腈水合酶/酰胺酶体系制备蛋氨酸羟基类似物

Preparation of Methionine Hydroxy Analogue by Nitrile Hydratase/Amidase System

摘要:

西洼湖戈登氏菌CGMCC 4.2184含有一个高效的腈水合酶/酰胺酶体系。本文利用该菌株全细胞中的腈水合酶催化2-羟基-4-甲硫基丁腈（HMTBN）水合为2-羟基-4-甲硫基丁酰胺（HMTBAm），利用酰胺酶以原位串联的方式将2-羟基-4-甲硫基丁酰胺水解为2-羟基-4-甲硫基丁酸（HMTBA，蛋氨酸羟基类似物）。考察了底物浓度、细胞浓度、温度、pH、反应时间对腈水合酶和酰胺酶活性的影响，对腈水合酶、酰胺酶的热稳定性和pH稳定性进行了测定。结果表明，最优的反应条件为：50mmol/L HMTBN，3g/L干重细胞，温度30℃，pH8.0，反应时间30min。在最优反应条件下，采用分批补料策略，细胞可使用23批次，HMTBA的累积量在44h内达到164g/L，产率95%。

Abstract:

A highly active nitrile hydratase/amidase system was found in Gordonia sihwensis CGMCC 4.2184, which was capable of converting 2-hydroxy-4-methylthiobutanenitrile (HMTBN) to 2-hydroxy-4-methylthiobutanamide (HMTBAm) by nitrile hydratase and then 2-hydroxy-4-methylthiobutanoic acid (HMTBA, methionine hydroxy analogue) followed by amidase in in-situ cascade manner. The effects of substrate concentration, cell concentration, temperature, pH and reaction time on nitrile hydratase and amidase activity were investigated. The thermal stability and pH stability of nitrile hydratase and amidase were determined. The results showed that the optimal reaction conditions were: 50mmol/L HMTBN, 3g/L dry weight cells, 30℃, pH8.0, 30min. Under the optimal reaction conditions, the cells could be used in 23 batches, and the cumulative amount of HMTBA reached 164g/L in 44h, with a yield of 95%.