酒香酵母外切-β-1,3-葡聚醣酶之生化特性與結構分析

Abstract

外切-β-1,3-葡聚醣酶 (exo-β-1,3-glucanase, EC 3.2.1.58) 參與酵母菌細胞壁之代謝，主要功能為水解 β-1,3-glucan 與 β-1,6-glucan ，可將多醣從非還原端分解為葡萄糖。先前研究發現酒香酵母 (Dekkera bruxellensis) 對於羅漢果皂苷之轉化與其他種類酵母菌相比有其特殊性，並且確認 D. bruxellensis EXG1基因所產生之 exo-β-1,3-glucanase (DbExg1) 能夠專一性的將羅漢果五醣皂苷 (mogroside V，MG V) 轉化為四醣皂苷 (siamensoide I，SI) 。為了對 DbExg1 有更深的瞭解，因此本研究以 DbExg1 為研究對象，對其進行生化特性分析與蛋白結構模擬。實驗使用 E. coli BL21 (DE3) 為宿主表現重組蛋白並經過純化來獲得 DbExg1 。在酵素特性的部分， DbExg1 在酸性環境下有較好的酵素活性，最適反應 pH 值為 6.0 。在溫度方面則是 30-50℃ 之間可維持 50% 以上的酵素活性，最適反應溫度為40℃ 。而熱穩定性試驗指出 DbExg1在 pH 5-6 有較高之 Tm 值 (melting temperature) ，說明 DbExg1 在酸性溶液中蛋白結構較穩定。此外，酵素動力學的結果顯示 DbExg1 對於 laminarin (β-1,3-glucan) 之催化效率遠高於 pustulan (β-1,6-glucan) ，符合本身酵素特性。對於 MG V 之催化效率則介於兩者之間。在結構分析的部分，為採用同源模擬法 (homology modelling) 建立 DbExg1 之三級結構，模擬結果顯示 DbExg1 為 (β/α)8-TIM-barrel 之構形，並且針對可能影響 MG V 轉化差異之胺基酸進行定點突變，雖然並未找到關鍵胺基酸，實驗卻意外發現突變位置 G325S 造成 DbExg1 酵素活性之下降。綜合研究結果，我們對於 DbExg1 之基本酵素特性有更多的了解，並透過模擬建立 DbExg1 立體結構，而突變蛋白 DbExg1 G325S 酵素活性之降低，則推論可能與結構空間障礙相關。

Exo-β-1,3-glucanase (EC 3.2.1.58) is involved in the metabolism of yeast cell wall. It can hydrolyze β-1,3-glucan and β-1,6-glucan by breaking down polysaccharides from non-reducing ends. Compared to other yeast species, previous studies have shown the unique property of Dekkera bruxellensis on mogroside bioconversion and confirmed that exo-β-1,3-glucanase produced by the EXG1 gene from D. bruxellensis (abbreviated as DbExg1) can specifically transform mogroside V (MG V) into siamensoide I (SI). To get a better understanding of DbExg1, analysis on biochemical characterization and protein structure modelling was therefore performed. E. coli BL21 (DE3) was used to express recombinant proteins. Regards to the enzyme characteristics, DbExg1 shows better enzyme activity in acidic environments. Its optimal pH is 6.0 and optimal temperature is 40°C. More than 50% enzyme activity of DbExg1 remains under 30-50°C treatment. In thermal shift assay, DbExg1 has a higher Tm value at pH 5-6, indicating that DbExg1 is more stable in acidic solution. In addition, the results of enzyme kinetics showes that the catalytic efficiency of DbExg1 for laminarin is much higher than for pustulan.The results agrees with the natural properties of the protein. Interestingly, the efficiency of DbExg1 for MG V is between those of laminarin and pustulan. In the structural analysis, the tertiary structure of DbExg1 built by homology modeling showed (β/α)8-TIM-barrel folding. Moreover, the residues which may make a difference in MG V conversion was mutated. We surprisingly observed the decrease in enzyme activity of DbExg1 G325S mutant. Taken together, the basic enzyme properties and modeling structure of DbExg1 were studied. DbExg1 G325S mutant has low enzyme activity which may be related to form a spatial barrier in structure and lead to the reduced biotransformation.