纤维素酶基因工程菌构建及水稻秸秆原位糖化发酵产乙醇
发布时间：2019-01-08 17:35
【摘要】：由纤维素和木聚糖等构成的木质纤维素是地球上存量最大的可再生资源,木质纤维素糖化及生物乙醇的开发对于降低对化石能源依赖,保护环境具有重大意义。提高纤维素酶活性以及乙醇发酵工艺改进仍然是第二代生物乙醇的主要研究内容。本研究将具有β-内切葡聚糖酶、β-外切葡聚糖酶和木聚糖酶活性的福寿螺纤维素酶基因sestc,通过遗传手段分别整合入黑曲霉和酿酒酵母细胞中,研究sestc基因在细胞中表达及特性,采用同步糖化发酵技术研究纤维素原位糖化和乙醇生产,主要结果如下:(1)黑曲霉基因工程菌的构建及纤维素酶研究。通过限制性内切酶酶切和T4 DNA连接酶连接的方法构建整合型真核表达载体gpF-sestc,此表达载体包含gpd-Fvs启动子和潮霉素B抗性筛选标记hpt,通过原生质体和PEG/CaCl2介导的方法将目的基因整合进入黑曲霉中;经过PCR筛选、透明圈筛选和SDS-PAGE检测,筛选出产酶活最高的基因工程菌A.niger No.17,经摇瓶液态发酵,A.niger No.17产纤维素酶的滤纸酶活、内切葡聚糖酶活、外切葡聚糖酶活和木聚糖酶活分别达到1.736±0.051 U/mL、16.225±0.91 U/mL、17.727±0.851 U/mL、30.165±0.54 U/mL,比野生型菌株提高了1.21、1.37、1.25和1.3倍;比较不同碳源对酶活的影响,产酶效率最高的为经碱性氧化预处理的水稻秸秆,发酵4 d后产FPA达到1.476±0.021 U/mL。(2)酿酒酵母基因工程菌的构建及纤维素酶表达。分别将含有gpd-Fvs和gpd-Shi两种启动子的表达载体,通过原生质体法转化入酿酒酵母基因组中,通过PCR筛选、RT-PCR和透明圈筛选获得产纤维素酶活最高的工程菌株S.cerevisiae No.14,Fvs-gpd启动子驱动表达效果高于Shi-gpd启动子;以麸皮为底物摇瓶发酵时,转化子No.14发酵48 h后的总纤维素酶活(FPA)达到最高,为1.1 U/mL,比野生菌提高27.5倍;No.14分泌β-内切葡聚糖酶活、β-外切葡聚糖酶活和木聚糖酶活分别达到378 U/mL、1.44 U/mL和164 U/mL;No.14纤维素酶粗酶液酶学性质研究表明,最适反应温度和pH分别为50℃和pH 5,不同的金属离子及其浓度对于酶活有不同程度的抑制或激活作用。(3)黑曲霉和酿酒酵母基因工程菌协同生产乙醇。A.niger No.17固态发酵水稻秸秆,工程菌降解底物能力高于野生菌,经预处理过的水稻秸秆发酵产纤维素酶活力较高且更容易被降解;将工程菌A.niger No.17和S.cerevisiae No.14结合,通过原位产酶及同步糖化发酵工艺,乙醇产量为9.71 g/100g水稻秸秆,比野生菌提高16.8%。整合sestc基因的黑曲霉和酿酒酵母基因工程菌整体纤维素酶性能得到提高,协同依次发酵有利于实现木质纤维素底物水解和发酵的同步,提高乙醇产率,简化纤维素乙醇生产工艺。本研究从纤维素酶基因工程菌的结合以及协同依次发酵方面为第二代生物乙醇(纤维素乙醇)开发可行性提供参考,同时,也为农产品加工副产物生物质原料的应用提供一个开发方向。
[Abstract]:Lignocellulose, composed of cellulose and xylan, is the largest renewable resource on earth. The saccharification of lignocellulose and the development of bioethanol are of great significance in reducing the dependence on fossil energy and protecting the environment. The improvement of cellulase activity and ethanol fermentation process is still the main research content of the second generation bioethanol. In this study, the cellulase gene sestc, which has the activities of 尾 -endoglucanase, 尾 -exoglucanase and xylanase, was integrated into Aspergillus Niger and Saccharomyces cerevisiae cells by genetic means, respectively. To study the expression and characteristics of sestc gene in cells, simultaneous saccharification and fermentation were used to study in situ saccharification of cellulose and ethanol production. The main results were as follows: (1) the construction of genetic engineering strain of Aspergillus Niger and the study of cellulase. Construction of an integrated eukaryotic expression vector gpF-sestc, by restriction endonuclease digestion and T4 DNA ligase ligation. The expression vector contains gpd-Fvs promoter and hygromycin B resistance screening marker hpt, The target gene was integrated into Aspergillus Niger by protoplast and PEG/CaCl2. After PCR screening, transparent loop screening and SDS-PAGE detection, the genetically engineered strain A.niger No.17, which produces the highest enzyme activity, was screened by liquid fermentation in shaking flask, and the cellulase-producing filter paper enzyme activity and endoglucanase activity of A.niger No.17 were obtained. The exoglucanase activity and xylanase activity were 1.736 卤0.051 U / mL and 16.225 卤0.91 U / mL respectively, 17.727 卤0.851 U / mL, 30.165 卤0.54 U / mL respectively, which were 1.21U 1.371.25,1.3 times higher than wild-type strains. Comparing the effects of different carbon sources on enzyme activity, the highest enzyme production efficiency was obtained from rice straw treated with alkaline oxidation, and the FPA production reached 1.476 卤0.021 UmL after fermentation for 4 days. (2) the construction of genetically engineered Saccharomyces cerevisiae and the expression of cellulase. The expression vectors containing gpd-Fvs and gpd-Shi promoters were transformed into Saccharomyces cerevisiae genome by protoplast method and screened by PCR. RT-PCR and transparent loop screening showed that the driving expression effect of S.cerevisiae No.14,Fvs-gpd promoter was higher than that of Shi-gpd promoter in the engineering strain with the highest cellulase-producing activity. When the wheat bran was used as the substrate, the total cellulase activity (FPA) reached the highest level after 48 h fermentation with No.14, which was 1.1U / mL, 27.5 times higher than that of wild bacteria. 