滑菇营养生理研究

本文研究了滑菇在木屑-麦麸基质上生长期间,菌体对基质的转化效率和基质中主要组分的降解规律。实验结果表明:子实体绝对生物学效率为12.43%,产量系数为16.94%。子实体阶段的木素、半纤维素和纤维素的降解量以及非木质纤维素组分的减少量占子实体阶段基物中干物质减少量的百分比值分别为:5.85、17.54、49.13和27.48%。由此可见,纤维素是子实体生长阶段的主要碳源。滑菇在菌丝生长和子实体生长阶段分别有纤维素酶和半纤维素酶的活性高峰出现。进一步证明了:子实体阶段这两种酶的活性增加与子实体形成有密切关系     

曲酸对斑玉蕈子实体形成过程中木质纤维素酶的影响研究

为了探究曲酸增加子实体产量的机制,首先考察了搔菌后外源添加曲酸对不同菌丝培养时间出菇的影响。研究发现当菌丝培养时间过短或者过长添加曲酸都得不到很好的增产效果,菌丝培养时间在60-80d之间增产效率最高,并且后熟期60d的增产效率大于80d的增产效率。进一步研究发现添加曲酸可以提高菌丝利用基质中木质纤维素的利用率。更深入地研究发现,基质中的漆酶和纤维素酶活性在斑玉蕈的不同发育时期受到曲酸调控。漆酶活性在最初的菌丝恢复期和转色期酶活性低于对照组,但是在原基期、钉头期和子实体期酶活性显著地高于对照组;纤维素酶活性在整个发育周期中曲酸组都高于对照组,在子实体发育后期酶活性被提高3.16倍。最后,从分子水平上分析了漆酶基因和纤维素酶基因的表达量,研究显示添加曲酸后漆酶基因和纤维素酶基因在不同程度上被上调,这个结果与酶活的结果相一致。这些结果说明外源添加曲酸通过提高生殖生长阶段的菌丝利用培养基质中的漆酶和纤维素酶活性,进而提高菌丝利用木质纤维素,为斑玉蕈子实体生长发育提供更多的能源,实现增加子实体产量的目的。     

培养基中添加海藻糖对大球盖菇、斑玉蕈菌丝生长的影响

【背景】大球盖菇和斑玉蕈是食药兼用且具有开发潜力的珍稀食用菌,培养基对菌丝生长及子实体发育具有重要作用,优化培养基显得尤为重要。【目的】筛选出最适合培养大球盖菇、斑玉蕈的新型培养基。【方法】使用添加海藻糖的新型培养基,对不同培养基培养的大球盖菇及斑玉蕈菌株的菌丝生长状况、生长速度和生物量及纤维素酶、漆酶活性进行测定与分析。【结果】相较于PDA培养基,添加海藻糖的培养基能够提高菌丝生长速度、增加生物量,海藻糖添加的比例对纤维素酶和漆酶的影响较大,对大球盖菇及斑玉蕈菌丝的生长产生显著的促进作用,但不会改变其蛋白质的组成。【结论】大球盖菇最适合选用PTA-5培养基,斑玉蕈的最佳培养基是PDTA培养基。     

毛木耳降解木质纤维素的研究

测定了毛木耳“沪毛一号”菌株瓶栽0—60天期间,其木屑基质中木素纤维素的降解和木质纤维分解酶活性的变化。结果表明,该菌能够同时降解木素、半纤维素和纤维素,尤以分解木素的能力为强(以各自相对百分含量减少计算),故毛木耳为白腐真菌。该菌的多酚氧化酶、羧甲基纤维素酶、滤纸纤维素酶和木聚糖酶的活性高峰均出现于菌丝体发育阶段,在子实体原基发生时(22天),羧甲基纤维素酶、滤纸纤维素酶、木聚糖酶的活性明显下降,在子实体发生后,该三种酶活性又复上升,直到头批耳成熟酶活仍处于较高水平。     

