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为实现色氨酸酶高效、低成本催化合成L-色氨酸,利用p ET30a为载体在宿主细胞E.coli BL21(DE3)中重组表达了产气肠杆菌(Enterobacter aerogenes)来源的色氨酸酶,以丙酮酸、吲哚和氨为底物,探究其酶学性质,考察了反应温度、起始p H、底物摩尔比对酶促反应的影响,并利用丙酮酸发酵液为底物酶法合成L-色氨酸。结果表明,色氨酸酶重组表达成功,色氨酸酶最佳反应条件为:温度35℃,起始p H=9.0,底物摩尔比n(吲哚)∶n(丙酮酸)=0.6∶1,底物丙酮酸浓度为0.17 mol/L。利用重组色氨酸酶全细胞催化100 m L浓度为0.57 mol/L丙酮酸发酵液,流加浓度为4.27 mol/L吲哚酒精溶液6.5 m L,反应28 h后,L-色氨酸浓度达0.25 mol/L,吲哚摩尔转化率达91.8%。
To achieve efficient and low-cost catalytic synthesis of L-tryptophan by tryptophanase, tryptophan derived from Enterobacter aerogenes was recombinantly expressed in host cell E. coli BL21 (DE3) using p ET30a as a vector Enzyme, pyruvic acid, indole and ammonia as substrates, to explore its enzymatic properties, the effects of reaction temperature, initial pH, substrate molar ratio on the enzymatic reaction was investigated, and pyruvate fermentation broth was used as a substrate Enzymatic Synthesis of L-Tryptophan. The results showed that the tryptophanase recombinant expression was successful. The optimal reaction conditions of tryptophanase were as follows: temperature 35 ℃, initial p H = 9.0, substrate molar ratio n (indole): n (pyruvate) = 0.6: 1, pyruvate concentration of substrate was 0.17 mol / L. The recombinant tryptophan whole cell was used to catalyze the fermentation of 100 mL pyruvate fermentation broth at a concentration of 0.57 mol / L and the concentration of 4.27 mol / L indole alcohol solution was 6.5 mL. After 28 h of reaction, L-tryptophan concentration Up to 0.25 mol / L, the conversion rate of indole mole reached 91.8%.