内切菊粉酶基因在毕赤酵母中的高水平表达研究

High Level Expression of the Endo-inulinase Gene in Pichia pastoris

低聚果糖(Fructooligosaccharides,FOS)具有改善胃肠道环境、刺激和加强机体免疫反应、抑制肠道特定肿瘤的发生等生物活性.内切菊粉酶能够水解菊粉获得低聚果糖.为了实现内切菊粉酶的高表达,从无花果曲霉(Aspergillus ficuum ATCC16882)中克隆了内切菊粉酶编码基因INU2,并实现了在毕赤氏酵母中的重组分泌表达,摇瓶表达的酶活力为570.4U/mL.去除内切菊粉酶的内源性信号肽序列后,其重组表达水平显著提高,摇瓶表达的酶活力为1013.8U/mL,提高幅度77.7%.TLC分析表明:毕赤酵母重组表达的内切菊粉酶(不含内源性信号肽序列)能将2%长链菊粉水解为2～3个聚合度的低聚果糖.

Fructooligosaccharides（FOS） can improve gastrointestinal conditions,inhibit the growth of intestinal cancer and stimulate immune response.FOS can be obtained by hydrolyzing inulin with endo-inulinase.In the current study,endo-inulinase gene INU2 was cloned from Aspergillus ficuum ATCC16882 genome by Polymerase Chain Reaction（PCR） and the recombinant was heterogeneously expressed in Pichia pastoris GS115.The enzyme activity of endo-inulinase expressed in flask was 570.4 U/mL.The effect of molecular structure of endo-inulinase on its expression was then studied.The deletion of endogenous signal peptide of endo-inulinase could efficiently increase its expression in Pichia pastoris.The enzyme activity of endo-inulinase without endogenous signal peptide was 1013.8 U/mL,77.7% higher than the previous one.TLC analysis showed that heterogeneously expressed endo-inulinase without endogenous signal peptide can hydrolyze long chain inulin into fructooligosaccharides.