Geobacillus sp. 嗜熱細菌之分離及其耐熱酸酯酵素之研究

Abstract

本研究利用細菌之外泌酵素粗萃取液，測試其酯解酵素(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-nitrophenylpalmitate, 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℃，且能在60℃下受熱2小時，而保有完整活性；但在70℃下受熱15分鐘，酵素活性則盡失。在p-nitrophenyl acetate測試及酯解酵素活性染色結果中，此酵素能在pH2~8下，具有活性作用，其最大活性表現在pH3與6~7。當酵素液與pH3~9緩衝溶液混合，於4℃下靜置24小時後，仍可保留100%活性。在化學試劑測試中，phenylmethylsulphonyl fluoride(PMSF)明顯抑制酵素活性；在清潔劑(detergent)測試中，Sodium dodecyl sulfate(SDS)、Triton X-100、Tween20對酵素活性具抑制作用。此酯解酵素對15%(v/v)乙醇(ethanol)、丙酮(acetone)等有機溶劑，其耐受性差，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(pH2~3)有不錯活性，實為其他脂解酵素或酯解酵素少有的特性。

Several bacteria isolated from a hot spring in Yang Ming Mountain, National Park of Taiwan having the 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 l6S rDNA and partial physiological characterized; and classified as Geobacillus sp. 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 DC. The enzyme retained its original activity at 60°C for a 2 h incubation period. However, enzyme activity was fully lost at 70°C for a 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 mixed with the buffer (PH 3-9) and incubated for 24h at 4°C, it was stable with a ratio of 100% compared to its original activity. Among the various metal ions influence tests, esterase activities were not significantly affected. The chemical reagent, PMSF could cause a significant inhibition in the enzyme activity. The activity of the enzyme was also inhibited by SDS, Triton X-100 and Tween 20. Last, the enzyme was inhibited a 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 are interested and warrant for further researches on its production and molecular characterizations.