| The industrial development of lignocellulosic ethanol is limited by the high cost of cellulase and low hydrolysis efficiency.?-xylosidases,as the essential component of hemicellulases,can effectively hydrolyze xylo-oligosaccharides produced from the hemicellulose degradation in the process of hydrolysis of lignocellulose,which can alleviate the inhibition of xylanase and cellulase activitives and promote the degradation of lignocellulose by cellulase,resulted in decreasing the total enzyme loading.Therefore,?-xylosidases are considered to be one of the core enzymes of cellulase.But P-xylosidase activity is deficient in most cellulase complex secreted by filamentous fungi,and there aren't available filamentous fungi that could produce cellulase cocktails with high-level production of ?-xylosidase.And it was mainly compensated by adding ?-xylosidases to cellulase cocktails or heterologously expressing ?-xylosidase in S.cerevisiae strain during simultaneous saccharification and fermentation.Howover,the domestic reports on ?-xylosidase resources are very limited,which cannot meet the application requirements of P-xylosidases in different industries.Therefore,discovery of novel ?-xylosidases with excellent properties,such as high catalytic efficiency,good thermal stability,high sugars tolerance,and good synergy with xylanase,etc.,which not only enriches the domestic P-xylosidase resources,and it is also of great significance for the optimization of cellulase system and construction of cellulase-producing strain with high ?-xylosidase activity.Penicillium oxalicum is an industral cellulase-producing fungus.Compared to Trichoderma reesei,it has the advantages of more perfect enzyme compositions and proportions,and more complete hemicellulase system,especially contains more kinds of ?-xylosidases in the lignocellulosic-degrading enzymes.Hyper-cellulolytic strain P.oxalicum RE-10 is a genetically engineered fungus with high-level expression of cellulase and xylanase,but productivity of ?-xylosidase is lower,which affected the cellulase efficiency for degradation of lignocellulose,resulted in increasing the cellulase dosage and cost used in the bioconversion process.Based on the above background,this thesis had systematically studied the resources excavation and functional research of xylosidases from P.oxalicum,and construction of the cellulolytic strain with high productivity of ?-xylosidase.The main research contents and results are as follows:1.Potential application and heterologous expression of xylosidases from Penicillium oxalicum in Pichia pastoris X-33Seven xylosidase-encoding genes were successfully cloned from P.oxalicum and were heterologously expressed in P.pastoris X-33.And the correct recombinant strains were obtained.The results of SDS-PAGE analysis showed that recombinant proteins such as the GH43 Xyl43A,XyI43C,Xyl43B,and Abf43D were efficiently secreted in P.pastoris,while the extracellular protein production of GH3 Xyl3C,Xyl3D and GH31 Xyl31A could be ignored.The enzymatic properties showed that Abf43D was a bifunctional ?-xylosidase/?-arabinofuranosidase,and Xyl43B was a alkali-tolerant GH43 ?-xylosidase,while Xyl3D was a multifunctional?-glucosidase/?-xylosidase/a-arabinofuranosidase,mainly presented ?-glucosidase activity,as well as Xyl31A and Xyl43A were(3-glucosidases.Results of enzymatic hydrolysis using the above-mentioned glycosidases and previously expressed GH3?-xylosidase Xyl3A showed that the addition of Xyl3A,Xyl3D and Abf43D to xylanase effectively promoted the hydrolysis of xylan under the condition of pH 4.8,especially Xyl3A,whereas Xyl43B was mixed with xylanase to degrade xylan at pH 7.0,the yield of xylose was increased by about 9 folds.2.Characterization of GH43 ?-xylosidases Xyl43B and Abf43D and its synergistic action on hemicellulose with xylanaseThe recombinant strain with high-level production of ?-xylosidase Xyl43B or Abf43D was successfully obtained by resistance screening with different gradient concentrations.Without fermentation optimization,the yields of Xyl43B and Abf43D reached approximately 92.8 mg/L and 65.8 mg/L,respectively,which were much higher than that of GH43 ?-xylosidase(0.22 mg/L)which was expressed in P.pastoris.The results of enzymatic properties indicated that the purified Xyl43B was a alkali-tolerant GH43 ?