培养温度和侧耳子实体形成对胞外纤维分解酶活性的影响

研究了培养温度及糙皮侧耳子实体形成对基物中胞外CMC酶、FP酶、半纤维素酶活性的影响。实验结果表明,于25℃培养。0—100天期间,基物上无子实体形成,基物中FP酶活力为0—1u,CMC酶和半纤维素酶的活力为0—8u左右。于11—13℃培养,基物上有子实体形成,并且在子实体迅速生长阶段基物中有该3种酶的活性高峰出现,高峰期FP酶、CMC酶和半纤维素酶的活力分别为7u,60u和70u左右。由此可见,上述3种酶活性的增加与培养温度和子实体形成有十分密切的关系。     

构菌栽培过程中对木质纤维素的降解和几种多糖分解酶活性的变化

本文分析了构菌在木屑-麦麸基物上生长发育期间,基物主要组分的降解和几种多糖分解酶活性的变化。结果表明,菌丝体迅速生长期间呼吸强度最大,在菌丝长满基物到子实体发生期间的呼吸强度很小,子实体发育期呼吸强度再次升高。在以新鲜木屑为主要基物时,构菌对基物中的纤维素和半纤维紊的分解作用较弱,并且构菌仅有很徽弱的木紊分解能力。胞外羧甲基纤维素酶、滤纸纤维素酶,木聚糖酶、果胶酶和淀粉酶的活性存在于构菌整个栽培过程,在菌丝体迅速生长期淀粉酶活力较高,在菌丝长满基物后活力明显下降,其它四种酶的活性变化规律与淀粉酶活性变化规     

生物垃圾好氧处理中的纤维素降解菌生长规律研究

目的:研究了蔬菜垃圾好氧处理过程中,纤维素降解菌和半纤维素降解菌(细菌和真菌),纤维素酶活和半纤维素酶活,和有机物降解之间的变化规律。方法:用添加纤维素和半纤维素的牛肉膏蛋白胨培养基和查式培养基,分别培养计数纤维素降解细菌、真菌和半纤维素降解细菌、真菌;马福炉灼烧测有机物含量。结果:好氧处理的初始阶段中,前4d有机物日均降解率5.2%,后3d日均降解率2.2%。结论:半纤维素降解菌的数量比纤维素降解菌的多,半纤维素酶活力,也高于纤维素酶活力;微生物的变化情况为前6d产两种酶的微生物主要有细菌和真菌;从第6d开始真菌快速生长;至第7d真菌纤维素酶和半纤维素酶活力显著升高。     

纤维素分解混合菌的扩繁条件研究

目的:开发纤维素分解混合菌制剂。方法:以菌数、纤维素酶活性及木聚糖酶活性做为评价指标,对具有开发潜力的菌群进行了培养条件研究。结果:该菌群的最适培养温度为35℃;最适接种量为1%;培养基的初始pH值以7.0-7.5为好;在培养基中添加一定量的FeSO4,有利于菌的生长;适当控制氧气浓度,可以确保混合菌群中纤维素酶产生菌的正常繁殖。结论:获得了纤维素分解混合菌的扩繁条件。     

贝叶多孔菌营养生理生化特性的研究

本文对贝叶多孔菌(Polyporus frondosus)在柞树木屑——麦麸基物上生长期间基物的降解特性和培养物中胞外纤维素酶、半纤维素酶和淀粉酶在培养过程中的活性变化规律及其它一些生物化学性质进行了研究。认为贝叶多孔菌是白腐型木腐真菌。在培养初期主要利用基物中可溶性糖类为碳源,在子实体发育期,碳源基本由降解基物中木质纤维素所提供。     

