N+离子注入诱变选育纤维素酶高产菌

纤维素酶产生菌Rhizopus sp.TCl经过N+离子注入、紫外线、5-氟尿嘧啶(5-Fu)等诱变剂诱变,选育得到突变株Rhizopus sp.TCl653,发酵终点时滤纸酶活力(FPA)和羧甲基纤维素酶活力(CMC)分别为22.6 IU/g、224.7 IU/g干物质,较出发菌株分别提高了276.7%和214.3%...     

5种优势腐生真菌降解华山松针叶的酶活测定

【目的】分析腐生真菌降解华山松落针过程中各种酶活性变化与酶间关系,探索真菌对华山松落针的降解能力。【方法】通过形态学观察和转录间隔区ITS序列分析,鉴定分离自华山松凋落物的5种优势菌株。以此为供试菌株,华山松落针为底物,通过发酵纯培养方法,测定底物总有机物质(Total organic matter,TOM)质量损失率及在发酵过程中产生的内切葡聚糖酶(Endoglucanase,EG)、木聚糖酶(Xylanase,Xyl)、木质素过氧化物酶(Lignin peroxidase,Li P)、锰过氧化物酶(Manganese peroxidase,Mn P)和酸性磷酸酶(Acid phosphatase,AP)活性。【结果】5种菌株分别为Mucor sp.、Pestalotiopsis sp.、Allantophomopsis sp.、Phoma sp.和Hypocrea sp.。5种菌引起的针叶TOM质量损失率在6.63%-15.77%之间,Pestalotiopsis sp.具有最高的AP酶活性,且EG酶、Xyl酶和Li P酶3种酶活性较高。Allantophomopsis sp.的Li P酶活性最高,并具有很高的Mn P酶活。Hypocrea sp.分泌的EG酶、Xyl酶活性低,但能产生Li P酶且有较高的AP酶活。相关性分析表明菌株分泌的AP酶活性与TOM质量损失率负相关,EG酶、Xyl酶及AP酶3种酶之间存在协同作用,特别是EG与AP之间。【结论】5种腐生真菌对华山松针叶均有降解作用,降解能力:Pestalotiopsis sp.>Allantophomopsis sp.>Hypocrea sp.>Phoma sp.>Mucor sp.。酶活大小及酶协同作用共同影响针叶降解,Pestalotiopsis sp.、Allantophomopsis sp.和Hypocrea sp.能产生木质纤维素降解酶并能引起较高的质量损失率,因此这3株菌为木质纤维素降解真菌。     

不同碳氮源对花脸香蘑胞外酶活性的影响

以花脸香蘑Lepista sordida为材料,研究其分别在9种碳源和11种氮源液体培养条件下不同阶段pH值和葡萄糖浓度的变化,以及不同碳氮源对其所分泌的木质素过氧化物酶、羧甲基纤维素酶、锰过氧化物酶和漆酶活性的影响。结果表明,pH值在不同碳源培养后期变化显著（P<0.05）,而在不同氮源培养阶段无明显变化（P>0.05）,葡萄糖浓度和菌丝量在不同碳氮源中也无显著差异（P<0.05）。不同碳源和氮源培养基对花脸香蘑木质素过氧化物酶、羧甲基纤维素酶、锰过氧化物酶和漆酶活性均具有影响（P<0.05）。木糖和米糠有利于花脸香蘑分泌羧甲基纤维素酶（P<0.05）,红糖和牛肉浸膏有利于其分泌漆酶（P<0.05）,白砂糖和豆粉有利于其分泌锰过氧化物酶（P<0.05）,木糖和尿素有利于其分泌木质素过氧化物酶（P<0.05）。本研究为选择合适培养基以提高花脸香蘑生物转化效率提供了理论基础。     

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 ...     

