Purification and Characterization of Carboxymethyl Cellulase from Sinorhizobium Fredii

羧基甲基纖維素分解酶（carboxymethyl cellulose, CMCase）為根瘤菌與豆科植物根部進行共生固氮作用時，根瘤菌分解植物根部細胞壁之關鍵酵素。本研究利用羧基甲基纖維素（carboxymethyl cellulose）為基質，經目標酵素CMCase作用後，輔以BCA（2,2’-bicinchoninic acid）溶液反應3小時後，測定反應夜之呈色變化（波長570nm），以決定酵素活性。本研究以Sinorhizobium fredii CCRC 15769作為生產CMCase之菌株，為了能有效得到大量菌體以供純化CMCase之用，首先探討培養基對菌體生長之影嚮，以便能夠應用於醱酵槽之大規模培養。結果發現於BIII培養中添加0.3％肌醇（myo-inositol）作為碳源，可以獲得較佳之菌體產量及酵素比活性。經3.5公升醱酵槽大量培養後，連續離心可收集約22.4g濕重之菌體。菌體懸浮於PCA（potassium phosphate-citric acid）緩衝溶液中以超音波震盪法破菌取得粗酵素液，再以硫酸銨分劃收集飽和度區間40％－60％之沈澱物，置於Tris-HCI緩衝溶液中透析，離心後上清液通入DEAE Sepharose陰離子交換管柱層析，收集具活性之劃分，經透析除鹽後，再進行Phenyl Sepharose疏水性管柱層析。所得具活性之劃分經透析除鹽後，SDS-PAGE活性染色分析，確定具有CMCase活性，在94kDa處有一具CMCase活性之色帶。純化後之酵素經特性分析其最適反應溫度為35°C，最適反應Ph值為7.0，純化倍率約為9.08倍，回收率為26.4％，酵素比活性為3.822 U/mg。

關鍵字

羧基甲基纖維素分解酶；根瘤菌；蛋白抽出體

並列摘要

Carboxymethyl cellulase (CMCase) is the key enzyme used by bacteria to decompose the plant root hair-wall during symbiosis. In this study, CMCase activities were routinely assayed using carboxymethyl cellulose (CMC) as a substrate. Coupled with the catalytic reaction of BCA (2,2'-bicinchoninic acid) solution, the color developed at 570 nm was measured to determine the enzyme activity. Sinorhizobium fredii CCRC 15769 was utilized to produce CMCase for this study. The optimum growth conditions of the cultural medium were studied in order to obtain sufficient cells for further CMCase purification, and further, for large-scale fermentation production. BIII medium containing 0.3% myo-inositol proved to be a suitable carbon source for bacterial cell growth and specific activity of CMCase. A 22.4 g wet weight of cells can be harvested from 3.5 liters of fermented medium through continuous centrifugation. The cells were suspended in 50 mM potassium phosphate-citric acid (PCA) buffer at pH 5.2 and disintegrated through ultra-sonication to obtain crude extract. Then, certain precipitates were collected at 40-60% ammonium sulfate saturation via ammonium sulfate fractionation of the crude extract. The supernatant obtained following centrifugation was loaded onto a DEAE Sepharose anion-exchange column, and the active fractions were collected and dialyzed against 10 mM Tris-HCl buffered at pH 7.4. For further purification, the dialyzed fraction was loaded onto a Phenyl-Sepharose column. The active fractions were dialyzed and then assayed by SDS-PAGE activity stain to confirm that it contained CMCase activity at the fraction of 94 kDa. The characterizations of CMCase were demonstrated as follows: the optimal temperature and pH were 35°C and 7.0, respectively. The purification fold was 9.08, and the recovery yield was 26.4%, and the specific activity was 3.822 U mg-1.

並列關鍵字

Carboxymethyl cellulase ； Sinorhizobium fredii ； Protein extracts

國際替代計量

Purification and Characterization of Carboxymethyl Cellulase from Sinorhizobium Fredii

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