-xylosidase with unique substrate specificity.The optimum pH of Xyl43B was 7.0,and it exhibited high catalytic activity in the range of pH 6.0 to 8.0.Abf43D was a bifunctional ?-xylosidase/a-arabinofuranosidase that maintained more than 60%residual activity in the range of pH 4.0-6.0.Moreover,both Abf43D and Xyl43B showed higher tolerance to xylose with the Ki values of 35.77 mM and 28.09 mM,respectively,which are beneficial to obtain higher concentration of xylose during enzymatic hydrolysis of lignocellulosic biomass.The analysis of kinetic parameters showed that the substrate affinity of Xyl43B and Abf43D was higher than that of most reported ?-xylosidases.Enzymatic hydrolysis of different sources of isolated xylan and alkaline-pretreated corn stover showed that both Abf43D and Xyl43B,produced from recombinant P.pastoris,showed good synergy with xylanase and effectively promoted the degradation of different sources of isolated xylan and hemicellulose contained in alkaline-pretreated corn stover,resulted in improving the yield of xylose.Abf43D can also boost the degradation of arabinose side chains in hemicellulose of alkaline-pretreated corn stover.The above results indicate that Abf43D and Xyl43B have good potential for industrial application.3.Construction of ?-xylosidase Xyl3A hyper-production cellulolytic P.oxalicum mutant and its application in saccharification and fermentation of cellulosic substratesThe P-xylosidase gene xy13A by using the constitutive promoter gpdA from A.nidulans or original inducible promoter was overexpressed in P.oxalicum RE-10.The obtained mutants RXyl,RGXyl-1 and RGXyl-2 significantly increased the production of P-xylosidase in the fermentation broth,especially RGXyl-1 which expressing the xyl3A by using the constitutive promoter.It showed the highest ?-xylosidase activity of 15.05 ± 1.79 IU/ml in extracellular cellulase complex,about 29 folds higher than native strain RE-10.In addition,it also found that other enzymatic activities in the fermentation broth produced from mutants RGXyl-1,RGXyl-2 and RXyl were also increased compared to the native strain RE-10,in which,the filter paper activity of mutants RGXyl-1,RGXyl-2 and RXyl was increased by 20.01%,8.43%and 18.57%respectively,and the xylanase activity was also increased by 16.12%,15.54%and 16.83%,probably due to the random insertion of the gene xyl3A expression cassette into different positions in the genomic DNA,which may interfere or disrupt other genes' expression.The cellulase produced by the liquid fermentation of the engineered strain RGXyl-1 was used instead of the cellulase produced by the starting strain RE-10 to semi-synchronous saccharification and fermentation of two different alkali-pretreated corn stover.It was found that the concentration of xylobiose in the reaction system was significantly decreased,and was maintained at less than 1 mg/mL during saccharification and fermentation.And the degradation rate of cellobiose was also enhanced.Thus,the conversion rates of cellulose and xylan in the alkali-pretreated corn stover were improved and ethanol production was increased by 26.27%and 31.91%,respectively.Compared to the industrial cellulase-producing strain P.oxalicum RE-10,the cellulase produced from mutant RGXyl-1 had higher?-xylosidase activity,FPA and xylanase activitives,and overcame the deficiency of?-xylosidase activity in cellulase cocktails,avoiding addition of ?-xylosidase to cellulase,resulted in increasing the hydrolysis efficiency of cellulase to cellulosic substrate,and improving the yield of cellulosic ethanol,which is beneficial to decrease total enzyme loading and cost in the cellulosic ethanol production,and to reduce the steps and complexity of the enzymatic hydrolysis process.4.Co-expression of ?-xylosidase and ?-glucosidase in P.oxalicum and its application in saccharification and fermentation of cellulosic substratesBoth ?-xylosidase and ?-glucosidase are important enzymes in the cellulase system and play an important role in the complete degradation of hemicellulose and cellulose in lignocellulosic feedstocks to produce fermentable sugars.The co-expression cassette of the ?-xylosidase Xyl3A(or Xyl3A and Abf43D)and?-glucosidase Bgll by using the Red/ET recombination technique was successfully constructed,and subsequently was transformed into P.oxalicum RE-10 for improving the production of ?-xylosidase and ?