1株红树林来源枝孢属真菌的分离鉴定及纤维素酶性质分析

利用水解圈法从三亚红沙河红树林区分离得到1株纤维素降解真菌SCSIO 43503。分子生物学鉴定表明,该菌ITS序列与菌株Ochrocladosporium frigidarii CZ549(FJ755255)的相似度为96%,β微管蛋白(β-tubulin)序列与Shiraia bambusicola(AB355003)的相似性为86%,钙调蛋白(calmodulin)与Paramyrothecium viridisporum CBS 873.85(KU846278)序列相似性为89%;综合其形态学特征,推测其为枝孢属(Gladaxporism)真菌,命名为Gladaxporism sp.SCSIO 43503(KY224732)。进一步对该菌株分泌的纤维素酶进行分析,结果显示该菌株维素酶活性的最适反应温度为50℃,在45~50℃范围内活性较高;最适pH为5.0,在3.0~7.0范围内具有较高的活性,最适发酵时间为3 d。在最优条件下,酶活力达到最高,为23.46 U。本研究发现了一株纤维素降解真菌,对纤维素酶的生产和利用具有重要价值。     

贵州地区茯砖茶“金花菌”的分离和分子鉴定

本研究对贵州地区茯砖茶中的"金花菌"进行了分离鉴定。通过PCR扩增及测序技术获得转录间隔区序列及部分核糖体大亚基(ITS+LSU)、β-微管蛋白(β-tubulin)、钙调蛋白(Ca M)和RNA聚合酶Ⅱ(RPB2)4个基因的序列,然后构建多基因系统树,并对其形态特征进行观察,将其鉴定为冠突曲霉Aspergillus cristatus。     

东方肉座菌EU7-22纤维素酶基因的克隆及序列分析

东方肉座菌EU7-22与XC-9、里氏木霉、康宁木霉、黑曲霉、斜卧青霉进行产纤维素酶比较,结果表明菌株EU7-22具有较高的产纤维素酶能力及完整的纤维素酶系.根据里氏木霉和绿色木霉的外切葡聚糖酶,内切葡聚糖酶及β-葡萄糖苷酶相关基因序列,设计引物PCR扩增出菌株EU7-22 cbhⅠ、cbhⅡ、egⅠ、egⅡ及bgl Ⅰ.基因序列经NCBI Blast分析表明,cbhⅠ与绿色木霉cbh1基因(FJ871063)同源性最高达99％;cbhⅡ与康宁木霉cbh2基因(DQ504304)同源性最高达99％;eg Ⅰ与长枝木霉egl基因(GU144298)同源性最高达99％;egⅡ与绿色木霉eg2基因(EF602036)同源性最高达99％;bglⅠ与菌株Trichoderma sp.SSL bgl基因(FJ040193)同源性最高达100％.5种纤维素酶基因编码的相应氨基酸序列与其他木霉纤维素酶的氨基酸序列相似性也非常高.对上述纤维素酶基因编码的相应蛋白的分子量、等电点、N-糖基化位点、信号肽序列进行分析;对纤维素结合区及糖基水解酶家族特征结构区进行了定位;用SWISS-Model模拟了酶蛋白的三级结构.     

一株耐冷玫红假裸囊菌HD1031的鉴定及其所产纤维素酶的研究

[目的]以纤维素为唯一碳源,从四川省阿坝自治州黄龙沟的高山低温环境中分离筛选产纤维素酶的耐冷菌,并研究菌株的产酶特征.[方法]根据菌株的ITS序列分析及形态特征,对菌株进行鉴定.利用DNS法测定纤维素酶酶活性.[结果]从四川省阿坝自治州黄龙沟的高山腐殖土中筛选出一株产纤维素酶的耐冷菌HD1031,经鉴定该菌为玫红假裸囊菌(Pseudogymnoascus roseus).该菌可在4℃-25℃生长,最适生长温度为16℃-17℃.该菌在以微晶纤维素和玉米芯粉为碳源、硫酸铵和Tryptone为氮源的培养基中,17℃、160 r/min摇瓶发酵8d后产生纤维素酶,其中内切葡聚糖酶酶活为366.67 U/mL,滤纸酶酶活87.6 U/mL,β-葡萄糖苷酶酶活90.8 U/mL,酶最适反应pH为6.0,最适反应温度为50℃.[结论]筛选获得一株产纤维素酶的耐冷菌HD1031,此菌株所产纤维素酶在20℃-40℃下活性较高,对热敏感,具有低温纤维素酶的特点.     

