【摘要】：甘蔗是我國重要經(jīng)濟植物,將甘蔗渣轉化為能源是緩解能源危機有效途徑之一。本文建立以三種重組畢赤酵母為基礎的統(tǒng)合生物工藝(consolidated bioprocessing,CBP)。選取里氏木霉eg3、綠色木霉cbh2和bgl1基因,分別與表達載體pGAPZαA連接,整合到畢赤酵母基因組,構建三株重組酵母。重組蛋白EG3、CBH2和BGL1酶活分別在72 h、96 h和168 h達到最高值0.82 U/mL、0.05 U/mL和5.91 U/mL,研究重組酶活最適溫度、pH和金屬離子影響。對甘蔗渣進行堿預處理優(yōu)化,確定優(yōu)化條件為堿濃度4%(w/v)NaOH、37℃、處理48 h、200目甘蔗渣、固液比1:10。優(yōu)化前后堿處理產(chǎn)生總酚濃度由高于0.84 mg/mL降為0.47 mg/mL。用酸對堿預處理樣品中和再生,用X射線衍射(X-ray diffraction,XRD)測算樣品結晶度系數(shù)(crystallinity index,CrI),原材料CrI為51.59%,無機酸再生樣品CrI均下降,其中硫酸再生CrI最低為39.12%。有機酸再生樣品CrI反而升高。用掃描電鏡(scanning electron microscope,SEM)觀察,無機酸再生樣品結構變得松散和雜亂無章,更有利纖維素酶接觸和水解。對發(fā)酵體系進行優(yōu)化,最適溫度30℃,三種重組酵母接種比1:3:3(P.pastoris-eg3、:P.pastoris-cbh2:P.pastoris-bgl1),與等比例接種相比,均在發(fā)酵108 h乙醇產(chǎn)量達到最高為6.58 g/L和5.20 g/L,前者比后者乙醇產(chǎn)量提高26.53%。添加非離子表面活性劑PEG和Tween能提高酶解效率,其中PEG提高乙醇產(chǎn)量約5%。優(yōu)化發(fā)酵體系乙醇產(chǎn)量達到理論值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 偽 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，

本文編號：2385020