海洋微生物产纤维素酶及其应用研究进展

纤维素是地球上最古老、最丰富的天然高分子,是天然可再生资源。纤维素酶广泛存在于自然界的生物体中,细菌、真菌和动物体内都能产生纤维素酶。微生物产纤维素酶已有较多报道,并在食品、医药、饲料、洗涤、纺织和造纸工业等领域有广阔的应用前景。海洋是一个巨大的资源库,海洋微生物产纤维素酶已经受到了广泛的关注。对产纤维素酶海洋微生物的种群、来源及基因筛选、海洋微生物产纤维素酶的酶学特性,以及纤维素酶的应用领域等方面的研究进展进行了简要综述,并对海洋微生物产纤维素酶的研究进行了展望。     

土壤和空气中纤维素酶高产菌株的筛选

目的:分别从土壤和空气中分离纤维素分解菌株,以提高纤维素利用率。方法:将分离得到的菌株进行纯培养,并用刚果红染色,找出能出现透明圈的菌株,测定透明圈直径与菌落直径比,筛选产纤维素酶的菌株。结果:通过对比,从分离到的6株菌株中选出产纤维素酶活力高的2株,并对其进行鉴定,初步确定一株为曲霉属真菌,一株为放线菌。结论:本研究为提高纤维素利用率提供了微生物资源,为相关后续研究提供了物质和实验基础。     

一株降解纤维素放线菌的产纤维素酶基因克隆与表达

【背景】纤维素在自然界中储量丰富,但天然纤维素的难降解性成为广泛应用纤维素资源的壁垒,近年来利用微生物来降解纤维素成为热点研究。【目的】筛选分离得到一株具有降解纤维素功能的放线菌菌株Lb1,通过全基因组测序确定其产纤维素酶关键基因5676,对基因5676进行克隆转化,使其在大肠杆菌中进行表达。【方法】通过基因工程技术将产纤维素基因连接到表达质粒上并导入表达菌株,对其降解纤维素生成葡萄糖的能力进行探究。【结果】将Lb1菌株的16S rRNA基因进行比对,确定菌株Lb1属于链霉菌属,命名为Streptomyces sp. Lb1。成功构建出纤维素酶表达载体,并且导入表达菌株大肠杆菌BL21(DE3),重组菌株的产纤维素酶能力大于空载菌株。【结论】通过基因工程技术成功克隆出产纤维素酶基因,从而表达纤维素酶,为今后利用微生物降解纤维素的大规模应用提供参考。     

从白蚁中分离到具有纤维素酶活的贪噬菌

以黄胸散白蚁Reticulitermes flaviceps后肠为材料,分离培养具有降解纤维素能力的微生物,以进一步了解白蚁后肠微生物的种类。通过以羧甲基纤维素钠(CMC-Na)为唯一碳源的富集及选择培养基培养、筛选,获得一株具有纤维素酶活的菌株R3063。形态学鉴定、革兰氏染色观察及16S rDNA基因序列分析表明该菌株属于贪噬菌(Variovorax sp.)。目前尚未见贪噬菌具有纤维素酶活的报道。     

白蚁栖息环境中蜡状芽孢杆菌的分离及其纤维素酶活性分析

【目的】了解白蚁栖息环境中有无降解纤维素的微生物。【方法】以羧甲基纤维素钠为唯一碳源,利用刚果红染色,根据透明圈大小进行筛选。通过显微形态、革兰氏染色及16S rRNA基因序列分析对菌株进行鉴定。DNS法测定菌株产纤维素酶与生长周期的关系,并进一步分析纤维素酶性质。【结果】从台湾乳白蚁(Coptotermes formosanus Shiraki)栖息环境中筛选到一株具有较高纤维素酶活性,革兰氏阳性菌株TT15,16S rDNA序列分析鉴定为蜡状芽孢杆菌(Bacillus cereus Gd2T)。菌株培养前12 h没有纤维素酶活性,随着培养时间的增加,纤维素酶活性逐渐增大;当生长达到稳定期(48 h),酶活性达到最大并保持稳定。菌株TT15纤维素酶活性的最适pH和最适反应温度分别为5.0和50°C。【结论】从白蚁栖息环境中分离到一株具有较高纤维素酶活的蜡状芽孢杆菌TT15,可作为产细菌纤维素酶的优良菌株。     