-glucosidase.Two mutants RXB-2 and RXB-3 with co-expression of Bgll and Xyl3A,as well as three recombinant strains RAXB-1,RAXB-3 and RAXB-4 with co-expression of Abf43D,Bgll and Xyl3A,were screened.The results of enzyme activity assay showed that the filter paper activity(FPA),?-xylosidase and ?-glucosidase enzymatic activities of all mutants were improved to different extents.The ?-glucosidase and ?-xylosidase activities in the fermentation broth of the engineering strain RXB-2 were improved by 11 folds and 6 folds higher than that of starting strain RE-10 at 6 days of fermentation,and the?-glucosidase and ?-xylosidase activities in the fermentation broth of mutant RAXB-4 were also increased by 20 folds and 11 folds,respectively.The filter paper activitives of mutants RXB-2 and RAXB-4 were increased by 33.3%and 52.1%compared to that of RE-10.In addition,the a-arabinofuranosidase activitives in the fermentation broth from the mutants RAXB-1,RAXB-3 and RAXB-4 were also increased by 28.4%,60.1%%and 36.1%.Desired target enzymes in the cellulase complex were effectively improved by co-expression of different glycosidase genes,resulted in optimizing the proportion of cellulase components,and also avoiding the complicated process of gradual single enzyme component expression.Compared to the crude cellulases produced from the starting strain RE-10,the engineered strains RGXyl-1 and RBB1-10(overexpression of Bgll in the strain RE-10),the cellulases obtained from the mutants RAXB-4 and RXB-2 effectively eliminated the accumulation of large amounts of cellobiose and xylo-oligosaccharides during the semi-synchronous saccharification and fermentation of alkali-pretreated corn stover for ethanol production,due to the enzymatic activities of P-xylosidase and?-glucosidase in the cellulase cocktails were improved.Thus,the saccharification efficiency of cellulase complex was enhanced and bioconversion rates of cellulose and xylan in alkali-pretreated corn stover were also improved,resulted in increasing the production of ethanol and decreasing the cellulase usage.5.Characterization of the multifunctional glycosylase Gax3A from P.oxalicum and its promoting effect on enzymatic hydrolysis of cellulosic substratesBased on the successful heterologous expression of the ?-xylosidase gene xyl3D from P.oxalicum in P.pastoris,analysis of substrate specificity of Xyl3D showed that the enzyme is a multifunctional ?-glucosidase/?-xylosidase/a-arabinofuranosidase.Because of the relatively low activitives of the above three enzymes in most current commercial cellulase preparations,the multifunctional enzyme is undoubtedly more advantageous than the monofunctional enzyme in optimizing the cellulase system.To further investigate the enzymatic properties of this multifunctional enzyme for promoting its application,homologous expression of the gene xyl3D was achieved in Penicillium oxalicum.And the Xyl3D was successfully isolated and purified from the fermentation broth by using the gel filtration chromatography.Xyl3D had?-glucosidase/?-xylosidase/a-arabinofuranosidase activitives,but predominantly presented ?-glucosidase activity,therefore the enzyme was renamed as Gax3A.The enzymatic properties showed that the specific activity of Gax3A ?-glucosidase was 61.53 IU/mg,and the enzymatic activity of ?-xylosidase and ?-arabinofuranosidase was 17.94 IU/mg and 1.25 IU/mg,respectively.The ?-glucosidase activity of Gax3A was most active onpNPG at 70?,and it was stable at 70? for 30 min,which still retained about 53%residual activity,indicating that Gax3A exhibited high thermo-stability.The Gax3A had good resistance to most metal ions,especially for Fe3+,Zn2+,Mn2+,K+.Gax3A had higher affinity for substrates and higher catalytic efficiency compared to most of the reported multifunctional enzymes.Results of hydrolysis experiments using sulfite-pretreated wheat straw and NaOH-pretreated corn stover as substrates showed that combination of Gax3A and cellulase RE-10 effectively improved the yields of glucose,xylose and arabinose in the hydrolysate.Compared to the monofunctional ?-glucosidase Bgl1,the addition of Gax3A not only promoted degradation of cellulose,but also further promoted the hydrolysis of hemicellulose in lignocellulose to xylose and arabinose,which can be used to optimize the cellulase system produced from Penicillium oxalicum. |
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