白腐真菌对稻草秸秆的降解及其有关酶活性的变化

以稻草秸秆加20%棉籽壳为培养基质,接种侧耳Z17、921、1024菌株,在不同生长阶段。测定培养物的主要化学成分和有关酶活的变化。结果表明,从接种到子实体形成,所试菌株培养物的纤维素、木质素等呈持续不断的下降趋势,水份、粗蛋白含量却逐渐升高,基质中漆酶酶活性在菌丝生长初期呈迅速上升趋势,后稍降低,而愈创木酚酶活性在菌丝上长初期及子实体形成时达到高峰,后有所降低或消失。     

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。     

滑菇子实体阶段胞外CMC酶和HC酶酶活性增加与培养温度和子实体形成的关系

本文研究了滑菇(Pholita nameko)子实体阶段培养物中胞外羧甲基纤维素酶(CMC酶)和半纤维素酶(HC酶)的活性增加与培养温度和子实体形成的关系。实验结果表明:(1)低温(9—11℃)培养,菌丝体能分化形成子实体,并且在子实体生长阶段培养物中胞外CMC酶和HQ酶的活性迅速增加,在子实体成熟期左右,有酶活性高峰出现,酶活力为40—100u左右。(2)在23—25℃培养,培养物上无子实体形成,在相应的子实体生长期间(以低温培养组为对照),培养物中CMC酶和HC酶活性很低(0—4u左右)     

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。     

漆酶同工酶基因在斑玉蕈生长发育过程中的表达分析

为了探明漆酶在斑玉蕈生长发育过程中的功能,对斑玉蕈转录测序预测的13个漆酶基因序列进行分析、鉴定和构建分子系统发育树;检测了不同生长发育时期漆酶的活性和漆酶基因表达水平。研究结果显示：13个基因片段中有10个是漆酶基因。不同的漆酶同工酶之间进化关系存在明显差异,大多数漆酶与木腐菌（金针菇Flammulina filiformis和侧耳属Pleurotus）进化关系较近。对斑玉蕈不同生长发育时期的酶活检测结果显示,从斑玉蕈的菌丝恢复期到钉头期,漆酶活性逐渐升高,而在子实体形成后期酶活逐渐降低。对培养40d、60d和80d的菌丝样品以及不同生长发育时期的样品进RT-qPCR检测,结果显示在菌丝营养生长时期,大多数漆酶基因在第40-60天表达量持续增加1-3倍,而在第60-80天时表达量出现降低的情况。而在生殖生长时期,大多数漆酶基因在转色期或者原基期相对表达量达到最大值,并在子实体期出现降低,这与漆酶活性的检测具有一致性。lcc3、lcc7、lcc8和lcc9在斑玉蕈生殖生长过程中相对表达量出现了10-100倍的上调。这说明从菌丝培养到菌丝扭结形成子实体和子实体发育的过程中,不同的漆酶可能发挥着不同的作用,表达量较高的漆酶基因可能对基质降解和子实体形成起主要作用。     

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     

培养温度对滑菇子实体阶段胞外纤维素酶活性的影响

本文研究了培养温度对滑菇子实体形成期间胞外羧甲基纤维素酶活性的影响。实验结果表明:1,低温(9—11℃)培养,菌丝体能分化形成子实体,并且在子实体担孢子释放期左右,培养物中有CMC酶活性高峰出现,酶峰期活力为40—100u左右。2、在23—25℃培养300天期间,培养物不能形成子实体,在相应于子实体生长期间(以有子实体生长的低温培养组为对照),培养物中无明显酶峰出现,酶活力为0—2u左右。由此可见,培养温度对滑菇子实体形成和胞外CMC酶活性有显著的影响。     

