以基因突變技術轉換地衣聚醣酶成為醣類合成□

Abstract

地衣聚醣酵素（即lichenase ; EC 3.2.1.73）屬於醣類水解酵素第16家族的水解酵素，可專一性的水解β-1,3-1,4-葡聚醣上的β-1,4糖□鍵，本研究旨在利用其催化能力，配合燕麥所萃取之葡聚醣，可以取得大量寡醣；並且針對其突變株酵素之轉醣基化能力加以探討。

首先利用基因工程的方法將地衣聚醣酵素(lichenase)的人造基因建構在大腸桿菌表現系統ECOS21中，以IPTG誘導大量表現酵素；經過離子交換樹脂之管柱層析後，可以取得均質度達95%的酵素，其單體分子量約為25 kDa左右。另外，從燕麥中大量取得高純度的β-葡聚醣作為酵素受質，可進行酵素的活性測試分析。實驗顯示地衣聚醣酵素在pH值7-8之間較穩定，活性也最佳，當pH值高於8與低於4時，酵素反應活性漸降;在pH值約高於9.5之後，反應活性則是低於50%。 此外，本酵素為高耐熱性酵素，可穩定存在55℃的環境，並且保持最佳活性，具有高度的工業應用價值，但是高過此溫度之後，其活性及穩定性則遽降。由燕麥中取得之高純度β-葡聚醣除了可進行酵素的活性測試分析外，同時亦可以與地衣聚醣酵素進行催化水解反應，取得大量的纖維三醣、纖維四醣產物；並且用以合成纖維三醣/四醣氟化物之醣□受質，作為突變株地衣聚醣酵素lichenaseE134A轉醣基化反應之研究。

本研究亦將野生株的地衣聚醣酵素基因作定點突變，所得到之突變株酵素lichenaseE134A可催化進行轉醣基化反應，利用野生株地衣聚醣酵素與燕麥β-葡聚醣反應所得之寡醣產物，經有機合成得到α-form葡聚醣氟化物，以此作為醣□受質，藉由突變株酵素lichenaseE134A的催化，可以單步驟inverting mechanism進行轉醣基化反應，可得到更多樣性的寡糖產物。

Lichenase ( EC 3.2.1.73) is an endo-glycosidase that can hydrolyse linear β-glucans including β-1,3 and β-1,4 and has a strict cleavage specificity for β-1,4 glycosidic bonds on 3-O-substituted glucosyl residues. The bacterial enzymes are retaining glycosyl hydrolases of family 16 with a jellyroll β-sandwich fold and a substrate binding cleft composed of six subsites.

An artificial gene of lichenase was cloned into pRSET_A plasmid and the protein was successfully expressed in E. coli ECOS21. After applying a series of ion-exchange purification steps, recombinant lichenase with 95% homogeneity could be obtained. The crude enzyme solution was purified by 3.2-fold with an apparent homogeneity and a recovery yield of 67.5%. The purified enzyme showed as a single protein band on SDS-PAGE with a molecular mass of 25 kDa. According to temperature-dependent and pH-dependent studies of the purified enzyme, the optimal temperature of lichenase was 55℃ and the the optimal pH value was about 7-8. The enzyme could hydrolyze barley β-D-glucan to yield tri-saccharide and tetra-saccharide as the main products. These products could also be made to 3-O-β-cellobiosyl-α-D-glucopyranosyl fluoride and 3-O-β-cellotriosyl-α-D-glucopyranosyl fluoride that could be used as a donor for β-glycosynthase reaction.

Besides, removal of the catalytic nucleophile Glu134 of the retaining lichenase by mutation to alanine yielded an enzyme with no glycosidase activity. The mutant was able to catalyze the glycosynthase reaction of α-form glycosyl fluoride with 4-methylumbelliferyl β-D-glucopyranoside as acceptors through a single-step inverting mechanism.