摘要
以ClostridiumparaputrificumM21染色体DNA为模板,经PCR扩增获得编码β N 乙酰氨基葡萄糖苷酶的基因nag3A,与质粒pQE30T所构建的表达质粒pNAG3A在EscherichiacoliM15中表达良好.从重组E.coliM15中纯化得到的Nag3A以4 MU GlcNAc为底物时,最适作用温度和最适作用pH分别为50℃和7.0;在30℃以下和pH6～9之间该酶活性稳定.对4 MU GlcNAc的Km和Vmax分别为7.9μmol和21.8μmol/(min·mg).Nag3A可以水解几丁寡糖和几丁质,作用方式是从非还原端逐一水解β 1,4 D N 乙酰氨基葡萄糖苷键,产生N 乙酰氨基葡萄糖,对几丁二糖具有较高的水解活性.
The recombinant plasmid pNAG3A was constructed from the plasmid pQE30T and nag3A gene coding for βNAcetylglucosaminidase, and the nag3A was amplified by PCR using the chromosomal DNA of Clostridium paraputrificum M21 as template. The recombinant plasmid expressed well in E. Coli M15. The Nag3A was purified from Escherichia coli M15 harboring pNAG3A. When 4MUGlcNAc was used as the substrate, the optimum temperature and optimum pH of the purified enzyme were 50 ℃ and 7.0, respectively. The enzyme was stable below 30 ℃ and within pH 6～9. The Km and Vmax for 4MUGlcNAc were 7.9 μmol/L and 21.8 μmol/(min·mg), respectively. Nag3A hydrolyzed chitooligosaccharides and ballmilled chitin from the nonreduced end to produce Nacetylglucosamine as the product. Nag3A had high activity on chitobiose.
出处
《无锡轻工大学学报（食品与生物技术）》
CSCD
北大核心
2003年第3期41-45,52,共6页
Journal of Wuxi University of Light Industry
基金
日本文部科学省大学共同利用基金项目(12794004)资助课题.
