N-乙酰氨基葡萄糖苷酶的异源表达及甘油糖苷的酶法合成

Heterologous Expression of N-Acetyl-Glucosaminidase and Enzymatic Synthesis of Glyceryl N-Acetyl-Glucosamine

从杆菌状链霉菌中克隆了一个N-乙酰氨基葡萄糖苷酶(N-acetyl-glucosaminidase,NAGase)的编码基因sbnag2550,并将其在大肠杆菌BL21(DE3)中进行了异源表达。利用镍离子亲和层析得到纯酶后对SbNag2550的酶学性质进行了研究。该酶的最适温度为50℃,在45~60℃均表现出90%以上的酶活力。SbNag2550还表现出优良的热稳定性,在55℃孵育60 h仍能保留将近90%的酶活力。利用SbNag2550催化N-乙酰氨基葡萄糖(N-acetyl-glucosamine,NAG)和甘油发生逆水解反应,合成了甘油-N-乙酰氨基葡萄糖苷(glyceryl N-acetyl-glucosamine,GNAG)。在最适条件下,反应24 h时转化率达到28.20%,反应72 h时转化率为34.82%。本研究中首次通过酶法合成了GNAG,为其功能研究和应用开发奠定了基础。

A gene(sbnag2550)encoding N-acetyl-glucosaminidase(NAGase)was cloned from Streptomyces bacillaris and expressed in Escherichia coli BL21(DE3).The enzymatic properties of SbNag2550 were studied using the purified enzyme obtained by nickel ion affinity chromatography.The optimal temperature of SbNag2550 was 50℃,and it could show more than 90%activity at temperature ranging from 45℃to 60℃.The enzyme also showed excellent thermostability,retaining nearly 90%activity after incubating at 55℃for 60 h.SbNag2550 was used to catalyze the reverse hydrolysis reaction of N-acetyl-glucosamine(NAG)and glycerol,realizing the synthesis of glyceryl N-acetyl-glucosamine(GNAG).Under optimal conditions,the conversion rate reached 28.20%and 34.82%at 24 h and 72 h,respectively.GNAG was firstly synthesized by enzymatic catalysis in this study,which laid a foundation for its functional research and application development.