篩選大腸桿菌耐酒精菌株與表現外切纖維水解酵素

Abstract

摘要 生質酒精在經濟與環保方面是極具潛力之再生能源，目前普遍使用的基因工程酒精發酵菌株分別為Escherichia coli、Saccharomyces cerevisia、Zymomonas mobilis及Clostridum sp. 等均有相關研究。 E. coli KO11基因工程菌株可發酵五碳與六碳糖混合液為酒精，產率達理論值之90％，酒精耐受力約為35 g L-1，相較Saccharomyces cerevisiae能耐受120 g L-1酒精濃度，E. coli在提高酒精耐受力方面仍有努力空間。本研究進行菌株改良及篩選提高E coli KO11之酒精耐受力與生產穩定性之菌株。利用NTG(N-methyl-N-nitrosoguanidine)及UV.紫外燈照射進行突變篩選，並以2 L發酵槽系統進行篩菌培養，篩得可耐6 %酒精濃度之E82突變菌株，與KO11原始菌株比較除具有高耐酒精能力，其發酵葡萄糖(glucose)原始菌株相近，而發酵木糖(xylose)產率之能力較原始菌株高出39 %。 纖維素(Cellulose)水解醣化由內切纖維水解酵素(endo-β-glucanase)、外切纖維水解酵素(exo-β-glucanase)及纖維二醣水解酵素(β-glucosidase) 之協同作用後產生葡萄糖以利於生質酒精之發酵生產，因此高效率酵素活性為纖維素水解之主要關鍵。Cellulomonas fimi 所產生的Cex [exoglucanase(EC.2.3.91)]為雙功能(bifunction)蛋白可水解纖維素之非還原端產生纖維二醣，也具有木聚糖酶(xylanase)功能，可水解木聚糖(xylan)產木寡糖(oligoxylose)與木糖(xylose)。以pET系統於E. coli中成功表現Cex(cex-his-p23)並具有水解1號濾紙(no.1filter paper)、硝基苯纖維二糖苷(pNPC)、木聚糖及羧甲基纖維素(CMC)等活性。進一步利用Bacillus sp.之PgsA膜蛋白與Cex進行蛋白融合(Cex-his-pgsA)，成功呈現於E. coli 表面(surface display)，並具水解活性。結合E82耐酒精突變菌株與Cex-his-pgsA外切纖維水解酵素表面呈現系統，構築出具有表面纖維水解與高耐酒精能力之cex-his-pgsA/E82重組菌株，作為未來微生物直接轉化纖維素之程序(direct microbial conversion,DMC或consolidated bioprocressing,CBP)。

Abstract Bioethanol is a potential and renewable energy in the economy and the environmental protection. Escherichia coli, Saccharomyces cerevisia, Zymomonas mobilis and Clostridum sp. are generally used for genetic engineer of ethanol fermentation. In previous study, E. coli KO11 can metabolize hexose and pentose into ethanol which the yield is up to 90%. Nevertheless, ethanol tolerance in Saccharomyces cerevisiae is 120 g L-1 better than that of 35 g L-1 in E. coli. In this study, the E. coli mutant of ethanol tolerance was successfully screened by NTG (N-methyl-N-nitrosoguanidine) and UV light. The ethanol tolerance of mutant E82 was 6 % higher than wild type KO11. Moreover, the ethanol production in E82 was 39% more than KO11 by xylose fermentation while it was the same by glucose fermentation. Cellulose is hydrolyzed by the synergism of endo-β-glucanase, exo-β-glucanase and β-glucosidase for ethanol fermentation. Therefore, it is important to improve the enzyme activity. Cex from Cellulomonas fimi is a bifunctional protein which can hydrolyze non-reduced end of cellulose into cellobiose and degrade xylan into oligoxylose and xylose. Cex (cex-his-p23) was successfully expressed in E. coli by pET system. The protein contained the activities for hydrolysis of no.1filter paper, pNPC and CMC. Cex with a fusion of membrane protein PgsA from Bacillus sp. was also expressed on the surface display in E. coli. Furthermore, the direct microbial conversion or consolidated bioprocressing can be carried out by the cex-his-pgsA introduced into the mutant E82 of ethanol tolerance.