| 摘要: | α-半乳糖苷酶已用於食品工業、造紙工業、醫藥工程等產業上，但在許多工業應用上，α-半乳糖苷酶面臨著不同pH值與產品熱處理過程中的活性問題，進而影響到產品之製成率，因此高熱穩定性、化學穩定性之α-半乳糖苷酶是目前提高產品製成率的解決方法。
本研究利用選殖至大腸桿菌DH5α之嗜熱菌Thermus thermophilus HB8 melA基因，於培養後大量表現耐熱性α-半乳糖苷酶，純化後測試此酵素在不同緩衝液的最佳pH值，並測試不同濃度之金屬離子、Glycerol、BSA、KCl、SDS與儲存溫度對其活性之影響，不同溫度之酵素動力學常數。在對反應液條件的探討結果顯示，純化的 α-半乳糖苷酶在磷酸鹽緩衝溶液中，在pH 7.4 時有最佳之酵素活性；在10 mM 的金屬離子Zn2+與Cu2+存在下，會顯著抑制α-半乳糖苷酶的活性；於反應液中含有0.1%、0.5%、1% 的Glycerol 則有相對較高之活性，但高於 3% 的Glycerol時，α-半乳糖苷酶活性隨著濃度之增加而減少；於反應液中含有濃度 0.5% 至10% 的 BSA可增加酵素之活性，但含有濃度 0.1% 至 1% 的 KCl 對α-半乳糖苷酶活性都無明顯之影響；於反應液中添加 SDS 會使酵素活性劇烈下降；在酵素特性的探討結果顯示，其酵素活性於 25℃、70℃、80℃ 與 90℃ 下半衰期大約分別為607天、71小時、46小時與20小時。Vmax 值、KM 值與 kcat 值於溫度 25℃、70℃ 與 90℃ 分別為2.1、44.8與322.6 μmole/mg∙min；KM值為 0.17、1.71與 2.71 mM；kcat 值為 7.5、161.4與1161.3 sec-1。分析結果顯示出Thermus thermophilus HB8之α-半乳糖苷酶能於高溫下展現高穩定性的特性。
α-Galatosidase (α-GAL) has been applied in various industries, including food industry, paper-making industry and medical application. However, there are some problems concerning the enzyme activity needed to be solved, for example the enzyme need to perform its activity under the broad range of pH and at high temperature, or it may reduce the productivity in industrial applications. Therefore, it is attempted to find a good α-GAL which has the high stability at different temperature and pH to have high productivity.
In this study, the Thermus thermophilus HB8 melA gene was cloned into E. coli DH5α to overexpress T. thermophilus α-GAL. The purified α-GAL was used to examine the enzyme activity with different buffers, different metal ions, and different concentration of glycerol, BSA, KCl, and SDS. According to the results, α-GAL showed the highest activity in sodium phosphate buffer of pH 7.4, and the activity of α-GAL was found to be suppressed by 10 mM Zn2+ and Cu2+ ion in the assay reaction. The activity of α-galatosidase was found to be enhanced when the assay reaction was supplied with glycerol in the final concentration from 0.1% to 1%, but the activity was suppressed when supplied with glycerol in the final concentration of higher than 3%; while the activity was also enhanced when supplied with BSA in the final concentration from 0.5% to 10%. The activity of α-GAL was found to have no influence when the assay reaction supplied with KCl in the final concentration from 0.1% to 1%, but the activity of α-GAL was found to be reduced when the assay reaction containing SDS. The stability of enzyme was also invertigated. Under the optimal assay conditions, the purified enzyme was used to analyze kinetic parameters at different temperature. The half-life of enzyme was found to be 607 days, 71 hours, 46 hours, 20 hours at the storage temperature of 25℃, 70℃, 80℃, 90℃, respectively. The kinetic parameters, Vmax was determined to be 2.1、44.8 and 322.6 μmol/mg．min at 25℃、70℃ and 90℃, respectively; KM was 0.17、1.71 and 2.71 mM at 25℃、70℃ and 90℃, respectively; kcat was 7.5、161.4 and 1161.3 sec-1 at 25℃、70℃ and 90℃, respectively. In this study, the best condition for the activity of T. thermophilus HB8 α-GAL was determined with a good stability in high temperature. |
