【作者】 程艳飞；

【导师】 曲音波；

【作者基本信息】 山东大学 ， 发酵工程学， 2009， 博士

【摘要】 随着资源短缺、环境污染和能源危机的加剧,寻找和开发可以代替化石能源的新能源就成了亟待解决的问题。纤维素是地球上分布最广、含量最丰富的可再生资源,纤维素酶可以将可再生的木质纤维素转化为可发酵的多糖、单糖等混合糖浆,进一步发酵生产为乙醇等清洁能源物质或者其它高值生物化学产品。在第二代生物乙醇工业或中,其中纤维素酶的成本,是纤维素乙醇商品化的一个重要制约因素。斜卧青霉JU-A10是我们实验室由斜卧青霉114-2通过多轮诱变得到的抗阻遏突变株,其本身除了纤维素酶外也可以产生大量的半纤维素酶,对降解结构复杂的木质纤维素有很好的应用前景。但是其纤维素酶生产能力仍不能满足工业要求。而目前,我们对斜卧青霉的遗传背景和纤维素酶合成机制不够清楚,也没有合适的遗传改造工具,所以通过理性设计改造菌株在操作上有一定的难度。本文通过近几年发展起来并广泛应用的基因组重排技术对其进行改造,主要获得了以下结果:1、建立了高效的纤维素双层平板筛选方法优化了双层平板中培养基的组成,确定了使用球磨微晶纤维素和葡萄糖作为筛选纤维素酶高产突变株的选择压力,建立了水解圈大小和纤维素酶活正相关的高效的纤维素双层平板筛选方法。JU-A10只可以在2%球磨微晶纤维素和低于1%葡萄糖的双层平板上产生纤维素水解圈,所以在筛选过程中,逐步提高纤维素和葡萄糖的浓度可进一步筛选到纤维素降解能力更强或是抗阻遏能力更高的突变株。2、建立了斜卧青霉原生质体制备、再生和双亲灭活原生质体融合方法建立了高效的斜卧青霉原生质体制备和再生的方法:在优化后的菌丝培养基中培养菌丝24h后,经2mg ml^-1溶壁酶、4mg ml^-1纤维素酶、4mg ml^-1蜗牛酶三种酶酶解菌丝后,得到的原生质体经纯化后,原生质体的再生比率可达90%以上。建立了双亲本灭活原生质体融合方法:原生质体在紫外照射30min或50℃热灭活50min后,失去再生能力。将这两种方法灭活后的原生质体混合在一起,于35%PEG、10mM Ca²⁺、35℃的条件下进行融合。经这两种不同灭活方法处理过的原生质体,会在融合过程中通过互补,再生出融合子。3、通过基因组重排技术得到了纤维素酶活和产量显著提高的融合子使用紫外-EMS复合诱变和N⁺离子注入诱变两种方法,经过纤维素双层平板初筛和摇瓶复筛,得到了4株相对于出发株JU-A10各有优势的突变株,满足了基因组重排所要求的初始亲本库的多样性。然后,我们以这四株突变株作为第一轮基因组重排的亲本,进行重排操作,在筛选平板SM2（2%纤维素、2%葡萄糖）上挑选能够快速产生水解圈的突变株,通过复筛后得到6株酶活进一步提高的融合子。以此这6株菌作为第二轮基因组重排的亲本,通过在筛选平板SM3（5%纤维素、2%葡萄糖）的初筛和摇瓶复筛,最终获得3株纤维素酶显著提高的融合子:GS2-15、GS2-21、GS2-22,它们可以在复筛培养基中产生相当于JU-A10 200%、209%、194%的纤维素酶活。4研究并分析了融合子GS2-15、GS2-21、GS2-22与出发株JU-A10差异通过对融合子和出发株进行了固体平板培养时菌落、孢子、液体培养时菌丝的形态学观察,结果表明:与出发株相比,融合子的菌落变小,孢子体积变大,孢子壁变薄;在液体培养时菌丝变细,在培养后期菌丝更易断裂成小段,初步推测其在液体培养时形态学的变化,有利于纤维素酶的大量分泌。通过优化随机扩增多态性DNA（RAPD）反应体系和程序,建立了斜卧青霉RAPD反应体系,对3个融合子和JU-A10的RAPD指纹图谱进行了分析。3个融合子大部分RAPD条带与JU-A10相同,但是它们之间及它们和JU-A10之间也出现了一些特异性条带。通过遗传相似系数的计算和聚类分析,融合子和出发株以遗传相似系数0.87为域阀值,分为三类:第一类群由出发株JU-A10和融合子GS2-22组成;第二类群、第三类群分别为GS2-15和GS2-21。这反映出融合子在分子水平上发生了变异。特别研究了融合子和出发株在以木糖渣为碳源时的胞外和胞内纤维素酶活、蛋白浓度和生物量等特征,结果表明:融合子生长旺盛,并且生物量略高于出发株,其蛋白浓度和纤维素酶活也明显高于出发株;同时发现融合子的胞内纤维素酶在发酵初期就大量合成。推测融合子的纤维素酶活提高可能是由于生长旺盛,更早的纤维素酶合成和更高的蛋白分泌能力等因素综合作用所致。此外,在阻遏条件下（2%葡萄糖为唯一碳源）,融合子比出发株表现更优越。融合子基本上一天就可以耗尽葡萄糖,并且生物量和纤维素酶活高于出发株。分析和比较了基因组重排后融合子和出发株的胞外蛋白SDS-PAGE和内切酶活性电泳图谱,发现不论是在诱导还是阻遏情况下,融合子的胞外蛋白与出发株大体相似,但是有个别蛋白条带的增减,某些内切酶活性条带表达增强。这些蛋白表达的差异反映了融合子在蛋白水平发生了变化。5、研究了融合子利用木质纤维素为碳源及5-L发酵罐产酶情况以木质纤维素玉米秸粉、麦秸粉、甘蔗渣作为底物,研究了基因组重排后融合子和出发株的产酶情况,发现融合子在较短的时间内就可以产生大量的酶,并且其纤维素酶活和木聚糖酶活与出发株相比都有显著提高。进行5-L发酵罐实验时,融合子的纤维素酶合成更快,并且酶活进一步提高,但是在后期pH会上升到中性从而导致酶活迅速下降,不利于实际生产。为解决此问题,我们以融合子GS2-15为实验菌株优化了500ml摇瓶中缓冲剂的种类和剂量,其中以0.08%CaCO₃作为缓冲剂成本最低,效果最好。通过进行5-L发酵罐的验证实验,发现发酵液pH和酶活在整个发酵中后期保持稳定,滤纸酶活可以达到15.04 FPU ml^-1,达到国际先进水平;胞外蛋白浓度达到5.81mg ml^-1;纤维素内切酶最高为133.12 IU ml^-1;β-葡萄糖苷酶酶活最高为5.18 IU ml^-1;木聚糖酶酶活最高为411.34 IU ml^-1。6、β-葡萄糖苷酶高产菌株斜卧青霉Peni-1的β-葡萄糖苷酶基因的克隆,酶的纯化和酶学性质研究Peni-1是本实验室筛选到的一株β-葡萄糖苷酶高产的斜卧青霉菌株。我们克隆了Peni-1的β-葡萄糖苷酶的基因,基因含有5个内含子,基因编码区全长2586,编码861个氨基酸,与GS2-15相比有三个氨基酸序列的差异,分别是第2位的Lys→Arg,482位的Gly→Ser,489位的Val→Ile。纯化了Peni-1的β-葡萄糖苷酶,并研究了其酶学性质,其最适反应温度为70℃;在60℃保温12h后,酶活能保留80%以上;最适作用pH为5;比活为129.6 IU mg^-1;以水杨素为底物时Km值为2.6mmol L^-1;以pNPG为底物时Km值为0.2mmol L^-1。与GS2-15的胞外蛋白SDS-PAGE相比,Peni-1有些蛋白条带消失,有些蛋白条带表达加强。Peni-1的β-葡萄糖苷酶本身性质变化不大,所以我们推测Peni-1可能是由于β-葡萄糖苷酶合成调控机制发生了变化,导致可以分泌表达大量的β-葡萄糖苷酶蛋白。7、斜卧青霉滤纸酶活与β-葡萄糖苷酶的最适比例的确定配比不同体积的GS2-15和Peni-1的粗酶液,使滤纸酶活与β-葡萄糖苷酶比例为:1:1、1:2、1:3、1:4、1:5和1:6,并通过比较混合前后滤纸酶活的变化,初步推断滤纸酶活与β-葡萄糖苷酶的最适比为1:1.89,现有的斜卧青霉纤维素酶系组成需要进一步的调整。更多还原