日耳曼曲霉(Aspergillus germanicus)—曲霉属焦曲霉组一个我国新记录种

【目的】鉴定并报道我国一个曲霉新纪录种,即日耳曼曲霉。【方法】采用形态学性状及钙调蛋白和β-微管蛋白基因部分序列及核糖体DNA ITS1-5.8S-ITS2序列进行多相系统分类学分析。【结果】根据与日耳曼曲霉Aspergillus germanicus模式菌株CBS 123887的形态学和分子性状的比较分析,两株分离自中国山东泰山地区土壤的曲霉菌(AS3.15303和AS3.15304)被鉴定为日耳曼曲霉A.germanicus。【结论】参考我国迄今已报道的曲霉物种,确定日耳曼曲霉A.germanicus是曲霉属焦曲霉组的一个我国新记录种。     

黑茶砖茶中两种产生“金花”的曲霉菌

"金花"是曲霉属Aspergillus真菌在黑茶后发酵过程中,在砖茶内部形成的黄色闭囊壳。从广西和湖南产的黑茶砖茶中分离获得2株"金花菌",均能在培养基上形成闭囊壳。根据分离菌株的培养特征和微观形态特征及β-微管蛋白基因(Ben A)、钙调蛋白基因(Ca M)及RNA聚合酶Ⅱ基因(RPB2)的系统发育分析,参照Hubka最新的曲霉属曲霉组Aspergillus section Aspergillus分类系统,将分离菌株分别鉴定为假灰绿曲霉A.pseudoglaucus及冠突曲霉A.cristatus。另外,通过扫描电镜对这2株"金花菌"进行了形态观察,并记录了菌株A672闭囊壳的发育过程。在广西产砖茶中分离鉴定出的"金花菌"假灰绿曲霉为国内首次报道。通过比较,将过去湖南产砖茶中广泛报道的"金花菌"冠突散囊菌修订为冠突曲霉A.cristatus。黑茶茶砖中"金花菌"的分离与鉴定对于黑茶品种的鉴别和质量评价具有重要指导意义。     

水稻恶苗病菌(Fusarium fujikuroi)β-微管蛋白基因克隆及与多菌灵抗药性关系

【目的】揭示水稻恶苗病菌(Fusarium fujikuroi)对多菌灵的抗药性与其β-微管蛋白基因的相关性。【方法】结合形态学和TEF-1α基因序列对分离菌株进行鉴定;根据近源种拟轮枝镰孢菌(Fusarium verticillioides)核基因组测序菌株7600的β-微管蛋白核苷酸序列设计引物,采用PCR方法克隆并比对分析了F.fujikuroi对多菌灵不同敏感性表型的5个菌株的β-微管蛋白基因全序列;利用实时定量技术(qRT-PCR)分析了β-微管蛋白基因在上述5个菌株中的表达特性。【结果】F.fujikuroi的β-微管蛋白基因核苷酸序列(GenBank登录号:JQ026022)全长1671 bp,包含4个内含子,编码447个氨基酸残基;2个敏感性菌株和3个抗药性菌株的β-微管蛋白基因核苷酸序列同源性100%;在无药剂处理下该基因在2个敏感性菌株中的表达水平显著高于3个抗药性菌株(p=0.05),且对同一菌株而言,药剂处理能够显著提高β-微管蛋白基因表达水平(p=0.05),但在相同药剂处理条件下,菌株间差异不显著。【结论】F.fujikuroi对多菌灵的抗药性机制与β-微管蛋白无关,有待进一步研究。     