产木质纤维素降解酶真菌的筛选及产酶特性

【背景】利用微生物处理秸秆引起研究者的广泛关注。【目的】筛选生长速度快、木质纤维素降解酶活性强的真菌菌株,用于植物秸秆降解和高效利用。【方法】从自然界采集的样品中分离纯化真菌菌株,利用PDA-愈创木酚和PDA-羧甲基纤维素钠平板初筛,再经过液体发酵检测漆酶酶活、羧甲基纤维素酶酶活及菌丝生长速率复筛目的菌株,通过内转录间隔区(internal transcribed spacer,ITS)测序法对目的菌株进行鉴定,对目的菌株产漆酶和羧甲基纤维素酶活力进行测定及酶学性质研究。【结果】从样品中分离纯化到18株真菌,通过初筛筛选出9株产木质纤维素降解酶真菌菌株,再经过复筛,筛选出一株产漆酶、羧甲基纤维素酶活力高、菌丝生长快的菌株M1,经过分子生物学鉴定M1为糙皮侧耳(Pleurotus ostreatus),其漆酶酶活为(243.59±1.11)U/mL,羧甲基纤维素酶酶活为(36.03±0.63) U/mL。在5 d的培养期内,菌丝生长速率为(9.43±0.32) mm/d。对菌株M1的发酵粗酶液的酶学性质进行了检测分析,结果表明,所产的漆酶在pH5.0-6.5相对酶活为90%以上,在pH ...     

造纸废液氧化塘纤维素酶产生菌群的分析与产酶菌株的筛选

【目的】解析造纸废液氧化塘中产纤维素酶微生物的群体组成和结构;筛选并获得一批纤维素酶产生菌,丰富菌株资源,并为纤维素酶的工业应用和环境污染的生物处理奠定基础。【方法】基于16S rRNA基因序列信息,系统考察了造纸废液氧化塘环境中产纤维素酶细菌的群体组成和结构,并通过测定纤维素酶在不同pH条件下酶活变化考察所产纤维素酶的特性。【结果】造纸废液氧化塘中产纤维素酶微生物具有丰富的多样性。在分类上分属于Firmicutes、Actinobacteria、Alpha-proteobacteria和Gamma-proteobacteria 4个门(亚门)15种。来自泥液混合样和黑液排污口泥样的产纤维素酶细菌群体多样性最为丰富,由6-7个种的细菌组成;而来自强碱性的黑液下层样品中微生物的多样性则较为贫乏,主要由来自Bacillus类细菌组成。分离菌株除酸性纤维素酶产生菌外,碱性纤维素酶和中性纤维素酶产生菌也较为丰富,且其分布与样品来源有紧密的关系。【结论】对造纸废液氧化塘产纤维素酶微生物群体组成和结构的研究,不仅有利于对新菌株资源的挖掘,也可为特殊环境的微生物学研究提供参考。     

微生物酶解法产生阿拉伯糖——菌种筛选与初步鉴定

使用半纤维素平板法从土壤分离菌中筛选到产半纤维素酶活较高的真菌。将这些菌株在含玉米芯半纤维素的培养基中发酵,鉴定发酵液中还原糖产量,从中筛选出3株产还原糖较多的菌。将其发酵产物经柱前衍生,HPLC检测,发现其中DHC菌株的发酵产物以木糖和阿拉伯糖为主,而培养基中的葡萄糖大部分被利用,可用于发酵法联产阿拉伯糖和木糖。对DHC菌株通过菌落形态、孢子形态以及ITS区基因序列测序分析,初步确定该菌为亮白曲霉(Aspergillus candidus)。对于亮白曲霉产生的半纤维素酶国内外尚较少研究报导,可为开发利用新的微生物资源奠定基础。     

