"探討台灣本土嗜熱菌Meiothermus taiwanensis WR220中endo-β-1,4-glucanase之生化特性"

Abstract

在自然界中，細菌及真菌或是一些昆蟲的體內常含有纖維素水解酵素(例如：內切型纖維素分解酵素，endo-β-1,4-glucanase)，這些水解酵素可以幫助分解植物細胞壁，以提供生物體內的碳來源。在工業應用上，纖維素分解酵素可用於再生能源的生產或是用於造紙業，也常做為家畜的飼料或酒類發酵的添加物。經由對Meiothermus taiwanensis WR220的全基因體解序，我們發現一個很少存在於Thermus species，註解為內切型纖維素分解酵素的酵素(Mta-glucanase)。從sequence alignment的結果中發現了一段低保留度的未知區域(269-379)，其在已知的纖維素分解酵素中表現出極低同源性。因此，為了闡明這個未知區域的功能，我們利用基因轉殖技術做出了GlucanaseWT與GlucanaseΔ269-379這兩個重組蛋白，經由圓二色光譜儀(CD)的分析，我們發現雖然GlucanaseWT與GlucanaseΔ269-379有相似的二級結構，但GlucanaseWT中含有比GlucanaseΔ269-379略高的β-sheet比例；而經由熱穩定性實驗與活性實驗，我們發現GlucanaseWT不論在熱穩定性或是受質水解活性都比GlucanaseΔ269-379來的好。為了更加深入探討他們對β-1,4-glucan(例如：CMC)的酵素機制，我們比較了兩個蛋白的酵素動力學參數，根據兩個蛋白的Km與kcat，我們認為這段未知區域的主要功能可能和受質的結合力有關，藉由homology modeling結果，我們發現這段區域可能是可以與carbohydrate結合的lectin-like構型。經由恆溫滴定微卡計(VP-ITC)實驗結果，我們驗證了這段未知區域可能是與醣受質(例如：CMC)結合的區域。綜合以上的實驗結果，GlucanaseWT中的269-379這段未知區域不但可以增加蛋白的熱穩定性與受質水解活性，也可以增加蛋白與受質的結合力，這些特性都顯示Mta-Glucanase在日後工業應用上具有更好的發展潛力。

In nature, bacteria, fungi and insects often utilize some cellulases (e.g., endo-β-1,4-glucanase), to acquire their carbon sources from hydrolyzing plants’ cell walls. Industrially, these enzymes are often applied to renewable energy production or paper industry, and can also be used as additives for animal feeds and alcohol fermentation. From whole genome sequence of Meiothermus taiwanensis WR220, we found a protein annotated as endo-β-1,4-glucanase (Mta-Glucanase), which is less commonly observed in Thermus species. Interestingly, the result from sequence alignments, an uncommon region (269-379) was identified, sharing no homology to any other known glucanases. Therefore, in order to elucidate the function of that extra region, we constructed both wild type (GlucanaseWT) and deletion mutant (Glucanase269-379). Based on circular dichroism, although they shared similar CD spectrum, GlucanaseWT contained more percentage of beta-sheet than that of Glucanase269-379. Not only the thermal stability but also the activity of GlucanaseWT is better than those of the mutant. To gain more insight into the mechanism underlying β-1,4-glucan (e.g., CMC), we compared the basic enzyme kinetic parameters of them. According to their Km and kcat, we thought the function of that extra region seems to play a role in binding to the substrate. By homology modeling, we found that region might be similar to the domain in lectin-like proteins responsible for carbohydrate-binding. Together with the results from isothermal titration calorimetry (VP-ITC), we demonstrated the extra region as a sugar-binding domain essential for CMC binding. Taken together, the extra region of GlucanaseWT plays important roles in not only thermal stability but also the affinity to the substrate. These findings are of vital importance in future industrial applications.