Substrate specificity of laccase from Lentinus edodes

In previous studies, the white-rot basidiomycete Lentinus edodes, strain SC-495, was proved to be a “selective” lignin degrader and its extracellular crude preparations arising from solid-state cultures were successfully employed in biopulping experiments on annual plants. This fungus produced extracellular laccase as the predominant phenoloxidase when growing in solid-state fermentation on corn stalks. Laccase from this strain was purified and partially characterized, as an initial approach towards the study of its ligninolytic complex. Laccase was purified 69.6-fold by anion-exchange chromatography and two affinity-chromatography steps with an overall yield of 7.45%. The native enzyme exhibited a molecular mass of 74 kDa, an isoelectric point of 3.42 and a carbohydrate content of 7.5%. The absorption spectrum of laccase showed a maximum at 605 nm, typical of blue-copper oxidases. The optimum pH and temperature for the activity of laccase were 4.0–4.2 and 50°C, respectively. Kinetic experiments, performed with a wide range of phenolic compounds, showed that the reaction rate and the substrate affinity greatly varied depending on the nature of substituents and their reciprocal positions on the aromatic ring. In particular, the enzyme showed high affinity to phenolic compounds bearing methoxyl or methyl groups, but no affinity to those bearing the nitro group directly attached to the benzene ring, nor to non-phenolic lignin-related compounds, such as trans-cinnamic acid or 3,4-dimethoxycinnamic acid. The huge differences in terms of reactivity of the enzyme towards phenolic compounds suggests that a preliminary systematic screening should be advisable when using laccase in effluent treatment applications.     

Effect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Lentinula (Lentinus) edodes

Abstract Lentinula (Lentinus) edodes , strain LS4, produces manganese-dependent peroxidase (MnP) and laccase, but not lignin peroxidase, when grown on a defined medium with glucose as sole carbon source. MnP production is suppressed by nitrogen whereas highest levels of laccase were observed when the fungus was grown under high nitrogen (26 mM) conditions. Both the titre and time of appearance of MnP were affected by the concentration of Mn in the culture medium with highest enzyme levels recorded in cultures supplemented with 1.1 ppm Mn. Purified MnP from L. edodes LS4 has an apparent M _r of 59000 and a p I of 5.6, and differs in several respects from a MnP isolated from L. edodes grown on a commercial wood substrate.     

Adapting liquid spawn Lentinus edodes to oak wood

Summary Lentinus edodes (Berk.) Sing. is a commercially important edible mushroom cultivated on oak (Quercus spp.) logs or wood particles. Inoculum (spawn) of this white-rot saprophyte, made by homogenizing cultures grown on a chemically defined liquid medium, dies when transferred to oak wood. This study shows that an aqueous extract of the oak wood is inhibitory to growth. Inoculum produced by homogenizing stationary-grown cultures was particularly sensitive to the extract. Three different methods decreased the sensitivity of inoculum: (1) use of intact (nonhomogenized) cultures as inoculum, (2) use of homogenized cultures produced from shaken rather than stationary cultures as inoculum, or (3) use of cultures grown in the presence of wood extract as inoculum. With the use of shaken cultures adapted to oak wood extract, homogenized-culture inoculum successfully colonized an oak wood medium. Adaptation of the fungus to extracts made from an intended substrate may be an inexpensive way of producing liquid spawn specifically suited for the inoculation of the substrate.This article was written and prepared by U.S. Government employees on official time, and it is therefore in the public domain (i.e., it cannot be copyrighted).Research carried out while T. J. Griffin was a volunteer worker at Forest Products Laboratory     

PEG介导下香菇的转化

表达载体p301-bG1含有香菇（Lentinus edodes (Berk.)Sing)三磷酸甘油醛脱氢酶启动子驱动下的gus基因和除草剂抗性基因。利用PEG法实现了p301－bG1对香菇原生质体的转化。香菇原生质体与经PEG纯化的质粒DNA混合,用PEG处理后培养于含40ug/mL除草剂的CYM再生平板上,得到了抗除草剂和有GUS活性的转化菌株。虽然这种方法转化效率较低,但不需要昂贵的仪器和限制性内切酶,为蘑菇的分子育种研究提供了一种简便经济的转化方法。     