低温产纤维素酶菌株的筛选、鉴定及纤维素酶学性质

[目的]筛选一株低温产纤维素酶菌株并进行鉴定,初步探索其酶学性质,为微生物肥料生产筛选菌种资源.[方法]常温条件下,采用CMC-刚果红染色法初筛纤维素降解菌株.采用低温条件诱导的方法,筛选耐低温且产纤维素酶能力最强的菌株,经形态学、生理生化特征试验、ITS序列等方面分析系统分类地位.单因素试验确定温度、pH及金属离子对纤维素酶活力的影响.[结果]从秸秆还田土壤中分离出一株在13℃低温环境下高效分解纤维素的真菌M11,鉴定M11为草酸青霉(Penicillium oxalicum).发酵试验表明:以玉米秸秆粉为唯一碳氮源,13℃、200 r/min摇床发酵培养9d时,纤维素酶活力最高为33.08 U/mL.对其酶学性质初步研究表明:该酶最适pH为5.0,最适反应温度为20℃,在5℃-20℃间酶活力仍能保持在90％以上.[结论]Penicillium oxalicum M11是一株高效的纤维素降解菌株,在低温条件下可分泌纤维素酶且活性显著,具有潜在的开发价值.     

一株高产纤维素酶真菌的选育与鉴定

采用微生物常规分离筛选方法,以蘑菇培养基质、牛瘤胃内容物、落叶堆和腐木等为材料,从中分离筛选得到一株高产纤维素酶的真菌Z90;进一步对该菌株进行紫外诱变育种,获得产酶能力更强且遗传稳定的突变菌株Z90-19,突变菌株Z90-19产CMCase和FPA的活性分别达到11.61±0.17U·mL-1和2.15±0.14U·mL-1;对该菌株进行鉴定,确定菌株Z90为核茎点霉(Phoma putaminum)。     

海洋细菌Bacillus sp．HN07的分离鉴定及所产碱性纤维素酶性质研究

从渤海海域（121°30′00″E,40°25′00″N）海泥中筛选到一株产碱性纤维素酶的海洋细菌。通过形态观察、生理生化特性及16SrDNA序列分析,鉴定该菌株属于芽孢杆菌属（Bacillus）,命名为Bacillussp．HN07。研究表明：HN07最适生长温度30℃,适宜在pH7．0～8．0、含2．0％～3．0％NaCl的培养条件下生长和产酶,并且具有较好的遗传稳定性。酶学性质初步研究显示,HN07所产碱性纤维素酶最适反应温度为45℃,最适pH为8．0,在碱性条件下具有较好的稳定性,具有潜在的工业应用价值。     

Penicillium verruculosum WA 30 a new source of cellulase

Summary Of fungi 110 strains were screened for extracellular cellulase production in shake flask experiments. Twelve strains produced the enzyme in significant quantity. Since the enzyme activity was assayed by different methods (liberation of reducing sugar from cotton, filter paper, carboxymethylcellulose and cellobiose), the estimation of the productivity of the strains differed according to the substrate used. The best cotton degrading activity per fermentation volume as well as per mg of secreted soluble protein was achieved by Penicillium verruculosum WA 30, a wild-type strain, for which the cellulase productivity has not yet been published. The cotton degrading (so-called C₁) activity was successfully enhanced nearly threefold in medium experiments. Analyses of saccharification digests showed that glucose was the predominant product, with negligible amounts of cellobiose. The pH and temperature optima for WA 30 cellulase complex were pH 4.2 and 60°C.     

