球毛壳菌降解天然木质纤维素能力差异及酶系基因分析

目的:以不同植物中分离到的4株内生球毛壳菌NK102、NK103、NK104和NK105为对象,研究不同生态来源球毛壳菌降解木质素和纤维素的能力。方法:首先采用羧甲基纤维素和纤维素刚果红平板检测各菌株的纤维素降解能力,并利用Bavendamm平板反应检测各菌株的木质素降解能力;将4株菌分别培养在以微晶纤维素、杨树叶和木屑为惟一碳源的液体培养基中,通过检测培养液中纤维素酶和漆酶的酶活力,比较各菌株分解利用天然木质纤维素材料的能力,连续培养12 d后检测培养液中次级代谢产物的合成情况;利用已测序的球毛壳菌CBS148.51的基因组信息,寻找编码木质纤维素降解酶类的基因,为球毛壳菌分解利用木质纤维素提供分子生物学依据。结果:NK102、NK103、NK104和NK105在羧甲基纤维素培养基和纤维素刚果红培养基上都能够生长并形成水解圈;Bavendamm平板反应显示4株菌降解木质素的能力由强到弱依次是NK103、NK102、NK105和NK104。4株菌都能分解利用微晶纤维素、杨树叶和木屑,分泌纤维素酶和漆酶,其中NK102在以木屑为碳源的培养基上纤维素酶活力最强,达到0.76 U/mL发酵液,NK103在以杨树叶为碳源的培养基上漆酶活力最强。与此同时,4株菌在发酵培养过程中都能够稳定地合成球毛壳甲素(ChA),ChA产量受到碳源影响,在以杨树叶为碳源的培养基上,NK104的ChA产量最高,可达到14.88 mg/L发酵液。利用已测序的球毛壳菌CBS148.51的基因组信息,寻找到119个编码纤维素半纤维素酶的基因、8个编码漆酶的基因和2个编码锰过氧化物酶的基因,球毛壳菌具有完整的降解纤维素半纤维素的酶体系,在木质纤维素降解真菌的开发过程中具有重要的研究价值。结论:本研究为球毛壳菌木质纤维素降解过程的研究及该菌种的开发利用奠定了基础。     

杀线虫天然产物Chaetoglobosin A合成的表观遗传调控证据

通过高效液相色谱(HPLC)、高分辨率质谱(HRMS)和~H和~(13)C核磁共振(NMR)证明1株球毛壳菌Chaetomiumglobosum NKl02产生的杀根结线虫(Meloidogyne spp.)成分为球毛壳甲素(Chaetoglobosin A)。C.globosum NK102能够利用微晶纤维素、杨树叶和木屑合成ChA。在12 d发酵液中,3种培养基获得的ChA产量由高到低的顺序为PDA、杨树叶、微晶纤维素和木屑,产量分别为26.49、9.38、4.47和0.47 mg/L。通过在微晶纤维素培养基中添加表观遗传沉默抑制药物表明:100μmol/L 5′-AZA的添加使得ChA的产量提高至原来的3.6倍,100μmol/L SAHA将ChA的产量提高到原来的2.4倍,DMSO使得ChA的产量提高到原来的1.7倍。这个结果预示ChA的合成的某些基因受到表观遗传调控。     

球毛壳菌(Chaetomium globosum)NK102 纤维素酶基因及其表达条件

[目的]生物质的利用是当前生物技术研究的一个热点.本小组分离到一株高效降解纤维素球毛壳菌(Chaetomium globosum)NK102,本文拟探索研究此菌的纤维素酶表达系统并寻找影响酶基因表达的关键因素.[方法]通过对NK102测序,本文界定了球毛壳菌NK102的主要纤维素酶编码基因,使用数字基因表达谱升级版(RNA-Seq)的方法得到纤维素酶基因的表达差异,然后观察了营养、物理条件下纤维素酶基因表达和酶活性变化的情况.[结果]发现随着培养时间的延长,纤维素酶基因整体上表达量升高.在所选基因中,外切葡聚糖酶、纤维二糖脱氢酶和内切葡聚糖酶基因(cbh1,cdh和egl1)的表达量最高.糖代谢的负调控因子ACE I和CreA的随时间表达量均降低,而Hap2/3/5复合体的表达量反而升高.之后检测了不同碳源培养基对纤维素酶基因表达量和酶活性的影响,发现葡萄糖为强阻遏因子,纤维二糖为其诱导物,而山梨醇没有影响.特别是,我们发现光照也影响纤维素酶基因的表达,黑暗条件明显抑制酶基因的表达.[结论]转录组学的方法可以初步探索纤维素酶表达的规律,酶基因的表达受到营养、物理条件的影响.本研究为揭示球毛壳菌降解纤维素分子机理和阐释生物质糖代谢途径提供了有用参考.     

