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

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84 h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8.2 IU/mL 和9.9 IU/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12 h.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46 kDa、47 kDa,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.%A n expressioncD N A library was constructed from Rhizopus stolonifer var. reflexes TP-02 with high capacity for hydrolysis of celluase , and then two novel β-glucosidase genes bgl1 and bgl2 were isolated and efficiently expressed in Pichia pastrois. The recom bined Pichia pastrois were culrured in the M M medium,using l℃ methanol to induce the expression of recom binant gene . The results showed that the maxinum activity of recom bined β-glucosidases were obtained at 84 h , 12 h earlier than that of the onginal strain Rhizoupus stolonifer var. reflexus T P-02 , and were 8 .2 IU /m L and 9 .9 IU /m L , 1.0-fold and 1.41-fold higher than that from the original strain . The exprPssed protein were purified from the ferm ented supernatant using Sephadex G 100 column and determ ined by SDS-PAGE . The results of SD S-PA G E also showed that the molecular weights of these two novel enzym es were 46 kD a and 47 kD a, respectively .