- 學位論文
以酵母菌表達瘤胃真菌的纖維分解酵素基因之研究
Expression of cellulase gene from rumen fungi by yeast
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摘要
木質纖維素是地球上最豐富的有機廢棄物之一，可以轉化為酒精等生物燃料，具有很高的經濟價值。在這個轉化過程中，酵素是一種有效且安全的方法，特別是纖維分解酵素。先前的研究從瘤胃真菌Orpinomyces sp. Y102中獲得了一個纖維分解酵素基因celC16，並利用大腸桿菌生產重組酵素。這些重組酵素可以將纖維素水解成葡萄糖，啤酒酵母則可以將葡萄糖發酵成酒精。本研究進一步探討了celC16的生化特性，並利用CBP製程構建能表現重組酵素的啤酒酵母。結果顯示，利用細胞表面呈現技術表達重組酵素的酵母轉型株具有較高的纖維分解酵素活性，能夠分解CMC、β-葡聚醣和前處理過的稻草並發酵產生酒精。然而，HPLC結果顯示，rcelC16對水解纖維二糖的能力較弱，因此從瘤胃真菌Neocallimastix patriciarum J11中選殖出一個名為JBG的β-葡萄醣苷酵素基因。rJBG酵素能水解4-nitrophenyl-β-D-glucopyranoside，對CMC也有水解活性。從文獻得知其重組酵素也可以直接將β-葡聚醣水解成葡萄糖。利用細胞表面呈現技術構建能表現JBG的啤酒酵母，結果顯示這些酵母轉型株能夠發酵β-葡聚醣產生酒精。最後，將celC16和JBG基因的酵母轉型株共同發酵β-葡聚醣，結果顯示共同發酵產生的酒精量達到4.53g/L，比單獨發酵高出249%以上。這種同時表現外切性纖維素水解酵素和β-葡聚醣水解酵素活性的基因，以及同時表現β-葡萄醣苷酵素和β-葡聚醣水解酵素活性的基因，使得利用酵素和酵母轉化木質纖維素為生質酒精的過程更加簡化和低成本。
並列摘要
Lignocellulose is one of the most abundant organic wastes on Earth and can be converted into biofuels such as ethanol, which has high economic value. In this conversion process, enzymes provide an efficient and safe method, particularly cellulolytic enzymes. Previous research obtained a cellulolytic enzyme gene, celC16, from the rumen fungus Orpinomyces sp. Y102 and produced recombinant enzymes by E. coli. These recombinant enzymes can hydrolyze cellulose into glucose, which can then be fermented into ethanol by Saccharomyces cerevisiae. This study further investigates the biochemical properties of celC16 and constructs S. cerevisiae expressing recombinant enzymes by CBP process. The results show that yeast transformants expressing recombinant enzymes using cell surface display technology have higher cellulolytic enzyme activity and can hydrolyze CMC, β-glucan, and pretreated straw to produce ethanol. However, HPLC results indicate that rcelC16 has a weaker ability to hydrolyze cellobiose. Therefore, a β-glucosidase gene named JBG was cloned from the rumen fungus Neocallimastix patriciarum J11. The rJBG enzyme can hydrolyze 4-nitrophenyl-β-D-glucopyranoside and also has hydrolytic activity on CMC. Literature indicates that its recombinant enzyme can also directly hydrolyze β-glucan into glucose. Using cell surface display technology, S. cerevisiae expressing JBG was constructed. The results show that these yeast transformants can ferment β-glucan to produce ethanol. Finally, co-fermentation of β-glucan using yeast transformants expressing celC16 and JBG genes resulted in ethanol production of 4.53 g/L, which is over 249% higher than single fermentation. This approach, which simultaneously expresses genes with exoglucanase and β-glucanase activities, as well as genes with β-glucosidase and β-glucanase activities, simplifies and reduces the cost of the typical process of converting lignocellulose to bioethanol using enzymes and yeast.
參考文獻
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