Analysis Of The Lignocellulose-degrading Enzyme System And Investigation Of The Major Protein Components Secreted By Penicillium Oxalicum

With the irreversible depletion of the fossil resource, exacerbating energy crisis and other environmental issues, there has been a world-widely interest in exploiting alternative sources of energy. Lignocellulosic material is the most abundant renewable resource in the world. However, the renewable resource has not been sufficiently exploited, even causing environmental problems. Utilization of the cellulase from microorganism to bioconversion of cellulose materials into liquid fuels and chemicals has been considered as the most promising sustainable strategy for resolving the resource, energy and environmental problems.Natural lignocellulolytic enzyme systems from filamentous fungi are generally deficient in terms of certain components and thus require enzyme supplementation to hydrolyze complex lignocellulosic materials completely. Therefore, the composition of these enzyme systems should be modified on the basis of biomass materials. The composition of cellulase mixtures should also be optimized to enhance hydrolytic efficiency. However, the design of cost-effective lignocellulolytic enzyme cocktails is limited by the lack of knowledge on whole enzyme systems and exact quantities of individual cellulolytic proteins secreted by lignocellulose-degrading microorganisms. Indeed, lignocellulolytic enzyme complexes should be optimized on the basis of proteomic analysis to develop efficient bioconversion processes.Penicillium oxalicum is a fast-growing cellulolytic fungus with good balanced enzyme system. The hypercellulolytic mutant P. oxalicum JU-A10 has been used for industrial-scale production of cellulase preparation in China since 1996. However, commercial enzyme mixtures produced by this strain at industrial scale have yet to be characterized in detail, and their proteinaceous components have yet to be elucidated. Proteomic methods have been employed to comprehensively evaluate enzymes involved in lignocellulosic biomass degradation, would help in understanding the properties of this enzyme system.In this study,we analyzed the dynamics of enzyme activities and the proteome of the cellulase preparations based on the genome and transcriptome studies of P. oxalicum. "Core" cellulase and auxiliary protein of P. oxalicum were studied to optimize the cellulase system. We functionally characterized the endoglucanase (Cel5B), exoglucanase (Cel7A-2), superoxide dismutase (SOD) and swollenin. These results provide the guidances in P. oxalicum engineering for improved cellulase production.The main results of the research are as follows:1. The cellulase from Penicillium oxalicum and Trichderma reesei showed the different saccharification capability for pretreated biomasses.As byproducts of xylose industry, both corncob residues (CCR) and delignified corncob residues (DCCR) contain abundant cellulose, so they are potential substrates in bioethanol field. In this study, the hydrolytic performances of three commercial cellulase preparations (SP from P. oxalicum JU-A10, ST from T. reesei SN1, and Celluclast 1.5L from Novozymes) on CCR and DCCR were compared. The cellulolytic and xylanolytic activities of an SP enzyme system prepared from P. oxalicum JU-A10 were comparatively analyzed. Results indicated that DCCR could be hydrolyzed by SP to a greater extent than CCRwhen using the equal protein loding. When using the 20mg protein/g glucan loading, the glucan conversion reached up to about 90% using the DCCR as substrate, while the glucan conversion is only 40% using the DCCR as substrate. However, the glucan conversion using ST (the cellulase from T. reesei) using the DCCR as substrate is 72%, and is higher than that SP (the cellulase from P. oxalicum). The results suggested that cellulase could be by adsorpted by lignin, so further affects the saccharification of biomass. This effect is more significant on SP.P. oxalicum possesses a complete cellulolytic-xylanolytic enzyme system. The cellobiohydrolase-and xylanase-specific activities of this system were higher than those of two other enzyme systems, i.e. ST from T. reesei SN1 and another commercial preparation Celluclast 1.5L. The cellobiohydrolase and xylanase acitivities of SP were 2.30 and 2.71 times higher than that of ST, but has a lower the β-glucosidase activity. Beta-glucosidase (BG) supplemented in SP increased the ability of the system to hydrolyze DCCR and CCR.The conversion significant improved by addition of β-glucosidase using the corn cob residues as substrates. Add a ratio of BG (FPA:BG=1:4) with SP cellulase loadings of 9 FPA/g glucan, the conversion reached to 92% using DCCR as substrate. This strategy resulted in a 64% decrease in enzyme dosage with the same glucose yield than the using the SP alone. So BGL is the bottleneck in the saccharification of biomass by the SP.The behaviors of the enzyme components in the hydrolysis of CCR werefurther investigated by monitoring individual enzyme dynamics. The total protein concentrations and cellobiohydrolase (CBH), endoglucanase (EG), and filter paper activities in the supernatants significantly decreased during saccharification. These findings were more evident in SP than in the other enzyme systems.There was a synergistic effect between P. oxalicum and T. reesei, which could improve the hydrolysis efficiency of the cellulase system of P. oxalicum. Thus, we further separated the individual enzyme component, and then add into the SP. In this study, we found that the protein which is from ST promoted the saccharification efficiency of SP. Together, the findings could be contributed to the strain improvement of P. oxalicum.2. The results of proteomics showed that the composition of the enzyme system was different between P. oxalicum and