Studies On The Synthesis Regulation Of Lignocellulose-degrading Enzymes In Penicillium Decumbens

At present,lignocellulose-degrading enzymes have been widely used in many industries such as energy,textile,dyeing,pulp and paper,detergent,food,feed and brewing.Therefore,they have huge market potentials.With the intensification of resource shortage,environment pollution and energy crisis,liquid fuel derived from renewable resource such as lignocellulose are becoming an alternative that can dramatically boost economic growth,alleviate energy crisis,and protect environment. As is well known,low production yield and specific activity,and unbalanced composition of enzyme system,are always the important factors that restrict the actual application of lignocellulose-degrading enzymes.To our understanding, lignocellulose,biomass-degrading enzymes and microorganisms form a dynamic and interactional system,and studying each factor and revealing their relationship will help us to improve biomass energy production and alleviate the energy crisis.For the improvement of microorganisms,most attentions have been focused on the selection and breeding of fine strains.Current methods,such as traditional physical or chemical mutation,genome shuffling,cell fusion and molecular mutation belong to random strategy for strain improvement,which are always time consuming and aimless.Therefore,directional engineering renovation of microorganisms is of high interest recently.But it is on the premise that the regulation mechanisms of enzyme synthesis are well clarified.So understanding the synthesis and regulation of lignocellulose-degrading enzymes in microorganisms is not only important for theoretical studies,but also helpful to enhance the production and application of lignocellulose-degrading enzymes by the improvement of nutritional strategy, bioprocess designing or directional engineering renovation of cellulolytic microorganisms.Lignocellulose-degrading enzymes from different sources often have different compositions and regulation mechanisms of synthesis.However,Current research is mainly around the synthesis regulation of Trichoderma and Aspergillus,few research regarding the regulation of cellulase synthesis in other filamentous fungi especially Penicillium is available.For the sake of improving the dynamic systems consisting of lignocellulose,biomass-degrading enzymes and microorganisms,in the thesis,the regulation mechanisms of the synthesis of lignocellulose-degrading enzymes in P. decumbens were studied.That would be helpful to deepen our understanding in related fields and provide reliable experimental data and theoretical basis for bioprocess designing and directional engineering renovation of cellulolytic fungi. The main results of the thesis are as follows;1 Cloning of creA and acel encoding cellulase regulation factors in P.decumbens and the mutation mechanism of strain JU-A10.creA and acel encoding cellulase regulation factors in P.decumbens were cloned by TAIL-PCR and RT-PCR.DNA sequencing results showed that creA had an open reading frame of 1251 bp without introns and encoded a polypeptide of 417 amino acids with relative molecular weight of 44956.072 Da and isoelectric points(pI)of 9.735.acel was composed of 2836 bp long containing 3 introns(129 bp,165 bp and 58 bp)and encoded a polypeptide of 827 amino acids with relative molecular weight of 89871.29 Da and isoelectric points(pI)of 5.685.Transcriptional analysis showed that creA and acel were expressed at constitutive level in P.decumbens.By comparing the sequences and transcription of creA and ace1 in P.decumbens 114-2 with its mutant JU-A10,we found that the catabolite repression-resistant characteristics of strain JU-A10 were not caused by the mutation of catabolite repressors CRE A or ACE I.Based on previous knowledge and analysis,it is proposed that the catabolite repression-resistant characteristics of strain JU-A10 are caused by the change of energy-yielding metabolism.2 The composition of basal cellulases and their difference from induced celluloses of P.decumbens.Enzyme activity determination and activity staining of P.decumbens extracellular cellulase under induction and repression conditions showed that the basal cellulase existed extracellularly.Basal cellulase was composed of endoglucanase, cellobiohydrolase andβ-glucosidase.The compositions of basal and induced endoglucanases were different,and partial induced endoglucanases were synthesized only under induction condition.Transcripts of the main cellulase genes cbh1,cbh2, egl1,egl2 and bgl1 were detected in P.decumbens cells grown with glucose or cellulose,whereas the transcriptional levels of the five cellulase genes and especially that of cbhl and egll were lower under glucose-repression conditions.With the exceptions