【Abstract】 With the concerns of increasing energy demands,consuming away of fossil fuels, and environment pollution,the search for sustainable supplies of energy and renewable materials are becoming more and more important in the coming years. About 35%-50%dry weight of plant are cellulose,which are the most abundant renewable resource in the world.Available cellulosic feedstock from agriculture and other sources can be transformed to the fermentable syrup composed of polysaccharides and monosaccharides which can be fermented to the clean liquid biofuels and other value-added products alternative to fossil fuels,becoming the research focus of many countries.Generally,the biorefinery from lignocellulose to bioethanol is envisioned to comprise four major sections:pretreatment of feedstock, production of cellulase,enzymatic hydrolysis,and sugar fermentation to ethanol by Saccharomyces cerevisiae,and recover of ethanol.As a key problem for production on commercial scale,conversion of cellulosic biomass requires using large amounts of cellulases,which makes the process costly.Penicillium decumbens JU-A10,a catabolic-repression resistant mutant from the wild strain P.decumbens 114-2 suffered several rounds of mutagenesis,could produce abundant cellulase and hemicellulase,which were enzyme complexes hydrolyzing lignocelluloses.But the productions and activities of cellulase were not sufficient for industrial demands,and the strain needed to be further improved.As the genetic background of P.decumbens is not clear yet and there is no suitable genetic tool,we still have difficulties in designing rational methods for its cellulase improvement.Genome shuffling has been demonstrated as an effective method for the rapid improvement of cellular phenotypes in recent years.In this paper,genome shuffling was used to improve cellulase activities of P.decumbens JU-A10,and the main results were as follows:1.Establishment of an effective two-layer cellulose screening plate The ingredients in the two-layer cellulose screening plate were optimized.To detect halos of cellulose hydrolysis and improve efficiency of each screening plate, the upper layer was supplemented with sodium deoxycholate,reaching a final concentration of 0.2%to restrict colonies extension.The ball-milled microcrystalline cellulose and glucose were used as selection pressure to choose the mutants out,and the volume of each upper medium is 7 ml.The cellulase activity was mainly positive related to the size of cellulose hydrolysis halo in the plate.JU-A10 could only form cellulose hydrolysis halo in the two-layer plate containing 2%ball-milled microcrystalline cellulose and less than 1%glucose.The mutants with better cellulase production or resistance to catabolite repression could be selected when the concentration of ball-milled microcrystalline cellulose and glucose gradually increased during screening.2.The protocols of protoplast preparation,protoplasts regeneration and protoplasts fusion of double parents inactivatedProtoplasts were prepared from the mycelia harvested after 24-h culture in mycelia culture medium.Protoplasts were isolated using an enzyme combinations composed of 4 mg ml^-1 Snailase,4 mg ml^-1 cellulase,and 2 mg ml^-1 Lywallzyme in pH 6.5 citric acid buffer,supplemented with 0.6 M NaCl as an osmotic stabilizer.A maximum number of 10⁶/ml protoplasts were obtained when treated at 35℃for 1 h. The final population of protoplasts in total cells was 95%,and a high regeneration ratio of 90%was occurred on the regeneration medium.The parent protoplasts purified could lose their ability to regenerate after irradiation of UV for 30 minutes or incubation at 50℃for 50 minutes.The protoplasts were gathered together after the previous two kinds of inactivation,and fused in the system composed of 40%PEG;35℃,10 mM Ca²⁺.Large