Cellulases from two Penicillium sp. strains isolated from subtropical forest soil: production and characterization

AIMS: To isolate new fungal strains from subtropical soils and to identify those that produce high cellulase activity. To select microbial strains producing thermostable cellulases with potential application in industry. METHODS AND RESULTS: The new strains Penicillium sp. CR-316 and Penicillium sp. CR-313 have been identified and selected because they secreted a high level of cellulase in media supplemented with rice straw. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focussing and zymography showed that the studied strains secreted multiple enzymes that hydrolyse cellulose. Cellulase activity of Penicillium sp. CR-316, the strain showing higher production, was analysed. Optimum temperature and pH of carboxymethyl cellulase activity were 65 degrees C and pH 4.5, respectively. Activity remained stable after incubation at 60 degrees C and pH 4.5 for 3 h. CONCLUSIONS: Fungal strains that secrete high levels of cellulase activity have been characterized and selected from soil. The isolated strains have complex sets of enzymes for cellulose degradation. Crude cellulase produced by Penicillium sp. CR-316 showed activity and stability at high temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: Two fungal strains with biotechnological potential have been isolated. The strains secrete high levels of cellulase, and one of them, Penicillium sp. CR-316, produces a thermostable cellulase, that makes it a good candidate for industrial applications.     

Mutants of the white-rot fungus Sporotrichum pulverulentum with increased cellulase and β-d-glucosidase production

This paper reports the isolation of mutants of the white-rot fungus Sporotrichum pulverulentum and the results of a survey of enzymic activity among these mutants. The strains were screened for extracellular cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) production in shake flask experiments. Apart from strain 63-2, strains 6, 63, 9, L5, E-1 and UV-18 showed equal or higher endo-1,4-β-d-glucanase (cellulase), filter paper-degrading and β-d-glucosidase activities than S. pulverulentum. The cellulase activity obtained, measured as filter paper activity, was comparable to that reported for Trichoderma reesei QM9414. However, the β-d-glucosidase activity was about six times higher than for the QM9414 strain. The pH and temperature-activity profiles of crude β-d-glucosidase preparations from the various strains were determined and were found to be identical. The thermal stability at pH 4.5 and 40°C was 5 days for all these preparations.     

Mutants of the white-rot fungus Sporotrichum pulverulentum with increased cellulase and β- -glucosidase production

This paper reports the isolation of mutants of the white-rot fungus Sporotrichum pulverulentum and the results of a survey of enzymic activity among these mutants. The strains were screened for extracellular cellulase [see 1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] and β- -glucosidase (β- -glucoside glucohydrolase, EC 3.2.1.21) production in shake flask experiments. Apart from strain 63-2, strains 6, 63, 9, L5, E-1 and UV-18 showed equal or higher endo-1,4-β- -glucanase (cellulase), filter paper-degrading and β- -glucosidase activities than S. pulverulentum. The cellulase activity obtained, measured as filter paper activity, was comparable to that reported for Trichoderma reesei QM9414. However, the β- -glucosidase activity was about six times higher than for the QM9414 strain. The pH and temperature-activity profiles of crude β- -glucosidase preparations from the various strains were determined and were found to be identical. The thermal stability at pH 4.5 and 40°C was 5 days for all these preparations.     

低温纤维素降解菌的筛选及其酶学性质初步研究

从青藏高原冰川雪样恢复出的4株细菌中筛选出1株降解纤维素能力比较高的菌株LHG-C-9。经16SrDNA序列分析,初步鉴定为假单胞菌属。对该菌所产纤维素酶的性质进行了初步研究,其最适作用温度为30℃;对热敏感;最适pH8．0;属碱性温酶。该低温纤维素酶在纺织、造纸、环保、医药和饲料等行业可望有广泛的应用前景。     

Isolation and partial characterization of novel genes encoding acidic cellulases from metagenomes of buffalo rumens

