N-乙酰-反式-4-羟脯氨酸的酰基转移酶法催化合成

Synthesis of N-Acetyl-trans-4-hydroxyproline Catalyzed by Acyltransferase

摘要:

【目的】 建立N-乙酰-反式-4-羟脯氨酸（N-acetyl-trans-4-hydroxyproline，N-ATHOP）的生物合成工艺过程。 【方法】 利用大肠杆菌（Escherichia coli，E. coli）BL21（DE3）作为表达宿主，异源表达耻垢分枝杆菌（Mycobacterium smegmatis）来源的酰基转移酶（MsAcT），并结合半理性设计得到能催化合成N-ATHOP的突变体MsAcT（S11C）。 【结果】 突变体MsAcT（S11C）的最适反应条件为：pH为6.9、反应温度为49 ℃、反式-4-羟脯氨酸（trans-4-hydroxyproline，THOP）初始浓度为315.5 mmol/L、乙酸乙烯酯体积分数为10%。在此条件下，MsAcT（S11C）催化12 h后合成的N-ATHOP产量达4.71 g/L，对应转化率为10.95%。 【结论】 突变体MsAcT（S11C）成功实现由THOP到N-ATHOP的微生物酶法生物合成。

Abstract:

[Objective] This study aims to establish the biosynthesis process of N-acetyl-trans-4-hydroxyproline (N-ATHOP). [Method]Escherichia coli (E. coli) BL21 (DE3) was selected as the host to heterologously express the acyltransferase (MsAcT) derived from Mycobacterium smegmatis. A semi-rational design was employed to construct the mutant MsAcT(S11C) capable of catalyzing the synthesis of N-ATHOP. [Result] The catalytic reaction conditions of the mutant MsAcT(S11C) were optimized as pH 6.9, reaction temperature 49 ℃, initial trans-4-hydroxyproline (THOP) concentration of 315.5 mmol/L, and vinyl acetate volume fraction of 10%. Under the optimized conditions, the yield of N-ATHOP synthesized under the catalysis of MsAcT(S11C) reached 4.71 g/L, with a conversion rate of 10.95% after 12 h of reaction. [Conclusion] The mutant MsAcT(S11C) successfully achieved microbial enzymatic biosynthesis from THOP to N-ATHOP.