一种筛选胞外纤维素酶产生菌的快捷、灵敏、有效的方法

【目的】建立一种能快捷、灵敏、有效筛选高β-葡萄糖苷酶活性的胞外纤维素酶产生菌的方法,用于胞外纤维素酶产生菌检测筛选。【方法】将以微晶纤维素(Avicel)或羧甲基纤维素(CMC)为底物的胞外纤维素酶产生菌平板筛选法常用的刚果红或碘液浸泡染色改为碘液熏染,减少碘液的消耗和对环境的污染;建立以对硝基苯酚-β-1,4-葡萄糖苷(pNPG)为底物的β-葡萄糖苷酶发色底物平板筛选法;将两方法串联用于高β-葡萄糖苷酶活性的胞外纤维素酶产生菌的筛选。【结果】建立了分别以CMC和Avicel为底物结合碘液熏染的胞外纤维素酶产生菌平板筛选法和以pNPG为底物的β-葡萄糖苷酶发色底物平板筛选法,从56株真菌中筛选出了8株纤维素酶活性水平为++++的胞外纤维素酶产生菌,从后者筛选出4株高β-葡萄糖苷酶活性的胞外纤维素酶产生菌。【结论】将以CMC和Avicel为底物结合碘液熏染的平板筛选法和以pNPG为底物的发色底物平板筛选法串联,可快捷、灵敏、有效地用于高β-葡萄糖苷酶活性的胞外纤维素酶产生菌的筛选。     

不同碳源诱导下牦牛瘤胃厌氧真菌Orpinomyces sp. YF3的产酶机制

为探究体外发酵牦牛瘤胃源厌氧真菌Orpinomyces sp. YF3在不同碳源诱导下的产酶机制，本研究利用厌氧培养管在10 mL基础培养基中分别添加不同碳源复杂度的葡萄糖(glucose, Glu)、滤纸(filter paper, Flp)、微晶纤维素(avicel, Avi)各8 g/L作为唯一碳源进行体外发酵，检测发酵液中的纤维降解酶活性和挥发性脂肪酸，并利用转录组学探究Orpinomyces sp. YF3的产酶机制。结果表明葡萄糖诱导下的发酵液中羧甲基纤维素酶、微晶纤维素酶、滤纸酶和木聚糖酶的活性，及乙酸的比例显著升高(P＜0.05)，丙酸、丁酸、异丁酸的比例显著降低(P＜0.05)。进一步分析发现与纤维降解酶相关的差异表达基因(differentially expressed genes, DEGs)在Glu组中显著上调。基因本体论(gene ontology, GO)功能富集显示DEGs主要集中在木聚糖酶、纤维素酶、葡萄糖和碳水化合物等的分解代谢过程及相关酶活性，京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)通路分析富集到的纤维降解酶相关的差异通路主要是淀粉和蔗糖代谢途径、其他聚糖降解途径。以上结果表明，以葡萄糖为碳源底物的Orpinomyces sp. YF3可增加纤维素降解酶活性，提高乙酸比例，通过调控纤维降解酶基因的表达及相关代谢通路来提高对底物的降解能力，提高能量利用效率。这为Orpinomyces sp. YF3在实际生产中的应用提供了理论基础。     

有机复合物H对纤维素酶促反应动力学特性的影响

采用3,5-二硝基水杨酸(DNS)为显色剂,滤纸和CMC为底物,测定纤维素酶的酶活.考察了在不同温度、pH值、反应时间、底物浓度等反应条件下,有机复合物H对纤维素酶滤纸酶活(FPA)和CMC酶活(CMCA)的影响.结果表明,有机复合物H对纤维素酶的FPA活性和CMCA活性均有一定的促进作用,且在不同条件下的促进效果有较大差异.     

