Changes in phenol oxidases and superoxide dismutase during fruit-body formation of <Emphasis Type="Italic">Pleurotus </Emphasis>on sawdust culture

 Peroxidase and laccase activities increased rapidly up to the formation of primordia and then declined throughout the entire stage of fruiting. In the case of Pleurotus ostreatus, the level of Mn-dependent peroxidase was very low in primordia and fruiting stages but gradually increased with the growth of the fruit-body, whereas no activity was detected in Pleurotus sajor-caju during all growth stages. Superoxide dismutase activity was observed mainly at the fruiting stages. These results show that changes in concentration of lignin-related enzymes are associated with the fruiting process. Received: December 11, 2000 / Accepted: March 28, 2002     

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。     

Changes in phenol oxidases and superoxide dismutase during fruit-body formation of Pleurotus on sawdust culture

Peroxidase and laccase activities increased rapidly up to the formation of primordia and then declined throughout the entire stage of fruiting. In the case of Pleurotus ostreatus, the level of Mn-dependent peroxidase was very low in primordia and fruiting stages but gradually increased with the growth of the fruit-body, whereas no activity was detected in Pleurotus sajor-caju during all growth stages. Superoxide dismutase activity was observed mainly at the fruiting stages. These results show that changes in concentration of lignin-related enzymes are associated with the fruiting process.     

Effect of Nutritional Parameters on Laccase Production by the Culinary and Medicinal Mushroom, <Emphasis Type="Italic">Grifola frondosa</Emphasis>

Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 μM Cu over the basal level (1.6 μM Cu) and peak levels observed at 300 μM Cu were 7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu²⁺. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M _r 70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct laccase protein band (M _r 45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. Using 2,2′-azino-bis(ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate, the optimal temperature and pH values for laccase activity were 65°C and pH 2.2, respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.     

Effect of nutritional parameters on laccase production by the culinary and medicinal mushroom, Grifola frondosa

Summary Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 (M Cu over the basal level (1.6 mM Cu) and peak levels observed at 300 mM Cu were ∼ ∼7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu²⁺. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M _r ∼ ∼70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct second laccase protein band (M _r␣∼ ∼45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. The optimal temperature and pH values for laccase activity were 65 °C and pH 2.2 (using 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) {ABTS} as substrate), respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.     

Production and characterisation of lignocellulases of Panus tigrinus and their application

This study on the lignocellulases in broth cultures of the basidiomycete Panus tigrinus indicates that laccase and xylanase enzymes are constitutive and cellulase is inducible. In stationary culture at 28°C, the greatest laccase and xylanase activity was observed after growth for approximately nine days. Laccase production was dependent on the presence, and the particular brand, of malt extract in the growth medium. While production of laccase was enhanced by growth at 37°C and 42°C, xylanase was not. Raising the pH of the growth medium from pH 5.6 to pH 7.0 did not affect xylanase production, but laccase production was reduced at the higher pH. In shake culture, growth was pelleted and biomass lower than in stationary culture, and synthesis of both enzymes was strongly inhibited. Cultures of P. tigrinus decolourised Poly R-478 and the toxic triphenyl methane dye, crystal violet. It was also shown to degrade a natural lignocellulosic waste, sawdust.     

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。     

Spatial and temporal distribution of the alkaloid sanguinarine in Sanguinaria canadensis L. (Bloodroot)

Acclimated and non-acclimated potted plants of Sanguinaria canadensis L. were harvested at early and late dormancy, anthesis, and immature and mature fruiting stages. Sanguinarine content and concentration were determined for rhizomes (distal, proximal, and middle sections), roots, leaves, flower, and fruit. Rhizomes had highest sanguinarine content and concentrations, and exhibited decreasing concentration gradients from the distal to proximal third. Concentrations in roots were a tenth of rhizome concentration. Concentrations in leaves, flowers, and fruit were one-thousandth of rhizome Sanguinarine content in whole acclimated plants was constant. Content in whole nonacclimated plants increased as the plant became physiologically active, but was constant during fruit maturation: content in roots, leaves, and fruit did not change. The substantial increase in whole-plant dry weight coupled with the unchanging sanguinarine content during fruit maturation suggests either a shift in photosynthate allocation from defense to growth, or a constant turnover of sanguinarine.     

