弧菌屬分離株所產纖維素分解酶之純化與特性探討

Abstract

本研究從新北市瑞芳的磅磅仔附近海水中分離出具有纖維素分解能力之細菌。經16S rRNA 定序後，建立分離株之親緣關係樹，結果顯示為弧菌屬 (Vibrio)。本研究對分離株所產生的纖維素分解酶進行分離、純化並探討該酵素的一般性質，以及評估工業上的應用。結果顯示：分離株使用250 mL具凹槽錐形瓶，添加0.5% CMC、pH 6、培養液50 mL，在30 °C 下，震盪培養72 小時後，纖維素分解酶的活性最佳。粗酵素液經由硫酸銨沉澱、透析、並以陰離子交換管柱層析 (DEAE-FF)分離出分子量約60 kDa、36 kDa 及 20 kDa三種纖維素分解酶。此纖維素分解酶在pH 6、50˚C時有最佳活性；在 pH 4~9 以及4~40 ˚C有良好的穩定性。本酵素受界面活性劑的影響，在1% SDS及 1% Tween 80 中，活性皆僅剩下約原本的25 % ；而1% 的 Tween 20使其活性降低約 55 %。另一方面，Ethylenediaminetetraacetic acid (EDTA) 和phenylmethanesulfonylfluride (PMSF) 等酵素抑制劑皆降低了本酵素活性 30 ~ 40 %，而2-Mercaptoethanol (β-ME) 幾乎不造成影響。在工業用途評估測試中，本酵素對微晶纖維素、石蓴、紙張和甘蔗渣皆具有分解能力，其中對微晶纖維素的分解能力最好，對石蓴和紙張次之，對甘蔗渣最差。

In this study, an isolate from sea water of Rueifang, Xinbei, had been tested for its cellulase activity. After the 16S rRNA was sequenced to establish phylogenetic tree of this isolate, which belonged to the genus Vibrio. Cellulase of this isolate was purified and characterized. Optimal cellulase yielding conditions for the culture were determined. For the best cellulase production, the culture was incubated at 30 °C with 50 mL LB broth (pH 6) in 250 mL flask and shaken for 72 hours (75 rpm). CMC (1%) was added as an cellulase inducer in LB broth. The cellulase was purified by ion exchange chromatography after ammonium sulfate precipitation, dialysis, and desalting. The molecular mass of the cellulases determined by SDS-PAGE was approximately 60 kDa, 36 kDa and 20 kDa. The cellulase showed optimum activity at pH 6 and 50 °C, and was stable at pH 4-9 and 4-40°C. When the cellulase was treated with various surfactants, 70 % activity was inhibited by 1% (g/mL) SDS and 1% Tween 80 , and had a lost of 55 percent under the effect of Tween 20. The cellulase was inactivated by 5 mM phenylmethanesulfonylfluride (PMSF) and 5 mM ethylenediaminetetraacetic acid (EDTA), but was not influenced by 2-Mercaptoethanol. Finally, in the industrial application tests, the cellulase showed better ability to decompose microcrystalline cellulose than to Ulva and paper, but showed weaker ability to break down sugar cane bagasse than to any others substrates.