麝鼠肠道纤维素分解菌的分离鉴定及其酶学特性分析

本研究旨在从麝鼠(Ondatra zibethicus)肠道中分离出高效分解纤维素的菌株,为开发纤维素分解菌微生物制剂提供菌种资源。本研究利用以羧甲基纤维素钠(CMC-Na)为单一碳源的培养基,从麝鼠盲肠内分离出--株高效分解纤维素的菌株WJ-3,并对该菌株进行形态鉴定、生理生化鉴定和16S.rDNA分子鉴定。对菌株WJ-3所产羧甲基纤维素酶(CMCase)进行酶学特性实验,分析此纤维素酶的最佳反应pH和最佳反应温度,以及此纤维素酶对不同温度和不同酸碱度的耐受性。结果表明,菌株WJ-3属于空气芽孢杆菌(Bacillus aerius),并将其命名为Bacillus aerius WJ-3。菌株WJ-3所产羧甲基纤维素酶在pH 4.0~6.0的范围内反应时,酶活性随pH值升高而增加,其最佳反应pH为6.0,且此纤维素酶在pH4.0~8.0范围内保存30min后均能保持80%以上的相对酶活性:菌株WJ-3所产羧甲基纤维素酶在温度30~50 ℃范围内反应时,随温度上升酶活性逐渐增加,在50 ℃时酶活性最高,之后随温度的升高酶活性逐渐下降,且纤维素酶在此温度范围内保存30 min后均能保持较高的酶活性。综上所述,菌株Bacillus aerius WJ-3所产羧甲基纤维素酶的酶活性较高,并且此纤维素酶的耐酸碱性及热稳定性良好,是具有一定利用价值的菌种资源。     

Some chemical and biochemical changes in straw constituents during growth of Pleurotus flabellatus (Berk & Br) Sacc

Summary The quantitative changes in constituents of rice straw during different stages of growth of the fungus Pleurotus flabellatus were investigated. Cellulose, hemicellulose(s), lignin, total carbon and total nitrogen showed a continuous decrease from inoculation until the end of fruit body harvesting, whereas free sugars, total ash and C/N ratio increased. As calculated on constant ash basis, 14 and 13.9% of cellulose, 6.6 and 7% of hemicellulose(s) and 4 and 1.5% of lignin were decomposed during the mycelial growth and fructification respectively. Total N decreased by 0.16 and 0.23% during the mycelial growth and fructification respectively. The progressive breakdown of cellulose and hemicellulose(s) was correlated to an apparent increase in the activities of celullase and hemicellulase(s). The trend in development of cellulases and -glucosidase activities in the substrate during different stages of its growth was demonstrated.     

川西高山林线交错带凋落物纤维素分解酶活性研究

以川西高山林线交错带3种典型植被类型(针叶林、高山灌丛、高山草甸)下两个层次(LF层: 新鲜凋落物层和发酵层; H层: 腐殖质层)的凋落物为研究对象, 分别模拟凋落物分解的前期和后期阶段, 对凋落物分解过程中的纤维素酶活性及凋落物质量进行了研究。结果表明, 凋落物分解前期的纤维素酶活性和纤维素含量均显著高于分解后期, 但植被类型对LF和H层的纤维素含量的影响都不显著。双因素方差分析结果表明, 凋落物分解阶段对纤维素酶活性和纤维素含量的影响比植被类型对纤维素酶活性和纤维素含量的影响更大。不同种类的纤维素酶活性在分解前期和分解后期受到不同因子的限制。凋落物分解前期, 微晶纤维素酶和β-葡萄糖苷酶活性可能受N、P含量的限制, 而羧甲基纤维素酶主要受底物纤维素含量控制; 凋落物分解后期, 羧甲基纤维素酶和β-葡萄糖苷酶可能受C、N含量的限制。生态化学计量学的理论预测, 底物质量比C:N > 27或C:P > 186时会限制微生物生长, 因此判断高山林线交错带凋落物微生物生物量和纤维素酶活性同时受到底物N、P的限制, 尤其是高山草甸上微生物生物量在凋落物分解前期受到底物N、P的限制比分解后期更显著, 这充分说明了底物质量调控着凋落物分解过程中的纤维素酶活性和微生物生物量。     

Degradation dynamics of lignocellulose materials by wood-rottingPleurotus fungi

When grown on wheat straw,Pleurotus decomposes both the lignin and the cellulose components of the substrate. The course of degradation differs during growth and fructification. The losses of dry mass during growth were about 20 %. The absolute amount of hemicelluloses, cellulose and lignin was decreasing. Hemicelluloses and lignin were degraded at a higher rate than cellulose. The total mass losses of the substrate after fructification were 32 to 45 %. Cellulose was consumed at a higher rate than lignin.     