一株木质纤维素降解菌的筛选及其全基因组分析北大核心CSCD

【目的】筛选和鉴定有木质纤维素降解能力的1株细菌,测定其相关酶活力并进行全基因组分析,为构建木质纤维素降解工程菌提供依据。【方法】采用3种木质素类似物(天青-B;酚红;愈创木酚)的脱色/染色法,从腐木和被枝叶覆盖的土壤中分离和筛选出1株具有较强木质纤维素降解能力的细菌。通过16S r RNA基因和全基因组序列分析对该菌进行种属鉴定。使用紫外分光光度法测定其锰过氧化物酶(Mn P)、漆酶(Lac)、羧甲基纤维素酶(CMCase)以及滤纸酶(FPA)活力,了解该菌相关酶活力大小在一定时间内的变化趋势。使用Illumina Miseq和454 GS Junior测序平台获取该菌的全基因组序列,将其全基因组序列经过注释的基因蛋白质序列提交COG和KEGG数据库进行BLASTp比对分析,确定该菌潜在的重要酶类和代谢途径,并对部分注释基因进行定量RT-PCR验证。【结果】筛选得到1株优势菌株S12,该菌经鉴定后命名为解鸟氨酸拉乌尔菌(Raoultella ornithinolytica)。在液体CMC-Na培养基中发酵28 h,菌体生长达到稳定期,纤维素降解相关酶活力也在此时达到峰值。生物信息学分析结果表明,菌株S12具有木质素降解通路中重要酶类的编码基因,如过氧化物酶、Fe-Mn型超氧化物歧化酶、邻苯二酚1,2-双加氧酶和原儿茶酸-3,4-双加氧酶等,这些基因在以碱性木质素为碳源的培养条件下表达量不同程度地高于以葡萄糖为碳源的培养条件。另外,菌株S12具备完整的纤维素降解和乙醇生成通路。【结论】本研究首次揭示了Raoultella ornithinolytica S12具备有效的木质纤维素降解性能,这对于推动木质纤维素应用产业的发展具有重要意义。     

不同碳源和氮源对金针菇降解木质纤维素酶活性的影响

以3株栽培的金针菇Flammulina velutipes为材料,研究它们在玉米芯和棉子壳以及不同碳源、氮源培养条件下纤维素、半纤维素和木质素降解酶活性的规律。结果表明,不同金针菇菌株的羧甲基纤维素酶、木聚糖酶和漆酶活力显著不同（P<0.001）,同时,培养条件对羧甲基纤维素酶、木聚糖酶和漆酶的活力都有显著影响（P<0.001）。在简单碳源存在的条件下,金针菇的羧甲基纤维素酶和木聚糖酶活力远远低于复杂碳源培养基（P<0.05）。全营养培养基上生长的金针菇的羧甲基纤维素酶和木聚糖酶活力低于缺乏碳源和氮源的培养基（P<0.05）。漆酶活力在无简单氮源培养基上低于全培养基（P<0.05）和无葡萄糖培养基（P<0.05）,即复杂碳源和氮源培养基上的漆酶活力低于简单碳源和氮源培养基（P<0.05）。     

鸡腿菇对棉籽壳的降解与转化

栽培在棉籽壳培养基中的鸡腿菇具有较强的木质纤维素降解能力和较高的绝对生物学效率;木质纤维素是子实体生长阶段的主要碳源;CMC酶、FP酶和HC酶的活性变化与纤维素、半纤维素的降解速率正相关,漆酶的活性变化与木质素的降解速率正相关,而过氧化物酶的活性变化与木质素的降解速率没有相关性;淀粉酶在菌丝生长阶段活性较高,蛋白酶的活性高峰出现在子实体生长发育期。     

