以大腸桿菌表現纖維素內切葡聚醣酶及酵素熱穩定性改良之研究

Abstract

用能源作物纖維素製造酒精是現階段最具潛力取代石化燃料之生質能源。現行纖維素產製酒精程序須依賴額外純化之纖維素水解酶(cellulase)分解纖維素成葡萄糖，再藉酒精發酵菌株發酵酒精。但酵素純化程序昂貴，因此大幅減少酵素純化成本為以纖維素製造酒精之發展重點。未來以纖維素製造酒精程序傾向同步水解及發酵程序(Consolidated Bioprocess, CBP)，CBP程序發展重點是將水解及發酵單元整合，並利用生物催化劑(biocatalyst)取代昂貴之酵素純化程序，大幅降低生產成本。內切葡聚醣酶(endoglucanase)是纖維素水解酶中重要成員，本研究利用菌體表面呈現系統在大腸桿菌表現Alicyclobacillus sp. 之內切葡聚醣酶CelA，並利用胺基酸序列排列比對方式改良酵素活性。Clostridium sp.之CelD與CelA同屬醣苷水解酶家族9 (Glycoside Hydrolase family 9, GH 9)，蛋白質序列相似度可達46.5%。先前研究顯示CelD之Asp-523突變為Ala後，活性提高為野生株之2.2倍，而在CelA可找到相對保留位置Asp-468，因此我們建構D468A突變株進行酵素活性改良。實驗結果顯示D468A之kcat/Km高於野生株1.2倍，且酵素半衰期在75℃提高為野生株約6倍時間。本研究進ㄧ步探討D468A熱穩定性改良因素，將Asp-468突變為三種不同化性之胺基酸，分別為Valine、Serine及Glutamate。結果顯示D468V半衰期在75℃較野生株高出約7倍時間，但D468S及D468E熱穩定性反而下降。因Alanine與Valine同為疏水性胺基酸，由此結果我們證明D468A熱穩定性提高可能是CelA之局部結構疏水性作用力增強所致。 由於纖維素為一大分子聚合物，無法直接進入菌體內利用，因此建構所需之生物催化作用需利用特殊菌體表面呈現系統將纖維素水解酶表現在菌體表面，以進ㄧ步將纖維素分解成可利用之小分子糖類。本實驗成功將~65kDa之CelA在大腸桿菌表現，測試全細胞活性(whole-cell activity)每毫升菌液最高可達約120mU/ml，同樣我們也將高熱穩定性D468A突變株進行表現，每毫升菌液活性也可達約118mU/ml，另外測試D468A在表面系統之酵素熱穩定性結果顯示，D468A仍維持較野生株高之熱穩定性。由以上結果證明我們能成功建構具有內切葡聚醣酶活性之大腸桿菌生物催化劑，並對於酵素熱穩定性改良提供更多資訊。

Cellulosic ethanol is one of the most potential biofuels in the world. However, current process of cellulosic ethanol production has to be improved. Purified cellulases for cellulose hydrolysis are high expenses and noneconomical. The future process for cellulosic ethanol is CBP (Consolidated BioProcess). The CBP process could reduce the costs because of the combination of “Hydrolysis” and “Fermentation” units which the biocatalysts displace the costly purified enzymes. Endoglucanase is an important component of cellulase. In this study, cell-surface display system was used to express the endoglucanase CelA from Alicyclobacillus sp. in Escherichia coli. Furthermore, the activity of CelA was improved by using the protein sequence alignment method. It shared 46.5% sequence similarity with CelD from Clostridium sp. that both enzymes belong to GH 9 (Glycoside Hydrolase family 9). In previous study, D523A mutant of CelD increases 2-fold of specific activity more than that of the wild type, and the Asp-523 in CelD has the same conserved site of Asp-468 in CelA. Therefore, the D468A mutant as a strategy was constructed to improve the enzyme activity. The results demonstrated that the kcat/Km of D468A had 1.2-fold more than that of the wild type. Interestingly, the half life of thermostability of D468A was 6-fold higher than that of wild type at 75oC. We further examined that the thermostability was affected by hydrophobicity. The Asp-468 was substituted for three different properties of amino acids which were valine, serine and glutamate. The results showed that the half life of D468V had 7-fold higher than that of wild type at 75oC. In contrast, the half life of D468S and D468E was decreased. These results suggested that the hydrophobic interaction of alanine and valine was important to increase thermostability in CelA. Because cellulose is a polymer that can not be taken into cell directly, in this study, we successfully displayed the CelA(~65kDa) on the surface of E. coli. The whole-cell activity had a maximum activity about 120mU/ml. D468A was also displayed and had a maximum activity about 118mU/ml. Moreover, the high thermostability was not be affected by using the surface-display system. These results led to the conclusions that we successfully constructed a biocatalyst with endoglucanase activity and provided more information on the thermostability improvement of the cellulosic enzymes.