Activity of cellulolytic enzymes in the contents of reticulorumen and caecocolon of roe deer (Capreolus capreolus)

Selective ruminants, which prefer easily digestible plants, cannot digest fibrous forage as well as grass eaters. Low enzyme activity or short retention time of ingesta particles in fermentation chambers appeared to be responsible for reduced cellulose breakdown. Seasonal activity of cellulolytic enzymes, cellulose concentration and protozoa population in reticulorumen (RR) and caecocolon (CC) of roe deer as a typical concentrate selector were investigated. Cellulase activities were lowest in winter when cellulose concentration in RR contents were highest. Highest enzyme activities and lowest cellulose concentration were measured in early spring. Cellulolytic activities were significantly correlated with the number of protozoa in RR. Only one entodinomorphic genus was identified in the RR. The enzyme activities in CC were far lower compared with those in RR. Low cellulose digestion in the RR cannot be compensated for by cellulose breakdown in the CC. The reduced cellulose digestion of roe deer may be attributed to the short retention time of food particles in spring and summer, whereas decreased colonisation of microorganisms in the rumen may be the main reason for low cellulose breakdown in winter.     

Analysis of the putative substrate binding region of hyperthermophilic endoglucanase from Pyrococcus horikoshii

A hyperthermophophilic beta-1,4 endoglucanase (family 5, cellulase) was identified in a hyperthermophilic archaeon Pyrococcus horikoshii and found to be capable of hydrolyzing crystalline cellulose at high temperatures. This hyperthermophilic enzyme has promise for applications in biomass utilization, but we have no information regarding the catalytic mechanism or structure of the enzyme. To determine its catalytic mechanism, we examined the roles of amino acids located in a loop near the speculative active site by the alanine scanning method. Ten mutants of the enzyme were constructed and expressed in Escherichia coli. The purified mutant enzymes were assayed for their hydrolytic activities on p-nitrophenyl cellobiose (pNG2), carboxylmethyl cellulose, and avicel. The results showed that His155, Arg156, and Ile162 play an important role in pNG2 binding capacity, and that H155 and I162 are important for catalysis.     

Bioconversion and biotransformation of coir pith for economic production of Pleurotus florida: chemical and biochemical changes in coir pith during the mushroom growth and fructification

Coir pith represents ∼50% of the waste from the coir industries and was tested for its potential in serving as a growth substrate for the production of species of oyster mushroom, Pleurotus florida. Due to its high lignin (∼48%) content and amorphous powdery nature, coir pith supported poor mushroom mycelial growth and yields were considerably low (∼25% bioconversion efficiency). Pre-treating coir pith with hot water did not prove economical to produce the mushroom yields. Acid swelling and alkali delignification of coir pith though served to change the structure of coir pith; the mushroom yields were not improved. Amendment of coir pith with rice (Oryza sativa) straw and horse gram (Dolichos biflorus) plant residue tended to greatly modify the physical characteristics of the inoculated mushroom bed. Such a supplementation of coir pith growth substrate resulted in production of mushroom yields with 110–125% bioconversion efficiency. Implications of supplementing coir pith with rice straw/horse gram plant residue in terms of holocellulose:lignin ratio are discussed. Sensorially, the mushrooms so produced did not differ from that on rice straw, the economic growth substrate recommended for production of the mushroom yields on commercial scale. Changes in cellulose, hemicellulose and lignin contents of coir pith amended with rice straw were studied. Cellulase, hemicellulase and protease enzyme activities in the amended coir pith substrate showed a continuous increase from inoculation till the end of fructification, whereas laccase activity decreased during fructification, in consonance with decreased lignin degradation during fructification.     

Effects of noncatalytic residue mutations on substrate specificity and ligand binding of Thermobifida fusca endocellulase cel6A.

