海洋耐冷細菌蛋白酶的特性探討及細菌分離株的鑑定

Characterization of protease from marine psychrotolerant bacteria and identification of the bacterial isolates

黃冠勳(Kuan Hsun Huang)

指導教授 ： 謝文陽

國立臺灣大學/理學院/海洋研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.02983

摘要

本研究從167株耐冷細菌分離株中篩檢出具蛋白酶活性的65株，其中屬於Vibrio 或 Pseudoalteromonas 屬的7株菌株活性較為顯著。進一步從這7株中選取活性最佳的菌株 D1 進行蛋白酶的純化及特性探討。經由膠體過濾層析管柱 (Sepharcyl S-200) 估算出分子量約為 168 kDa。此蛋白酶在 4-70℃下有活性，在 20-60℃ 活性較好，在 50℃時活性最好。最適 pH 值為 pH 7-8。會受到蛋白酶抑制劑 PMSF 及EDTA 所抑制。蛋白酶不受大部分的金屬離子影響而失去活性，但會受到 Hg2+ 所抑制，對界面活性劑也具有耐受性 (1-2% SDS, 0.5-2% Tween 20 及 0.5-2% Triton X-100)，經由以上的特性分析結果，可以得知分離株 D1 所產生的蛋白酶可適用於食品加工和清潔劑添加物。

關鍵字

耐冷細菌 ； 蛋白酶弧菌屬 ； 假交替單胞菌屬 ； 膠體過濾層析管柱 ； 蛋白酶抑制劑 ； 金屬離子 ； 界面活性劑

並列摘要

In this study, 167 isolates of psychrotolerant bacteria had been tested for their protease activity. 65 isolates were observed to possess positive protease activity, 7 isolates of these, with higher protease activity were identified as members of the genus Vibrio and Pseudoalteromonas. The bacterial isolate, strain D1, exhibiting the best protease activity was selected for protease purification and the purified protease was further used for characterization. Molecular weight of protease determined by gel filtration (Sephacryl S-200) was approximately 168 kDa. Protease was active over a wide temperature range of 4-70 ℃ and significant activity was observed between 20 to 60 ℃with maximum activity at 50 ℃. The optimal pH range for protease activity was pH 7-8. Enzyme activity was completely inhibited by phenylmethyl sulphonyl fluoride (PMSF) and ethylene diamine tetra acetic acid (EDTA). Apparantly, no remarkable effects were observed of metal ions on the protease activity, except Hg2+, which completely inactivated the protease. Protease was stable in presence of surfactants SDS (1-2 %), Tween 20 (0.5-2 %) and Triton (0.5-2 %). According to the results, protease obtained from strain D1 is suitable for food industry and detergent additives.

並列關鍵字

psychrotolerant bacteria ； protease ； Vibrio ； Pseudoalteromonas ； gel filtration ； protease inhibitor ； metal ions ； surfactant

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