外生菌根真菌与丝状真菌混合对红松凋落物降解效能的影响

实验以原始红松林中高频出现的丝状真菌(链格孢Alternarria sp.)和两种外生菌根真菌(细粉绒牛肝菌.Xerocomus Pulverulentus、大杯伞Clitocybe maxima)为供试菌株,以粉碎的新鲜红松凋落物为分解底物,发酵培养后测定底物的质量损失率,酶活性以及营养元素的变化情况。结果表明,AX处理(Alternaria sp.和X.Pulve Rrulentus混合菌株组合)在60天内质量损失率最高,为18.33%,而混合菌AC处理(Alternaria sp.和C.maxima混合菌株组合)由于产生拮抗作用质量损失较低,为13.27%;20天时,X处理(X.Pulverulentus)产生漆酶活性较高为0.093 U·mL^-1,C(C.maxima)和AX处理的酶活性的最高值分别为0.063 U·mL^-1和0.047 U·mL^-1,而A(Alternaria sp.)处理的纤维素酶活性为0.59 U·mL^-1,AC处理为0.57 U·mL^-1,AX为0.53 U·mL^-1,但是AX处理在50天时再次出现高峰,为0.48 U·mL^-1:AX处理底物的营养元素N含量减少幅度最为明显,减少百分率为12.21%,同时,P元素的减少幅度也最大,减少百分率25.82%。研究结果进一步揭示了森林生态系统中不同功能真菌类群对林木有机凋落物降解过程中的作用机制,及真菌类群间的相互作用关系。     

秸秆纤维素分解菌的酶活力测定

目的:测定秸秆纤维素分解菌的酶活力。方法:从土壤中分离出具有分解纤维素能力的菌株,采用刚果红染色法进行粗选,得到7株透明圈较大的菌株。将这7株菌株液体发酵培养6d,再分别用滤纸分解度观察、羧甲基纤维素酶活法(CMC)、滤纸酶活法(FPA)和天然纤维素酶活法测定其酶活力。结果:在7株菌株中,F-1、F-2、F-3、F-5的酶活力测定结果与其溶解圈的测定结果、滤纸分解结果基本相同。且天然纤维素酶活力高的菌株,其CMC酶活、FPA酶活也高,滤纸分解效果也比较明显。结论:CMC法、FPA法和天然纤维素酶活法适于测定秸秆纤维素分解菌的酶活力。     

纤维素酶产生菌的筛选、鉴定及发酵产酶条件优化

以腐烂木材、腐殖土等材料作为菌源,从中分离出180个菌株,以期得到具有纤维素酶活性的菌株。采用革兰氏碘液染色法进行定性初筛,获得了44个纤维素酶产生菌株。将此44个菌株发酵培养后,使用滤纸酶活性测定法进行定量复筛,滤纸酶活性最高的是菌株J1-3-1。通过对J1-3-1菌株的16S r RNA的序列测定分析,将J1-3-1鉴定为鞘氨醇杆菌(Sphingobacterium sp.)。经对J1-3-1进行产酶发酵条件优化,分别确立了产生最高滤纸酶(FPase)活性、内切葡聚糖酶(CMCase)活性和β-葡萄糖苷酶(β-glucosidase)活性的发酵条件。在最优发酵产酶条件下,菌株J1-3-1的滤纸酶、内切葡聚糖酶和β-葡萄糖苷酶的最高酶活性分别为8.76、28.04和7.02 U/m L。     

Involvement of lignocellulolytic enzymes in the decomposition of leaf litter in a subtropical forest

The involvement of ligninolytic and cellulolytic enzymes, such as laccase, lignin peroxidase, manganese peroxidase, carboxymethylcellulase (CMCase), and filter paper activity (FPA), in the decomposition process of leaf litter driven by 6 soil-inhabiting fungi imperfecti was studied under solid-state fermentations. All the tested fungi exhibited varied production profiles of lignocellulolytic enzymes and each caused different losses in total organic matter (TOM) during decomposition. Based on the results, the 6 fungi could be divided into 2 functional groups: Group 1 includes Alternaria sp., Penicillium sp., Acremonium sp., and Trichoderma sp., and Group 2 includes Pestalotiopsis sp. and Aspergillus fumigatus. Group 1, with higher CMCase and FPA activities, showed a higher decomposition rate than the fungi of Group 2 over the first 16 d, and thereafter the cellulolytic activities and decomposition rate slowed down. Group 2 showed the maximum and significantly higher CMCase and FPA activities than those of the Group 1 fungi during the later days. This, combined with the much higher laccase activity, produced a synergistic reaction that led to a much faster average mass loss rate. These results suggest that the fungi of Group 1 are efficient decomposers of cellulose and that the fungi of Group 2 are efficient decomposers of lignocellulose. During cultivation, Pestalotiopsis sp. produced an appreciable amount of laccase activity (0.56+/-0.09 U/ml) without the addition of inducers and caused a loss in TOM of 38.2%+/-3.0%, suggesting that it has high potential to be a new efficient laccase-producing fungus.     

