【摘要】：生物柴油是一種清潔可替代綠色能源,己引起人們極大的關注。脂肪酶催化制備生物柴油,具有反應條件溫和、無污染等優(yōu)勢,是生物柴油技術的發(fā)展方向。隨著酶固定化方法的日臻完善,脂肪酶催化應用領域越來越廣泛。脂肪酶表面展示技術集分離、純化和固定化于一體,不僅可以提高脂肪酶的穩(wěn)定性,而且還能降低酶固定化操作成本。為此,我們采用a凝集素Aga2作為錨定蛋白,以釀酒酵母為宿主菌,表面展示米根霉脂肪酶(ROL),構建全細胞催化劑,研究了全細胞催化劑的酶學特性,初步探討了全細胞催化劑的發(fā)酵成本,以及在生物柴油中的應用。主要研究成果如下：1.借助生物信息學方法,確定了錨定蛋白、宿主菌和目的脂肪酶蛋白表面展示單元的匹配規(guī)律,為表面展示全細胞催化劑的構建奠定了理論基礎。分析4種不同種類的脂肪酶和錨定蛋白結構信息,其中ROL為目標展示酶時,結構中具有分布較均勻的疏水性和柔性；催化三聯(lián)體由S242,D301,H354組成,陰離子氧洞為T213,L267,活性中心離C端較近。因此,選擇N端錨定的a凝集素Aga2-Agal比較合適。2.以a凝集素Aga2-Agal為錨定蛋白,成功地將ROL展示在釀酒細胞表面,獲得了具有水解橄欖油活性的全細胞催化劑。該全細胞催化劑水解橄欖油活力為78.9 U·g-1,高于相關文獻報道。同時,研究了ROL全細胞催化劑的酶學特性,最適溫度為40 C,最適pH為7.5,具有良好的穩(wěn)定性和有機溶劑耐受性。3.初步探討了ROL全細胞催化劑發(fā)酵生產成本和催化生物柴油應用。結果表明,2L發(fā)酵菌絲體生物量大于3g,粗略計算,發(fā)酵成本為68.9元g-1。海藻酸鈉固定化ROL全細胞催化劑催化制備生物柴油得率為81.2%,重復利用7批次后的轉酯率仍保持80%以上
[Abstract]:Biodiesel is a clean alternative to green energy, which has attracted great attention. The preparation of biodiesel catalyzed by lipase has the advantages of mild reaction conditions and no pollution. It is the development direction of biodiesel technology. With the improvement of enzyme immobilization methods, lipase catalytic applications are more and more extensive. Lipase surface display technology can not only improve the stability of lipase, but also reduce the cost of enzyme immobilization. Therefore, we used a lectin Aga2 as anchor protein and Saccharomyces cerevisiae as host strain to construct a whole cell catalyst with lipase (ROL), from Rhizopus oryzae on the surface. The enzymatic properties of the whole cell catalyst were studied. The fermentation cost of whole cell catalyst and its application in biodiesel were discussed. The main research results are as follows: 1. By means of bioinformatics, the matching rules of surface display units of anchoring proteins, host bacteria and target lipase proteins were determined, which laid a theoretical foundation for the construction of surface display whole-cell catalysts. The structure information of four kinds of lipases and anchored proteins were analyzed. When ROL was used as the target display enzyme, the hydrophobicity and flexibility of the structure were more evenly distributed, and the catalytic triad was composed of S242D301H354. The anion oxygen hole is T 213N L 267, and the active center is close to C terminal. Therefore, the N-terminal anchoring of a lectin Aga2-Agal is more suitable. 2. 2. Using a lectin Aga2-Agal as anchoring protein, ROL was successfully displayed on the surface of wine making cells, and a whole cell catalyst with hydrolytic olive oil activity was obtained. The activity of the whole cell catalyst for the hydrolysis of olive oil was 78.9 U g -1, which was higher than that reported in related literatures. At the same time, the enzymatic properties of ROL whole cell catalyst were studied. The optimum temperature was 40 C and the optimum pH was 7.5, which had good stability and organic solvent tolerance. The production cost of ROL whole cell catalyst and the application of catalytic biodiesel were discussed. The results showed that the biomass of 2L fermentation mycelium was more than 3g, and the fermentation cost was 68.9 yuan / g ~ (-1). The yield of biodiesel prepared by immobilized ROL catalyst catalyzed by sodium alginate was 81.2%, and the transesterification rate of 7 batches of re-use remained above 80%.
【學位授予單位】：北京化工大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：TE667;Q814

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相關期刊論文 前1條

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本文編號：2285718