乙酰氨基葡萄糖合成关键酶氨基葡萄糖-6-磷酸乙酰转移酶的筛选及酶学性质分析

Screening of Glucosamine-6-phosphate N-acetyltransferase for N-acetylglucosamine Production and Enzyme Kinetics Analysis

DOI：10.3969/j.issn.1673-1689.2019.12.011

中文关键词: 重组氨基葡萄糖-6-磷酸乙酰转移酶 N-乙酰氨基葡萄糖 枯草芽孢杆菌

英文关键词: Glucosamine-6-phosphate N-acetyltransferase,N-acetylglucosamin,Bacillus subtilis

基金项目:

摘要点击次数: 7

全文下载次数: 10

中文摘要:

N-乙酰氨基葡萄糖（N-acetylglucosamine，GlcNAc)是氨基葡萄糖（Glucosamine，GlcN)的衍生物，在保健食品和医药领域具有广泛应用。氨基葡萄糖-6-磷酸乙酰转移酶（glucosamine-6-phosphate N-acetyltransferase，GNA）是N-乙酰氨基葡萄糖合成途径中的关键酶。本研究在前期构建的产GlcNAc枯草芽孢杆菌（Bacillus. subtilis，B. subtilis）工程菌的基础上，通过过量表达不同来源的GNA，筛选出来源于秀丽隐杆线虫（Caenorhabditis elegans-）的氨基葡萄糖-6-磷酸乙酰基转移酶（Cegna1）最适于加强GlcNAc合成途径。在B. subtilis工程菌中表达Cegna1，摇瓶水平发酵GlcNAc产量达到7.31 g/L，与过量表达来源于酿酒酵母（Saccharomyces cerevisiae，S. cerevisiae）的GNA的对照菌株相比，GlcNAc的产量提高了24.51%。在3 L发酵罐中进行分批发酵，GlcNAc产量达到24.23 g/L，相较于对照菌株GlcNAc的产量提高了24.69%。进一步研究发现重组Cegna1对GlcN-6P和乙酰辅酶A（Ac-CoA）有着较高的亲和力和催化效率，最后对重组Cegna1的基本酶学性质进行了考察，发现底物GlcN-6P对重组Cegna1的抑制作用明显，下一步可采用定向进化策略改造Cegna1，解除底物GlcN-6P对重组Cegna1的抑制作用。本研究结果为应用定向进化策略改造GNA的实现提供了理论依据。

英文摘要:

N-acetylglucosamine（GlcNAc），derivative of glucosamine（glucosamine，GlcN），is widely used in the field of health food and pharmaceutical. Glucosamine-6-phosphate N-acetyltransferase（GNA） is one of the crucial enzymes in the GlcNAc synthesis pathway. On the basis of recombinant GlcNAc-producingBacillus subtilis，we overexpressed glucosamine-6-phosphate N-acetyltransferase，originates fromCaenorhabditis elegans（Cegna1），in the engineeredB. subtilis，which enhanced GlcNAc titer to 7.31 g/L. Compared with the engineered B. subtilis with overexpression of glucosamine-6-phosphate N-acetyltransferase derived fromSaccharomyces cerevisiae，the GlcNAc yield of the recombinantB. subtiliswith Cegna1 increased by 24.51%. By fed-batch fermentation in 3 L bioreactor，GlcNAc titer further increased to 24.23 g/L，which is 24.69% higher than the reference strain. Further investigation results showed that the recombinant Cegna1 has higher affinity and catalytic efficiency for GlcN-6P and Ac-CoA. Finally，it is found that the substrate GlcN-6P has obvious inhibition on the the recombinant Cegna1. Identified substrate inhibition of Cegna1 provides future directions for improvement of GlcNAc production by directed evolution of Cegna1 for alleviating substrate inhibition.

查看全文 查看/发表评论 下载PDF阅读器