利用大腸桿菌表現β-glucosidase/xylosidase及其特性研究

Abstract

纖維素水解酶分成三種型態，為exoglucanase、endoglucanase與β-glucosidase。從Dickeya dadantii中選殖出具有β-glucosidase與β-xylosidase的雙功能酵素(BgxA)，此酵素屬於Glycosy hydrolase family 3 (GH3)。β-glucosidase與β-xylosidase皆會作用於雙醣(cellobiose與xylobiose)的非還原端，產生單醣(glucose與xylose)。本研究發現，BgxA之β-glucosidase最高活性為678 mU/μg，其最佳反應條件為40 ℃及pH 5；而β-xylosidase最高活性為8.77 mU/μg，最佳反應條件為37℃及pH 5；顯示BgxA對於六碳雙醣活性大於五碳雙醣。利用E.coli大量表現蛋白，經0.1 mM IPTG誘導後，在25℃下培養3小時後，可得到最大量的BgxA蛋白量。金屬離子會影響BgxA活性方面，Co2+、Cu2+、Zn2+、Ni2+、Mn2+、Mg2+皆會影響β-glucosidase以及β-xylosidase活性，使兩者活性降低。利用序列比對及點突變方法確認BgxA的活性部位胺基酸為E203及D360，與基質結合部位胺基酸包含E144、R227、K267及H268。進一步根據Consensus sequence方法，選取同屬Dickeya中的β-glucosidase具差異性之胺基酸，突變成其他不同微生物物種中conserved且帶電性的胺基酸，可增加salt bridge與氫鍵來穩定整個蛋白質結構，實驗結果發現N456D、N487K、Q509R、H530E、N607E及A614K六個突變蛋白質的Tm值皆有所提升。

The cellulase contains the exoglucanase, endoglucanase and β-glucosidase. BgxA, encoding β-glucosidase and β-xylosidase, is a bifunctional enzyme from Dickeya dadantii and classified as a member of glycosy hydrolase family 3(GH3). Both β-glucosidase and β-xylosidase hydrolyze glycosidic bond and release non-reducing terminal glucosyl residues (glucose and xylose) from disaccharides (cellobiose and xylobiose), respectively. In this study, the characterization of BgxA was carried out. The β-glucosidase and β-xylosidase activity of BgxA were 678 mU/μg and 8.77mU/μg, respectively. The optimal temperature and pH of β-glucosidase were determined at 40℃and pH 5, while β-xylosidase was 37℃ and pH 5. BgxA protein can be largely obtained by addition of 0.1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) at 25℃ for 3 h. Based on the effect of metal ion on BgxA, the result indicated that Co2+, Cu2+, Zn2+, Ni2+, Mn2+ and Mg2+ inhibited the BgxA activity. According to the sequence alignment with GH3 family, we identified the catalytic site of BgxA which located on E203 and D360. The other binding residues including E144, R227, K267 and H268 was identified by site directed mutagenesis. In addition, the thermostable protein was obtained by consensus sequence aligment. The eleven residues of BgxA revealed as variable hot-spot residues in the Dickeya genus. The residuces were substituted for conserved and charged residus by comparing various β-glucosidases from different microorganisms. The result showed that the melting temperature of N456D, N487K, Q509R, H530E, N607E and A614K mutants were higher than wild-type BgxA. The results clearly demonstrated that consensus sequence can promote the melting temperature.