尾 -endoglucanase activity was secreted by No.14, 尾 -exoglucanase activity and xylanase activity reached 378U / mL1.44 U/mL and 164U / mL, respectively. The study on the enzymatic properties of No.14 cellulase crude enzyme solution showed that the optimum reaction temperature and pH were 50 鈩，
本文编号：2404917
[Abstract]:Lignocellulose, composed of cellulose and xylan, is the largest renewable resource on earth. The saccharification of lignocellulose and the development of bioethanol are of great significance in reducing the dependence on fossil energy and protecting the environment. The improvement of cellulase activity and ethanol fermentation process is still the main research content of the second generation bioethanol. In this study, the cellulase gene sestc, which has the activities of 尾 -endoglucanase, 尾 -exoglucanase and xylanase, was integrated into Aspergillus Niger and Saccharomyces cerevisiae cells by genetic means, respectively. To study the expression and characteristics of sestc gene in cells, simultaneous saccharification and fermentation were used to study in situ saccharification of cellulose and ethanol production. The main results were as follows: (1) the construction of genetic engineering strain of Aspergillus Niger and the study of cellulase. Construction of an integrated eukaryotic expression vector gpF-sestc, by restriction endonuclease digestion and T4 DNA ligase ligation. The expression vector contains gpd-Fvs promoter and hygromycin B resistance screening marker hpt, The target gene was integrated into Aspergillus Niger by protoplast and PEG/CaCl2. After PCR screening, transparent loop screening and SDS-PAGE detection, the genetically engineered strain A.niger No.17, which produces the highest enzyme activity, was screened by liquid fermentation in shaking flask, and the cellulase-producing filter paper enzyme activity and endoglucanase activity of A.niger No.17 were obtained. The exoglucanase activity and xylanase activity were 1.736 卤0.051 U / mL and 16.225 卤0.91 U / mL respectively, 17.727 卤0.851 U / mL, 30.165 卤0.54 U / mL respectively, which were 1.21U 1.371.25,1.3 times higher than wild-type strains. Comparing the effects of different carbon sources on enzyme activity, the highest enzyme production efficiency was obtained from rice straw treated with alkaline oxidation, and the FPA production reached 1.476 卤0.021 UmL after fermentation for 4 days. (2) the construction of genetically engineered Saccharomyces cerevisiae and the expression of cellulase. The expression vectors containing gpd-Fvs and gpd-Shi promoters were transformed into Saccharomyces cerevisiae genome by protoplast method and screened by PCR. RT-PCR and transparent loop screening showed that the driving expression effect of S.cerevisiae No.14,Fvs-gpd promoter was higher than that of Shi-gpd promoter in the engineering strain with the highest cellulase-producing activity. When the wheat bran was used as the substrate, the total cellulase activity (FPA) reached the highest level after 48 h fermentation with No.14, which was 1.1U / mL, 27.5 times higher than that of wild bacteria. 尾 -endoglucanase activity was secreted by No.14, 尾 -exoglucanase activity and xylanase activity reached 378U / mL1.44 U/mL and 164U / mL, respectively. The study on the enzymatic properties of No.14 cellulase crude enzyme solution showed that the optimum reaction temperature and pH were 50 鈩，
本文编号：2404917
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