選殖自牛瘤胃真菌Orpinomyces sp. Y102的一株纖維素水解酶之表現與生化特性分析

Expression and Characterization of a Cellulase Cloned from a Bovine Rumen Fungus Orpinomyces sp.Y102

陳又嘉(Yo-Chia Chen) ； 顏書品(Shu-Pin Yen) ； 劉政成(Jeng-Chen Liu) ； 劉佳威(Chia-Wei Liu) ； 鄭雪玲(Hsueh-Ling Cheng)

《中華生質能源學會會誌》 31卷1&2期 (2012/06) Pp. 7-19

https://doi.org/10.6385/JBESC.201206.0007

摘要

纖維素水解酶催化纖維素中β-1,4 糖苷鍵的水解，可應用於相當廣泛的領域，包括生質能源開發。欲將纖維素完全水解需要至少三種纖維素水解酶的協同作用，即內切型纖維素水解酶(endo-β-1,4-glucanase, EC3.2.1.4)、外切型纖維素水解酶(cellobiohydrolase,EC3.2.I.91)以及β -葡萄糖苷酶（β-glucosidase, EC3.2.1.21) 。證據顯示，牛的瘤胃真菌是纖維素水解酶的豐富來源，但目前被發表者並不多。本研究的目的是分析一株選殖自台灣黃牛瘤胃真菌sp. Y102的纖維素水解酶CelC6，嘗試其大量表現於大腸桿菌的條件，並分析其催化活性。由其一級結構預測，CelC6可能是一株內切型纖維素水解酶，屬於醣類水解酶第9 家族。以6x-His為融合籤進行CelC6大腸桿菌中的表達，發現其表達量低，且為不可溶形式。再以maltose binding protein為融合籤進行大腸桿菌中的表達，則表達量明顯提高，且有可溶性重組蛋白出現。以amylose親合管柱純化出重組蛋白後分析其生化特性，發現其最佳反應條件為45 °C，pH 7.0 。耐熱範圍為50°C以下；酸鹼耐受範圍為pH5〜10。對soluble barley β-glucan的比活性為12.62 units/mg，對 carboxymethyl cellulose, xylan及pachyman則無活性。

關鍵字

瘤胃真菌 ； 纖維素水解酶 ； maltose binding protein ； cellulosome

並列摘要

Cellulase, which catalyzes the hydrolysis o f β-1, 4-glycosidic bonds in cellulose, can be applied in various fields, including bioenergy production. The complete hydrolysis of celluose needs at least three types o f cellulase, i.e. endo-β-1, 4-glucanase (endocellulase; EC3.2.1.4), ccllobiohydrolase (EC3.2.1.91), and β-glucosidase (EC3.2.1.21). Evidence suggested that bovine rumen fungi are a rich source for cellulase, whereas a limited number o f cellulases cloned from rumen fungi were reported. The aim o f this study was to characterize a cellulase previously cloned from a bovine rumen fungus Orpinomyces sp. Y102, named CelC6. Analysis o f the primary structure predicted CelC6 an endocellulase belonging to family 9 of the glycoside hydrolase superfamily. Using 6x-His as the fusion tag, the expression o f CelC6 in E. coli was low and insoluble. Instead, using maltose binding protein as the fusion tag, the recombinant protein became soluble, and the expression level was obviously elevated. The optimal reaction condition of the affinity-purified enzyme was found to be 45°C, pH 7.0. The enzyme was stable at temperatures ≦50℃, and in pH 5〜10. The recombinant enzyme displayed a specific activity of 12.62 units/mg to soluble barley β-glucan, but was inactive to carboxymethyl cellulose, xylan and pachyman.

並列關鍵字

Rumen fungi ； Cellulase ； Maltose binding protein ； Cellulosome