【作者】 孙宪昀；

【导师】 曲音波；

【作者基本信息】 山东大学 ， 微生物学， 2007， 博士

【摘要】 目前,木质纤维素降解酶系已广泛地应用于能源、纺织、印染、造纸、洗涤、食品、饲料、酿酒等多个行业,显示了巨大的市场潜力。随着资源短缺、环境污染和能源危机的加剧,利用廉价的木质纤维素类可再生资源生产液体燃料和生物化工产品,不仅能够节粮代粮,缓解能源危机,同时还可以保护环境,促进全球经济的可持续发展。但木质纤维素酶系的产量和酶的比活力低,以及酶系组成不合理一直是制约木质纤维素酶系实际应用的重要原因。就我们的理解,木质纤维素、生物质降解酶和微生物这三个要素构成一个动态互作系统,深入研究每个要素及其之间的关系是提高生物质能源的生产能力、缓解能源危机的便捷之路。在这三个要素中,人们对微生物的关注主要集中于优良菌株的选育和改良,包括菌株的筛选和诱变。目前用到的方法有常规理化诱变、基因组重排、细胞融合和分子插入诱变等,但这些方法都属于随机的菌株改良策略,不仅工作量大,而且没有针对性。具有针对性的菌株的定向工程改良近来受到人们的重视,但其前提是弄清酶的合成调控机制。因此,研究微生物木质纤维素酶系的合成与调控不仅具有重要的理论意义,而且有助于通过改进营养策略和生物过程设计或通过定向工程改良,提高酶的生产和应用。研究表明,不同来源的木质纤维素酶系的酶系组成及合成调控机理往往存在差异,而目前国内外的研究主要围绕木霉和曲霉的合成调控展开的,对其它丝状真菌尤其是青霉木质纤维素酶系合成调控的研究较少。本论文从改进木质纤维素、生物质降解酶和微生物这一动态互作系统的角度出发,以我们实验室选育的斜卧青霉为实验材料,研究了其木质纤维素酶系的合成调控机理,丰富了对相关领域的认识,同时为菌株的定向工程改良和生物过程设计,提供了可靠的实验数据和理论依椐。本论文的主要研究结果如下;1克隆了青霉木质纤维素酶系合成调控因子基因creA与acel,初探了菌株JU-A10的抗降解物阻遏突变机理利用TAIL-PCR和RT-PCR技术克隆了青霉木质纤维素酶系合成调控因子编码基因creA与acel。creA基因全长为1251bp,不含有内含子,编码417个氨基酸。CRE A蛋白理论分子量和等电点分别为44956.072 Da和pI 9.735。acel基因全长为2836 bp,含有3个内含子,大小分别为129 bp、165 bp和58 bp。acel基因共编码827个氨基酸。ACE I蛋白的理论分子量和等电点分别为89871.29 Da和pI 5.685。转录分析表明斜卧青霉的creA与acel基因是组成性表达的。通过对斜卧青霉114-2及其突变菌株JU-A10的creA与acel基因的序列和转录比较发现,两菌株的creA与acel基因的序列和转录情况相同。因此,菌株JU-A10的抗降解物阻遏特征不是由于碳源代谢抑制因子CRE A或ACE I的突变而引起的。结合已有的知识和分析,菌株JU-A10的脱阻遏特征应该是由产能代谢发生了变化引起的。2揭示了斜卧青霉基础性与诱导性纤维素酶的组成及区别通过对诱导与阻遏条件下斜卧青霉胞外纤维素酶活力测定和活性染色进行分析,发现斜卧青霉的胞外存在基础性纤维素酶,基础性纤维素酶由内切葡聚糖酶、外切葡聚糖酶和β-葡萄糖苷酶组成。其中基础性与诱导性表达的内切葡聚糖酶组分是不同的,部分诱导性内切葡聚糖酶组分只有在诱导条件下才合成。转录分析表明,斜卧青霉的主要纤维素酶基因cbh1、cbh2、egl1、egl2和bgl1均存在低水平的基础性转录,除了β-葡萄糖苷酶和外切葡聚糖酶外,只有部分内切葡聚糖酶基因转录后进行了翻译并分泌到胞外,这部分组分才是真正的基础性内切葡聚糖酶。