褐藻胶裂解酶基因的克隆表达及重组酶酶学性质

发布时间：2019-03-24 11:24

【摘要】：【目的】克隆交替假单胞菌(Pseudoalteromonas sp.)BYS-2的褐藻胶裂解酶基因,实现其在大肠杆菌细胞中异源表达,对分离纯化的重组酶进行酶学性质研究。【方法】以交替假单胞菌BYS-2菌株基因组DNA为模板,克隆得到褐藻胶裂解酶基因alg738,构建重组基因工程菌BL21(DE3)/p ET22b-alg738,诱导表达,表达产物通过Ni-NTA树脂纯化后进行酶学性质研究。【结果】重组酶的最适反应p H为8.0,在p H 6.0-9.0范围内37°C保温1 h仍能保持84%以上的相对酶活力,具有较好的p H稳定性;最适反应温度为45°C,热稳定性实验显示在37°C下保温60 min其残余酶活力仍达66.6%;在5 mmol/L浓度下,Na~+、Mg~(2+)、Mn~(2+)对该酶具有明显的促进作用,Ni~(2+)、Co~(2+)、Cu~(2+)、Hg~(2+)、Zn~(2+)、EDTA、β-巯基乙醇、SDS具有明显的抑制作用。动力学参数Km、Vmax分别为1.11 g/L和0.011 g/(L·min),底物特异性分析表明该重组酶为偏好聚甘露糖醛酸钠(Poly M)裂解作用的双功能酶。【结论】重组褐藻胶裂解酶具有良好的酶学特性,为褐藻胶裂解酶的开发应用打下基础。
[Abstract]:[objective] to clone the alginate lyase gene of Pseudomonas alternatus (Pseudoalteromonas sp.) BYS-2 and realize its heterologous expression in E. coli cells. The enzymatic properties of the purified recombinant enzyme were studied. [methods] using genomic DNA of Pseudomonas alternans BYS-2 strain as template, the alginate lyase gene alg738, was cloned to construct recombinant gene engineering strain BL21 (DE3) / p ET22b-alg738,. The expression product was purified by Ni-NTA resin and the enzymatic properties were studied. [results] the optimal reaction pH of the recombinant enzyme was 8.0, In the range of pH 6.0 and 9.0, 37 掳C kept 84% of the relative enzyme activity for 1 h, and had good stability of pH. The optimum reaction temperature was 45 掳C, and the thermal stability test showed that the residual enzyme activity was still up to 66.6% at 37 掳C for 60 min. At the concentration of 5 mmol/L, Na~, Mg~ (2) and Mn~ (2) promoted the enzyme obviously. Ni~ (2), Co~ (2), Cu~ (2), Hg~ (2), Zn~ (2), EDTA, 尾-mercaptoethanol), SDS had obvious inhibitory effect. The kinetic parameters Km,Vmax were 1.11 g / L and 0.011 g / (L 路min), respectively. The substrate specificity analysis showed that the recombinant enzyme was a bifunctional enzyme favoring the cleavage of (Poly M) by sodium polymannose. [conclusion] the recombinant alginate lyase has good enzymatic properties, which lays a foundation for the development and application of alginate lyase.
【作者单位】：福建省海洋酶工程重点实验室福州大学;
【基金】：国家海洋局海洋公益性行业科研专项项目(No.201305015) 福建省企业技术创新项目福州市科技计划项目(No.2016X0005)~~
【分类号】：Q55;Q78

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