The availability of a high-resolution structure of the Thermobifida fusca endocellulase Cel6A catalytic domain makes this enzyme ideal for structure-based efforts to engineer cellulases with high activity on native cellulose. In order to determine the role of conserved, noncatalytic residues in cellulose hydrolysis, 14 mutations of six conserved residues in or near the Cel6A active-site cleft were studied for their effects on catalytic activity, substrate specificity, processivity and ligand-binding affinity. Eleven mutations were generated by site-directed mutagenesis using PCR, while three were from previous studies. All the CD spectra of the mutant enzymes were indistinguishable from that of Cel6A indicating that the mutations did not dramatically change protein conformation. Seven mutations in four residues (H159, R237, K259 and E263) increased activity on carboxymethyl cellulose (CM-cellulose), with K259H (in glucosyl subsite -2) creating the highest activity (370%). Interestingly, the other mutations in these residues reduced CM-cellulose activity. Only the K259H enzyme retained more activity on acid-swollen cellulose than on filter paper, suggesting that this mutation affected the rate-limiting step in crystalline cellulose hydrolysis. All the mutations lowered activity on cellotriose and cellotetraose, but two mutations, both in subsite +1 (H159S and N190A), had higher kcat/Km values (6.6-fold and 5.0-fold, respectively) than Cel6A on 2,4-dinitrophenyl-beta-D-cellobioside. Measurement of enzyme : ligand dissociation constants for three methylumbelliferyl oligosaccharides and cellotriose showed that all mutant enzymes bound these ligands either to the same extent as or more weakly than Cel6A. These results show that conserved noncatalytic residues can profoundly affect Cel6A activity and specificity.     

Characterization of the extracellular cellulase from a mesophilic clostridium (strain C7).

An extracellular, 700,000-Mr multiprotein complex that catalyzed the hydrolysis of crystalline cellulose (Avicel) was isolated from cultures of Clostridium sp. strain C7, a mesophile from freshwater sediment. In addition to cellulose (Avicel, ball-milled filter paper), the multiprotein complex hydrolyzed carboxymethylcellulose, cellodextrins, xylan, and xylooligosaccharides. Hydrolysis of cellulose or cellotetraose by the complex yielded cellobiose as the main product. Cellopentaose or cellohexaose was hydrolyzed by the complex to cellotriose or cellotetraose, respectively, in addition to cellobiose. Xylobiose was the main product of xylan hydrolysis, and xylobiose and xylotriose were the major products of xylooligosaccharide hydrolysis. Activity (Avicelase) resulting in hydrolysis of crystalline cellulose required Ca2+ and a reducing agent. The multiprotein complex had temperature optima for Avicelase, carboxymethylcellulase, and xylanase activities at 45, 55, and 55 degrees C, respectively, and pH optima at 5.6 to 5.8, 5.5, and 6.55, respectively. Electron microscopy of the 700,000-Mr enzyme complex revealed particles relatively uniform in size (12 to 15 nm wide) and apparently composed of subunit structures. Elution of strain C7 concentrated culture fluid from Sephacryl S-300 columns yielded an A280 peak in the 130,000-Mr region. Pooled fractions from the 130,000-Mr peak had carboxymethylcellulase activity but lacked Avicelase activity. Except for the inability to hydrolyze cellulose, the 130,000-Mr preparation had a substrate specificity identical to that of the 700,000-Mr protein complex. A comparison by immunoblotting techniques of proteins in the 130,000- and 700,000-Mr preparations, indicated that the two enzyme preparations had cross-reacting antigenic determinants.     

棉秸秆降解高温菌株的筛选及产酶分析

从新疆地区分离具有降解棉秸秆纤维素功能的菌株,得到4株耐高温真菌(50°C)。纤维素酶学性质分析表明,该4株菌的纤维素酶具有良好的耐酸性(最适pH为4.5)和耐高温性(最高达60°C)。以羧甲基纤维素钠(CMC-Na)、微结晶纤维素、棉花、滤纸、淀粉、果胶为底物测定酶活力,滤纸酶活力(FPA)最高达2.63 U/mL、淀粉酶活力最高达6.17 U/mL、果胶酶活力最高达5.86 U/mL。4株真菌酶学特性分析表明,该系列菌株在秸秆生物质利用方面有很大的应用潜力。     

Properties of a genetically reconstructed Prevotella ruminicola endoglucanase.

A pUC19-derived plasmid was constructed that coded for a hybrid cellulase with the Thermomonospora fusca E2 cellulose-binding domain at its C terminus joined to the Prevotella ruminicola 40.5-kDa carboxymethyl cellulase (CMCase). The hybrid enzyme was purified and characterized enzymatically. It bound tightly to cellulose, and its specific activities on carboxymethyl cellulose, amorphous cellulose, and ball-milled cellulose were 1.5, 10, and 8 times that of the 40.5-kDa CMCase, respectively. Furthermore, the modified enzyme gave synergism with an exocellulase in the degradation of filter paper, while the 40.5-kDa CMCase did not.