白灵侧耳栽培过程中胞外酶和呼吸酶活及离子流速的研究

研究了白灵侧耳Pleurotus nebrodensis栽培过程中生长发育各阶段的漆酶、羧甲基纤维素酶（CMC酶）、半纤维素酶和淀粉酶等4种胞外酶及苹果酸脱氢酶、葡萄糖-6-脱氢酶和6-磷酸葡萄糖酸脱氢酶等3种呼吸酶的活性变化,同时测定了菌丝表面H+、K+、Ca2+离子流速。结果表明,整个生育期的胞外酶活性变化具有明显的阶段性。漆酶酶活在菌丝生长阶段第28天时达到最大值,纤维素酶和半纤维素酶酶活在子实体生长阶段第107天时达到最大值,子实体收获后活性下降,而淀粉酶活性在各时期均比较低,说明白灵侧耳对木质素类物质利用最早。苹果酸脱氢酶、葡萄糖-6-脱氢酶和6-磷酸葡萄糖酸脱氢酶酶活在后熟期、温差刺激及子实体生长期出现3个峰值。整个栽培周期的菌丝表面H+、K+、Ca2+离子流速各个阶段有不同的变化规律,Ca2+在菌丝生长阶段内流,从后熟期开始外排,K+和H+在整个周期外排;C/N和温度对离子的流速有一定的影响。     

芬娜冬菇子实体发育过程及胞外酶活性

分离并鉴定了采自北京妙峰山的1株野生大型真菌——芬娜冬菇Flammulina fennae,对该菌株进行了子实体发育过程研究,并对其最适生长条件进行了正交实验,测定并分析了菌株液体发酵及子实体生长过程中的酶活变化。结果表明:芬娜冬菇在原基期就完成了菌柄菌盖的分化;正交实验结果显示,不同氮源和p H对菌株生长影响显著,不同碳源则影响不显著;菌株液体发酵过程中未检测到过氧化氢酶和酸性木聚糖酶活性,而子实体发育过程中,漆酶、纤维素酶、超氧化物歧化酶、过氧化氢酶、酸性木聚糖酶活性较高。在原基形成期和子实体发育后期5种酶活性较高,均呈现先升后降的变化趋势,超氧化物歧化酶、过氧化氢酶、纤维素酶活性峰值出现在原基形成期和子实体发育后期,酸性木聚糖酶活性峰值出现在原基发育期,漆酶活性峰值出现在子实体发育后期。     

锰离子Mn(Ⅱ)对糙皮侧耳木质素降解酶活性和原基形成的影响

木质素降解酶是糙皮侧耳Pleurotus ostreatus分解木质素的关键酶。本文以糙皮侧耳栽培菌株New831为试验材料,通过固体培养法和液体培养法测定了不同浓度锰离子Mn(II)对其菌丝生长、偶氮染料橙黄II降解率、木质素降解酶活性和原基形成的影响。结果表明,添加Mn(II)可促进糙皮侧耳菌丝生长,GP-Orange液体培养时的最适Mn(II)浓度为50μmol/L,GP-Urea-Orange固体培养时的最适Mn(II)浓度为70μmol/L;添加Mn(II)可加速橙黄II的降解,Mn(II)浓度为70μmol/L时,橙黄II降解率最高、脱色圈最大;漆酶和锰过氧化物酶活性均随Mn(II)浓度增加呈现"先升后降"的趋势,而木质素过氧化物酶活性则随Mn(II)浓度的增加而降低;添加Mn(II)可诱导原基形成,Mn(II)浓度为70μmol/L时,原基数量最多。此外,平板培养结果揭示木质素降解酶活性与菌丝生物量之间无相关性,其主要是在营养限制条件下表达。     

Enzymes of the xylotrophic basidiomycete Lentinus edodes F-249 in the course of morphogenesis

It has been shown that the fungus Lentinus edodes grown on a solid wort agar substrate produces intracellular enzymes, including Mn-dependent peroxidase, laccase, and tyrosinase as a family of isoforms. The composition of the complex (containing one to four forms of each enzyme) varied during the basidiomycete life cycle. The activity of oxidases was maximal at the stage of nonpigmented mycelium and at the stages of a brown mycelial mat and a fruit body. The activity of tyrosinase increased in the course of mycelium pigmentation and had two maxima: at the stage of a brown mycelial mat and at the stage of a fruit body. Laccase and tyrosinase activities were shown to increase sharply upon addition of oak sawdust extract to the culture medium as compared with the enzyme activities of mycelium grown on wort agar alone. It was established that the effect of phenol oxidase substrates on the growing mycelium consists in a twofold acceleration of the process of morphogenesis in the fungus L. edodes.     

