摘要
[目的]对来自羊鲍肠道中弧菌(Vibrio sp. YBCH18)的褐藻胶裂解酶基因val1进行异源表达并研究其酶学性质。[方法]采用PCR技术获得褐藻胶裂解酶基因val1,分析基因序列,构建重组质粒pET-VAL1并在大肠杆菌中实现异源表达,经纯化后对其酶学性质进行研究。[结果]重组酶rVAL1最适温度为30℃;最适pH为8.5,在pH 4.5~10.0范围内保温30 min仍能保持50%以上的相对酶活力;对海藻酸钠具有良好的底物特异性;Mn^(2+)和Co^(2+)对rVAL1酶活具有明显促进作用,而Fe^(2+)、Fe^(3+)、SDS和EDTA对rVAL1活性抑制作用明显;rVAL1动力学参数Km值为2.23 mg/mL,Kcat为3.28 s^(-1),Vmax为21.70 mg/(mL·min);薄层层析分析显示,VAL1降解终产物主要是单糖组分,推测其属于外切型褐藻胶裂解酶。[结论]成功对褐藻胶裂解酶基因val1进行外源表达,研究发现VAL1是一个PL7家族中具有适冷性的外切型褐藻胶裂解酶,最适条件下比酶活为6.6 U/mg。
[Objective]A new alginate lyase gene from Vibrio sp. YBCH18 isolated from the intestine of abalone,Haliotis ovina,designated as val1,was heterologously expressed in Escherichia coli,and the enzymatic properties of the recombinant VAL1(rVAL1) were characterized.[Method]The val1 gene was obtained by PCR amplification,and its coding sequence was analyzed using bioinformatics tools. A recombinant plasmid pET-VAL1 containing the val1 gene was constructed and expressed in E. coli. The enzymatic properties of the purified recombinant enzyme were examined. [Result]The optimum temperature and pH of rVAL1 were 30℃ and 8. 5,respectively. The enzyme remained stable in pH range 4. 5-10. 0,and its relative activity maintained above 50% after 30 min. Sodium alginate was found to be the good substrate for the enzyme compared to other tested substrates. The activity of rVAL1 was significantly enhanced in the presence of Mn^(2+)and Co^(2+),while obviously inhibited by Fe^(2+),Fe^(3+),SDS and EDTA. The kinetic parameters Km and Vmax of rVAL1 for degradation of sodium alginate were 2. 23 mg/mL and 21. 70 mg/(mL·min),respectively. Thin-layer chromatography(TLC) analysis of end-products showed that rVAL1 degraded sodium alginate into monosaccharides,suggesting that the enzyme is an exo-type alginate lyase. [Conclusion]The alginate lyase gene val1 was successfully expressed in vitro. Our data suggest that VAL1 is a cold-adapted exo-type alginate lyase belonging to the PL7 family,and the specific enzyme activity was 6. 6 U/mg under the optimal conditions.
作者
陈琳
邵宗泽
易恩
杨江科
周梅先
CHEN Lin;SHAO Zong-ze;YI En;YANG Jiang-ke;ZHOU Mei-xian(Department of Biological and Pharmaceutical Engineering,Wuhan Polytechnic University,Wuhan 430023,China;Key Laboratory of Marine Genetic Resources,Third Institute of Oceanography,Ministry of Natural Resources of PR China,Xiamen 361005,China.)
出处
《生物技术》
CAS
2021年第2期121-128,207,共9页
Biotechnology
基金
国家自然科学基金面上项目(41676149)。
关键词
外切型褐藻胶裂解酶
海洋弧菌
异源表达
酶学性质
Exo-type alginate lyase
Marine Vibrio
Heterologous expression
Enzymatic properties
