鱼腥藻N-乙酰-D-葡萄糖胺2-差向异构酶基因的克隆及其在大肠杆菌中的高效表达

Cloning and high expression of N-acyl-D-glucosamine 2-epimerase gene from Anabaena sp.in Escherichia coli

目的从鱼腥藻7120(Anabaenasp.PCC 7120)中克隆N-乙酰-D-葡萄糖胺2-差向异构酶(N-acyl--Dglucosamine 2-epimerase,AGE)基因,并在大肠杆菌中表达。方法利用PCR技术从鱼腥藻染色体组扩增AGE基因,通过基因工程方法构建表达质粒pLY-11,在大肠杆菌中表达,并利用Ni-NTA柱分离纯化。利用柱前衍生化的方法,通过HPLC检测表达产物转化N-乙酰-D-甘露糖胺(N-acyl-D-mannosamine,ManNAc)的活性,并联合N-乙酰神经氨酸裂合酶(N-acetylneuraminatelyases,NAL),进行双酶转化生成N-乙酰神经氨酸(N-acetylneuraminic acid,Neu5Ac)实验。结果AGE基因在大肠杆菌中表达,可溶性产物蛋白占总可溶性蛋白质量的18%,经纯化后得到1.6 g.L-1产物蛋白。双酶转化实验中,转化终产物Neu5Ac质量浓度为14.8 g.L-1。结论鱼腥藻7120 AGE基因在大肠杆菌中的表达产物具有活性,为双酶转化制备Neu5Ac进行了初步的探索。

Objective To clone the gene of N-acyl-D-glucosamine 2-epimerase （AGE） and express it in E. coli. Methods The gene of AGE was cloned from the genome of Anabaena sp. PCC7120 by PCR technique. An expression plasmid pLY-11 was constructed through genetic engineering method and expressed in E. coli BL21 （DE3）. The soluble recombinant AGE was purified by the Ni-NTA column, and the activity of AGE was detected using PMP derivatization method and N-acyl-D-mannosamine （ManNAc） as substrate. Subsequently, a preliminary test for dual-enzymatic synthesis of N-acetylneuraminic acid（Neu5Ac）was established. Results The AGE gene was expressed resulting in 18% of the total soluble protein, and 1.6 g· L^- 1 protein was obtained after purification. In the dual-enzymatic synthesis system, the final concentration of transformation product, Neu5Ac, was 14.8 g· L^- 1. Conclusions AGE expressed in E. coli is active, and the dual-enzymatic synthesis for Neu5Ac is conducted for preliminary exploration.