评价ITS、BenA和CaM序列分析在曲霉菌种鉴定方面的应用

目的评价内转录间隔区(ITS)、β-微管蛋白基因(BenA)和钙调蛋白基因(CaM)序列分析技术对曲霉的鉴定能力。方法对169株曲霉临床分离株分别进行ITS、BenA和CaM序列测定,并在Genbank数据库中进行比对分析以获得其菌种鉴定信息。结果 169株曲霉经ITS、BenA和CaM序列分析分别有52.7%、66.3%、97.6%菌株鉴定至种水平,47.3%、33.7%、2.4%菌株鉴定至属水平,3个序列对曲霉均不存在无法鉴定情况。结论 ITS、BenA和CaM均可用于曲霉菌种鉴定,其中以CaM的鉴定能力最强。     

类开费曲霉Aspergillus keveioides——曲霉属焦曲霉组一新种

报道分离自中国山东泰山土壤的曲霉属焦曲霉组的一个新种——类开费曲霉Aspergillus keveioides.基于形态学及钙调蛋白、β-微管蛋白基因部分序列和核糖体DNAITSl-5.8S-ITS2序列分析的证据支持新种的建立.     

禾谷镰孢菌β-微管蛋白基因克隆及其与多菌灵抗药性关系的分析

应用3对引物,从禾谷镰孢菌(Gibberella zeae)对多菌灵(MBC)的敏感菌株(MBC^R)和田间及室内诱导抗药性菌株(MBC^R)中扩增β-微管蛋白基因。该基因全长1631bp,包含3个内含子,编码447aa,与其他常见植物病原丝状真菌β-微管蛋白基因的氨基酸同源性达95．12％～99．30％。MBC^R和MBC^R菌株核苷酸序列分析表明,MBCR菌株未发生任何位点的突变,说明G．zeae对MBC的抗药性机制并非像其他丝状真菌一样由β-微管蛋白198位氨基酸突变所致。     

Correlation between X-ray diffraction measurements of cellulose crystalline structure and the susceptibility to microbial cellulase

Chemical and physical treatments of cotton cellulose have been studied in order to elucidate the relationship between the degree of crystallinity of cellulose and the susceptibility of cellulose to cellulase. Cotton cellulose powder was treated with the following solvents: 60% H₂SO₄, Cadoxen, and DMSO-p -formaldehyde. The dissolved celluloses were recovered at high yield of over 97% by addition of nine volumes of cold acetone. X-ray diffraction for measurements of relative crystallinity showed that the crystalline structure of cellulose declined in quantity and perfection by the dissolving treatment and changed to an amorphous form that is highly susceptible to enzymatic hydrolysis. These reprecipitated celluloses were hydrolyzed almost completely within 48 hr by Aspergillus niger cellulase containing mainly 1,4-β-glucan glucanohydrolase (EC 3.2.1.4), without action of 1,4-β-glucan cellobiohydrolase (EC 3.2.1. 91). On the other hand, cryo-milled cellulose (below 250 mesh) still had a crystalline structure, was resistant to cellulase, and gave a low percentage of saccharification. These results indicate that in pure cellulose there are good correlations between x-ray diffractograms and susceptibility to microbial cellulase.     

Effects of substrate pretreatment and water activity on lipase-catalyzed cellulose acetylation in organic media

Lipase-catalyzed acetylation of cellulose solubilized in the dimethyl sulfoxide/paraformaldehyde organic solvent system was conducted with lipase A12 from Aspergillus niger. The accompanying side cellulase activity of the A. niger lipase partly accounted for the enhanced acetylation mediated by the enzyme, via facilitating the partial degradation of cellulose substrate as evidenced by high-performance size exclusion chromatograph analysis. The enzymatic cellulose acetylation was improved by substrate pretreatment with cellulase or ultrasound by 18 and 14%, respectively, as a result of the reduced substrate molecular size. Additionally, the ultrasound-pretreated cellulose as the starting substrate was beneficial for the cellulose solution preparation due to the increased accessible surface of cellulose as evidenced by its increased sedimentation volume and SEM micrographs. The effect of thermodynamic water activity (aw) on lipase catalytic activity in organic media was also investigated. The maximum acetylation extent (nearly 11 wt %) occurred at aw = 0.52, which was improved by 51% relative to the enzymatic reaction with no control of water activity. The much larger extent to which the lipase-catalyzed cellulose acetylation was enhanced by water activity optimization than by substrate pretreatment further supported the predominant role played by the major lipase activity of the A. niger lipase over its side cellulase activity in catalyzing cellulose ester synthesis in organic media.     

