低温纤维素酶菌株CNY086选育及发酵培养基优化(Ⅱ)

自渤海湾海泥中分离21株低温纤维素酶产生菌。其中菌株CNY01为绿色木霉(Trichoderma viride), 酶活力为67.30 U/mL。以该菌株为出发菌株, 经UV、DES等诱变, 选育出高产突变菌株CNY086, 酶活力为92.17 U/mL。该突变菌株低温纤维素酶发酵具有遗传稳定性。通过单因素和正交实验确定突变菌株CNY086低温纤维素酶发酵最适培养基: 秸秆粉1.20%、麸皮0.70%、硫酸铵0.50%、磷酸二氢钾0.55%, 上述条件下CNY086菌株酶活力达到108.55 U/mL。     

微波诱变结合化学诱变选育纤维素酶高产菌的研究

以纤维素酶产生菌TP1202为出发菌株,通过微波诱变和硫酸二乙酯、氯化锂诱变剂进行诱变,选育到1株纤维素酶高产菌株AS5。在适宜条件下,其产生的羧甲基纤维素酶活力(CMC),滤纸酶活力(FPA)和棉花糖酶活力分别是出发菌株的107%、152.4%和140.5%。     

人工锌指蛋白介导调控的里氏木霉纤维素酶生产

里氏木霉Trichoderma reesei Rut-C30是目前研究最广泛的纤维素酶生产菌,选育高产纤维素酶的里氏木霉菌株有助于提高木质纤维素资源生物炼制的经济性。利用人工锌指蛋白文库转化T.reeseiRut-C30,筛选获得了两株高产纤维素酶的突变株T. reesei M1和M2,与出发菌株比较,突变株M1和M2滤纸酶活分别提高100%和53%,且M1突变株外泌蛋白量提高69%,M2内切纤维素酶活提高64%。实时定量PCR分析结果表明,与对照菌株相比,突变株M1和M2中主要纤维素酶基因转录均上调,但不同酶基因在两株菌中有不同的变化特征。此外,纤维素酶抑制转录因子基因ace1在两株突变株中都转录下调,而纤维素酶正调控转录因子基因xyr1仅在M1突变株中上调。以上结果表明,不同人工锌指蛋白对纤维素酶活性的影响具有多样性。对这些突变体中人工锌指蛋白靶基因进行深入分析,为进一步深入探究里氏木霉纤维素酶合成调控的机理,以及利用代谢工程选育更高效的产酶菌株提供了基础。     

木质纤维素降解酶生产菌株的遗传改造及应用

通过纤维素酶将木质纤维素向生物新能源的转化对经济社会的可持续发展具有重要意义,被用于纤维素酶制剂工业化生产的微生物大多属于丝状真菌,但丝状真菌的遗传操作困难,且纤维素酶诱导机制尚未阐明,严重制约了纤维素酶高产菌株选育与应用。本文综述了近年来纤维素酶高产菌株遗传操作方法的进展,重点论述了丝状真菌合成纤维素酶过程中的信号感应、信号传导、转录调控的研究,通过理性改造以提高纤维素酶生产菌株的产酶能力,并且总结展望了丝状真菌在工业生产中的应用。     

斜卧青霉纤维素酶和木聚糖酶高产菌株的选育

以纤维素酶高产菌株斜卧青霉A50为出发菌株,通过紫外诱变原生质体获得1株木聚糖酶活力提高80%而纤维素酶活力没有改变的6号菌。蛋白质电泳和酶谱检测结果显示,纤维素酶谱基本无差别,而木聚糖酶谱显示6号菌比A50多了一条带。6号菌优化后的产酶培养基组成为:麸皮7%、葡萄糖0.1%,该条件下,纤维素酶活为19.7IU/mL,木聚糖酶活力为215.4IU/mL。     