Aims:  To clone and characterize genes encoding novel cellulases from metagenomes of buffalo rumens.
Methods and Results:  A ruminal metagenomic library was constructed and functionally screened for cellulase activities and 61 independent clones expressing cellulase activities were isolated. Subcloning and sequencing of 13 positive clones expressing endoglucanase and MUCase activities identified 14 cellulase genes. Two clones carried two gene clusters that may be involved in the degradation of polysaccharide nutrients. Thirteen recombinant cellulases were partially characterized. They showed diverse optimal pH from 4 to 7. Seven cellulases were most active under acidic conditions with optimal pH of 5·5 or lower. Furthermore, one novel cellulase gene, C67-1, was overexpressed in Escherichia coli , and the purified recombinant enzyme showed optimal activity at pH 4·5 and stability in a broad pH range from pH 3·5 to 10·5. Its enzyme activity was stimulated by dl -dithiothreitol.
Conclusions:  The cellulases cloned in this work may play important roles in the degradation of celluloses in the variable and low pH environment in buffalo rumen.
Significance and Impact of the Study:  This study provided evidence for the diversity and function of cellulases in the rumen. The cloned cellulases may at one point of time offer potential industrial applications.     

一株低温玉米秸秆降解真菌的筛选、鉴定及降解特性

【背景】在我国北方地区玉米秸秆还田时期地温低、秸秆降解慢,如何加速玉米秸秆低温腐解成为研究热点。【目的】从冷凉地区土壤中筛选具有高效降解纤维素能力的低温菌株,为秸秆的有效利用奠定基础。【方法】在低温培养条件下,采用稀释涂布平板法、羧甲基纤维素钠(sodium carboxymethyl cellulose,CMC-Na)水解圈测定法、胞外酶活测定法、秸秆失重法进行低温秸秆降解菌株的初筛、复筛和秸秆降解性能的测定;根据菌株形态学特征及ITSrDNA序列分析对筛选菌株进行鉴定;利用3,5-二硝基水杨酸(3,5-dinitrosalicylic acid,DNS)法和秸秆失重法对菌株在不同接种量、培养基初始pH、温度情况下的纤维素酶活力和玉米秸秆降解能力进行研究。【结果】以16°C为筛选温度,获得一株在刚果红-羧甲基纤维素钠平板上D/d值为2.17、CMC酶活力为703 U/mL的高产纤维素酶低温真菌SDF-25;该菌株在4°C可以生长,10-16°C为最适生长温度,37°C条件下仍能生长;综合菌株的形态学和分子生物学测定结果,菌株SDF-25为草酸青霉菌(Penicillium oxalicum);该菌株最佳产纤维素酶的培养条件为接种量2%、初始pH为7.0、培养温度为10°C,在该培养条件下菌株SDF-25的CMC酶活为993.3 U/mL。失重法测定接种SDF-25于10°C培养15 d时秸秆降解率为39.5%,16°C时为44.9%。【结论】草酸青霉菌SDF-25可在低温条件下生长并具有较强的纤维素酶生产能力,在秸秆还田方面具有良好的应用前景。     

一株纤维素分解菌的筛选、鉴定及其对玉米秸秆的降解效果

为了筛选具有高效分解玉米秸秆纤维素能力的菌株,采集玉米秸秆还田土样作为样品,并于20℃条件下进行富集培养。利用以玉米秸秆纤维素为唯一碳源的固体分离培养基和刚果红染色法进行初筛,再将筛选到的菌株进行液体发酵培养并取上清液测定酶活,最终获得1株产纤维素酶能力较强的真菌SY-403。结合形态学特征与分子生物学鉴定结果得知,菌株SY-403为蓝状菌属（Talaromyces stollii）。对其所产纤维素酶酶学性质进行初步研究,结果表明,该酶最适反应pH为6.0,最适反应温度为20℃。在模拟室外条件（15℃）下进行秸秆降解试验,玉米秸秆经菌株SY-403处理40 d时,秸秆失重率及纤维素分解率分别达到42.67%、55.26%。利用傅里叶变换红外（Fourier transform infrared,FTIR）光谱技术对降解过程中官能团的变化进行分析,结果表明,纤维素相关谱峰（1 052~1 054 cm-1）相对强度减弱,而羟基相关谱峰（1 328~1 330 cm-1）相对强度增强,这说明纤维素已被分解为可利用的短链结构,即菌株SY-403可用于降解玉米秸秆。