Enzymes of the xylotrophic basidiomycete Lentinus edodes F-249 in the course of morphogenesis

It has been shown that the fungus Lentinus edodes grown on a solid wort agar substrate produces intracellular enzymes, including Mn-dependent peroxidase, laccase, and tyrosinase as a family of isoforms. The composition of the complex (containing one to four forms of each enzyme) varied during the basidiomycete life cycle. The activity of oxidases was maximal at the stage of nonpigmented mycelium and at the stages of a brown mycelial mat and a fruit body. The activity of tyrosinase increased in the course of mycelium pigmentation and had two maxima: at the stage of a brown mycelial mat and at the stage of a fruit body. Laccase and tyrosinase activities were shown to increase sharply upon addition of oak sawdust extract to the culture medium as compared with the enzyme activities of mycelium grown on wort agar alone. It was established that the effect of phenol oxidase substrates on the growing mycelium consists in a twofold acceleration of the process of morphogenesis in the fungus L. edodes.     

灵芝深层发酵优良菌株的筛选

灵芝种类多,种间与品种间的不同均可能引起有效成分在产量表达上的差异,为了能筛选适宜深层发酵法生产的灵芝菌株,以8个灵芝菌株为研究对象,比较不同菌株在固体培养基上的菌丝体及液体发酵的菌球萌发菌丝生长速度、液体发酵产物、子实体多糖等指标。结果表明:8个菌株菌丝体生长速度有一定差异,以灵芝G9、红芝早萌发且生长速度快,甜芝、紫芝、灵芝G8、血芝其次,灵芝5760、黑芝最慢。8个菌株深层发酵产物由于生物学特性不同而有差别,同时菌株菌丝体生物量与次生代谢产物多糖之间无必然的联系,甜芝菌丝体生物量最大但自溶速度最快,灵芝5760菌球细小,速度最慢,血芝次生代谢产物多糖最高。菌丝体多糖与子实体多糖比较,各个菌株的菌丝体多糖含量均高于子实体多糖含量。提取多糖工艺以超声波辅助破壁处理后,再以热水浸提法提取多糖的得率明显提高,紫芝、黑芝提高了2倍以上。     

Fruiting of Agaricus bisporus

Several enzymes were assayed in extracts from mycelium-colonised compost during growth and fruiting of Agaricus bisporus (Lange) Imbach. Comparison of changes of enzyme levels in axenic and nonaxenic cultures and in cultures of non-fruiting strains indicated that they were associated directly with the fungal mycelium. Large changes were found in the amounts of laccase and cellulase which were correlated with fruit body development. Laccase concentration increased during mycelial growth and then declined rapidly at the start of fruiting. Cellulase activity could be detected throughout growth but increased at fruiting. No such changes were observed in xylanase, alkaline protease, laminarinase and acid and alkaline phosphatases. Activities of laccase and cellulase were measured in axenic cultures arrested at various stages of fruiting development. Such cultures showed that the changes in concentration of laccase and cellulase were associated with the enlargement of fruit bodies.     