Spatial and temporal patterns of morel fruiting

The biotic and abiotic factors conditioning morel fruit body production are incompletely known. We examined spatial and temporal patterns of Morchella esculenta fruiting over five years in a wooded site in Missouri, USA. Fruiting onset was inversely correlated with spring air and soil temperatures, whereas abundance was positively correlated with rain events (>10 mm) during the 30 d preceding fruiting. The two years with the greatest fruiting had the shortest fruiting seasons (6–7 d). Fruiting season length was positively correlated with soil warming, suggesting that a narrow range of optimum soil temperatures favour the explosive production of fruit bodies. All woody stems of at least 1 cm diam were mapped and stem diameter and crown condition were noted. Morel fruit bodies were significantly closer to stems of Carya spp., Tilia americana and Ulmus americana than predicted by the frequencies of these woody species or their contribution to the total basal area on the site. Although intra-annual clustering of fruit bodies was often observed, inter-annual clustering was not. The spatial pattern of M. esculenta fruiting appears to be associated with vegetation pattern, whereas the onset and abundance of fruiting are determined by the interaction of spring temperatures with availability of supporting precipitation.     

The effect of bacteria on interspecific relationships between the euryhaline rotifer Brachionus rotundiformis and the harpacticoid copepod Tigriopus japonicus

Inconsistent results have been obtained on the population growth of Brachionus rotundiformis and Tigriopus japonicus, when results from single-species and two-species mixed cultures are compared. Bacteria growth was not regulated in these experiments, which could be the cause for this. In order to test this possibility, we conducted similar experiments under axenic and synxenic (with presence of one species of bacteria) conditions. The population growth of B. rotundiformis was suppressed by the presence of T. japonicus in axenic cultures. T. japonicus could not persist in axenic cultures, but its population increased when grown in synxenic cultures. T. japonicus used RT bacteria strain as a food source, while these bacteria were toxic to B. rotundiformis. These results suggest that bacteria can modify the interspecific relationship between B. rotundiformis and T. japonicus.     

Production of laccases by Pleurotus ostreatus in submerged fermentation in co‐culture with Trichoderma viride

Aims: To evaluate the production and stability of laccases by Pleurotus ostreatus in liquid co‐cultures with Trichoderma viride as a function of infection time and agitation rate. Methods and Results: Pleurotus ostreatus cultures were infected with T. viride spores at 30 and 48 h. Maximal laccase volumetric activity was seen after 48 h (control cultures) or 72 h (co‐cultures) of cultivation time. Only the cultures infected at 30 h showed an increased laccase volumetric activity compared to control cultures. After maximal laccase volumetric activity value was reached, a sharp decrease in it was observed in control cultures. Co‐cultures exhibited a comparatively lower loss of activity. The influence of P. ostreatus and/or T. viride on the stability of laccase volumetric activity and isoenzyme pattern was evaluated. Trichoderma viride induced changes in the laccase isoenzyme pattern. Agitated cultures increased biomass growth and specific productivity threefold and sevenfold, respectively, to the static cultures. Conclusions: The laccase volumetric activity is very likely the result of the balance between biosynthesis and degradation/biotransformation rates occurring during the cultures. The individual presence of P. ostreatus or T. viride in the culture negatively affected the volumetric laccase activity. Significance and Impact of the Study: The evaluation of culture parameters that could influence Trichoderma–basidomycetes interaction and laccase production during submerged fermentation has not been reported. This study showed how laccase production in co‐cultures of P. ostreatus and T. viride was influenced by the infection time and agitation/oxygenation conditions.     