Lectin activity of Lentinus edodes

The hemagglutinating activity of submerged mycelium and culture liquid for four strains of Lentinus edodes (Berk.) Sing [L. edodes (Berk.) Pegler] was studied in the search for lectins. The hemagglutinating activity of culture liquid was substantially higher, compared with mycelium. The carbohydrate-binding capacity of the agglutinins was established, and the lectin activity of extracts from mycelia grown on several agar media was elucidated in relation to fruiting. The lectin activity of L. edodes was examined at different morphogenetic steps: mycelium, brown mycelial film, primordium, and fruiting body. Hemagglutination titers at the brown film step were higher than in the mycelium, whereas activity at the primordial and fruiting bodies steps decreased. Lectins seem to be involved in the formation of hyphal aggregates of brown mycelial film.     

PEG-mediated Transformation of Lentinus edodes

Expression vector p301-bG1 contains a gus gene and a bialaphos resistance gene both driven by glyceraldehydes-3-phosphate dehydrogenase (GPD) gene promoter isolated from Lentinus edodes (Berk.) Sing. Using p301-bG1, PEG-mediated transformation of protoplast of L. edodes was studied. Mixed with PEG-purified plasmid DNA, the protoplasts of L. edodes were treated with PEG solution and cultured on CYM regeneration plate containing 40 μg/mL bialaphos. Bialaphos-resistant and GUS-positive transformants were obtained using this transformation system. Although the transformation efficiency was relatively low, the protocols release large expenses on expensive instrument and restriction enzymes, providing a simple and economical method for mushroom breeding at the molecular level.     

Molecular mechanism of macrophage activation by Exopolysaccharides from liquid culture of Lentinus edodes

Mushrooms are regarded as one of the well-known foods and biopharmaceutical materials with a great deal of interest. beta-Glucan is the major component of mushrooms that displays various biological activities such as antidiabetic, anticancer, and antihyperlipidemic effects. In this study, we explored the molecular mechanism of its immunostimulatory potency in immune responses of macrophages, using exopolysaccharides prepared from liquid culture of Lentinus edodes. We found that fraction II (F-II), with large molecular weight protein polysaccharides, is able to strongly upregulate the phenotypic functions of macrophages such as phagocytic uptake, ROS/NO production, cytokine expression, and morphological changes. F-II triggered the nuclear translocation of NF-kappaB and activated its upstream signaling cascades such as PI3K/ Akt and MAPK pathways, as assessed by their phosphorylation levels. The function-blocking antibodies to dectin-1 and TLR-2, but not CR3, markedly suppressed F-II-mediated NO production. Therefore, our data suggest that mushroom-derived beta-glucan may exert its immunostimulating potency via activation of multiple signaling pathways.     

Whey permeate as a growth medium for Pleurotus ostreatus and Lentinus edodes

Whey permeate, a dairy by-product, was studied as a growth medium for two white-rot Basidiomycetes, Lentinus edodes and Pleurotus ostreatus. Mycelial growth and phenoloxidase production appeared to be highly stimulated when the fungi were grown on the permeate instead of synthetic medium. No further increment in phenoloxidase production was observed when the permeate was supplemented with CuSO₄.     

HPLC法测定香菇中有机酸含量的检测技术研究

本实验建立了HPLC法同时测定香菇中酒石酸、苹果酸、乙酸和柠檬酸的方法。在酸提条件下,利用Agela Venusll MP C18色谱柱,以2%CH3OH-20 mM(NH4)2HPO4(pH 2.40)为流动相,流速为1.0 mL/min,柱温为35℃,检测波长为210 nm,进样量为10μL。四种有机酸均在8 min内出峰。本法准确、可靠、快速、简便,检出限较低,适合于大批量样品的分析与检测。     

