Geobacillus uzenensis 嗜熱細菌之分離及其耐熱耐酸酯解酵素之研究

Characterization of a novel thermo- and acido- stable esterase activity from the isolated thermophilic Geobacillus uzenensis.

張莉芳(Li-Fang Chang)

指導教授 ： 李銘亮 博士

國立臺灣師範大學/理學院/生命科學研究所/碩士(2007年)

摘要

自陽明山國家公園溫泉中，分離出數株有能力水解三丁酸甘油酯（tributyrin）的細菌，取水解能力最佳者，作為研究之對象。經16S rDNA定序分析，與部分生理生化測試，將此菌暫定為Geobacillus uzenensis。
本研究利用細菌之外泌酵素粗萃取液，偵測此酯解酵素（esterase）之特性。在受質特異性（substrate specificity）上，相較於長鏈酯質（p-nitrophenyl myristate，pNPC14 、 p-nitrophenyl palmitate，pNPC16），此酵素偏好水解短鏈酯質（p-nitrophenyl acetate，pNPC2）。酵素反應之最適溫度為60~70℃，能在60℃下受熱2小時，保有完整活性；但在70℃下受熱15分鐘，則酵素活性盡失。在p-nitrophenyl acetate測試及酯解酵素活性染色結果中，此酵素能在pH 2?8下，具有活性作用，其最大活性表現在pH 3與6?7。當酵素液與pH 3?9緩衝溶液混合， 於4℃靜置24小時後，仍可保留100％活性。酵素對鉀離子（K+）的存在，有活性下降之情形發生；在化學試劑測試中，phenylmethylsulphonyl fluoride（PMSF）明顯抑制酵素活性，erhylenediamine tetraacetic acid（EDTA）、dithiothreitol（DTT）則不影響酵素活性；在清潔劑（detergent）測試中，Sodium dodecyl sulfate（SDS）、Triton X-100、Tween 20對酵素活性具抑制作用，Tween 80、CHAPS（3-[(3-cholamidopropyl)- dimethylammonio]
-1-propanesulfonate）則無影響。此酯解酵素對15%（v/v）乙醇（ethanol）、丙酮（acetone）等有機溶劑，其耐受度差，但在15%（v/v）DMSO（Dimethyl sulfoxide）存在下，仍有80%的活性。總之，本研究發現一耐熱、耐酸的酯解酵素，且此酵素能在酸性環境下（pH 2?3）有不錯活性，實為其他脂解酵素或酯解酵素少有的特性。

關鍵字

酯解酵素 ； 熱穩定性 ； 酸穩定性 ； 三丁酸甘油酯

並列摘要

Several bacteria isolated from a hot spring in Yang Ming Mountain, National Park of Taiwan have capability to degrade tributyrin. The bacterium selected for this study showed the biggest clear zone on the tributyrin agar among all samples. This thermophilic esterase producing bacterium was analyzed using 16S rDNA and partial physiological characterization; and provisionally classified as Geobacillus uzenensis. In this study, the extracellular crude preparation was used to determine the characterization of esterase activity. The substrate specificity of the enzyme showed a preference for p-nitrophenyl acetate as compared with p-nitrophenyl myristate and p-nitrophenyl palmitate. The optimum temperature for the esterase was 60~70 oC. The enzyme was retained its original activity at 60 oC for 2 h incubation period. However, enzyme activity was fully lost at 70 oC for 15 min incubation period. In addition, the p-nitrophenyl acetate assay and esterase activity staining (zymogram) showed the enzyme was active over a pH range from pH 2 to 8; with two maximal activities peaked at pH 3 and 6~7 respectively. After the crude enzyme extract was mixed with the buffer (pH 3~9) and incubated for 24h at 4 oC, it was stable with a ratio of 100% compared to its original activity. Among the various metal ions influence tests, esterase activity was significantly inhibited only by K+. The chemical reagent, PMSF could caused a significant inhibition in the enzyme activity, whereas the esterase was unaffected by EDTA and DTT. The activity of the enzyme was also inhibited by SDS, Triton X-100 and Tween 20. However, Tween 80 and CHAPS had no effect. Last, the enzyme was inhibited with 15% (v/v) concentration of ethanol and acetone and it retained 80% activity in presence of DMSO at the same concentration of 15% (v/v) . Briefly, the esterase was stable in the acidic pH region. These properties support the interest of further research on its production and characterization.

並列關鍵字

esterase ； thermostable ； acidostable ； Geobacillus gen. nov. ； p-nitrophenyl acetate

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