三种白腐菌生物学特性与木质纤维素酶基因遗传多样性

以采自东北林业大学帽儿山实验林场的3种白腐真菌——木蹄层孔菌(Fomes fomentarius)、鲍姆鲍姆木层孔菌(Phellinus baumiibaumii)和火木层孔菌(Phellinus igniarius)为材料,用菌落直径测量法比较3种白腐菌在马铃署葡萄糖固体培养基上的生长速度,采用菌丝体干重法比较其在马铃署葡萄糖液体培养基中的生物量变化。结果显示:马铃薯葡萄糖固体培养基上3种白腐菌均为快速生长类型,其生长速度木蹄层孔菌火木层孔菌鲍姆鲍姆木层孔菌;马铃署葡萄糖液体培养基中生物量增长速度木蹄层孔菌鲍姆鲍姆木层孔菌火木层孔菌。用比色法测量其木质纤维素酶活性,结果显示:木蹄层孔菌产锰过氧化物酶和漆酶量较高,鲍姆鲍姆木层孔菌和火木层孔菌产木质素过氧化物酶量较高;木蹄层孔菌、鲍姆鲍姆木层孔菌和火木层孔菌3种白腐菌的3种主要木质素酶(锰过氧化物酶、漆酶和木质素过氧化物酶)的表达量,种间差异显著(F=3.75*、5.20**、3.01*),白桦木屑诱导处理与对照间差异显著(F=3.84*、4.19*、5.28*);两种主要纤维素酶(葡聚糖内切酶、葡聚糖外切酶)的表达量,种间差异不显著,受碳源影响作用显著(F=3.99*、4.04*)。筛选29对引物组合,对3种白腐菌几种主要木质纤维素酶基因进行TRAP-PCR分子标记检测,比较3菌种间遗传差异,扩增总条带数为357条,多态性条带数为255条,多态性条带的比例为71.43%,其中木质素降解酶基因总多态位点比率为73.77%,纤维素降解酶基因总多态位点比率为68.97%。3种白腐菌的木质纤维素降解酶基因在种间均存在较高的遗传差异。因此,特定基因的TRAP分子标记可以用于木腐菌的遗传变异分析。     

碳源和氮源对彩绒革盖菌Coriolus versicolor木质纤维素酶和木质素酶分泌的影响

研究了彩绒草盖菌在不同碳源和氮源培养基中生长时,对纤维素酶、半纤维素酶、木质素酶（漆酶、多酚氧化酶、愈创木酚氧化酶）分泌的影响。结果表明不同碳源和氮源对酶类的分泌影响很大,富含淀粉的物质能明显促进木质素酶的分泌,而专一性底物（纤维素和半纤维素）对纤维素酶和半纤维素酶有诱导作用,麸皮也能诱导半纤维素酶的产生。     

嗜热厌氧细菌Caldicellulosiruptor bescii降解木质纤维素研究进展

嗜热厌氧菌Caldicellulosiruptor bescii具有强大的木质纤维素降解能力,能以多种模式植物细胞壁多糖如微晶纤维素Avicel和木聚糖,甚至未经预处理的木质纤维素如柳枝稷作为唯一碳源快速生长,该菌还具有少见的厌氧降解木质素的能力。对基因组注释发现,该菌所编码的蛋白大多为多结构域双功能酶,即在多肽链的N端和C端分别是不同家族的糖苷水解酶,间隔以2-3个碳水化合物结合结构域。该菌降解纤维素相关的酶基因多集中于一个植物细胞壁多糖降解利用的基因簇,例如纤维素酶/木聚糖酶、纤维素酶/甘露聚糖酶和纤维素酶/木葡聚糖酶等。C.bescii的木聚糖酶主要属于GH10家族,该家族的酶底物特异性较为宽泛,氨基酸序列的同源性在18.7%-59.5%间。Caldicellulosiruptor属细菌进化出了一系列的机制使得糖苷水解酶和底物、细菌和木质纤维素能更好的吸附在一起,从而有利于木质纤维素的酶解。C.bescii有12个含SLH结构域的蛋白,以及新发现的黏附蛋白Tāpirin,可能参与了木质纤维素的吸附与利用。综述了近年来对C.bescii降解植物细胞壁的糖苷水解酶的基因资源挖掘方面和降解分子机制方面的研究进展,对高效、多功能高效木质纤维素降解酶的设计和优化具有积极的意义。     

不同木质纤维素基质上白腐菌降解特性的研究

通过测定木质素、纤维素、半纤维素和漆酶分泌的变化,研究白腐菌在稻草、木屑、粗纤维素、滤纸、黑液木素基质上的降解特性。结果表明,除黑液木素上白腐菌不能生长外,在前25d,各基质中纤维素、半纤维素和木质素含量呈持续下降趋势,之后,降解速率减少,其中木质素的降解速率大于纤维素和半纤维素的降解速率。漆酶分泌在生长初期呈快速上升趋势,第10d酶活达到最大,第10～20d快速下降,其后基本不变,基质中酶活大小顺序为稻草基质、木屑基质、粗纤维和滤纸基质,显示了木质素存在对漆酶分泌的诱导作用。     