Decolorization of synthetic dyes by solid state cultures of Lentinula (Lentinus) edodes producing manganese peroxidase as the main ligninolytic enzyme

The ability of the white-rot fungus Lentinula (Lentinus) edodes to decolorize several synthetic dyes was investigated using solid state cultures with corn cob as substrate. Cultures, containing amido black, congo red, trypan blue, methyl green, remazol brilliant blue R, methyl violet, ethyl violet and Poly R478 at 200 ppm, were completely decolorized after 18 days of incubation. Partial decolorization was observed in the cultures containing 200 ppm of brilliant cresyl blue and methylene blue. High manganese peroxidase activity (2600 U/g substrate), but very low lignin peroxidase (<10 U/g substrate) and laccase (<16 U/g substrate) activities were detected in the cultures. In vitro, the dye decolorization was markedly decreased by the absence of manganic ions and H2O2. These data suggest that manganese peroxidase appear to be the main responsible for the capability of L. edodes to decolorize synthetic dyes.     

香菇谷氨酰胺转氨酶的分离和动力学研究

采用超滤,DEAE-纤维素和SephadexG-100层析,研究了香菇谷氨酰胺转氨酶的制备方法,对酶反应动力学的最适温度、最适pH、酶的稳定性和酶催化激活剂等进行了研究。结果表明,获得了香菇谷氨酰胺转氨酶,经SDS-PAGE电泳检测,为一均一性蛋白;香菇谷氨酰胺转氨酶酶动力学研究结果显示,最适温度为40℃,酶促反应的Vmax为0.020 4 mg/(mL.min),米氏常数Km为1.520 mg/mL。Na 、Ca2 、Pb2 、K 、Mg2 、Cu2 等离子对酶活影响甚微,为非Ca2 依赖性酶。该酶由2个亚基组成,分子量分别为53 ku和27 ku,pI为5.33。     

Cellulase hyperproducers constructed from polyploids of Lentinus edodes

A mycelial mat of Lentinus edodes was treated with 0.01% (w/v) colchicine solution for 240 h at 26 degrees C and autopolyploidization occurred. The mycelia were treated subsequently with the haploidizing reagent, benomyl, and fanshaped sectors were produced from colonies. Among such sectors, cellulase hyperproducers could be selected. The cellulase productivity of the hyperproducer, L1, did not decrease through five generations.     

香菇蛋白的分级纯化和结构分析

香菇粉经10℃pH 10的水提取制备香菇蛋白,得率13.1%,其蛋白含量47.5%,多糖含量24.2%.香菇蛋白经DEAE Sepharose CL-6B柱层析分级得5个级分,收集级分F1、F2、F3、F4,它们都是由蛋白和多糖构成的复合物.Sepharose CL-6B凝胶色谱显示,F2和F4的分子量分布较为均匀,且以蛋白为主,多糖含量很低;F3主要由两个分子量不同的蛋白级分构成,含有一定的多糖;F1中多糖含量较高,蛋白含量较少,且多糖分子量分布均匀.香菇蛋白的分子量主要集中在20 kDa～40 kDa之间.F1、F3、F4都属于酸性蛋白质,含有除色氨酸之外的7种必需氨基酸,除蛋氨酸含量较低外,其余必需氮基酸含量接近,且赖氨酸含量较高.红外光谱分析表明,香菇蛋白的二级结构主要为α-螺旋和无规卷曲.     

Mannitol Metabolism in Lentinus edodes, the Shiitake Mushroom

Mannitol metabolism was evaluated in fruiting bodies of Lentinus edodes. Cell extracts were prepared from fruiting bodies, and key enzymes involved in mannitol metabolism were assayed, including hexokinase, mannitol dehydrogenase, mannitol-1-phosphate dehydrogenase, mannitol-1-phosphatase, and fructose-6-phosphatase. Mannitol dehydrogenase, fructose-6-phosphatase, mannitol-1-phosphatase, and hexokinase activities were found in extracts of fruiting bodies. However, mannitol-1-phosphate dehydrogenase activity was not detected. Mycelial cultures were grown in an enriched liquid medium, and enzymes of the mannitol cycle were assayed in cell extracts of rapidly growing cells. Mannitol-1-phosphate dehydrogenase activity was also not found in mycelial extracts. Hence, evidence for a complete mannitol cycle both in vegetative mycelia and during mushroom development was lacking. The pathway of mannitol synthesis in L. edodes appears to utilize fructose as an intermediate.     

