選殖自瘤胃真菌根囊鞭菌屬sp.Y102的纖維素水解酶A7的異源表達與分析

Heterologous expression and characterization of cellulase A7 cloned from a rumen fungus Orpinomyces sp. Y102

巫岱玲(Rahmah utami budiandari)

指導教授 ： 鄭雪玲

國立屏東科技大學/農學院/生物科技研究所/碩士(2015年)

https://doi.org/10.6346/NPUST.2015.00297

摘要

纖維素，D-葡萄糖的聚合物，是自然界中最豐富的碳源。纖維素需要至少三種類型的酶才能完全降解，即內切纖維素水解酶，外切型纖維素水解酶及β葡糖苷酶。瘤胃真菌已被證明高活性纖維素梅的來源。於前期研究中，基因celA7，由瘤胃真菌根囊鞭菌屬sp.Y102（NCBI編號KM114221）選殖出來，並預測可能是一株外切型纖維素水解酶。本研究的目的是嘗試使用麥芽糖結合蛋白質(MBP)作為融合籤以進行celA7的異源表現於大腸桿菌，並分析其生化特性。結果，MBP-celA7可在大腸桿菌BL21（DE3）中被大量表現，且只需要使用一個步驟，即DEAE-sepharose離子交換樹脂，即可純化出來。MBP-celA7的最佳反應條件為50℃和pH 7.0。MBP-celA7於pH具有專一性，5.0-8.0和25-50℃之間較為穩定。它對葡萄糖β-1,4鍵結的聚合物產物為纖維二糖，故證實為外切型纖維素水解酶。MBP-celA7的Vmax為780.6 Vmole/min/mg，Kcat為3.3 1170.9Sec-1，而Km為3.3 mg/ml。MBP-CelA7的活性明顯高於選殖自Trichoderma商業外切型纖維素梅，後者的比活性1.82Mmol/min/mg。

關鍵字

纖維素 ； 大腸桿菌 ； 瘤胃真菌 ； 外切型纖維素水解酶 ； 麥芽糖結合蛋白

並列摘要

Cellulose, a polymer of D-glucose, is the most abundant carbon source in the nature. The complete degradation of cellulose requieres at least three types of enzymes, that is endocellulase, exocellulase and β-glucosidase. The rumen fungi have been proved to be a source for higlhy active cellulases. A gene designated celA7, was cloned from the rumen fungus Orpinomyces sp.Y102 (NCBI number KM114221) and was predicted to encode an exocellulase. In this study, the heterologous expression of celA7 was tried using maltose-binding protein as a fusion tag, and the biochemical properties of the resulting fusion protein was characterized. Consequently, MBP-CelA7 was highly expressed in E.coli BL21(DE3). The recombinant protein was purified by one-step purification using DEAE-Sepharose column chomatography. The optimal reaction condition of MBP-CelA7 was 50°C and pH 7.0. MBP-CelA7 is stable between pH 5.0-8.0 and 25-50°C. It was specific to glucose β-1,4 polymers and was confirmed to be a cellobiohydrolase with cellobiose as the reaction product. MBP-celA7 displayed a Vmax of 780.6 µmole/min/mg with Km of 3.3 mg/ml and kcat of 1170.9 sec-1 to barley β-glucan. The activity of
MBP-CelA7 is obviously higher than the commercial enzyme cellobiohydrolase I cloned from Trichoderma reesei with a specificactivity 1.82 µmole/min/mg to soluble barley β-glucan

並列關鍵字

cellulose ； rumen fungi ； Orpinomyces sp.Y102 ； maltose-binding protein

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