Lentinus edodes and Pleurotus species lignocellulolytic enzymes activity in submerged and solid-state fermentation of lignocellulosic wastes of different composition

Lentinus edodes and Pleurotus species from various origins were compared for the first time for their ability to produce lignocellulolytic enzyme in solid-state (SSF) and submerged (SF) fermentation of various plant raw material. Fungi cultivation in identical culture conditions revealed wide differences among both species and strains of the same species. The yields of CMCase (62.3Uml(-1)), xylanase (84.1 U ml(-1)), FPA (5.9 U ml(-1)), and laccase (4103 Ul(-1)) are the best so far obtained with the strains of oyster mushrooms. The study pointed out that the nature of lignocellulosic material and the method of fungi cultivation are factors determining the expression of lignocellulolytic potential of fungi as well as the ratio of individual enzymes in enzyme complex. SSF of tree leaves is favorable for laccase and MnP secretion by the majority L. edodes and Pleurotus strains, whereas SF provides better production of hydrolytic enzymes.     

Characterization of cellulases of fungal endophytes isolated from Espeletia spp.

Endophytes are microorganisms that asymptomatically invade plant tissues. They can stimulate plant growth and/or provide defense against pathogen attacks through the production of secondary metabolites. Most endophyte species are still unknown, and because they may have several applications, the study of their metabolic capabilities is essential. We characterized 100 endophytes isolated from Espeletia spp., a genus unique to the paramo ecosystem, an extreme environment in the Andean mountain range. We evaluated the cellulolytic potential of these endophytes on the saccharification of the oil palm empty fruit bunch (OPEFB). The total cellulolytic activity was measured for each endophyte on filter paper (FPA). In addition, the specific carboxymethyl cellulase (CMCase), exoglucanase, and β-glucosidase activities were determined. We found four fungi positive for cellulases. Of these fungi, Penicillium glabrum had the highest cellulolytic activity after partial purification, with maximal CMCase, exoglucanase and β-glucosidase enzyme activities of 44.5, 48.3, and 0.45 U/ml, respectively. Our data showed that the bioprospection of fungi and the characterization of their enzymes may facilitate the process of biofuel production.     

苹果霉心病微生态研究

对苹果心室及霉心病病果心室的微生物区系,霉心病的发病特点及生态防治做了系统研究,认为霉心病及健康果实心室存在多种微生物,霉心病的发生是微生态系统失调引起的。霉心病具有复合侵染、潜伏侵染特性。一些芽孢杆菌(Bacillus.sp)对病原菌有拮抗作用。生态防治可以应用于霉心病的防治。     

Fungal endophytes from Acer ginnala Maxim: isolation, identification and their yield of gallic acid

Aims:  The aim of the study was to isolate the endophytic fungi from Acer ginnala and screen isolates rich in gallic acid.
Methods and Results:  After epiphytic sterilization, 145 fungal endophytes were isolated from the stem, annual twig and seed of Acer ginnala . The endophytes were grouped into ten different taxa, Phomopsis sp., Neurospora sp., Phoma sp., Epicoccum sp., Penicillium sp., Alternaria sp., Fusarium sp., Trichoderma sp., Cladosporium sp. and a species of Pleosporales Incertae Sedis , by their morphological traits and ITS-rDNA sequence analysis. The content and yield of gallic acid of 141 isolates were determined by HPLC. On average, the species of Pleosporales Incertae Sedis had the highest content and yield of gallic acid (13·28 mg g⁻¹ DW; 119·62 mg l⁻¹), while Alternaria sp. had the lowest.
Conclusions:  Of 141 fungal endophytes from A. ginnala , Phomopsis sp. isolate SX10 showed both the highest content and the highest yield of gallic acid (29·25 mg g⁻¹ DW; 200·47 mg l⁻¹).
Significance and Impact of the Study:  Endophytic fungi isolated from A. ginnala may be used as potential producers of gallic acid and other compounds with biological activities, or functioned as elicitors to produce natural compounds.     

