Improvement Of The Efficiency Of Cellulase System From Penicillium Oxalicum In Degradation Of Lignocelluloses

The use of biomass to produce biofuels and bulk chemicals is one of the most important ways to solve the current energy and environmental crisis.As the only renewable resource in large-scale,biomass resources,especially lignocellulosic materials,have not been fully exploited.Among them,the high cost of cellulase becomes the key bottleneck to restrict the comprehensive utilization of lignocellulose,especially the industrial conversion of lignocellulose to ethanol.How to improve the degradation efficiency of lignocellulose and reduce the dosage of cellulase has become the key problem.As an important cellulase-producing strain,cellulase from Penicillium oxalicum has many advantages,such as complete enzyme system and high stability.It is one of the most widely studied strains in this research field in the world.Cellulase production from Penicillium oxalicum has been studied since 1980s,through mutagenesis and genetic engineering.Lignocelluloses could be degraded with cellulase efficiently,but not fulfilled the demand of industrialized production.How to improve the biodegradation efficiency of cellulase,especially at high solid content,and how to reduce cellulase dosage and process cost,are critical to the industrialization of cellulose ethanol.Based on the background above,cellulase from P.oxalicum was taken as the main research object.In this study,we analyzed respectively process optimization,composition of cellulase complex,cellulose non-degrading enzymes and degradation ability of enzyme components.The solving strategies were developed to improve the degradation efficiency of lignocellulosic feedstocks.The main research contents and results are as follows:1.Identifying and overcoming the effect of mass transfer limitation on decreased yield in enzymatic hydrolysis of lignocellulose at high solid concentrationsThere are lots of advantages with enzymatic hydrolysis at high solids concentrations such as higher product concentration and economic competitiveness.However,cellulose conversion decreased significantly with increasing solid concentrations during enzymatic hydrolysis of insoluble lignocellulosic materials.Mass transfer limitation due to high viscosities at high solid loadings instead of glucose inhibition was shown to be the potential determining factor in decreased yield at the solid concentration of delignified corncob residue up to 20%dry matter(DM)content.Two mass transfer efficiency-related factors,mixing speed and flask filling,were shown to correlate closely with cellulose conversions at solid loadings higher than 15%DM.The role of substrate characteristics in mass transfer performance was also significant,which was revealed by the saccharification of two corn stover substrates with different pretreatment methods at the same solid loading.Several approaches including premix,fed-batch operation,and use of horizontal rotating reactor were shown to be valid in facilitating cellulose conversion via improving mass transfer efficiency at solid concentrations higher than 15%DM.The horizontal rotating reactor was proved to be more efficient.2.Efficient enzymatic hydrolysis of cellulosic material with different pretreatment at high solids concentrations via optimized enzyme componentsCellulase complex include endoglucanase(EG),cellobiohydrolase(CBH)and?-glucosidase(BG).To improve the enzymatic hydrolytic efficiency of different cellulosic materials,especially at high solids concentrations,the mixture design was used to evaluate the optimal mixture of EGII,CBHI and BGI according to the maximum cellulose conversions.The final optimal mixture was distinctly different between substrates from different pretreatments,which was aggravated during hydrolysis at high solids concentration.CBHI and EGII was preponderant during hydrolysis of cellulosic materials with acid and sulfite pretreatment respectively,especially at high solids concentrations.This was resulted from the nonproductive adsorption by lignin in acid-pretreated material and change of crystalline structure in sulfite pretreated material.With the optimal mixture combination,the cellulose conversions could be significantly improved,especially at high solids concentrations.Replacement test showed that overexpression of the most critical components could effectively improve the hydrolytic efficiency and alleviate solids effect at high solids concentrations.3.Addition of chemicals and auxiliary proteins at high solid loading to improve enzymatic efficiency on substrates with different pretreatmentsHigh dosage of enzyme is required to achieve effective lignocellulose hydrolysis,especially at high solids loadings,which is a significant barrier to large-scale bioconversion of lignocellulose to fuels and chemicals.Here,we screened four chemical additives and three accessory proteins for their effects on the enzymatic hydrolysis of various lignocellulosic materials by cellulase preparation from P.oxalicum.The effects were found to be highly dependent on the composition and solids loadings of substrates.For xylan-extracted lignin-rich corncob residue,the enhancing effect of PEG 6000 was most pronounced and negligibly affected by solids content.More than half of enzyme demand could be reduced with PEG 6000 addition during the hydrolysis at 20%DM.Lytic polysaccharide monooxygenase enhanced the hydrolysis of ammonium sulfite wheat straw pulp,and its addition reduced about half of protein demand at the solids loading of 20%DM.Supplementation of the additives in the hydrolysis of pure cellulose and complex lignocellulosic materials revealed that their effects are tightly linked to pretreatment strategies.4.Analysis and overcoming of limiting factors for the degradation of crystallinecellulose by cellulase from P.oxalicumThe low efficiency of cellulases is a major bottleneck for industrial bioconversion of lignocellulosic materials.Commercial cellulase preparations are mainly produced by the fungus Trichoderma reesei.The cellulase mixturesecreted by T.reesei showed both higher rate and greater final yield in the hydrolysis of corncob residue than that of P.oxalicum,while the difference was negligible on ammonium sulfite wheat straw pulp.The two cellulase preparations also haddifferent performances on the hydrolysis of Avicel which consists of crystalline cellulose I,with about 56%of Avicel hard to degrade by P.oxalicum cellulases.CBHI was identified as a determinant of this disparity of hydrolysis efficiency between cellulase mixtures.The addition of T.reesei CBHI,but not those from P.oxalicum and Aspergillus niger,efficiently continued the hydrolysis of partially hydrolyzed Avicel that was less reactive to P.oxalicum cellulases.Further domain exchange experiment attributed the superior hydrolyzing and binding abilities ofT.reesei CBHI on Avicel to its interdomain linker and cellulose-binding domain CBM1.The results in part explained the superior performance of T.reesei cellulases on the degradation of native crystalline cellulose,and highlighted the important role of cellulose-binding region in determining the degree of hydrolysis by cellulase mixtures.