铜绿假单胞菌algL基因在毕赤酵母中的表达及其性质研究

Expression of Pseudomonas aeruginosa Alginate Lyase Gene (algL) in Pichia pastoris and Characterization of the Enzyme

【目的】通过构建毕赤酵母工程菌株,以期获得重组海藻酸裂解酶,并对其性质加以研究。【方法】采用PCR的方法,以铜绿假单胞菌(Pseudomonas aeruginosa)基因组DNA为模板,克隆出约1.0kb的海藻酸裂解酶基因algL的成熟蛋白编码序列,并将其插入巴斯德毕赤酵母(Pichia pastoris)表达载体pPIC9K中,获得重组质粒pPIC9K-algL。重组质粒线性化后用聚乙二醇(PEG)法导入毕赤酵母菌株GS115中表达。【结果】用甲醇诱导培养基进行摇瓶发酵,表达得到40kD的目的蛋白,酶活力达540U·ml-1左右。经测定,该重组酶的最适反应pH为8.5,最适反应温度为40℃,并且在20～45℃,pH3.0～12.0具有较好的稳定性。50mmol·L-1的Co2+和Ca2+对酶活力均有显著的促进作用,Zn2+、Cu2+和Fe2+在不同浓度下对酶活力均有不同程度的抑制作用。在重组酶的辅助作用下,抗生素的抑菌效果明显提高。【结论】用重组毕赤酵母可高效表达外源海藻酸裂解酶,为其今后在工农业中的应用奠定了基础。

[ Objective ] The purpose was to clone and express alginate lyase gene （algL） of Pseudomonas aeruginosa in Pichia pastoris expression system. [Method] Alginate lyase gene of P aeruginosa was amplified and inserted into pPICgK expression vector by adding EcoR I site to generate recombinant pPIC9K-algL construct. The positive construct was transformed to Pichia pastoris GSll5 cells by PEG method. The AlgL protein was over-produced in P. pastoris induced by methanol. [Result] The product of recombinant alginate lyase showed a molecular weight of 40 kD on SDS-PAGE with activity of 540 U.ml^-1 at an optimal temperature 40℃ and pH 8.5, respectively. The recombinant AlgL was stable below 45℃between pH 3.0 and 12.0. The recombinant AlgL was sensitive to some metal cations negatively including Zn^2＋, Cu^2＋ and Fe^2＋, but Co^2＋ and Ca^2＋ promoted it dramatically. The sterilization of ampicilin and kanamycin on P. aeruginosa increased with the help of AlgL. [Conclusion] P pastoris expression system is an efficient way of production of AlgL. The recombinant AlgL has potency as feed additive and antibiotic assistant.