食鹿角菜交替假单胞菌芳香基硫酸酯酶的克隆、表达及酶学性质

Cloning, Expression and Characterization of an Arylsulfatase from Pseudoalteromonas carrageenovora

目的:在大肠杆菌中高效表达食鹿角菜交替假单胞菌芳香基硫酸酯酶基因,并对重组酶的酶学性质进行研究,为开发酶法去除琼脂硫酸酯的技术奠定基础。方法:通过PCR克隆芳香基硫酸酯酶基因,将它克隆至表达载体pET-28a;表达产物用亲和层析纯化,并对重组酶的酶学性质进行研究。结果:从食鹿角菜交替假单胞菌克隆得到芳香基硫酸酯酶基因(987 bp),预测编码328个氨基酸残基。将该基因在大肠杆菌BL21(DE3)中表达和纯化,SDS-PAGE分析显示,重组酶的分子质量为35.1 ku。以对硝基苯硫酸钾(p-NPS)为底物,酶的比活力达26.7 U/mg,最适反应温度50℃和最适反应pH 7.0,Km为0.65 mmol/L,Vmax为19.99μmol/(mg·min)。除了SDS,重组酶对其他测试的抑制剂和去垢剂具有一定的抗性。重组芳香基硫酸酯酶和龙须菜粗多糖在40℃反应2 h,酶解产物凝胶强度提高2.1倍,可脱除多糖84.9%的硫酸基团。结论 :重组芳香基硫酸酯酶能有效脱除龙须菜粗多糖的硫酸基团,为开发环保、高效的酶法琼脂生产技术奠定基础。

Objective： The gene encoding a putative arylsulfatase from Pseudoalteromonas carrageenovora was cloned and expressed in Escherichia coli BL21 （DE3）, and then the recombinant enzyme was characterized. These will set up a theoretical tbundation for developing the enzymatic technique to remove agar sulfate groups. Methods： The arylsulfatase gene was amplified by PCR and cloned into pET-28a expression vector. The recombinant arylsulfatase was characterized after it was purified using affinity chromatography. Results： A gene （987 bp） encoding an arylsulfatase was identified from Pseudoalteromonas carrageenovora. The open reading frame of this gene encoded 328 amino acid residues. The gene was cloned and expressed in Escherichia coli BL21 （DE3）, and the target protein was purified to homogeneity. The purified recombinant enzyme presented a molecular mass of 35.1 ku by SDS-PAGE. The purified arylsulfatase was characterized using the synthetic substrate of phenolic ester, p-nitrophenyl sulfate （p-NPS）. The enzyme had a specific activity of 26.7 U/mg with the optimal temperature and pH at 50 ℃ and 7.0, respectively. The Km, and Vmax values of the recombinant enzyme were determined to be 0.65 mmol/L and 19.99 μmol/（mg,min）, respectively. Except SDS, the enzyme showed resistance in some degrees toward other tested inhibitors and detergents. After the recombinant arylsulfatase reacted with the crude polysaccharides of Gracilaria lemaneiformis at 40 ℃ for 2 h, the gel strength of the product increased by 2.1-fold. 84.9% of the sulfate in the polysaccharides had been removed. Conclusion： The recombinant arylsulfatase could effectively remove the sulfate in the crude polysaecharides of Gracilaria lemaneiformis, laying the foundation for the environmental and efficient technology development of agar production using enzyme.