纤维素酶高产菌种选育及酶活测定

一般从自然界筛选分离的野生型菌株产酶能力比较低．为了提高纤维素酶的活力,筛选和培育高产的菌种是关键。纤维素酶是多组分的复合酶．各个组分的底物专一性不同．并且不同来源的纤维素酶各组分的比例有差别。另一方面．纤维素酶作用的底物比较复杂,常常影响酶活力的表现,加上反应产物的不同．致使纤维素酶活力的测定方法多而繁杂。上期已对纤维素乙醇生产技术中的纤维质原料预处理技术进行了全面的综述．这期将围绕纤维素酶高产菌种选育及酶活测定进行介绍。     

微生物纤维素酶的应用研究

我国纤维素酶的应用研究近年来取得了很大进展。阐述了纤维素分解菌的选育 ,酶学性质以及在发酵、纺织和洗涤剂工业中的应用。     

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.     

谷氨酸高产菌株选育与发酵条件的初步研究

本文介绍了选育的谷氨酸高产菌与培养基成分、pH通气量等几个主要因素的关系,和对不同菌株谷氨酸高产的影响与调控方法。在控制好气体、pH、C/N等几个主要因素条件下,选育的谷氨酸高产菌产酸率可稳定在6.9％,比对照的平均产酸率高2—2.5倍。     

原生质体紫外诱变选育竹红菌甲素高产菌株

采用原生质体紫外诱变技术选育竹红菌甲素高产菌株。结果表明:以竹黄菌Shiraia sp.S8为出发菌株,当使用混合酶系(5 mg/mL纤维素酶和10 mg/mL蜗牛酶)在30℃处理菌丝2 h,获得菌丝原生质体3.24×106个/mL。以竹黄菌原生质体在距离15 W紫外灯30 cm处照射诱导,获得诱变菌株C6。其竹红菌甲素产量达到28.1 mg/L,比原始出发菌株提高了53.7%,且遗传稳定,具有较高的医药与工业应用价值。     

A novel screening method of cellulase-producing bacteria

Cellulase is the key to utilize the renewable and abundant cellulose resource, cellulase-producing microorganism is an important source of cellulase. The traditional screening method of cellulase-producing microorganism is low efficacy and not macroscopic. The screening method in this study was based on the interactive culture character between cellulase-producing bacteria and Phytophthora parasitica var. nicotianae on plates, the results indicated that the inhibition zone and cellulase activity of bacterial strains are conformity on the whole, so the screening method is very quickly and apparent.     

A Novel screening method of cellulase-producing bacteria based on <Emphasis Type="Italic">Phytophthora parasitica</Emphasis> var. <Emphasis Type="Italic">nicotianae</Emphasis>

Cellulase is the key to utilize the renewable and abundant cellulose resource, cellulase-producing microorganism is an important source of cellulase. The traditional screening method of cellulase-producing microorganism is low efficacy and not macroscopic. The screening method in this study was based on the interactive culture character between cellulase-producing bacteria and Phytophthora parasitica var. nicotianae on plates, the results indicated that the inhibition zone and cellulase activity of bacterial strains are conformity on the whole, so the screening method is very quickly and apparent     

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.     

Development of a simple cultivation method for isolating hitherto-uncultured cellulase-producing microbes

Although enrichment culture is typically employed to isolate cellulolytic microbes, this approach tends to favor fast-growing species and discriminates against all others. Therefore, efforts to prevent the overgrowth of fast-growing species are necessary to isolate novel cellulase-producing strains. In this study, we developed a simple culture method for isolating hitherto-uncultured microbes that possess cellulase activity, particularly exocellulase. In this method, the microbial source (a forest soil) was suspended in sterilized water and inoculated onto a mineral salts agar medium, which was then overlaid with filter paper to sandwich the microbial suspension between the agar surface and paper. The filter paper fibers served to immobilize the microbial cells and were the dominant carbon source. Following cultivation at 30°C for 2 weeks, emerging colonies were isolated based on their morphology and were then subjected to phylogenetic and enzyme analyses. Using this method, 2,150 CFUs/g dry soil were obtained, and the ratio of fungal to bacterial isolates was approximately 4:1. Phylogenetic analyses revealed that most fungal and bacterial isolates belong to ten and two genera, respectively. Notably, all isolates possessed exocellulase activity, and several strains showed strong activity that was comparable to Trichoderma cellulase. Many isolates also exhibited cellulase and xylanase activity, and several strains possessed laccase activity. It is expected that the culture method described here will be useful for the isolation of hitherto-uncultured cellulolytic microbes and the identification of novel cellulases.     

