菌種最適培養條件評估及發酵製程放大研究

Abstract

全球性的能源耗竭迫使人類不僅要節約能源，更要開發新的可持續性能源。然而，地球上產量最多的可利用再生能源-纖維素，仍未被充分使用。利用富含纖維素原料之農業廢棄物為生質酒精原料，不但不與糧食競爭且可協助解決環保問題。依目前生質酒精製程中, 除了一貫化生物轉化製程(Consolidated Bio-Process, CBP)外，皆需外加纖維素水解酵素，以分解纖維素至葡萄糖。因此，纖維素水解酵素於生質酒精製程中占相當重要的一個角色， 纖維素水解酵素活性的高低將影響製程中所需的用量，這對生質酒精生產的成本是關鍵之一;同時原物料的前處理過程中會產出某些化學物質，這些小分子的化學物質易與纖維素水解酵素結合，會抑制纖維素水解酵素的活性，本計畫將針對這些問題，結合微生物學、蛋白質化學、結構生物學及基因工程等等技術，以尋求對纖維素水解酵素活性及抑制物問題的根本解決之道;另外，如何提高纖維素水解酵素的產量亦是本計畫重點之一。計畫內容包含針對篩選之木黴菌生成之纖維素水解酵素，及具潛力之商業纖維素水解酵素進行酵素分離及純化，並建立酵素反應最佳條件；對這些纖維素水解酵素作蛋白質晶體繞射研究，以尋出這些關鍵酵素的結構特徵及其與酵素活性的關係，以及酵素與不同抑制物共結構關係；建立纖維素水解酵素的大量表現系統；最後再透過蛋白質工程的方法，改變某些纖維素水解酵素活性區域的關鍵胺基酸、提高受質與酵素結合的能力等方法，改變某些纖維素水解酵素活性區域的關鍵胺基酸、提高受質與酵素結合的能力。透過結合這些方法的研究，酵素的活性有可能提昇至少10倍以上，例如在Acidothermus cellulolyticus中的endocellulase Ce15A中只改變活性區域的一個氨基酸，其Ki值即提昇1,480%；對抑制物的耐受度也可同樣的大大提高，這對整個纖維酒精生產的成本降低，將有非常大的幫助。我们期望借由此一研究，於較短的時間內，能生產出高活性、高產量，且降低抑制物影響的纖維素水解酵素，運用於纖維酒精的生產。

Global energy shortage has forced us not only to decrease energy consumption but also to pursue renewable energy sources. However, the use of the most abundant bio-product, cellulose, as renewable energy has yet to be explored. Our research will use agricultural wastes that consist of high cellulose content as the source of bio-ethanol. This can avoid the competition with food which is a problem for current commercial bio-ethanol manufacture and help to deal with environmental problem for the treatment of agricultural wastes. All the current bio-ethanol producing processes, except consolidated bio-process (CBP), commercial cellulose degrading enzyme, cellulase, is required to degrade cellulose to glucose. Cellulase plays a very critical role in the bio-ethanol producing process. In the meantime, some kind of small molecules will be generated during the material pretreatment process and these small molecules could bound to cellulase and inhibit its activity. To deal with these problems, we integrated different techniques, i.e. microbiology, protein chemistry, structural biology and genetics engineering, in this proposal. This proposal includes the screening of suitable cellulase host (Trichoderma or Aspergillus species), separation and purification of cellulases and establishment of their optimal reaction conditions, resolution of celllulase crystal structure and understanding its structure-function relationship, establishment of mass production expression system of cellulase, and finally, protein engineering cellulase to produce novel enzymes. In some case, ten fold activity increments can be expected just for a single amino acid replaced in active domain or raise up the affinity between enzyme and substrate. It has been reported, the endocellulase Ce15A from Acidothermus cellulolyticus, the Ki value can be changed 1,480% when only single residue replaced. The tolerance of inhibition can be changed with the similar way. We expect to invent, in a short time, a new cellulose degrading enzyme with high cellulose-hydrolyzing activity and low affinity to cellulase inhibitors that can be used in bio-ethanol manufacture.