重组大肠杆菌全细胞催化合成4-羟基异亮氨酸

Whole-Cell Catalytic Synthesis of 4-Hydroxyisoleucine Using Recombinant Escherichia coli

摘要:

4-羟基异亮氨酸（4-HIL）是一种很有前景的药物，它具有促进胰岛素分泌、改善外周组织对胰岛素的抵抗性和调节血脂异常等作用，而L-异亮氨酸羟化酶（IDO）常用于4-HIL的生产。首先，克隆了苏云金芽孢杆菌来源的L-异亮氨酸羟化酶，实现了其在E. coli BL21（DE3）中的异源表达。其次，通过同源建模和蛋白质结构分析，本着将与底物氨基酸侧链结合的氨基酸残基从亲水性或长链疏水性结构突变成丙氨酸Ala的原则，对I156位点进行了定点突变，以增大底物结合口袋，扩宽底物通道进而提高4-HIL的产量。最后，对野生酶及突变酶的酶学性质、突变酶的羟基化反应体系进行研究，在最优催化条件下，分批补料转化底物进行4-HIL的生产。酶学性质结果显示，野生酶及突变酶I156A的最适温度均为25 ℃，最适pH均为7.0；突变酶I156A比酶活比野生酶提高了1.9倍，L-ILe转化率提高了28%。羟基化反应体系的最优转化条件为：20 mmol/L L-ILe，20 mmol/L α-酮戊二酸，8 mmol/L Fe²⁺，30 mmol/L抗坏血酸和HEPES（50 mmol/L，pH 7.0）缓冲液。在最优转化条件下，重组菌E. coli BL21/pET28a-idoI156A进行分批补料转化底物，时间间隔为4 h，32 h后得到77.3 mmol/L 4-HIL，底物最高转化率98.35%。

Abstract:

4-Hydroxyisoleucine(4-HIL) is a promising drug that promotes insulin secretion, improves peripheral tissue resistance to insulin, and regulates dyslipidemia. L-isoleucine hydroxylase (IDO) is commonly used in the production of 4-HIL. First, the L-isoleucine hydroxylase derived from Bacillus thuringiensis was cloned, and successfully expressed in E. coli BL21(DE3). Secondly, the site-directed mutation of I156 locus was achieved through homology modeling and protein structure analysis based on the principle of changing the amino acid residue bound to the supstrate side chain from the hydrophilic or long-chain hydrophobic structure to Ala. The mutation aimed at increasing the supstrate binding pocket and widening the supstrate channel to increase the yield of 4-HIL. Finally, the biochemical properties of wild-type and mutated enzymes were studied. The hydroxylation reaction system of mutated enzyme was studied. Under the optimal catalytic conditions, the supstrate was fed in batches to produce 4-HIL. The results of enzymatic properties showed that the optimum temperature and pH of wild-type enzyme and mutated enzyme I156A were 25 ℃ and pH 7.0, and the I156A mutated enzyme activity was 1.9 times higher than that of the wild-type enzyme with 28% increased L-ILe conversion rate. The optimal conversion conditions for the hydroxylation reaction system were: 20 mmol/L L-ILe, 20 mmol/L α-ketoglutarate, 8 mmol/L Fe²⁺, 30 mmol/L ascorbic acid, and HEPES (50 mmol/L, pH 7.0) buffer. Under the optimal transformation conditions, the recombinant E. coli BL21/pET28a-ido^I156A was fed in batches for a time interval of 4 h. After 32 h, 77.3 mmol/L 4-HIL was obtained, and the highest conversion rate of supstrate was 98.35%.