- 學位論文
瘤胃真菌纖維素水解酶C15於酵母菌的異源表現及其重組蛋白之特性分析
Heterologous expression and biochemical characterization of rumen fungus cellulase C15
摘要
由瘤胃微生物基因體中所選殖而來的纖維素水解酶經常具有高活性。團隊於前期研究中，由台灣黃牛的瘤胃真菌(Orpinomyces sp)中選殖出一個基因，經由序列比對及預測，推測可能是一個內切型纖維素水解酶(endoglucanase)，稱之為c15。本研究的目的是分析C15是否為一個內切型纖維素水解酶，並研究其催化活性及生化特性，以評估其應用於降解纖維素的潛力。方法是將c15表現於畢赤酵母(Pichia pastoris) SMD1168H，結果發現C15能成功大量表現於SMD1168H的胞外且純度大於85%。經過Ni-NTA 親和管柱純化後，分析其活性，發現可水解barley β-glucan達比活性(specific activity) 4762.05 units/mg。以薄層層析(TLC)分析其水解產物，發現可產生多種聚合度的寡糖(oligosaccharides)，證明C15為內切型纖維素水解酶，但其最小的基值(substrate)是纖維三糖。分析C15之反應條件，發現最佳反應溫度及pH值分別是50℃及pH 5.5 (citrate buffer)。分析C15的耐酸鹼性及耐熱性，發現C15可耐受pH 2.0 ~ pH 12.0，但55℃處理十分鐘以上即失去活性，所以C15有高酸鹼耐受性，但不是一株耐熱的酵素。C15於2%甲醇或乙醇存在下，可維持80%以上的活性；至甲醇或乙醇增加至7%，則活性降至50%以下。C15於0.5%SDS存在下活性為15.4 ± 2.1%；於1%或更高濃度之SDS中則完全失去活性。基質專一性分析顯示C15只催化glucose β-1-4鍵結的水解，對glucose β-1-3鍵結或α-1-4鍵結均無活性。對xylan也無活性。由酵素動力學的分析得知，C15的Vmax為0.88 ± 0.05 mM/min (相當於6603.64 ± 375.9 units/mg)，KM值為7.88 ± 0.75 mg/mL，kcat為6152.30 ± 343.00 s-1。以濾紙水解評估C15分解纖維素的能力，證實C15能明顯降解濾紙。綜合而言，C15是一株高活性的內切型纖維素水解酶，具有降解纖維素及寡糖的能力，可應用於纖維素分解的相關產業。
並列摘要
Cellulases cloned from rumen microorganisms were often demonstrated to possess high activities. Previously, a gene designated c15 was cloned from a bovine rumen fungus (Orpinomyces sp). Based on sequence analysis, it was speculated to be an endocellulase. Therefore, the purpose of this study is to characterize the biochemical properties of C15 and to evaluate its potential in degrading cellulose for industrial purposes. The open reading frame of c15 was transformed into Pichia pastoris SMD1168H. Consequently, C15 was successfully over-expressed by SMD1168H extracellularly. After purification with Ni-NTA affinity chromatography, the purified C15 was found to have a specific activity of 4762.05 units/mg using barley β-glucan as a substrate. The hydrolytic products of C15 were found to be oligosaccharides of different molecular weights when analyzed used thin-layer chromatography, suggesting that C15 is an endo-cellulase. The smallest substrate that can be digested by C15 cellotriose. Furthermore, the optimal reaction pH and temperature for C15 pH 5.5 (citrate buffer) and 50℃, respectively. C15 can tolerate pH 2.0 ~ pH 12.0, but lost activity at 55℃ or higher temperature for 10 min. Thus, C15 is not a thermophilic enzyme. C15 maintained over 80% activity in 2% methanol or ethanol, whereas in 7% methanol or ethanol, C15 maintained less than 50% activity. In 0.5% SDS, C15 remained 15.4 ± 2.1% activity, and completely lost its activity in 1% or higher concentrations of SDS. For substrate specificity, C15 could only catalyze the hydrolysis of glucose β-1-4 linkage, not glucose β-1-3 or α-1-4 linkage, and not xylan. Kinetic analysis revealed that the Vmax of C15 is 0.88 ± 0.05 mM/min ( 6603.64 ± 375.9 units/mg), KM is 7.88 ± 0.75 mg/mL, kcat is 6152.30 ± 343.00 s-1. Moreover, filter paper analysis demonstrated that C15 could hydrolyze filter paper, suggesting that it can hydrolysis cellulose. In summary, C15 is a highly active endo-cellulase that can hydrolysis cellulose and oligosaccharides. C15 may be applied in industries associated with cellulose degradation.
參考文獻
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