醣类吸附模组数量对纤维水解酵素特性分析.DOC

醣類吸附模組數量對纖維水解酵素特性分析 Effects of numbers of carbohydrate-binding modules on cellulases from Orpinomyces sp. Y102 研究生：王浩宇　Wang, Hao-Yu 　指導教授：陳又嘉　Chen, Yo-Chia 【摘要】 纖維素水解酵素可透過彼此之間的協同作用，將不具溶解性的纖維素水解成單糖，而使纖維素可被生物所利用。纖維素水解酵素根據其特性以及截切纖維素方式的不同可以分為外切性纖維素水解酵素、內切性纖維素水解酵素以及β-葡萄糖苷酶三大類。外切性纖維水解酵素(cellobiohydrase; CBH)，結構上往往包含催化區域 (catalytic domains; CD) 及非催化區域的醣類吸附模組 (carbohydrate-binding modules; CBM)。本實驗從瘤胃真菌Orpinomyces sp.中篩選出兩段纖維水解酵素基因celC-1CBM與celC-2CBM，其胺基酸序列最大的差別在於帶有不同數目之CBM。為了探討CBM的數量對於纖維水解酵素之催化活性和纖維水解酵素與纖維素結合之程度的影響力，將celC-2CBM與celC-1CBM基因序列加以插入或截短CBM序列，建構出帶有不同CBM數目的纖維素水解酵素。將此四段基因轉殖到大腸桿菌表現系統，並加以進行誘導、純化及特性分析。結果顯示出在最適pH 5和最適溫度40℃左右的環境時，帶有不同CBM數量之纖維素水解酵素對於羧甲基纖維素 (CMC)的水解活性較不具有CBM之纖維素水解酵素來的好。而CBM數目的增加可增強纖維素水解酵素與芹菜葉肉(厚角)細胞結合之程度。然而CBM的存在對於纖維素水解酵素的活性才有明顯的影響。由於CBM的數目越多相對的蛋白質結構越不穩定，並且在CBM的區域有斷掉的趨勢，因此如何提高附帶CBM蛋白的穩定性是值得被討論的重要課題。 關鍵字：carbohydrate-binding modules、catalytic domains、glycoside hydrolases、Orpinomyces sp. Y102 【Abstract】 Non-soluble cellulose can be hydrolyzed into simple sugars through synergistic effects of cellulases, and utilized by organisms. Cellulose degrading enzyme can be divided into three categories, including exo-1-4-β-D-glucanase, endo-1-4-β-D-glucanase and β-glucosidase according to their characteristics and different modes of action. Exo-1-4-β-D-glucanase (cellobiohydrase; CBH) generally are complex protein consisting of catalytic domaim (CD) and non-catalytic domain like carbohydrate-binding modules (CBMs). In this study, celC-1CBM and celC-2CBM were obtained from a rumen fungus Orpinomyces sp. Y102, the main difference between deduced amino acid sequences of celC-1CBM and celC-2CBM is the number of CBM. In order to examine the effects of CBM numbers on properities of hydrolytic and binding abilities to cellulose, CBM sequences was respectively added and deleted from celC-2CBM and celC-1CBM, and constructs of cellulase genes with different numbers of CBM were obtained. These four genes were cloned into pET21a and recombinantly expressed in E. coli BL21 (DE3).