Foreign protein production in plant tissue cultures

Foreign proteins synthesised by plants are now in the marketplace, and clinical trials for plant-derived therapeutic proteins are underway. Economic analysis of plant production systems has helped identify the types of protein that would be most suitable for manufacture using tissue culture methods. The major advantages associated with in vitro plant systems include the ability to manipulate environmental conditions for better control over protein levels and quality, the rapidity of production compared with agriculture, and the use of simpler and cheaper downstream processing schemes for product recovery from the culture medium.     

Improving alpha-tocopherol production in plant cell cultures

Suspension cell cultures of Helianthus annuus L. were previously established for the production of the most active component of vitamin E, alpha-tocopherol, by optimizing medium composition and culture conditions. In the present work, the possibility of enhancing alpha-tocopherol production by the addition of jasmonic acid to the culture medium was investigated both in sunflower and Arabidopsis cell cultures. A considerable increase (49% and 66%, respectively) of alpha-tocopherol production was obtained in both, after a 72-h treatment with 5 microM jasmonic acid. The modulation of alpha-tocopherol levels in plant cell cultures can provide useful hints for a regulatory impact on tocopherol metabolism.     

死谷芽孢杆菌对香菇栽培料中木霉菌的抑制研究

[目的]木霉是食用菌生产过程中侵染率较高的杂菌,通常使用化学药剂消杀,但这会带来农药残留问题,利用死谷芽孢杆菌防治木霉展开研究.[方法]首先确定死谷芽孢杆菌抑制木霉的成分是胞内还是胞外物质;其次有效物质经过提取,分析其理化性质的稳定性;最后研究在香菇栽培料中的初步应用效果.[结果]提取的死谷芽孢杆菌胞外分泌物属于脂肽类,能有效抑制木霉孢子萌发和菌丝生长,0.02 g/L即可达到62.8％的抑菌率,对香菇菌丝生长影响较小,且理化性质比较稳定.香菇栽培料中添加26.6％的36h死谷芽孢杆菌发酵液,对木霉菌丝抑制程度最高,防治效果较理想.[结论]死谷芽孢杆菌产生的胞外分泌物对木霉有良好的抑制作用,虽然对香菇的菌丝萌发也有一定的抑制作用,但从减少农药残留问题考虑,值得进一步研究.     

Characterization of Ribonucleases from Culture Medium of Lentinus edodes

Lentinus edodes (shiitake) cultivated in potato dextrose medium produced five RNases in the culture filtrate. The two major RNases (RNase Le37 and RNase Le45) were highly purified and their molecular masses, base specificities, N-terminal amino acid sequences, and amino acid compositions were analyzed and compared to RNase Le2 isolated from the fruit bodies of the same mushroom. RNase Le37 and RNase Le45 are base non-specific and adenylic acid preferential RNases like RNase Le2 and their N-terminal sequences are very similar to RNase Le2, but they are glycoproteins and their amino acid compositions are significantly different from that of RNase Le2. In addition to these enzymes, a guanylic acid-specific RNase with a molecular mass 13 kDa was partially purified. Since RNase Le2, which has very similar N-terminal sequence to RNase Le 37 and RNase Le 45, was not excreted from the mycelia, the analysis of the structures of these two excreted RNase may shade a light on the mechanism of excretion of RNases in this organism.     

花菇的冷冻干燥技术研究

实验研究用板层导热法研究了花菇的冻干特性,获得了新鲜花菇的冻干曲线,分析了花菇冻干过程,测定和比较了新鲜花菇和冻干花菇的营养成份。证实试验机的适应性并确定了花菇的冻干工艺,为工业生产提供了理论依据和参考价值。