超临界CO2流体对纤维素酶催化反应的影响

超临界二氧化碳流体预处理对纤维素超分子结构及纤维素酶催化反应有重要影响。一定含水量的微晶纤维素用SC-CO2在10MPa,50℃处理30min,其结构发生了有利于进一步被酶解的变化。上述超临界条件单独作用于纤维素酶时,并未造成酶催化活力的降低;但与纤维素共同进行SC—CO2处理时,纤维素酶则失去催化活性,但这种处理却能提高纤维素进一步被酶解的效率。一定范围内处理时的酶用量与酶解效率的增加正相关。纤维素的含水量对SC-CO2处理后的酶解效率有显影响。     

Engineering Aspergillus oryzae A-4 through the Chromosomal Insertion of Foreign Cellulase Expression Cassette to Improve Conversion of Cellulosic Biomass into Lipids

A genetic modification scheme was designed for Aspergillus oryzae A-4, a natural cellulosic lipids producer, to enhance its lipid production from biomass by putting the spotlight on improving cellulase secretion. Four cellulase genes were separately expressed in A-4 under the control of hlyA promoter, with the help of the successful development of a chromosomal genetic manipulation system. Comparison of cellulase activities of PCR-positive transformants showed that these transformants integrated with celA gene and with celC gene had significantly (p<0.05) higher average FPAase activities than those strains integrated with celB gene and with celD gene. Through the assessment of cellulosic lipids accumulating abilities, celA transformant A2-2 and celC transformant D1-B1 were isolated as promising candidates, which could yield 101%–133% and 35.22%–59.57% higher amount of lipids than the reference strain A-4 (WT) under submerged (SmF) conditions and solid-state (SSF) conditions, respectively. Variability in metabolism associated to the introduction of cellulase gene in A2-2 and D1-B1 was subsequently investigated. It was noted that cellulase expression repressed biomass formation but enhanced lipid accumulation; whereas the inhibitory effect on cell growth would be shielded during cellulosic lipids production owing to the essential role of cellulase in substrate utilization. Different metabolic profiles also existed between A2-2 and D1-B1, which could be attributed to not only different transgene but also biological impacts of different integration. Overall, both simultaneous saccharification and lipid accumulation were enhanced in A2-2 and D1-B1, resulting in efficient conversion of cellulose into lipids. A regulation of cellulase secretion in natural cellulosic lipids producers could be a possible strategy to enhance its lipid production from lignocellulosic biomass.     

大熊猫肠道纤维素分解菌的分离鉴定及产酶性质

【目的】从健康大熊猫新鲜粪便中分离具有纤维素酶活性的菌株,并对其进行菌种鉴定及产酶性质研究。【方法】利用羧甲基纤维素钠培养基分离纯化具有较高纤维素酶活性的菌株,根据形态学特征、生理生化特性以及16S rDNA分析对其进行分类鉴定,研究影响该菌株纤维素酶的产酶条件,以及对不同纤维素底物的降解情况。【结果】分离得到一株纤维素酶产生菌株P2,该菌株为好氧的革兰氏阳性细菌,生长温度范围20-50℃(最适温度37℃),pH范围6.0-9.0(最适pH7.0),NaCl浓度范围0%-15%(最适2%NaCl),培养24h达到产酶高峰。16S rDNA基因序列分析显示,菌株P2与解淀粉芽胞杆菌(Bacillusamyloliquefaciens)NBRC15535相似性为99.66%。该菌株对四种纤维素底物(滤纸、脱脂棉、秸秆、竹纤维)均有不同程度的降解,内切葡聚糖酶、外切葡聚糖酶、β-葡萄糖苷酶和总酶活具有不同的酶活变化。【结论】本研究首次从大熊猫粪便中分离出了好氧纤维素分解菌,并鉴定为解淀粉芽胞杆菌,对上述四种纤维结构均有一定的破坏和分解作用,为进一步研究大熊猫竹纤维消化机制提供了菌源。     

Expression, purification and characterization of two thermostable endoglucanases cloned from a lignocellulosic decomposing fungi Aspergillus fumigatus Z5 isolated from compost

