| Cellulose and hemicellulose are the most abundant polysaccharides in nature.Xylan and mannan are the major components of hemicellulose and their efficient degradation requires the synergy of various hemicellulases.Xylose as the major product from xylan degradation and can be further converted into value-added products such as ethanol.Mannose oligosaccharide(MOS)as the hydrolytic product of mannan is an important prebiotics.This study consists of three parts:first part comprised on the expression of the P-mannanase gene from Aspergillus sulphureus in Saccharomyces cerevisiae and applications of P-mannanase towards the production of MOS.The second part of study was attempted to elucidate the mechanism of a novel trifunctional enzyme Ttxy43 belongs to glycoside hydrolases(GH)43 and thirdly,construction of engineered S.cerevisiae strains responsible for the utilization of xylose.S.cerevisiae as a model eukaryote for studies and considered by the Food and Drug Administration(FDA)as safe(GRAS).For increased production of ?-mannanase,five different strains of S.cerevisiae were constructed by various strategies,including haploid or diploid,constitutive promoter or inducible promoter,?-mannanase gene in episomal vector or integrated into genome.P-mannanase expression were performed in both natural medium and synthetic medium.Haploid TIB with constitutive promoter was established through the integration of P-mannanase gene into the genome of S.cerevisiae.The production rate of P-mannanase in high-density culture was reached to 16 U/mL/day with the optimal pH and temperature of 3.2 and 60?,respectively.The locust bean gum(LBG)or konjac glucomannan(KGM)was used as substrate and Km were obtained as 24.13 mg/mL or 33 mg/mL respectively,while Vmax were recorded as 715 ?mol/min/mg or 625?mol/min/mg,respectively.The hydrolytic efficiency of KGM was obtained as 73.7%and liberated mannose,mannobiose,mannotriose,mannotetraose,mannoligosaccharides and mannohexaose in final hydrolysis product.A trifunctional enzyme Ttxy43 belongs to GH 43 was previously heterologously expressed in Pichia pastoris by Abdul Basit.Ttxy43 exhibits ?-xylosidase,a-L-arabinofuranosidase and endo-1,4-p-xylanase activity with the optimal pH of 6.0,7.0 and 7.0,respectively.The optimal temperature for the Ttxy43 were obtained as 50?,50?and 60 ?.To illustrate the catalytic mechanism of Ttxy43,five mutant strains D134A,E228A,E31A,E170A and E176A were constructed.Results showed that the trifunctional enzyme activity of D134A and E228 were completely abolished,and suggesting that 134Asp,228Glu are important catalytic sites for trifunctinal activities of Ttxy43.Moreover,?-xylosidase and ?-L-arabinofuranosidase activity of E176A decreased by 55%and 49%,and indicated that 176Glu involved in the assistance of in the enzyme activity.There are several hundred of long terminal repeats(LTRs)in S.cerevisiae retrotransposons known as delta(?)elements and can be used as integrated sites for heterologous genes.Xylose utilization couldn't be achieved directly by S.cerevisiae.By integration based on ? elements,three genes related to xylose utilization from Aspergillus niger,xylose reductase,xylitol dehydrogenase and xylulokinase were integrated into genome of auxotrophic S.cerevisiae strain BY4741,and strain RHS that can utilize xylose was constructed successfully.After knocked out conserved phosphatase gene PH013 and integrated with xylose reductase gene contain R277H,we obtained optimized strain RHS?PM.Under oxygen limited condition,40 g/L xylose was used up after 80 hours and 10 g/L ethanol was produced by RHS?PM when xylose was the solo carbon source.In conclusion,?-mannanase extended the application of mannan for various industrial practices.Site mutation study of Ttxy43 was helpful for clarifying the mechanism of Ttxy43,improving the efficiency of xylan converted to xylose.Moreover,the construction of RHS?PM that utilize xylose can be used for producing ethanol by xylose,and provide foundation for utilization of xylose and xylan. |
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