A Rapid and Easy Method for the Detection of Microbial Cellulases on Agar Plates Using Gram’s Iodine

Screening for cellulase-producing microorganisms is routinely done on carboxymethylcellulose (CMC) plates. The culture plates are flooded either with 1% hexadecyltrimethyl ammonium bromide or with 0.1% Congo red followed by 1 M NaCl. In both cases, it takes a minimum of 30 to 40 minutes to obtain the zone of hydrolysis after flooding, and the hydrolyzed area is not sharply discernible. An improved method is reported herein for the detection of extracellular cellulase production by microorganisms by way of plate assay. In this method, CMC plates were flooded with Gram's iodine instead of the reagents just mentioned. Gram's iodine formed a bluish-black complex with cellulose but not with hydrolyzed cellulose, giving a sharp and distinct zone around the cellulase-producing microbial colonies within 3 to 5 minutes. The new method is rapid and efficient; therefore, it can be easily performed for screening large numbers of microbial cultures of both bacteria and fungi. This is the first report on the use of Gram's iodine for the detection of cellulase production by microorganisms using plate assay.     

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。     

60Coγ射线诱变褐色高温单孢菌及其在粪便发酵中的应用研究

本试验以褐色高温单孢菌(Therm om onospora fusca)为出发菌株,通过60Coγ射线诱变孢子悬液,采用透明圈法初筛和摇瓶培养复筛的方法,获得了一株纤维素酶高产菌株AV5,与出发菌株相比,其产酶能力提高1.8倍。接种牛粪发酵后,牛粪中粗纤维含量降低率为32.95%,是接种出发菌株相对降解率的1.7倍。     

低温纤维素酶的研究进展

低温纤维素酶在低温下仍具有较高酶活力及较高催化效率,在应用中能够缩短处理工艺时间,节省加热或冷却费用,且易失活,有着中高温纤维素酶无法比拟的优势。现针对微生物低温纤维素酶的研究现状,包括菌种来源、分离鉴定、酶学性质、适冷结构及机理研究和基因工程等方面进行综述。     

真菌产纤维素酶培养基中刚果红转移机理研究

通过对产纤维素酶真菌在纤维素刚果红液体培养基中刚果红染料移动情况研究,表明刚果红染料进入真菌的机制为纤维素分解真菌首先分解纤维素物质为含有葡聚糖等结构的多聚糖类物质,多聚糖与刚果红形成多聚糖-刚果红复合物,复合物不仅破吸附到产纤维素酶活的菌丝外表面,而且能被进一步转运吸收至该部分菌丝内部,使菌丝体和菌落呈现红色。所以,纤维素刚果红培养基可作为分离、筛选纤维素分解直菌的特异性培养基。     

适冷海洋细菌交替假单胞菌(Pseudoalteromonas sp.)MB-1内切葡聚糖酶基因的克隆和表达

从黄海深海海底淤泥中筛选出一株产纤维素酶的适冷革兰氏阴性杆菌MB 1,克隆和分析了MB 1的 16SrDNA序列 (GenBank接受号 :AY5 5 132 1) ,经鉴定为交替假单胞菌 (Pseudoalteromonas) ,命名为Pseudoalteromonassp .MB 1。克隆了该菌适冷内切葡聚糖酶基因celA(GenBank接受号 :AY5 5 132 2 ) ,并在大肠杆菌 (Escherichiacoli)BL2 1中进行了表达。重组E .coli菌体破碎后 ,获取上清液 ,其中融合蛋白GST CelA浓度约为 78 5mg L。分析了融合酶GST CelA的性质 ,其最适反应温度为 35℃ ,最适反应pH值为 7 2 ,为中性适冷酶。实验结果为交替假单胞菌低温纤维素酶的基础理论和应用研究奠定了基础