Two genes encoding endoglucanase, designated as egl2 and egl3, were cloned from a lignocellulosic decomposing fungus Aspergillus fumigatus Z5 and were successfully expressed in Pichia pastoris X33. The deduced amino acid sequences encoded by egl2 and egl3 showed strong similarity with the sequence of glycoside hydrolase family 5. SDS-PAGE and western blot assays indicated that the recombinant enzymes were secreted into the culture medium and the zymogram analysis confirmed that both recombinant enzymes had endoglucanase activity. Several biochemical properties of the two recombinant enzymes were studied: Egl2 and Egl3 showed optimal activity at pH 5.0 and 4.0, respectively, and at 50 and 60°C, respectively. Egl2 and Egl3 showed good pH stability in the range of 4-7, and both enzymes demonstrated good thermostability ranging from 30 to 60°C. The K(m) and V(max) values using carboxymethyl cellulose (CMC, soluble cellulose, polymerized by β-1, 4-linked glucose residues) as the substrate at optimal conditions were determined. The activities of the enzymes on a variety of cello-oligosaccharide substrates were investigated, and Egl2 can hydrolyze cellotetraose and cellopentaose but not cellobiose and cellotriose, whereas Egl3 can hydrolyze all cello-oligosaccharides, except cellobiose.     

Fungi isolated from olive ecosystems and screening of their potential biotechnological use

This study investigated the fungi diversity of fresh olive (Olea europaea L.) fruits, olive paste (crushed olives) and olive pomace (solid waste) and screened and quantified enzymatic activities with biotechnological applications. Fungi were randomly isolated from olive cultivars from Castilla La Mancha region (Spain). Identification included comparison of their polymerase chain reaction (PCR) amplicons of the ITS1-5.8S-ITS2 ribosomal DNA region, followed by nucleotide sequence analysis. Fourteen different species with DNA sequences of different similarities were identified, belonging to seven different genera (Aspergillus, Penicillium, Rhizomucor, Mucor, Rhizopus, Lichtheimia and Galactomyces). Aspergillus fumigatus, followed by Galactomyces geotrichum, Penicillium commune and Rhizomucor variabilis var. regularior were the most frequent species. Specific enzyme screening was assayed on agar plates, using cellobiose, carboxymethylcellulose (CMC), polygalacturonic acid and CaCl(2)/Tween 80 as substrates for β-glucosidase, carboxymethylcellulase (CMCase), polygalacturonase and lipase, respectively. Species exhibiting the best activities were: Aspergillus fumigatus (for β-glucosidase, CMCase and lipase); Rhizopus oryzae (for β-glucosidase and lipase); Rhizomucor variabilis (for β-glucosidase, CMCase and polygalacturonase); Mucor fragilis (β-glucosidase, CMCase and lipase); Galactomyces geotrichum (for β-glucosidase, polygalacturonase and lipase) and Penicillium commune and Penicillium crustosum (for lipase). The species that had shown the best enzymatic activities were grown on hemicellulose, cellulose and pectin and some activities were quantified (xylanase, cellulase, β-glucosidase and pectinase). An isolate of A. fumigatus and one of A. niger showed the best cellulase and xylanase activities, while no species presented good pectinase and β-glucosidase activities. The selected species with potential enzymatic activities could be used for future applications of industrial interest.     

DNA sequences of three beta-1,4-endoglucanase genes from Thermomonospora fusca.

The DNA sequences of the Thermomonospora fusca genes encoding cellulases E2 and E5 and the N-terminal end of E4 were determined. Each sequence contains an identical 14-bp inverted repeat upstream of the initiation codon. There were no significant homologies between the coding regions of the three genes. The E2 gene is 73% identical to the celA gene from Microbispora bispora, but this was the only homology found with other cellulase genes. E2 belongs to a family of cellulases that includes celA from M. bispora, cenA from Cellulomonas fimi, casA from an alkalophilic Streptomyces strain, and cellobiohydrolase II from Trichoderma reesei. E4 shows 44% identity to an avocado cellulase, while E5 belongs to the Bacillus cellulase family. There were strong similarities between the amino acid sequences of the E2 and E5 cellulose binding domains, and these regions also showed homology with C. fimi and Pseudomonas fluorescens cellulose binding domains.