【摘要】：內(nèi)切纖維素酶Cel5A缺乏是限制纖維素酶制劑高效酶解天然纖維素的關(guān)鍵因素。本文嘗試構(gòu)建高效表達里氏木霉Cel5A的畢赤酵母重組菌株以彌補目前Cel5A的天然分泌不足,通過基因密碼子偏好性優(yōu)化里氏木霉Cel5A基因和構(gòu)建表達載體p PIC9K-eg2,并將其電轉(zhuǎn)入畢赤酵母GS115以構(gòu)建重組子,利用濃度梯度平板和搖瓶發(fā)酵篩選獲得一株高產(chǎn)畢赤酵母Pichia pastoris菌株GS115-EGⅡ。重組酶的酶學(xué)性質(zhì)分析顯示,該酶分子量50 k Da、最適p H(p H 4.5)略有降低及最適反應(yīng)溫度為60℃,專一性地作用于非結(jié)晶纖維素,與天然里氏木霉Cel5A并無明顯區(qū)別。通過搖瓶發(fā)酵的初步優(yōu)化,該菌搖瓶培養(yǎng)條件:培養(yǎng)溫度28℃、起始p H 5.0、接種量2%、每24 h添加甲醇1.5%(V/V)、每24 h添加山梨醇4 g/L及吐溫80添加4 g/L,發(fā)酵192 h重組酶酶活達到24.0 U/m L。進一步上罐(5 L)發(fā)酵180 h,該重組酶Cel5A酶活高達270.9 U/m L,蛋白含量達到4.16 g/L。重組畢赤酵母P.pastoris GS115-EGⅡ是一株適合于外源表達Cel5A的工程菌,該重組酶可替代天然分泌Cel5A適用于當(dāng)前酶基生物煉制模式下木質(zhì)纖維素基質(zhì)高效水解中。
[Abstract]:The deficiency of endonuclease Cel5A is the key factor to limit the efficient enzymatic hydrolysis of natural cellulose. In this paper, we try to construct a recombinant Pichia pastoris strain expressing Trichoderma Cel5A efficiently to make up for the deficiency of natural secretion of Cel5A, optimize the Cel5A gene of Trichoderma rimannii and construct the expression vector p PIC9K-eg2, through gene codon preference. The recombinant was constructed by electroporation of Pichia pastoris GS115. A high yield Pichia pastoris strain GS115-EG 鈪，

本文編號：2321024