酿酒酵母细胞表面展示米根霉脂肪酶制备全细胞催化剂及其特性研究
发布时间：2018-10-21 16:40
【摘要】：生物柴油是一种清洁可替代绿色能源,己引起人们极大的关注。脂肪酶催化制备生物柴油,具有反应条件温和、无污染等优势,是生物柴油技术的发展方向。随着酶固定化方法的日臻完善,脂肪酶催化应用领域越来越广泛。脂肪酶表面展示技术集分离、纯化和固定化于一体,不仅可以提高脂肪酶的稳定性,而且还能降低酶固定化操作成本。为此,我们采用a凝集素Aga2作为锚定蛋白,以酿酒酵母为宿主菌,表面展示米根霉脂肪酶(ROL),构建全细胞催化剂,研究了全细胞催化剂的酶学特性,初步探讨了全细胞催化剂的发酵成本,以及在生物柴油中的应用。主要研究成果如下：1.借助生物信息学方法,确定了锚定蛋白、宿主菌和目的脂肪酶蛋白表面展示单元的匹配规律,为表面展示全细胞催化剂的构建奠定了理论基础。分析4种不同种类的脂肪酶和锚定蛋白结构信息,其中ROL为目标展示酶时,结构中具有分布较均匀的疏水性和柔性；催化三联体由S242,D301,H354组成,阴离子氧洞为T213,L267,活性中心离C端较近。因此,选择N端锚定的a凝集素Aga2-Agal比较合适。2.以a凝集素Aga2-Agal为锚定蛋白,成功地将ROL展示在酿酒细胞表面,获得了具有水解橄榄油活性的全细胞催化剂。该全细胞催化剂水解橄榄油活力为78.9 U·g-1,高于相关文献报道。同时,研究了ROL全细胞催化剂的酶学特性,最适温度为40 C,最适pH为7.5,具有良好的稳定性和有机溶剂耐受性。3.初步探讨了ROL全细胞催化剂发酵生产成本和催化生物柴油应用。结果表明,2L发酵菌丝体生物量大于3g,粗略计算,发酵成本为68.9元g-1。海藻酸钠固定化ROL全细胞催化剂催化制备生物柴油得率为81.2%,重复利用7批次后的转酯率仍保持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%.
【学位授予单位】：北京化工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TE667;Q814
本文编号：2285718
[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%.
【学位授予单位】：北京化工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TE667;Q814
【参考文献】
相关期刊论文 前1条
1 苏国栋;张少平;尹钰;林影;;毕赤酵母展示表达南极假丝酵母脂肪酶B[J];生物技术通报;2012年08期
，本文编号：2285718
本文链接：https://www.wllwen.com/kejilunwen/shiyounenyuanlunwen/2285718.html
