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

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K_m和k_cat分别为530.8 mmol/L与19.9 s^-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。

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

Abstract:

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 K_m and k_cat values 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.

Key words:l-arabinose isomerase;rational design;d-galactose;d-tagatose;conversion rate