铜绿假单胞菌algL基因在毕赤酵母中表达及其性质研究.docVIP

铜绿假单胞菌algL基因在毕赤酵母中的表达及其性质研究 岳 明，丁宏标，乔 宇 （中国农业科学院饲料研究所生态饲料研究室，北京 100081） 摘要：【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40 kD的目的蛋白，酶活力达540 U·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℃，pH 3.0～12.0具有较好的稳定性。50 mmol·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。 关键词：海藻酸裂解酶；毕赤酵母；海藻寡糖；诱导表达 Expression of Pseudomonas aeruginosa Alginate Lyase Gene (algL) in Pichia pastoris and Characterization of the Enzyme Yue Ming, Ding Hong-biao, Qiao Yu (Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081) Abstract: 【Objective】The purpose was to clone and express lginate lyase gene (algL) of Pseudomonas aeruginosa in Pichia pastoris expression system. 【Method】Alginate lyase gene of P. aeruginosa was amplified and inserted into pPIC9K expression vector by adding EcoR I site to generate recombinant pPIC9K-algL construct. The positive construct was transformed to Pichia pastoris GS115 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 SDSwith 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 Zn2+, Cu2+ and Fe2+, but Co2+ and Ca2+ 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. Key words: Alginate lyase; Pichia pastoris; Alga oligosaccharides; In