2-酮基-L-古龙酸合成L-木糖的酶筛选及反应优化

Enzyme screening and reaction optimization for synthesis of L-xylose from 2-keto-L-gulonic acid

[目的]构建能够以2-酮基-L-古龙酸为底物合成L-木糖的工程菌。[方法]将马来酸醋杆菌(Acetobacter malorum)、驹形氏杆菌(Komagataeibacter pomaceti)、解脂耶氏酵母(Yarrowia lipolytica)、酸醋杆菌(Gluconacetobacter johannae)中的α-酮酸脱羧酶在大肠杆菌中重组表达,筛选功能性菌株,优化工程菌合成反应条件。[结果]构建了BL21(pET28a-AmDC)、BL21(pET28a-KpDC)、BL21(pET28a-YpDC)、BL21(pET28a-GjDC)四个工程菌,其中BL21(pET28a-YpDC)对100 mmol/L底物2-酮基-L-古龙酸的转化率>52%,产物L-木糖的纯度>99%。BL21(pET28a-YpDC)转化反应最适pH值为7.0,最适温度为37℃,最适Mg^(2+)浓度为5 mmol/L,最适TPP浓度为10 mmol/L。[结论]筛选得到能够催化合成L-木糖的工程菌BL21(pET28a-YpDC),该菌株对2-酮基-L-古龙酸的转化率>52%,L-木糖纯度>99%。

[Objective]To construct an engineering strain capable of synthesizing L-xylose from 2-keto-L-gulonic acid.[Method]α-keto decarboxylase gene was cloned from Acetobacter malorum,Komagataeibacter pomaceti,Yarrowia lipolytica and Gluconacetobacter johannae.The engineering strain was constructed and the functional strain was screened to optimize the reaction conditions of biosynthesis.[Result]Four engineering bacteria BL21(pET28 a-AmDC),BL21(pET28 a-KpDC),BL21(pET28 a-YpDC),BL21(pET28 a-GjDC)were constructed.The transformation rate of BL21(pET28 a-YpDC)to 2-keto-L-gulonic acid was more than 52%,and the e.e.value of L-xylose was more than 99%.The results showed that the optimal pH,temperature,Mg^(2+)concentration and TPP concentration were 7.0,37℃,5 mmol/L and 10 mmol/L,respectively.[Conclusion]An engineering strain BL21(pET28 a-YpDC)was constructed,which could catalyze the synthesis of L-xylose.The conversion rate of 2-keto-L-gulonic acid was more than 52%,and the purity of L-xylose was more than 99%.