Isolation of a Keratinase-Producing Bacterium and Purification of Its Keratinase

角蛋白酶之生產菌篩選、純化及其特性

殷儷容(Li-Jung Yin) ； 李京樺(Jein-Hwa Lee) ； 江善宗(Shann-Tzong Jiang)

《臺灣水產學會刊》 33卷4期 (2006/12) Pp. 377-390

https://doi.org/10.29822/JFST.200612.0008

摘要

由土壤中篩選可生產keratinase降解羽毛之Pseudomonas aeruginosa之菌種。樣品在羽毛培養基中於37℃、150 rpm下振盪培養54小時後，再取其培養液轉入另一羽毛培養基，重複三次後取其培養液在羽毛Agar上進行畫線分離，挑出可在羽毛Agar上產生透明環之菌落，進行培養及活性測定。再挑選出活性最高的Pseudomonas aeruginosa菌株放入羽毛培養基中於37℃、150 rpm下振盪培養54小時，收集胞外酵素液，經0.45 μm膜過濾後，測其蛋白酶活性約為141 unit。將粗酵素液經5 kDa超過濾濃縮後，進行sephacryl s-100 HR管柱層析可得到具有活性之角蛋白酶，其比活性為4068 units/mg，回收率為10.6%，純化倍率為6.79倍。經SDS-PAGE電泳分析確定純化之角蛋白酶分子量為39kDa。其最適pH為9，而在pH 5.5-9.0之間有較好的安定性。而最適溫度為60℃，在10℃-50℃之間有較好的溫度安定性。該角蛋白酶被金屬離子Hg(上标 +)、Cu(上标 2+)、Fe(上标 3+)和Ni(上标 2+)及蛋白酶抑制劑pCMB、IAA與NEM會抑制其活性，但在還原劑β-mercaptoethanol、dithiothreitol和glutathione影響下，能促進其活性，故推測此角蛋白為硫醇類蛋白質且其活化能為123.28 kcal/mole。

關鍵字

角蛋白酶 ； Pseudomonas aeruginosa ； 羽毛分解細菌 ； 角蛋白酶特性

並列摘要

The feather-degrading strain of Pseudomonas aeruginosa was isolated from soil under aerobic conditions at 37℃ using feather powder agar. After 54 h incubation at 37℃, the cells were removed by centrifugation and passing through a 0.45 μm membrane. The keratinase was purified to electrophoretic homogeneity after Sephacryl S-100 HR chromatography and up to 6.79 purification fold with 10.6% recovery. The MW of purified keratinase was 39 kDa, while the optimal pH and temperature were 9.0 and 60℃, respectively. It was stable at pH 5.5~9.0 and 10~50℃ and inhibited by Hg(superscript +), Cu(superscript 2+), Fe(superscript 3+) and Ni(superscript 2+). The purified keratinase was considered to be cysteine protease based on its sensitivity to pCMB, IAA and NEM, and strong activation by β-mercaptoethanol, dithiothreitol and glutathione.

並列關鍵字

Pseudomonas aeruginosa ； Feather-degrading bacteria ； Keratinase ； Characters