Alterations in the composition of membrane glycero-and sphingolipids in the course of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> surface culture development

Qualitative and quantitative characteristics of the alterations in the lipid composition of the membrane of the basidial fungus Flammulina velutipes in the course of surface culture development were investigated. Modifications of the lipid composition were shown to be timed to specific ontogeny stages, such as changes in the growth rate of the colonies, the appearance of differentiated vegetative cells, and the formation of generative structures. A slowdown of growth correlated with an alteration in the ratio of major classes of phospholipids, namely, with a decrease of phosphatidylcholine relative content and an increase in phosphatidylethanolamines. The differentiation of vegetative cells of the mycelium proceeded along with modifications of molecular composition of glycoceramides. In the course of the first week of growth, the surface culture of F. velutipes produced monohexosylceramides with epoxidized methyl sphingadienine as a sphingoid base. Later on, along with culture growth and specialization of mycelium cells, molecular species with methyl sphingadienine, common for basidiomycetes, start to prevail among the fungal glycoceramides. The formation of fruit bodies is accompanied by enrichment of molecules of phospholipids, mainly, the phosphatidylcholines, with unsaturated fatty acids.     

Profile of the extracellular lignocellulolytic enzymes activities as a tool to select the promising strains of Volvariella volvacea (Bull. ex Fr.) sing

Out of 26 strains of Volvariella volvacea used, 18 were of ‘typical’ type and possessed all the characteristics of a normal V. volvacea mycelium, while the rest 4 ‘atypical’ type strains showed completely distinct mycelial growth characteristics. The remaining 4 strains grew very slowly and exhibited growth characteristics of single spore isolates of V. volvacea. Strains varied in their extracellular lignocellulolytic activities and strains; OE-274, OE-272 and OE-210 with high ligninase enzymes (laccase and polyphenol oxidase) activities, gave highest mushroom yield on pasteurized paddy straw substrate. On the composted paddy straw substrate, additional two strains, OE-213 and OE-215 with lower activities of ligninases also gave higher mushroom yield. Mushrooms were harvested 3 to 4 d early from the composted substrate than on the pasteurized substrate. Activities of endoglucanase, laccase and polyphenol oxidase were found to be more crucial for mushroom yield on pasteurized substrate, while xylanase and β-glucosidase were more important for composted substrate. Strains also varied in their fruiting bodies quality and the substrate used for mushroom cultivation also affected the fruiting body quality. The superior yielding strains varied in shape, size, weight, colour and contents of sodium and potassium in their fruiting bodies; while contents of carbon, calcium and protein did not vary much with the strains.     

血红铆钉菇SN菌株生物学特性研究

采用组织分离法从野生血红铆钉菇子实体分离得到1株性状稳定的分离株SN,并对其生物学特性进行了研究.结果表明:最适合菌株生长的碳源为葡萄糖和蔗糖,最适宜的氮源为蛋白胨,菌丝生长的最适温度为22～24℃,最适pH为8,无光照培养时菌丝长势较好.培养料中添加20％腐殖质适合菌丝的生长.     

肥皂草素对斑玉蕈菌丝体抗氧化酶活性及相关基因表达量的影响

【目的】研究肥皂草素对斑玉蕈菌丝体的活性氧代谢、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性及其基因表达的影响。【方法】在摇瓶培养基中添加不同浓度的肥皂草素研究菌丝体在不同培养时间内活性氧代谢、SOD和CAT的活性及其基因表达的变化。【结果】添加肥皂草素后,在7?11 d内超氧阴离子及丙二醛(MDA)的含量整体较对照组有升高趋势,但是在13?15 d含量较对照又有所下降;肥皂草素处理的菌丝体SOD活性随着浓度的增加而逐渐增强(第9天除外),特别是在培养13?15 d更加明显;CAT活性同样随着浓度的增加而逐渐增强,特别是0.05 g/L实验组酶活性始终保持在较高的水平;不同浓度的肥皂草素都能使Mn-SOD基因、CAT基因的表达出现上调趋势,在0.05 g/L时,肥皂草素诱导Mn-SOD基因的表达与其活性的变化趋势基本一致,而CAT基因表达与其活性的变化并不一致,其活性可能受基因翻译后的修饰调控等因素的影响。【结论】肥皂草素的添加能够诱导SOD和CAT酶活性的增强及上调Mn-SOD、CAT基因的表达量,减缓菌丝培养后期活性氧和MDA的积累。