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

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。

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-producing Bacillus subtilis,we overexpressed glucosamine-6-phosphate N-acetyltransferase,originates from Caenorhabditis elegans(Cegna1),in the engineered B.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 from Saccharomyces cerevisiae,the GlcNAc yield of the recombinant B.subtilis with 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.