Some characteristics of ammonia fungi 1. In relation to their ligninolytic enzyme activities

Twenty-six species of ammonia fungi comprising 71 strains were screened for ligninolytic activity using agar plate tests. The tests comprised a wood powder plate test, the Bavendamm reactions, and a Remazol Brilliant blue R (RBBR) decolorization test. The wood powder plate test detected phenol oxidases of Coprinus spp., whereas this method obviously detected no activities from facultative mycorrhizal fungi, such as Hebeloma radicosoides and ectomycorrhiza: H. spoliatum and H. vinosophyllum. With quantitative assays of ligninolytic activity, Coprinus phlyctidosporus, C. echinosporus, Lyophyllum tylicolor, Lepista nuda, L. tarda, Calocybe leucocephala, and Crucispora rhombisperma, which grow on oak-leaf litter, the major phenol-oxidizing enzyme was a laccase. The concentration of urea affected laccase activity; however, urea was not the obligate nitrogen source for the laccase production.     

Screening of New Zealand Native White-Rot Isolates for PCP Degradation

From a pool of 367 white-rot fungi native to New Zealand (over 77 genera), isolates were screened for their bioremediation potential of pentachlorophenol (PCP). Fungi were tested for their ligninolytic activity (Poly R-478, 367 isolates; wood decay, 235 isolates), tolerance to temperature (261 isolates), resistance to PCP (253 isolates), and PCP degradation potential plus laccase expression (20 isolates). Of the isolates tested, 26% showed a discolouration in the polymeric dye assay, but all caused wood decay (5 to 169 mm) on willow cuttings. In the temperature tolerance tests, all isolates survived incubation from 0 to 30°C, however, 18% and 40% did not survive incubation at 35 and 40°C, respectively. In the PCP resistance tests, 23 isolates (9%) were able to grow on 200 mg/L PCP amended agar, of which 20 isolates were further studied for laccase expression and PCP degradation in vitro. All 20 isolates reduced (P < 0.05) PCP in the liquid fraction in the absence or presence of laccase and five of the isolates produced no detectable levels of PCP. None of the screening tests were predictive for PCP degradation in vitro. The requirements to build a database to select a superior white-rot fungal isolates for bioremediation is discussed.     

Reliability of Poly B-411, a Polymeric Anthraquinone-Based Dye, in Determining the Rot Type Caused by Wood-Inhabiting Fungi

In both Basidiomycotina and Ascomycotina, Poly B-411 decolorization was an excellent indicator of the ability to cause white rot: 109 of the 110 isolates of brown rot fungi tested definitely did not decolorize Poly B-411, and 392 of the 401 mainly active isolates of white rot fungi decolorized Poly B-411. The Bavendamm (tannic acid) reaction was a less reliable test: of 74 white rot isolates examined that could not decay wood, 6 decolorized Poly B-411, but 19 gave positive Bavendamm reactions. Of 80 isolates of Ascomycotina and Deuteromycotina that do not cause white rot, only 4 decolorized Poly B-411, but 17 gave a positive Bavendamm test.     

Role of antibiosis in the biological control of spot blotch (Cochliobolus sativus) of wheat by Chaetomium globosum

Chaetomium globosum Kunze, has been identified as a potential antagonist of Cochliobolus sativus (S. Ito & Kurib.) Deschler ex Dastur. (Syn = Drechslera sorokiniana). Production of antifungal compounds by Chaetomium globosum (Cg) and their role in suppression of spot blotch of wheat caused by this fungus under in vitro and in vivo has been evaluated. Interaction between Chaetomium globosum isolates and C. sativus showed mycoparasitism by isolates Cg 1 and Cg 6 whereas isolates Cg 2, Cg 3, Cg 4 and Cg 5 showed antibiosis. Syringe filtered culture extracts of Cg 2 completely inhibited mycelial growth of C. sativus in liquid broth. In vitro bioassays were undertaken by amending the medium with crude extracts and agar diffusion method in order to assess the fungistatic activity of crude extracts from culture filtrates of different isolates of Chaetomium globosum. Significant differences in antagonism between isolates were observed. Antifungal metabolite profiling, on TLC (Thin Layer Chromatography) plates identified 13 compounds in isolate Cg 2, 11 compounds in Cg 3 and 7 compounds in Cg 6. Isolate Cg 1 produced only two faint bands and Cg 5 produced two bands of the same Rf value but of higher intensity. The production of antifungal compounds by isolates was positively correlated with antagonism to C. sativus on seedlings in glasshouse studies. The results showed high antifungal metabolite production by isolate Cg 2, which also gave maximum bioefficacy under laboratory and glasshouse conditions.     