基础性内切葡聚糖酶由4个组分组成,分子量大小分别为43 kDa,39 kDa、36 kDa和34 kDa。其中34 kDa蛋白是最先表达的关键基础性内切葡聚糖酶。对纯化的34 kDa基础性内切葡聚糖酶和45 kDa诱导性内切葡聚糖酶的性质比较显示,斜卧青霉中基础性的与诱导性的内切葡聚糖酶不是由同一基因编码。3纯化了斜卧青霉的主要纤维素酶组分并构建了其肽质量指纹图谱库利用多种色谱分离技术相结合方法从斜卧青霉114-2发酵液中分离得到了1个纯β-葡萄糖苷酶和6个内切葡聚糖酶组分。β-葡萄糖苷酶的分子量大小为124kDa。其对底物水杨素的Km值为0.0007 mol/L。β-葡萄糖苷酶最适作用温度和pH分别为65℃和pH 4-5。6个内切葡聚糖酶的分子量大小分别约为100 kDa、60 kDa、49 kDa、45 kDa、34 kDa和24 kDa。其中45 kDa和34 kDa内切葡聚糖酶达到了电泳纯,二者对底物CMC-Na的Km值分别为0.7793 g/L和0.8365g/L。45 kDa与34 kDa内切葡聚糖酶的最适作用温度均为55-60℃,最适作用pH分别为pH 4.5和pH 5.0。这2种内切葡聚糖酶及β-葡萄糖苷酶在50℃以下及偏酸性的条件下能保持较好的稳定性。通过将纯化的纤维素酶进行SDS-PAGE后胶内酶解和MALDI-TOF质谱分析,并结合对斜卧青霉纤维素酶基因或基因片段编码蛋白序列的分析,构建了斜卧青霉纤维素酶的肽质量指纹图谱(PMF)库。本PMF库中包含1种β-葡萄糖苷酶、6种内切葡聚糖酶和2种外切葡聚糖酶的肽质量指纹图。4建立了斜卧青霉胞外差异蛋白组研究技术体系通过条件优化,建立了稳定可靠的斜卧青霉胞外蛋白的双向电泳实验方法。比较诱导与阻遏条件下斜卧青霉114-2胞外蛋白的双向电泳图谱发现,在诱导与阻遏条件下共同存在的蛋白点约23个,而在诱导条件下,斜卧青霉胞外蛋白的种类和数量较阻遏条件下明显增多,多出的蛋白点近40个。我们比较了三种蛋白鉴定方法;一种是采用MASCOT搜索引擎对蛋白的PMF在NCBInr数据库中检索;第二种是通过软件GPMAW 6.0或手工方法对蛋白的PMF在我们构建的斜卧青霉纤维素酶的PMF数据库中检索。第三种是采用MASCOT搜索引擎对串联质谱结果在NCBInr数据库中检索。通过比较,验证了通过纯化蛋白构建数据库用于蛋白鉴定的方法可行性。采用这种方法,我们共鉴定出2个β-葡萄糖苷酶、13个内切葡聚糖酶。其中2个β-葡萄糖苷酶和2个内切葡聚糖酶是基础性表达的。5分析了麦麸中各组分对斜卧青霉生物质降解酶系合成的影响,找出了促进纤维素酶合成的关键因子之一是可溶性的纤维类寡糖在不同成分含量的碳源培养条件下,通过测定斜卧青霉菌体生长量和胞外生物质降解酶活力发现;麦麸中的淀粉可以诱导斜卧青霉淀粉酶的合成,但不能提供菌体生长需要的生长因子,过量时会抑制纤维素酶与木聚糖酶的合成。麦麸中的蛋白能帮助斜卧青霉生长,但添加过量蛋白也会抑制斜卧青霉纤维素酶、木聚糖酶与蛋白酶的合成。麦麸中的木聚糖可以诱导斜卧青霉纤维素酶与木聚糖酶的合成,但对纤维素酶的诱导能力明显低于对木聚糖酶的。研究表明促进斜卧青霉纤维素酶合成的关键因子主要在麦麸汁中。通过对麦麸汁的成分分析及添加纤维寡糖实验,证明了麦麸中促进斜卧青霉纤维素酶合成的关键因子之一是其可溶性成分中的纤维类寡糖。因此,测定和控制培养基中淀粉、蛋白和纤维类寡糖含量是稳定和提高斜卧青霉纤维素酶产量的重要措施。更多还原

【Abstract】 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.更多还原