来源于葡萄球菌的N-乙酰神经氨酸裂合酶的基因克隆及性质

葡萄球菌Staphylococcus hominis来源的N-乙酰神经氨酸裂合酶基因shnal(GenBank Accession No.EFS20452.1)构建至pET-28a质粒并在大肠杆菌中得到表达.通过目的蛋白的纯化和酶学性质研究发现,ShNAL是一个四聚体,裂解方向的最适反应pH为8.0；合成方向的最适反应pH为7.5,最适反应温度为45℃.在45℃下孵育2h对ShNAL的活力基本无影响,高于45℃时,活力迅速下降.该酶在pH 5.0～10.0的环境中比较稳定,4℃下放置24 h酶的残余活力在70％以上.ShNAL对N-乙酰神经氨酸(Neu5Ac)、N-乙酰甘露糖胺(Man)和丙酮酸(Pyr)的Km值分别是(4.0±0.2) mmol/L、(131.7±12.1)mmol/L和(35.14±3.2) mmol/L,kcat/Km值分别为1.9 L/(mmol·s)、0.08 L/(mmol·s)和0.08 L/(mmol·s).

Molecular cloning and characterization of a N-acetylneuraminate lyase gene from Staphylococcus hominis

A N-acetylneuraminate lyase gene (shnal) from Staphylococcus hominis was cloned into pET-28a and expressed in Escherichia coli BL21 (DE3) host cells. The recombinant enzyme was purified and characterized. It is a homotetrameric enzyme with the optimum pH at 8.0 for the cleavage direction and the optimum pH and temperature were 7.5 and 45 °C for the synthetic direction. The activity of ShNAL is stable when incubated at 45 °C for 2 h but decreased rapidly over 50 °C. ShNAL showed high stability in a wide range pH from 5.0 to 10.0 with the residual activity being >70% when the enzyme was incubated in different buffers at 4 °C for 24 h. Its Km towards N-acetylneuraminic acid, pyruvate and ManNAc were (4.0±0.2) mmol/L, (35.1±3.2) mmol/L and (131.7±12.1) mmol/L, respectively. The kcat/Km value of Neu5Ac, ManNAc, and Pyr for ShNAL were 1.9 L/(mmol·s), 0.08 L/(mmol·s) and 0.08 L/(mmol·s), respectively.