假交替单胞菌JMUZ2重组κ-卡拉胶酶的异源表达和酶学性质

Enzymatic properties of the recombinantκ-carrageenase from Pseudoalteromonas sp.JMUZ2

将假交替单胞菌 JMUZ2的 κ-卡拉胶酶基因在大肠杆菌中异源表达,利用亲和层析对重组 κ-卡拉胶酶进行 分离纯化,表征重组酶的酶学性质,利用质谱分析 κ-卡拉胶的酶解产物,并进行产物的抗氧化活性分析。结果 表明,假交替单胞菌 JMUZ2的 κ-卡拉胶酶属于 GH16家族,能专一性降解 κ-卡拉胶,重组 κ-卡拉胶酶的最适反 应温度和 pH 分别为 50℃和 8.0,在 40℃条件下处理 1 h,保持约 80% 残余活力。重组酶对去垢剂 Tween 20、 Tween 80和 Triton X-100有良好的耐受性。LC-MS分析显示,重组酶水解 κ-卡拉胶的终产物为二糖和四糖。酶 解产物对·OH自由基、DPPH自由基、ABTS自由基具有一定清除作用,还具有良好的还原能力。重组 κ-卡拉胶酶 的酶学性质及酶解产物的分析为该酶用于 κ-卡拉胶寡糖绿色制备的应用奠定理论基础。

The k-carrageenase gene from Pseudoalteromonas sp.JMUZ2 was expressed heterologously in Escherichia coli.After the recombinantκ-carrageenase was purified,the properties of the recombinant enzyme were characterized and the antioxidant activity was explored for the enzymatic hydrolysis products ofκ-carrageenan.The results showed that the k-carrageenase from Pseudoalteromonas sp.JMUZ2 belonged to GH16 family.The recombinant enzyme could specifically degrade k-carrageenan.The optimal reaction temperature and pH of the recombinant k-carrageenase were 50℃and 8.0,respectively.The recombinant k-carrageenase maintained the residual activity of about 80%after incubation at 40℃for 1 h.The recombinant enzyme had good tolerance to the detergents of Tween 20,Tween 80 and Triton X-100.LC-MS analysis showed that the final products of k-carrageenan hydrolyzed by recombinant enzyme were disaccharides and tetrasaccharides.The hydrolysates had scavenging effects on·OH,DPPH and ABTS radicals,and also had good reducing ability.The enzymatic properties of the recombinantκ-carrageenase and the analysis of the enzymatic hydrolysates lay a theoretical foundation for the application of the enzyme in the green preparation of κ-carrageenan oligosaccharides.