纖維素酶基因工程菌構建及水稻秸稈原位糖化發(fā)酵產(chǎn)乙醇
發(fā)布時間：2019-01-08 17:35
【摘要】：由纖維素和木聚糖等構成的木質纖維素是地球上存量最大的可再生資源,木質纖維素糖化及生物乙醇的開發(fā)對于降低對化石能源依賴,保護環(huán)境具有重大意義。提高纖維素酶活性以及乙醇發(fā)酵工藝改進仍然是第二代生物乙醇的主要研究內容。本研究將具有β-內切葡聚糖酶、β-外切葡聚糖酶和木聚糖酶活性的福壽螺纖維素酶基因sestc,通過遺傳手段分別整合入黑曲霉和釀酒酵母細胞中,研究sestc基因在細胞中表達及特性,采用同步糖化發(fā)酵技術研究纖維素原位糖化和乙醇生產(chǎn),主要結果如下:(1)黑曲霉基因工程菌的構建及纖維素酶研究。通過限制性內切酶酶切和T4 DNA連接酶連接的方法構建整合型真核表達載體gpF-sestc,此表達載體包含gpd-Fvs啟動子和潮霉素B抗性篩選標記hpt,通過原生質體和PEG/CaCl2介導的方法將目的基因整合進入黑曲霉中;經(jīng)過PCR篩選、透明圈篩選和SDS-PAGE檢測,篩選出產(chǎn)酶活最高的基因工程菌A.niger No.17,經(jīng)搖瓶液態(tài)發(fā)酵,A.niger No.17產(chǎn)纖維素酶的濾紙酶活、內切葡聚糖酶活、外切葡聚糖酶活和木聚糖酶活分別達到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;比較不同碳源對酶活的影響,產(chǎn)酶效率最高的為經(jīng)堿性氧化預處理的水稻秸稈,發(fā)酵4 d后產(chǎn)FPA達到1.476±0.021 U/mL。(2)釀酒酵母基因工程菌的構建及纖維素酶表達。分別將含有gpd-Fvs和gpd-Shi兩種啟動子的表達載體,通過原生質體法轉化入釀酒酵母基因組中,通過PCR篩選、RT-PCR和透明圈篩選獲得產(chǎn)纖維素酶活最高的工程菌株S.cerevisiae No.14,Fvs-gpd啟動子驅動表達效果高于Shi-gpd啟動子;以麩皮為底物搖瓶發(fā)酵時,轉化子No.14發(fā)酵48 h后的總纖維素酶活(FPA)達到最高,為1.1 U/mL,比野生菌提高27.5倍;No.14分泌β-內切葡聚糖酶活、β-外切葡聚糖酶活和木聚糖酶活分別達到378 U/mL、1.44 U/mL和164 U/mL;No.14纖維素酶粗酶液酶學性質研究表明,最適反應溫度和pH分別為50℃和pH 5,不同的金屬離子及其濃度對于酶活有不同程度的抑制或激活作用。(3)黑曲霉和釀酒酵母基因工程菌協(xié)同生產(chǎn)乙醇。A.niger No.17固態(tài)發(fā)酵水稻秸稈,工程菌降解底物能力高于野生菌,經(jīng)預處理過的水稻秸稈發(fā)酵產(chǎn)纖維素酶活力較高且更容易被降解;將工程菌A.niger No.17和S.cerevisiae No.14結合,通過原位產(chǎn)酶及同步糖化發(fā)酵工藝,乙醇產(chǎn)量為9.71 g/100g水稻秸稈,比野生菌提高16.8%。整合sestc基因的黑曲霉和釀酒酵母基因工程菌整體纖維素酶性能得到提高,協(xié)同依次發(fā)酵有利于實現(xiàn)木質纖維素底物水解和發(fā)酵的同步,提高乙醇產(chǎn)率,簡化纖維素乙醇生產(chǎn)工藝。本研究從纖維素酶基因工程菌的結合以及協(xié)同依次發(fā)酵方面為第二代生物乙醇(纖維素乙醇)開發(fā)可行性提供參考,同時,也為農產(chǎn)品加工副產(chǎn)物生物質原料的應用提供一個開發(fā)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 鹵0.051 U / mL and 16.225 鹵0.91 U / mL respectively, 17.727 鹵0.851 U / mL, 30.165 鹵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 鹵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，
本文編號：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 鹵0.051 U / mL and 16.225 鹵0.91 U / mL respectively, 17.727 鹵0.851 U / mL, 30.165 鹵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 鹵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，
本文編號：2404917
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