Inhibition of cell wall degrading cellulase enzyme – the incitant of anthracnose disease caused by Colletotrichum capsici on capsicum fruit

Abstract

Cell wall degrading enzymes, namely cellulase, were detected in senescence and diseased macerated tissue of capsicum fruit. Their purported role in pathogenicity of Colletotrichum capsici was studied in detail. In situ production of cellulase concentration ranging from 80?–?360 μg was recorded in healthy capsicum fruit during storage. Production of this enzyme increased with an increase in the storage period. Increase in production of cellulase enzyme was recorded in infected tissue from seventh day after inoculation. Increased productions of this cell wall degrading enzyme coincide with disease manifestation and rapid progress of decay on capsicum fruit. Production of cellulase enzyme ranged from 80?–?360 μg. Conidial germination with advance of incubation period and further growth of C. capsici increased the production of cellulase enzyme. Conidia of C. capsici showed a gradual increase in production of cellulase enzyme with an increase in the incubation period. Treatment of capsicum fruit with different concentrations of calcium chloride, lactic acid and sorbic acid showed a 100%, 85.1% and 83.2% decrease in production of cellulase enzyme at the concentration of 1000 ppm, 500 ppm and 1000 ppm respectively.     

Effect of nitrogen source on lignocellulolytic enzyme production by white-rot basidiomycetes under solid-state cultivation

Summary The effect of additional nitrogen sources on lignocellulolytic enzyme production by four species of white-rot fungi (Funalia trogii IBB 146, Lentinus edodes IBB 363, Pleurotus dryinus IBB 903, and P. tuberregium IBB 624) in solid-state fermentation (SSF) of wheat straw and beech tree leaves was strain- and substrate-dependent. In general, the yields of hydrolytic enzymes and laccase increased by supplementation of medium with an additional nitrogen source. This stimulating effect of additional nitrogen on enzyme accumulation was due to higher biomass production. Only xylanase specific activity of P. dryinus IBB 903 and laccase specific activity of L. edodes IBB 363 increased significantly (by 66% and 73%, respectively) in SSF of wheat straw by addition of nitrogen source to the control medium. Additional nitrogen (20 mM) repressed manganese peroxidase (MnP) production by all fungi tested. The study of the nitrogen concentration effect revealed that 10 mM peptone concentration was optimal for cellulase and xylanase accumulation by P. dryinus IBB 903. While variation of the peptone concentration did not cause the change in MnP yield, elevated concentrations of this nutrient (20–40 mM) led to a 2–3-fold increase of P. dryinus IBB 903 laccase activity. About 10–20 mM concentration of NH₄NO₃ was optimal for cellulase and xylanase production by F. trogii IBB 146. However, neither the laccase nor the MnP yield was significantly changed by the additional nitrogen source.     

Laccase, cellulase and xylanase activities during growth ofPleurotus sajor-caju on sagohampas

Sagohampas, the fibrous pith residue left after starch extraction from sago palm, is abundant at sago-processing factories and can be used as a substrate for the production of laccase by solid substrate fermentation (SSF) withPleurotus sajorcaju, an edible mushroom. The fungus grown onhampas with an adjusted carbon : nitrogen ratio of 35:1, exhibited high laccase activity together with variable cellulase (0.3-2.8 U/g) and xylanase (0.9-10.1 U/g) activity. The maximum amount of laccase produced was approximately 17.7 U/g after 6 days of SSF using 4-week-old inoculum at a density of 10%. With the mature four-week inoculum, laccase activity increased 12-fold compared to that achieved with two-week-old inoculum. The optimum pH and temperature of the crude laccase were 6.0 and 50‡C, respectively. The apparent K_m and V_max values obtained were 0.073 mM and 0.962 U/min, respectively. The maximum laccase activity could be almost doubled after 6 days of fermentation by addition of 0.2 mM vanillin or ferulic acid; the cellulose to lignin ratio increased significantly during the 12 days of SSF, from 2.74 in the control to 3.3, when 0.2 mM of either vanillin or ferulic acid was added to the substrate.     