ofβ-glucosidase and cellobiohydrolase,only a subset of the endoglucanase transcripts was translated with the protein then being secreted from the cells,and these comprised the real basal endoglucanases.The basal endoglucanase contained four components with molecular weights of about 43 kDa,39 kDa,36 kDa and 34 kDa,and the 34-kDa protein was the first basal endoglucanase to be expressed.A comparison of two purified endoglucanase with molecular weights of 34 kDa(basal cellulase)and 45 kDa(induced cellulase), revealed that basal and induced endoglucanases were encoded by different genes.3 Purification of the main cellulose components from P.decumbens and construction of its peptide mass fingerprinting database.Oneβ-glucosidase and six endoglucanases were purified or partly purified from P. decumbens 114-2 fermentation broth by the combination of several steps of chromatographies.The molecular weight of theβ-glucosidase was 124 kDa,and the Km toward salicin was 0.0007 mol/L.The optima temperature and pH of theβ-glucosidase were 65℃and pH 4-5,respectively.The molecular weights of the six endoglucanases were 100 kDa,60 kDa,49 kDa,45 kDa,34 kDa and 24 kDa, respectively.The endoglucanases of 45 kDa and 34 kDa were purified to electrophoretic homogeneity for further study.The two endoglucanases had similar affinity toward CMC-Na with a Km of 0.8365 g/L and 0.7793 g/L respectively,and the same optimal temperature of 55℃to 60℃.The optimum pH of the two endoglucanases was pH 4.5 and pH 5.0,respectively.The activities of the two endoglucanases and theβ-glucosidase were stable when the temperature was below 50℃and in the acidic environment.By combining MALDI-TOF-MS analysis of the purified cellulases after SDS-PAGE,and sequence analysis of cellulase genes(or gene fragments)of P. decumbens,the peptide-mass-fingerprinting(PMF)database of cellulase from P. decumbens was constructed.The PMF database contains oneβ-glucosidase,six endoglucanases and two cellobiohydrolases.4 Establishment of the research system of extracellular differential proteomics of P.decumbens.Stable and reliable two-dimensional electrophoresis method of extracellular proteins of P.decumbens was established by optimization of conditions.By comparind of 2DE maps of the extracellular proteins of P.decumbens 114-2 under induction and repression conditions,it was showed that about 23 spots existed under both conditions,while under induction condition,the type and quantity of extracellular proteins increased obviously,with nearly 40 protein spots observed.Three methods for protein identification were compared,the first method was via searching the protein's PMF in NCBInr database using MASCOT search tool;the second method was via searching the protein's PMF in P.decumbens cellulase PMF database using GPMAW 6.0 search tool or manual method;the third method was via searching the results of tandem mass spectrometry in NCBInr database using MASCOT search tool.The method for protein identification through protein purification and database construction was proved to be feasible.By using this method,twoβ-glucosidase and 13 endoglucanases were found to be extracellular proteins of P.decumbens,in which twoβ-glucosidase and two endoglucanases were expressed at basal level.5 The effects of wheat bran composition and soluble cello-oligosaccharides on the production of biomass-hydrolyzing enzymes by P.decumbens.Determination of biomass and activities of extracellular biomass-hydrolyzing enzymes of P.decumbens cultured with different carbon-resource components showed that the starch content of wheat bran is an important,potentially negative factor.As high starch contents could stimulate amylase production,but could not maintain high levels of mycelial growth,excessive starch could inhibit cellulase or xylanase production.Similarly,although the addition of high amounts of wheat protein did not inhibit the growth of P.decumbens,it did result in a reduction in the levels of cellulase,xylanase and protease released to the medium.Xylan in wheat bran could induce the synthesis of cellulase and xylanase in P.decumbens,but induced less cellulase than xylanase.The significant factors inducing cellulase synthesis of P.decumbens most possibly originated from the wheat bran juice.Analysis of the wheat bran juice compositions and the experimental results of adding cell-oligosaccharides revealed that the soluble cello-oligosacchaddes present in wheat bran(concentrations that are much higher than in cellulose-only preparations)was one of most positive factors for cellulase production by P.decumbens.As a result,measuring and regulating the composition of starch,protein and cell-oligosaccharides in wheat bran used as a fermentation supplement may allow for improved induction of cellulase production by P. decumbens.