丁香假单孢菌重组褐藻胶裂解酶的酶学性质及酶解产物的抗氧化活性

Characterization of Recombinant Alginate Lyase from Pseudomonas syringae and Antioxidant Activity of Its Hydrolysates

目的:将丁香假单孢菌的褐藻胶裂解酶进行克隆表达和纯化,研究重组酶的酶学性质及酶解产物的抗氧化活性,为进一步研究该酶的结构与功能奠定良好的基础。方法:利用PCR扩增褐藻胶裂解酶基因,将它克隆至表达载体pET-28a;表达产物用亲和层析纯化,研究重组酶的酶学性质;通过测定酶解产物的还原能力和清除ABTS·+和·OH自由基的能力,分析酶解产物的抗氧化活性。结果:来自丁香假单孢菌的重组褐藻胶裂解酶的分子质量为40.8 ku。该重组酶专一性降解聚甘露糖醛酸,酶的最适反应温度和pH分别为30℃和7.5,温度低于50℃时稳定。除了SDS和DTT,重组酶对其它抑制剂和去垢剂具有较好的抗性。酶解产物对ABTS·+和·OH自由基的半数抑制剂量IC50分别为1.56 mg/mL和0.56 mg/mL,还原能力较强。结论:该重组褐藻胶裂解酶的酶解产物有一定的抗氧化活性,具有应用潜力。

Objective:The alginate lyase gene from Pseudomonas syringae was cloned and expressed in Escherichia coli BL21(DE3).After the recombinant alginate lyase was purified,the enzyme and the antioxidant activity of the enzymatic hydrolysates were characterized.These will set up a good foundation for further researches of structure and function of this enzyme.Methods:The alginate lyase gene was amplified by PCR and cloned into pET-28a expression vector.After the recombinant enzyme was purified using affinity chromatography,it was characterized.The antioxidant activities of the enzymatic hydrolysates were analysed by detecting the reducing ability and scavenging power on ABTS+and hydroxyl radicals.Results:The molecular weight of the recombinant alginate lyase from P.syringae was 40.8 kDa.The recombinant enzyme showed activity towards polymannuronate.The optimal temperature and pH of the enzyme were 30℃and 7.5,respectively,and it was stable at temperature below 50℃.In addition to SDS and DTT,the recombinant alginate lyase had good resistance to other detected inhibitors and detergents.The enzymatic hydrolysates had the reducing power.The half inhibitory concentration(IC50)values of scavenging ABTS+and hydroxyl radicals were 1.56 mg/mL and 0.56 mg/mL,respectively.Conclusion:The enzymatic hydrolysates exhibited the antioxidant activity and had potential as antioxidant products.