葡枝根霉TP-02cDNA文库中两种新的β-葡萄糖苷酶基因的克隆及其在毕赤酵母中的表达  

Cloning and expression of two novel β-glucosidase genes from Rhizopus stolonifer var.reflexus TP-02 cDNA library in Pichia pastrois

作　　者：汤斌[1] 丁丽霞[1] 潘海波[1] 汤文晶[1] 张凤琴[1] 

机构地区：[1]安徽工程大学微生物发酵安徽省工程技术研究中心,芜湖241000

出　　处：《工业微生物》2011年第4期72-76,共5页Industrial Microbiology

基　　金：安徽省自然科学基金(No.KJ2007A018)

摘　　要：从具有良好降解纤维素能力的葡枝根霉TP-02的cDNA文库中筛选获得两个新的β-葡萄糖苷酶基因bgl1和bgl2,并在毕赤酵母中高效表达。阳性克隆在MM培养基中发酵84h和1%的甲醇的诱导的情况下,产生的β-葡萄糖苷酶酶活达到峰值分别为8.2IU/mL和9.9IU/mL,分别较原菌株葡枝根霉的β-葡萄糖苷酶酶活提高了1倍和1.41倍,并且其发酵时间缩短12h。用G100分离纯化其发酵上清液后,用SDS-PAGE电泳方法,测得它们的分子量分别为46kDa、47kDa,而且条带清晰,所以用甲醇诱导的重组毕赤酵母发酵的上清液较大肠杆菌易于纯化。An expression cDNA library was constructed from Rhizopus stolonifer var. reflexes TP-02 with high capacity for hydrolysis of cellulase, and then two novel β-glucosidase genes bgll and bgl2 were isolated and efficiently expressed in Pichia pastrois. The recombined Pichia pastrois were cultured in the MM medium, using 1% methanol to induce the expression of recombinant gene. The results showed that the maximum activity of recombined β-glucosidases were obtained at 84 h, 12 h earlier than that of the original strain Rhizoupus stolonifer vat. reflezus TP-02, and were 8.2 IU/mL and 9.9 IU/mL, 1.0-fold and 1.41-fold higher than that from the original strain. The expressed protein were purified from the fermented supematant using Sephadex G100 column and determined by SDS-PAGE. The results of SDS-PAGE also showed that the molecular weights of these two novel enzymes were 46 kDa and 47 kDa, respectively.

关 键 词：Β-葡萄糖苷酶 毕赤酵母 表达 纯化 

分 类 号：Q933[生物学—微生物学]