Mycoflora and mycotoxin-producing fungi of air-dust particles from Egypt

Using the dilution-plate method, 27 genera and 64 species were collected from 20 air-dust samples on glucose — (24 genera and 57 species) and cellulose — (21 genera and 45 species) Czapek's agar at 28 °C. There are basic similarities between the mycoflora of air-dust on the two media and the most prevalent species were Aspergillus niger, A. flavus, A. ochraceus, A. terreus, A. versicolor, Penicillium chrysogenum, P. funiculosum, Alternaria alternata, Cladosporium herbarum, Fusarium oxysporum, Rhizopus stolonifer and Trichoderma viride. Chaetomium globosum, Stachybotrys chartarum, Humicola grisea and Arthrobotrys oligospora were common only on cellulose agar plates.Extracts of mycelium from 25 isolates were tested with brine schrimp (Artemia salina); of these 23 displayed varying degrees of toxicity. Thin layer Chromatographic analysis of 12 isolates of Aspergillus flavus revealed that 4 strains were producing detectable aflatoxin. Zearalenone production was noted for 3 out of 5 strains of Fusarium oxysporum and 2 out of 5 strains of F. solani.     

Endophytic fungi in rose (Rosa hybrida) in Bogota, Colombia

We have investigated the presence of endophytic fungi associated with rose plants (Rosa hybrida) in Colombia. Endophytic fungi were isolated from healthy leaves of ten ornamental roses plants from gardens cultured in malt extract, peptone, yeast extract agar plates (MPY). We sampled 560 leaves fragments, 56 per sample. Endophytic fungi comprised 92 isolates (16.4%); of these isolates, 41 were classified as sterile mycelium (without reproductive structures that allowed their identification), 31 isolates were identified to genus or to species, and 20 isolates could not be identified at all. The identified endophytic fungi were as follow: Nigrospora oryzae, Aureobasidium spp, Acremonium spp. The fungi Nodulisporium sp, Gliocladium virens, Cladosporium sp, Alternaria sp, Phoma sp and Chaetomium globosum were represented by one isolate each. Since the endophytic fungi are known for their capacity to produce metabolites with biological activity, it is possible that the microorganisms found in this study have potential as antagonist of rose pathogens.     

Wood biodeterioration control potential of Acalypha hispida leaf phenolic extract in combination with Trichoderma viride culture filtrate

The phenolic extract of Acalypha leaves inhibited growth of Gloeophyllum sepiarium and Pleurotus sp. (test wood-rot fungi) in potato dextrose agar, starch agar, starch glucose agar, carboxyl methyl cellulose agar and carboxyl methyl cellulose glucose agar. Fungicidal or fungistatic concentration of the extract (10–14 mg/ml) depended on the medium. However a lower concentration of the extract (8–10 mg/ml) in combination with Trichoderma viride culture filtrate caused a similar inhibitory pattern. Degradation of obeche (Triplochiton scleroxylon), mahogany (Khaya ivorensis) and walnut (Lovoa trichilioides) by the test fungi was limited or prevented by extract treatment of 8–10 mg/g wood. A similar inhibitory effect again occurred when a combination of T. viride filtrate and lower extract concentration (6–8 mg extract per gram of wood) was used. On-going wood decay was limited or halted by a combined treatment involving 8–12 mg extract per gram of wood depending on the fungal residence period. Treated stakes exposed to 6 months of tropical wet season retained resistance to fungal attack including soft rot. The phenolic extract of A. hispida may prove useful in an integrated chemical and biological approach to wood treatment.     

The role of antibiotics in the decomposition of sawdust; inhibition of the growth of cellulose decomposing fungi

The action of the antibiotics present in deal sawdust on the growth on Czapek-Dox agar of cellulose-decomposing fungi has been examined. Stachybotrys atra and Chaetomium indicum were strongly inhibited by substances in cold-water extracts, but C. globosum only slightly so. The extracts also contained material which stimulated the growth of C. globosum but not that of the other two fungi. The formation of perithecia by C. indicum and C. globosum was also stimulated by the extract. There was no marked inhibition or stimulation of the growth of Aspergillus terreus, A. fumigatus , or three species of Penicillium by the extracts.