numbers of multi-cellular aggregates were observed in the fusion system described above under phase contrast microscope.Although the parent cells might be nonviable after inactivation treatment, they could form viable recombinants when fused with the protoplasts treated by another method.3.Improvement of the ability to produce cellulase after two rounds of genome shufflingFour mutants were selected after primary two-layer screening and secondary fermentation:UE-5 and UE-6 were obtained after UV and EMS mutagenesis;NII-5 and NII-8 were chosen after 30 key N⁺ ion implantation.The initial parents’ pool was composed of four developed mutants with different advantages respectively.Two successive rounds of protoplast fusion were carried out,and after each round,the concentration of ball-milled cellulose or glucose in the plates used for selection was increased.The four mutants were used as parents of the first round for genome shuffling,then the colonies quickly regenerating and forming cellulose hydrolysis halos on screening plate SM2（2%ball-milled cellulose,2%glucose） were chosen for further fermentation assay.The 6 strains with further improvements were selected as the parents for the second round of shuffling.After screening on plates SM3（5% ball-milled cellulose,2%glucose） and secondary fermentation assay,three fusants: GS2-15,GS2-21,GS2-22 with significant improvement were acquired.They could produce cellulase as much as 190%of the parent strain JU-A10.4.The reasons why the fusants GS2-15,GS2-21,GS2-22 produce more cellulaseFirstly,some morphological characteristics of the fusants changed during the process of genome shuffling.The width of mycelia and the colony diameter of the fusants were smaller than that of JU-A10,while the volume of spores became larger. The mycelia of the fusants were more easily fragmented in the late fermentation stage （90 h） comparing with JU-A10.There may be somewhat relationship between the improved cellulase production and morphological changes,and it is probably that the easy fragmentation of mycelia is helpful to the release of cellulase,and the work is underway in our lab now.Secondly,the randomly amplified polymorphic DNA（RAPD） system in P. decumbens was established after detailed optimization of reaction parameters.Most of RAPD bands from the three fusants were the same with JU-A10,but some of them were distinct.The genetic similarity varied from 0.842 to 0.905 indicating variability among the three fusants and JU-A10.The dendrogram showed three distinct clades by the genetic similarity 0.87,the first containing JU-A10 and GS2-22,the second and the third containing GS2-15 and GS2-21,respectively.The present work shows the usefulness of RAPD molecular markers for genetic characterization to establish phylogenetic relations,and RAPD would therefore be a suitable tool to discriminate the diversity of the fusants and parent strain.Thirdly,the extracellular and intracellular cellulase,protein concentration,and biomass when they were cultured with the corncob residue as carbon sources were studied.The fusants had robust growth,higher protein concentration and cellulase activities compared with JU-A10.Interestingly,the fusants synthesized sharply intracellular cellulase in very early fermentation stage.The improvements of the fusants were possibly due to their enhanced growth rates,earlier cellulase synthesis and higher secretion of extracellular proteins.Fourthly,the fusants had better performances on