木霉属纤维素酶基因在毕赤酵母表达及在统合工艺中应用

发布时间：2018-12-18 00:37

【摘要】：甘蔗是我国重要经济植物,将甘蔗渣转化为能源是缓解能源危机有效途径之一。本文建立以三种重组毕赤酵母为基础的统合生物工艺(consolidated bioprocessing,CBP)。选取里氏木霉eg3、绿色木霉cbh2和bgl1基因,分别与表达载体pGAPZαA连接,整合到毕赤酵母基因组,构建三株重组酵母。重组蛋白EG3、CBH2和BGL1酶活分别在72 h、96 h和168 h达到最高值0.82 U/mL、0.05 U/mL和5.91 U/mL,研究重组酶活最适温度、pH和金属离子影响。对甘蔗渣进行碱预处理优化,确定优化条件为碱浓度4%(w/v)NaOH、37℃、处理48 h、200目甘蔗渣、固液比1:10。优化前后碱处理产生总酚浓度由高于0.84 mg/mL降为0.47 mg/mL。用酸对碱预处理样品中和再生,用X射线衍射(X-ray diffraction,XRD)测算样品结晶度系数(crystallinity index,CrI),原材料CrI为51.59%,无机酸再生样品CrI均下降,其中硫酸再生CrI最低为39.12%。有机酸再生样品CrI反而升高。用扫描电镜(scanning electron microscope,SEM)观察,无机酸再生样品结构变得松散和杂乱无章,更有利纤维素酶接触和水解。对发酵体系进行优化,最适温度30℃,三种重组酵母接种比1:3:3(P.pastoris-eg3、:P.pastoris-cbh2:P.pastoris-bgl1),与等比例接种相比,均在发酵108 h乙醇产量达到最高为6.58 g/L和5.20 g/L,前者比后者乙醇产量提高26.53%。添加非离子表面活性剂PEG和Tween能提高酶解效率,其中PEG提高乙醇产量约5%。优化发酵体系乙醇产量达到理论值66.19%。
[Abstract]:Sugarcane is an important economic plant in China. Converting bagasse into energy is one of the effective ways to alleviate the energy crisis. An integrated biological process (consolidated bioprocessing,CBP) based on three recombinant Pichia pastoris was established. The cbh2 and bgl1 genes of Trichoderma eg3, were selected and ligated with the expression vector pGAPZ 伪 A, respectively, and integrated into the genome of Pichia pastoris to construct three recombinant yeast strains. The activity of recombinant protein EG3,CBH2 and BGL1 reached the highest values of 0.82 U / mL0. 05 U/mL and 5. 91 U / mL at 72 h, 96 h and 168 h, respectively. The optimum temperature of recombinant enzyme activity, the effect of pH and metal ions on the activity of recombinant protein were studied. The optimum conditions of alkali pretreatment for bagasse were determined as follows: alkali concentration was 4% (w / v) NaOH,37 鈩，

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