿拉伯糖异构酶对D-半乳糖的活性及其在生物转化中的转化率，对一株来源于CGMCC2921的L-阿拉伯糖异构酶进行了重组表达并应用于生物转化。此外，对其底物结合口袋进行了合理设计，以提高对D-半乳糖的亲和力和催化活性。我们发现，变体F279I对D-半乳糖的转化率比野生型酶提高了1.4倍。通过叠加突变获得的双突变体M185A/F279I的Km和kcat值分别为530.8 mmol/L和19.9 s，催化效率比野生型提高了8.2倍。当以400 g/L乳糖为底物时，M185A/F279I的转化率达到了22.8%的高水平，这表明其在乳糖酶法生产塔格糖方面具有巨大的应用潜力。
