Saccharomyces cerevisiae에서 Thermomonospora fusca Exoglucanase의 발현 및 Cellulose 분해에의 응용
Expression of Thermomonospora fusca Exoglucanase in Saccharomyces cerevisiae and Its Application to Cellulose Hydrolysis
0
조회
0
다운로드
2
인용
0
참고문헌
초록
효과적이고 강력한 효모 생균제를 개발하기 위해,Saccharomyces cerevisiae 균주들 에서 섬유소 분해효소를유전공학적 방법으로 생산하였다. Thermomonospora fusca유래의 exoglucanase 유전자(E3)를 구성적 ADH1 promoter하류에 subcloning하여 구성적 발현계인 plasmid pVTTExo(8.8 kb)를 제작하였고, 이를 S. cerevisiae 숙주세포SEY2102에 형질전환 시켜, YPD에서 배양한 결과, 총 생산된 exoglucanase의 avicelase 활성은 190 unit/l에 도달하였고, 분비효율은 50%, plasmid 안정성은 91%로 나타났다. 재조합 exoglucanase의 양은 Clostridium endoglucanase(CelA)와 Trichoderma endoglucanase(C4) 보다 더 높은 avicel 결합능을 나타냈다. 각 혼합비에 대한 avicel 분해에 대한 상승효과와 당 생성은, E3와 CelA의 혼합비가 4:1일 때 가장좋은 포도당 생성이 관찰되었으며, endoglucanase(CelA)와exoglucanase(E3)의 혼합물은, 단독으로 처리했을 때보다, 포도당의 생산은 2.5배 향상되었고, avicelase 활성은 결과적으로 3.2배 증가하였다.
Abstract
To develop effective and powerful probiotic, Saccharomyces cerevisiae strains producing cellulolytic enzymes were genetically breeded. For the production of exoglucanase, the plasmid pVT-TExo (8.8 kb) was constructed, in which Thermomonospora fusca exoglucanase gene (E3) was under the control of ADH1 promoter, and introduced into S. cerevisiae SEY2102. When the transformant, S. cerevisiae SEY2102/pVT-TExo, was cultivated on YPD medium, the total expression level of avicelase reached about 190 unit/l. The secretion efficiency and plasmid stability were about 50% and 91%, respectively. Recombination exoglucanase enzyme bound to avicel better than Clostridium endoglucanase (CelA) and Trichoderma endoglucanase (C4) enzymes. The mixing ratio of E3 and CelA displaying the best synergistic hydrolysis for avicel was observed at 4:1. The mixture of endoglucanase (CelA) and exoglucanase (E3) resulted in 3.2-fold increase of avicelase activity and 2.5-fold enhanced production of sugar production from avicel, compared to the single enzyme treatment.
참고문헌 (24)
- "Cloningand amplification of the gene encoding an extracellular β-glucosidase from Trichoderma reesei evidence for improvedrates of saccharification of cellulosic substrates", pp.562~567 (1991)
- "Production ofcellulase on sugar cane bagasse by fungal mixed culturesolid substrate fermentation", 19, pp.665~667 (1997)
- "Secretion of anenzymatically active Trichoderma harzianum endochitinaseby Saccharomyces cerevisiae", pp.404~409 (1996)
- "Production of Trichoderma cellulase inlaboratory and pilot plant", pp.67~76 (1991)
- "Synergistic action of exo-type cellulase in thehydrolysis of cellulase with different crystallinities", pp.300~306 (1997)
- "Activity studies of eight purified cellulase", pp.1002~1013 (1993)
- "Transformation of intact yeast cells treated with alkalications", pp.163~168 (1983)
- "Adsorptionbehaviours of two cellobiohydrolase and thier core proteinsfrom Trichoderma reesei on avicel PH101", 19, pp.893~897 (1997)
- "Product inhibition of therecombinant CelS an exoglucanae component of theClostrdium thermocellum cellulosome", 44, pp.399~404 (1995)
- "Regulation of geneexpression in industrial fungi", 60, pp.515~522 (2003)
- "Structuralcharacterization of N-linked oligosaccharides fromcellobiohydrolase I secreted by the filamentous fungusTrichoderma reesei RUTC 30", pp.617~625 (1997)
- "Hydrolysis of microcrystalline cellulose by cellobiohydrolaeI and endoglucanase II from Trichoderma reesei and synergism of the enzyme", pp.621~634 (1998)
- "Measurement of carboxymethyl cellulase activity", pp.127~132 (1960)
- "Constitutive expression of Clostridium thermocellumendoglucanase gene in Saccharomyces cerevisiae", 12, pp.430~437 (1997)
- "Secretion andlocalization of invertase and inulinase in recombinantSaccharomyces cerevisiae", pp.1049~1504 (1993)
- "Expression of two Trichoderma reeseiendoglucanase in yeast Saccharomyces cerevisiae", pp.175~185 (1987), Lehtovaara
- "Expression and secretion of Trichoderma endoglucanase inSaccharomyces cerevisiae", 26, pp.406~412 (1998)
- "Secretion of abacterial cellulase by yeast EXPRESSION OF EXOGLUCANASE IN YEAST AND CELLULOSE HYDROLYSIS 273", pp.958~230 960 (1985)
- "1998 Optimum condition ofcellulose hydrolysis reaction with mixed enzymes ofcellulase and β-glucosidase", 13, pp.20~25
- "Adsorption of Thermomonospora fusca E", suvajitta(nt), pp.12~18 (2001)
- "Homologous domains in Trichodermareesei cellulolytic enzymes", pp.43~52 (1983), Lehtovaara gene sequence and expression ofcellobiohydrolase II
- "Directed evolution for engineeringpH profile of endoglucanase III from Trichoderma reesei", 22, pp.89~94 (2005)
- "Wood hydrolysis by Cellulomonas fimi endoglucanaseand exoglucanase coexpressed as secreted enzymes inSaccharomyces cerevisiae", 6, pp.713~719 (1988)
- "purification and properties of someendoglucanase components with special reference to theiraction on cellulose when action along in synergism with thecellobiohydrolase", 171, pp.61~72 (1978)