古银杏内生真菌的分离及其抑菌活性

采用组织分离法从古银杏健康组织中分离得到55株内生真菌, 其中28个分离菌株在PDA培养基上不产孢子, 占总分离菌株的50.9%, 其它菌株根据其在PDA培养基上的培养特征, 10株被鉴定为青霉、6株为曲霉、4株为交链孢霉、3株为简梗孢霉, 另外酵母、毛霉、小单头孢霉、镰孢霉各1株。考察内生真菌培养上清对7种受试指示菌的抑制作用, 共筛选得到23株至少对一种指示菌的生长有抑制作用的菌株, 其中11株为不产孢真菌, 占活性菌株的47.83%。对活性最强的一株菌进行形态学和分子生物学鉴定, 将其确定为Xyla     

Detection of fungal infectous agent of wheat grains in store-pits of Markazi province, Iran

Wheat is an economic and important crop that provides approximately 20% of food calorie in the world. It is first crop in Iran and cultivated in the most areas of this country. Store-pit fungi make undesirable changes in quality and appearance of wheat grains. Even, some fungi produce different mycotoxins which are toxic to human and livestock's that use wheat grains as source of food. In this study, several samples were randomly collected from each of five store-pits located in different areas of Markazi Province including: Arak, Mahallat, Khomein, Saveh and Sarband. Grains were treated on PDA, and blotter, agar and washing test also used for isolating and detection of fungi. At least 100 grains per each sample were randomly used for each test and treatment. The fungi that determined in this study were Cochliobolus australiensis, Cladosporium herbarum, Epicoccum sp., Tilletia leavis, Aspergillus flavus, A. niger, A. fumigatus, Alternaria alternata, Alternaria sp., Penicillium italicum, P. digitatum, Fusarium sp., Rhizopus sp., Ustilago tritici, Scytalidium sp. Among these fungi the most isolates were belonged to Cladosporium, Alternaria, Rhizopus and Fusarium. Cladosporium herbarum was the most common in different sampling areas. Tilletia laevis and Ustilago tritici were just recovered in washing test. This study revealed that different fungi are associated with wheat grains in store-pits in Markazi Province. Some of them like Aspergillus flavus normally produce aflatoxin, a very toxic and carcinogenic mycotoxin that is harmful for human.     

Screening of tetrachlorodibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxin-degrading fungi capable of producing extracellular peroxidases under various conditions

Forty-six pulp-bleaching fungi were screened for production of key enzymes for conversion of polychlorinated dibenzo-p-dioxins—lignin peroxidase (LiP), manganese peroxidase (MnP), and manganese-independent peroxidase (MiP)—under various conditions that would allow their utilization in the environment. Of 38 MnP-producing strains with MiP activity, 22 produced LiP. Three of the new isolates, Bjerkandera sp. strains MS191, MS325, and MS1167, were the best producers of the three different peroxidases, and had reasonable growth rates. The most promising Bjerkandera sp. strain, MS325, exhibited significant levels of LiP and MnP activities under various conditions, e.g., nutrient nitrogen-sufficient or -limited conditions, conditions with or without Mn(II), and changes in temperature (15–37°C). Furthermore, the ability of this strain to degrade 1,3,6,8-tetrachlorodibenzo-p-dioxin was confirmed. The results presented here indicate that utilization of Bjerkandera sp. strain MS325 on a practical scale in the environment has several advantages over many white rot fungi, which produce extracellular peroxidases only under specific conditions such as nutrient limitation.