褐藻胶裂解酶基因的克隆表达及重组酶酶学性质

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)/pET22b-alg 738,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pH为8.0,在pH 6.0-9.0范围内37℃保温lh仍能保持84％以上的相对酶活力,具有较好的pH稳定性;最适反应温度为45℃,热稳定性实验显示在37℃下保温60 min其残余酶活力仍达66.6％;在5 mmol/L浓度下,Na+、Mg2+、Mn2+对该酶具有明显的促进作用,Ni2+、C02+、Cu2+、Hg2+、Zn2+、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.%[Objective] The alginate lyase gene of Pseudoalteromonas sp.BYS-2 was cloned and expressed in Escherichia coli,and the properties of recombinase were characterized.[Methods] The alginate lyase gene alg738 was cloned from the genomic DNA ofPseudoalteromonas sp.BYS-2 and the recombinant strain BL21(DE3)/pET22b-alg738 was constructed.The recombinase was purified with Ni-NTA resin and the enzymatic properties were studied.[Results] The optimum pH of recombinase was 8.0.It was stable in the pH range of 6.0 to 9.0 and could maintain more than 84％ of its relative enzyme activity after incubation at 37 ℃ for 1 hour.The optimum temperature of recombinase was 45 ℃ and 66.6％ of enzyme activity was remained after incubation at 37 ℃ for 60 minutes.At the concentration of 5 mmol/L,Na+,Mg2+ and Mn2+ had significant stimulation on the enzyme activity,while Ni2+,Co2+,Cu2+,Hg2+,Zn2+,EDTA,β-mercaptoethanol and SDS had inhibitory effects on the enzyme.The kinetic parameters Km and Vmax of rAlg738 were 1.11 g/L and 0.011 g/(L·min),respectively.Moreover,this recombinase was a bifunctional enzyme which prefers sodium polymannuronate (Poly M).[Conclusion] The properties of the recombinase rAlg738 has laid a good foundation for its future development and application.