A new role for veratryl alcohol: regulation of synthesis of lignocellulose-degrading enzymes in the ligninolytic ascomyceteous fungus, Botryosphaeria sp.; influence of carbon source

A new physiological role for veratryl alcohol in fungi important in the biodegradation of the lignified plant cell wall is presented. Botryosphaeria sp., grown on starch, pectin, cellulose or xylan produced amylase, pectinase, cellulase, xylanase and laccase, whereas glucose and xylose repressed the synthesis of cellulase and xylanase, but not laccase. When cultured on each of these substrates in the presence of veratryl alcohol, laccase activity increased but the activities of amylase, pectinase, cellulase and xylanase significantly decreased. Basal medium containing softwood kraft lignin in the presence of veratryl alcohol induced laccases above constitutive levels. Ethyl alcohol also stimulated laccase production.     

Extracellular Enzymes Produced by the Cultivated Mushroom Lentinus edodes during Degradation of a Lignocellulosic Medium

Although the commercially important mushroom Lentinus (= Lentinula) edodes (Berk.) Sing. can be rapidly cultivated on supplemented wood particles, fruiting is not reliable. This study addressed the problem by developing more information about growth and development on a practical oakwood-oatmeal medium. The study determined (i) the components degraded during a 150-day incubation at 22 degrees C, (ii) the apparent vegetative growth pattern, (iii) the likely growth-limiting nutrient, and (iv) assays that can be used to study key extracellular enzymes. All major components of the medium were degraded, lignin selectively so. The vegetative growth rate was most rapid during the initial 90 days, during which weight loss correlated with glucosamine accumulation (assayed after acid hydrolysis). The rate then slowed; in apparent preparation for fruiting, the cultures rapidly accumulated glucosamine (or its oligomer or polymer). Nitrogen was growth limiting. Certain enzyme activities were associated with the pattern of medium degradation, with growth, or with development. They included cellulolytic system enzymes, hemicellulases, the ligninolytic system, (gluco-)amylase, pectinase, acid protease, cell wall lytic enzymes (laminarinase, 1,4-beta-d-glucosidase, beta-N-acetyl-d-glucosaminidase, alpha-d-galactosidase, beta-d-mannosidase), acid phosphatase, and laccase. Enzyme activities over the 150-day incubation period with and without a fruiting stimulus are reported. These results provide a basis for future investigations into the physiology and biochemistry of growth and fruiting.     

Dependence of the maximum temperature for growth of Saccharomyces cerevisiae on nutrient concentration

Anacystis nidulans (Synechococcus) was maintained in a medium of low phosphate concentration (0.1 mM) and grew with a normal doubling time of 5 hrs at 30°C. Such cultures ahd a normal pigment composition and alkaline phosphatase was detectable at low specific activities only.The onset of phosphate-limited growth occurred when the phosphate concentration in the medium fell to a value below 4 M (the limit of accurate determination by the assay method used) and resulted in increases in alkaline phosphatase activity, reaching a final 10 to 15 fold increase in specific activity after a period of several hours. Marked changes in the overall pigment composition occurred in this period of growth restriction. The addition of phosphate to such cultures resulted in a halt in synthesis of the enzyme and the restoration of normal pigmentation before growth resumed at the normal rate.Several organic phosphate esters could replace inorganic phosphate for growth and were also hydrolyzed by the partially purified enzyme, but growth rates were characteristically lower and the specific activity only 3 to 4 fold higher than in cultures grown in phosphate excess.Studies with the partially purified enzyme suggested that it differed in some of its properties from other alkaline phosphatases described in the literature.Abbreviations Used pNP pnitrophenol - pNPP pnitrophenylphosphate     

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。