海洋弧菌中褐藻胶裂解酶Alg的克隆表达及酶学性质  

Cloning Expression and Characters of Alginate Lyase Gene Alg from Marine Vibrio

作　　者：李云涛 张齐 汪立平 黄宇良 LI Yun-tao;ZHANG Qi;WANG Li-ping;HUANG Yu-liang(College of Food Science and Technology,Shanghai Engineering Research Center of Aquatic-Product Processing&Preservation/Shanghai Ocean University,Shanghai 201306,China;College of Food Science and Technology,Engineering Research Center of Food Thermal-processing Technology/Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学食品学院,上海水产品加工及贮藏工程技术研究中心,上海201306 [2]上海海洋大学食品学院,食品热加工工程技术研究中心,上海201306

出　　处：《山东农业大学学报（自然科学版）》2018年第4期659-666,共8页Journal of Shandong Agricultural University：Natural Science Edition

基　　金：上海市科学技术委员会工程中心建设(11DZ2280300)。

摘　　要：褐藻胶裂解酶是制备功能性寡糖的关键酶,以基因工程手段构建重组褐藻胶裂解酶,为实现褐藻胶裂解酶向商业酶转化奠定重要基础。以海洋弧菌SS-1总DNA为模板,运用PCR技术克隆得到褐藻胶裂解酶基因Alg,构建重组表达菌株E.coli BL21(DE5α)/p ET28a(+)-Alg,进行诱导表达,产物通过Ni-NTA resin亲和层析柱纯化,研究其酶学性质及酶解产物。结果表明,重组酶的最适pH为8.0,在pH 4.0~9.0范围内,30℃条件下保温2 h,仍能保持60%以上的相对酶活力;最适温度30℃,热稳定实验显示在高于30℃保温2 h其残余酶活力损失40%以上;在添加浓度1 mmol/L离子K^+、Na^+和Mg^(2+)显示对重组酶有明显的促进作用,Cu^(2+)、Fe^(3+)、Ba^(2+)、Fe^(2+)、Al^(3+)、SDS和EDTA等对该酶抑制作用明显;动力学参数Km为5.93 mg/mL,Vmax为826.44 mg/(mL·min);电离喷雾质谱(ESI-MS)分析其降解褐藻胶产物主要为小分子寡糖。重组酶是具有良好的酶学特性及有效的作用poly(M)降解产生低聚寡糖的双功能酶,可用于褐藻寡糖的制备。Alginate lyase was a key enzyme for the preparation of functional oligosaccharides,the recombinant alginate lyase was constructed by genetic engineering,which lays an important foundation for the conversion of alginate lyase to commercial enzyme.Algase gene of alginate lyase was cloned by PCR using total DNA of marine Vibrio sp.SS-1 as template.The recombinant expression strain E.coli BL21(DE5α-pET28a(PET-Alg)was induced and expressed.The enzymatic properties and enzymatic hydrolysis products were studied by Ni-NTA resin affinity chromatography column purification.The results showed the optimum pH of the recombinant enzyme were 8 that in the range of pH 4.0~9.0.It heat preservation at 30℃for 2 h,that relative enzyme activity could maintain more than 60%.The optimum temperature was 30℃,and the thermal stability test showed the residual enzyme activity loss more than 40%when the temperature was higher than 30℃for 2 h.At the concentration of 1 mmol/L,K+,Na+and Mg2+had significant promoting effects on the recombinant enzyme,while Cu2+,Fe3+,Ba2+,Fe2+,Al3+,SDS and EDTA had obvious inhibitory effects on the enzyme.The kinetic parameters Km was 5.93 mg/mL,and Vmax was 826.44 mg/(mL·min).The degradation of alginate by ionization mass spectrometry(ESI-MS)was mainly composed of small molecular oligosaccharides.Recombinant enzyme was a bifunctional enzyme with good enzymatic properties and effective effect on the degradation of oligosaccharides,which could be used in the preparation of brown algae oligosaccharides.Recombinant enzyme was a bifunctional enzyme with good enzymatic characteristics and effective action of poly(M)degradation,which could be used to prepare brown algal oligosaccharides.

关 键 词：褐藻胶裂解酶 海洋弧菌 克隆与表达 电离喷雾质谱 酶学性质 

分 类 号：Q819[生物学—生物工程]