果糖基转移酶AoFT在毕赤酵母中的表达与纯化及其酶学性质研究
Expression and purification of fructosyl transferase AoFT in Pichia pastoris and study on its enzymatic properties
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摘要: 利用基因工程技术方法，将构建的重组质粒pPICZαA-AoFT线性化后，经电转化导入毕赤酵母X33（Pichia pastoris X33）中，构建重组毕赤酵母工程菌pPICZαA-AoFT/X33，实现果糖基转移酶AoFT在毕赤酵母中的表达，进而研究纯化后的果糖基转移酶AoFT的酶学性质.结果表明：pPICZαA-AoFT/X33随发酵时间的增加而快速繁殖，菌体OD600值于发酵时长120 h时达到峰值，此时发酵液胞外总蛋白质量浓度为1.64 mg/mL；纯化后的果糖基转移酶AoFT的比活力为71.4 U/mg，产率达70.6%；PNGase F酶切反应证实纯化后的果糖基转移酶AoFT发生了N-糖基化反应；果糖基转移酶AoFT的最适反应温度为45℃，最适缓冲体系为醋酸钠缓冲液（pH值为4.5~6.0）；金属离子Mn2+，Ca2+对果糖基转移酶AoFT具有激活作用，而Al3+，Fe2+，Ni2+，Zn2+和EDTA对果糖基转移酶AoFT具有抑制作用；表面活性剂SDS，吐温20和Triton X-100对果糖基转移酶AoFT的酶活性影响较小，还原剂DTT对果糖基转移酶AoFT的酶活性具有明显的抑制作用，酶抑制剂PMSF低浓度时对果糖基转移酶AoFT的酶活性无显著影响，高浓度时可使酶活性降低；果糖基转移酶AoFT对有机溶剂乙醇、二甲基亚砜、二甲基甲酰胺、正丁醇、正己烷和甲苯具有较强的抗性.Abstract: Using genetic engineering techniques,the recombinant plasmid pPICZαA-AoFT was linearized,and then electroporated into Pichia pastoris X33 to construct recombinant Pichia pastoris engineering strain pPICZαA-AoFT/X33 and achieve the recombinant fructosyl transferase AoFT in Pichia pastoris.Furthermore,the enzymatic properties of the purified fructosyl transferase AoFT were studied.The results showed that the pPICZαA-AoFT/X33 multiplied rapidly with the increase of fermentation time.The OD600 value of the cell reached a peak at 120 h,and the total protein concentration of the extracellular protein in the fermentation broth was 1.64 mg/mL.The specific activity of the purified fructosyl transferase AoFT enzyme was 71.4 U/mg,and the yield was 70.6%.The peptide N-glycosidase F digestion reaction confirmed that the purified fructosyl transferase AoFT had some N-glycosylation sites.The optimal reaction temperature of fructosyl transferase AoFT was 45 ℃,and the optimal buffer system was sodium acetate buffer (pH value was 4.5~6.0).Metal ions Mn2+ and Ca2+ could activate fructosyl transferase AoFT,while Al3+,Fe2+,Ni2+,Zn2+ and EDTA could inhibit fructosyl transferase AoFT.Surfactants (SDS,Tween-20 and Triton X-100) had little effect on the activity of fructosyl transferase AoFT,while the reductant DTT had obvious inhibition effect on the activity of fructosyl transferase AoFT.The low concentration of PMSF had no significant effect on the activity of fructosyl transferase,but the high concentration could reduce the activity.The activity of fructosyl transferase AoFT showed strong resistance to organic solvents (ethanol,DMSO,dimethylformamide,n-butanol,n-hexane and toluene).
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Key words:
- fructosyl transferase /
- Pichia pastoris /
- expression /
- purification /
- enzymatic property
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