釀酒酵母與畢赤酵母重組木酮糖激酶的活性分析

Abstract

石油能源危機日益嚴重，積極發展再生能源尤為當務之急，其中生 質酒精之開發是相當重要的一環。酒精生產原料以纖維素、半纖維 素、等植物纖維為主，纖維素與半纖維素經過生物、物理或化學處理 後，分別降解成以葡萄糖為主的六碳糖與木糖(xylose)為主的五碳 糖，而參與木糖發酵途徑的主要酵素包括木糖醇脫氫酶（xylitol dehydrogenase ），木糖還原酶（xylose reductase）和木酮糖激酶 （xylulokinase）。本研究自釀酒酵母(Saccharomyces cerevisiae)和畢赤酵母(Pichia stipitis)以聚合脢連鎖反應分別增幅選殖木酮糖激脢基因，在大腸桿菌中大量表現並進一步純化此重組的木酮糖激酶，期以活性比較分析作為改殖的依據，最終使木酮糖激脢活性在酒精生產發酵過程中不因溫度升高或過酸環境而失活。 首先，我們根據生物資訊工具分析釀酒酵母木酮糖激脢(Sc-XK) 胺基酸的亂度變化，再以定點突變法將第88個胺基酸由絲氨酸(serine)轉變為纈氨酸（valine）。然而，Sc-XK和Sc-XK(S88V)在非原宿主中表現不具活性，我們推測此重組蛋白可能需要經過後修飾作用或修正活性測試的方法。另一方面，在畢赤酵母的木酮糖激脢(Ps-XK)選殖基因過程，我們意外得到一突變株在胺基酸序列中第55的位置由丙胺酸(alanine)突變為穌胺酸(threonine)。進一步比較Ps-XK和Ps-XK(A55T)重組蛋白代謝木酮糖的活性顯示Ps-XK (A55T)於25℃、pH 7.8的活性比Ps-XK提高三倍，有趣的是，此單一氨基酸的改變不僅使Ps-XK最佳活性的溫度升為42℃也影響了其最適反應之pH值。

The development of biomass ethanol as a renewable energy is currently a very important issue. The starting materials in the alcohol production process include cellulose and hemicelluloses from plant fibers. After the conversion via biodegradation, chemical or physical treatments, cellulose could be converted to six carbon sugars, mainly glucose, and hemicelluloses transformed to five-carbon sugars, mainly xylose. Xylitol dehydrogenase (XDH), xylose reductase (XR) and xylulokinase (XK) are three major enzymes involved in xylose fermentation pathway. In the study, we intend to clone the XK encoding gene using PCR from Saccharomyces cerevisiae and Pichia stipitis, and heterologously express the genes in Escherichia coli, and purify the recombinant proteins. After the activities comparatively analyzed, a recombinant XK that can tolerate high temperatures and acidic environments will be generated accordingly for practical use in the fermentation process. Based on the entropy analysis(http://sdse.life.nctu.edu.tw/index.cgi) of S. cerevisiae XK (Sc-XK), site-directed mutagenesis was firstly employed to generate a change from Ser to Val (S88V). However, Sc-XK and Sc-XK (S88V) expressed in E. coli appeared no enzymatic activity. If a post-transcriptional modification is required for Sc-XK activity remains to be investigated. On the other hand, a mutant with an amino acid change from Ala to Thr was obtained during the PCR-cloning of XK from P. stipitis (Ps-XK) and named Ps-XK (A55T). Interestingly, activity analysis revealed Ps-XK (A55T) had a 3-fold higher specific activity than Ps-XK at 25℃, pH 7.8. Moreover, single amino acid change of XK affected not only its suitable temperature, which increased to 42℃, but also caused the pH shift.