T. reesei.P. oxalicum has the potential to be used as a major industrial strain for the production of cellulase. The comparative proteomic analysis of the enzyme systems revealed that the SP cellulase preparation was composed of fourteen cellulase including three cellobiohydrolases (two CBH Ⅰ and one CBH Ⅱ), nine endo-b-1,4-glucanases (EGs). Eventhough, P. oxalicum possessed twoBGLs, the proportion of the secreted BGL are very low. Both SP and ST contained lytic polysaccharide monooxygenase (LPMO) and swollenin protein which were active in the hydrolysis of lignocellulose.The production of CBH, EG, BG, LPMO, chitinase, and amylase comprises about 37.15%,15.03%,0.66%,2.11%,0.02%, and 0.01% of total secreted proteins by ST, respectively. However, the corresponding enzyme ratios in SP were 39.00%,11.27%,0.25%,1.37%,0.19%, and 0.44% of total secreted proteins by SP.On the other hand, our data showed that ST highly likely employed strategies different from those of SP to gain access to biomass substrates. We speculated that the unique components xyloglucanase, Cip1,4-O-methyl-glucuronoyl methylesterase (Cip2), and hydrophobin in ST were capable of promoting substrate degradation by removing the hemicellulose-lignin obstacle. These enzymes have been reported to exhibit some synergistic activity with both LPMO and swollenin. These components may contribute to the high levels of CCR saccharification by ST. Collectively, the LPMOs, swollenin, Cipl, Cip2, and xyloglucanase could serve as desirable targets for the rational construction of an optimal cellulase system.A larger number of carbohydrate-binding modules 1 (CBM1) were also identified in SP than in ST. This difference might be linked to the greater adsorption to substrates and lower hydrolysis efficiency of SP enzymes than ST during lignocellulose saccharification, because CBM1 not only targets enzymes to insoluble cellulose but also leads to non-productive adsorption to lignin. Adsorption experiments showed that the content of LPMOs and swollenin are very low in the supernatant during the saccharification. It is suggested that these proteins exhibited the active hydrolysis of the cellulosic material. Meanwhile, the protein containing more CBMs caused the more unproductive adsorption, and impeded the hydrolysis of the complex cellulosic substrate.Our study provided comprehensive insights into the protein repertoire and their relative abundances in the secretome of the hypercellulolytic P. oxalicum. Our study also proposed targets to optimize enzyme systems and to facilitate the rational designing of highly effective enzyme cocktails for lignocellulose bioconversion.3. Characterizationof endo-β-1,4-glucanases(Cel5B) and cellobiohydrolase (Cel7A-2) and their effectson saccharification.The content of Cel5B orCel7A-2 was the first or eighth in cellulase from P. oxalicum separately. In orderto research "core" cellulase and their influence on saccharification, these two proteins had been expressed in P. oxalicum All Δ and purified, then were characterized for enzymatic properties.Compared with other endo-β-1,4-glucanases in P. oxalicum, rCel5B has better thermal stability and pH tolerance. It retained more than 90% of its initial activity after the incubation for 6h as the temperature was raised from 30 to 50 ℃. And a relative activity of 80% was detected at 60 ℃ after 6 h of incubation. The activity of cel5B could retain more than 90% when the pH was changed from 4.2 to 6.0. To a certain extent, Cel5B has hydrolytic ability to all the substrates, especially, carboxymethyl cellulose sodium salt.Cel7A-2 belongs to cellobiohydrolase andit was sensitive to temperature and pH. rCel7A-2 showed anoptimum temperature range of 55 ℃. It retained more than 90% of its initial activity after the incubation for 6h as the temperature was raised from 30 to 50 ℃. When the temperature was raised to 60 ℃, the activity was reduced to 60% after 6 h of incubation. The activity of rCel7A-2 could retain more than 90% when the pH was changed from 3.6 to 4.8, and the optimum pH value was 4.2. Its activity retained 70% at pH 3. When pH value was raised to 5.4, its activity was reduced to 20% rapidly, and the activity almost disappeared at pH 6.0 and 6.6.Adding rCel5B or rCel7 A-2 to SP could contribute to degradation lignocellulose to a certain extent. Adding Cel5B or Cel7 A-2 alone, the glucose yields were improved. And the conversion of glucan was 2% and 6%, respectively. We used central combination design to study the influence of cellulase from P. oxalicum to corncob residue (CCR) saccharifiation, the result showed the conversion of glucose was improved 33% when adding 13% Cel7A-2,21% Cel5B and 29% BG.4. The effect of swollenin and Cu/Zn-SOD on P. oxalicumCu/Zn-SOD and swollenin had higher content in the secretome of P. oxalicum, which were important "auxiliary protein" for lignocellulose conversion. We successfully constructed these two deletion mutants using high cellulase producing strain 4-1 as the parent strain. The result showed that the deficiency of Cu/Zn-SOD affected the enzyme activity and protein concentration. However, it had no direct function in biomass saccharifiation.We found that deleting swollenin had obvious effect on the activity of β-glucanase. The result of saccharifiation using equal enzyme dosage and equal filter paper activity showed that the deletion of swollenin had affected the saccharifiation effciency of CCR. It was found that knocking-out swollenin gene had no significant effect of phenotypic. We constructed over-expression strain of swollenin using expression plasmid. After purification, swollenin added to saccharification experiments and enhanced saccharification efficiency of P.oxalicum. So, the overexpression of swollenin in cellulase producer was successfully constructed to optimize the cellulase system of P.oxalicum.