the liquid fermentation culture containing 2%glucose as sole carbon source.They consumed the glucose more quickly,and the biomass and cellulase activities were also higher,comparing to JU-A10.The SDS-PAGE and CMCase activity staining showed that the fusants had their own new special protein bands,although in despite of most of the protein profiles were nearly the same as the JU-A10.Some protein bands showing CMCase activity expressed stronger.These differential protein patterns suggested that extracellular protein of the fusants had changed.5.Cellulases production of the fusants using lignocellulosics as carbon source and in 5-L fermentorThe cellulase production of the fusants and JU-A10 were investigated with corn stover,wheat straw,and bagasse as carbon sources.The fusants could synthesize more enzymes earlier,and both cellulases and xylanases were observably higher than that of JU-A10.The pH value is an important factor effecting cellulase production and is very important to keep the enzyme stability during the fermentation.In 5-L fermentor assay, the fusants produced cellulase more quickly,and the cellulase activities were further improved compared to that in 500-ml flask.However,the pH value in late fermentation stage rose up nearly to 7,resulting in losing much of enzymatic activities, which is not beneficial to industrial production.The performances of several kinds of buffering agent were tested in 500-ml flask by the fusant GS2-15,and the performance with addition of 0.08%CaCO₃ was the best one.The pH value and enzymatic activities were very stable in late fermentation stage in 5-L fermentor by GS2-15,and the highest FPase activity reached 15.04 FPU ml^-1,the concentration of extracellular reached 5.81 mg ml^-1,the CMCase activity reached 133.12 IU ml^-1,β-glucosidase reached 5.18 IU ml^-1,and xylanase activity reached 411.34 IU ml^-1.6.Cloning of aβ-glueosidase gene from P.decumbens Peni-1,and purification and characterization of theβ- glucosidaseAβ-glucosidase gene（bgl） in Peni-1 was cloned by PCR and RT-PCR.DNA sequencing results showed that bgl had an open reading frame of 2586 bp with 5 introns and encoded a polypeptide of 862 amino acids.There were three amino acids different from the fusant GS2-15:Lys→Arg（the second site）,Gly→Ser（the 482 site）, Val→Ile（the 489 site）.The protein ofβ-glucosidase was purified by gel filtration chromatography,and enzymatic characteristics were studied.The thermal stability of the enzyme purified was determined,and about 80%of enzyme activity remained after incubation at 60℃for 12 h.The optimum reaction temperature is 70℃.The pH optima with salicin as substrates were determined as 5.0.The Km ofβ-glucosidase toward salicin was 2.6 mmol L^-1,and was 0.2 mmol L^-1 toward pNPG.There were some differences between the extracellular protein profiles of Peni-1 and GS2-15.The regulation mechanism ofβ-glucosidase synthesis in Peni-1 might be different,which make it producing moreβ-glucosidase.7.The determination of best ratio of FPA andβ-glucosidaseEnzymatic broth from Peni-1 and GS2-15 were mixed in order to make the final ratio of FPA andβ-glucosidase at 1:1,1:2,1:3,1:4,1:5 and 1:6,and enzymatic activities before or after mixture were tested.The best ratio of FPA andβ-glucosidase was found to be 1:3 in P.decumbens cellulase system.Consequently,the cellulase system of P.decumbens IU-A10 or GS2-15 was still not optimal enough.This offered theoretical evidence for further strain improvement.更多还原