煙麴菌α-半乳糖苷酶的過量表現並利用基因工程改質為糖苷鍵合成酶

Abstract

摘要 本研究利用Picha Pastoris表現出一未確定功能的酵素，由序列預測是α-半乳糖苷酶，而後藉由活性測試證明其催化功能確實是水解α-半乳糖苷鍵，並且和β-甘露聚糖酶可共同作用在半纖維素的分解上。由於P.Pastoris 可大量表現高純度的胞外酵素，產量約為2 g/L，可不需純化就可測定蛋白質性質，並有利於工業用途。此蛋白質在pH 4~5的環境下有最高的催化活性，去除過度糖基化後可得知單體分子量約為47 kDa，和設計的基因符合。 檢測野生型酵素的各項特性後，為了進一步確認其反應必須胺基酸，並且嘗試將其改質為糖苷鍵合成酶，我們經由比對同功酵素的親核基位置後，決定將134D突變為A以及G。經過突變的酵素幾乎完全喪失其活性，可見134D在催化過程中扮演重要的角色，極有可能是親核基。突變後的酵素根據文獻報導有作為糖苷鍵合成酶的潛力，利用含氟半乳糖苷當作糖基的給予者，其他各種單糖及寡糖當作糖基的接受者，可以進行與水解反應相反的反應－糖苷鍵的合成。儘管在合成方面還有很多需要改進的地方，但是提供一個合成α-(1,6)鍵結的方式，未來有很大的應用空間。

ABSTRACT

This study is the first report to overexpression an unconfirmed enzyme, which is predicted being an α-galactosidase. Overexpress by P.Pastoris can produce a large amount of pure enzyme, up to 2g per liter; it’s profitable for industrial usage. This enzyme then assays by 4-nitrophenyl-galactopyranoside and prove that it’s an α-galactosidase. We mix α-galactosidase and β-endo-mannanase and galactomannan then observed the synergism between them . This recombination α-galactosidase is belongs to family 27, and is most active in pH4~5. After deglycosylation treatment can estimate the molecular weight by SDS-PAGE and this enzyme is about 47kDa. After finish protein characterization, we predict the nucleophile by multiple alignment with other well-known sequence. We found that 134D is a conserve amino acid with the nucleophile of other enzyme. When 134D is mutated to A or G, α-galactosidase almost lost all activity, we suppose that 134D should be one of the essential group of this enzyme. The mutant enzyme may alter it’s function to a glycosynthase, therefore we tried to use galactosyl fluoride as a donor and some other carbohydrate as an acceptor. Although there still many deficient in the method, we demonstrate a potential manner producing α-(1,6) linkage.