Effect of mannan oligosaccharide elicitor and ferulic acid on enhancement of laccases production in liquid cultures of basidiomycetes

The effects of mannan oligosaccharides preparation (MO), as elicitor, and ferulic acid inducer for enhancement in laccases production in liquid cultures of three strains of basidiomycetes, Pycnoporus sanguineus, Coriolopsis polyzona and Pleurotus ostreatus was studied using a full factorial 3² experimental design. MO, either individually or combined with ferulic acid, enhanced laccases levels in the three different strains of the white-rot fungi. The enhancement of laccases production was species specific with the highest increase in liquid cultures of P. sanguineus (88-fold) followed by P. ostreatus (3-fold) and C. polyzona (2-fold). Separate additions of 75 mg/l of MO to the cultures of P. sanguineus and P. ostreatus caused the increase while a combined addition of 150 mg/l of MO and 1 mM ferulic acid resulted in the optimal production of laccases in the cultures of C. polyzona.     

不同栽培基质对糙皮侧耳不同发酵方式下产漆酶活性的影响

为了比较糙皮侧耳栽培种在不同常规栽培基质上的漆酶活性,分析更适合糙皮侧耳生长的栽培基质,以1株糙皮侧耳Pleurotus ostreatus栽培菌株为研究材料,研究在固态和液态发酵条件下添加木屑、玉米芯和棉籽壳这3种栽培基质后,对其产漆酶活性的影响。结果表明,不同栽培基质对糙皮侧耳漆酶活性具有极显著的影响（P<0.001）;不同发酵方法对糙皮侧耳漆酶活性也具有极显著的影响（P<0.001）,仅第2天差异不显著。固体发酵与液体发酵条件下,糙皮侧耳在棉籽壳培养基上所检测到的漆酶活性均高于在木屑或者玉米芯培养基上,表明棉籽壳对提高糙皮侧耳漆酶活性的诱导能力更强。此外,糙皮侧耳在棉籽壳培养基上能够快速分泌漆酶,表明棉籽壳对缩短糙皮侧耳漆酶分泌时间的诱导能力更强。     

不同诱导培养基对糙皮侧耳液体发酵产漆酶活性的影响

利用1株糙皮侧耳Pleurotus ostreatus栽培菌株为材料,研究添加碱性木质素或者配合简单碳源或氮源后对其液体发酵产漆酶活性的影响。结果表明,不同诱导培养基对糙皮侧耳漆酶活性具有极显著的影响（P<0.001）,而且不同诱导培养基对糙皮侧耳菌丝生物量也产生了极显著的影响（P<0.001）。此外,只利用碱性木质素或者是再添加碳源葡萄糖均有利于糙皮侧耳产漆酶,既包括产漆酶酶活性的提升,同时也包括产漆酶时间的提前,但只利用碱性木质素诱导不利于菌丝生物量的积累;而富含简单碳/氮源的诱导培养基,无论是否含碱性木质素,均有利于菌丝生物量的积累,其中,富含简单碳/氮源的培养基中再添加碱性木质素后的菌丝生物量和漆酶活性均高于不添加碱性木质素时的菌丝生物量和漆酶活性。相比而言,含碱性木质素的培养基中测得的漆酶活性大部分时间下都要高于不含木质素的简单碳/氮源培养基,含碱性木质素的培养基对糙皮侧耳菌株产漆酶的诱导作用更强。     

Detection,quantification and identification of fungal extracellular laccases using polyclonal antibody and mass spectrometry

This study presents a combined method to analyze extracellular fungal laccases using a new anti-laccase antibody together with the identification of tryptic laccase peptides by mass spectrometry (nanoLC–ESI–MS/MS). The polyclonal anti-laccase antibody LccCbr2 was raised against peptides designed from the copper binding region II of fungal laccases using in silico data obtained from GenBank database. As a consequence, detection requires denaturation of the enzymes due to the stable conformation of the copper binding region II. The specificity of the antibody was shown with denatured laccase Lcc1 of Coprinopsis cinerea and laccase of Hypholoma fasciculare. LccCbr2 detected amounts as low as 5 ng of highly purified laccase, indicating a possible use of the antibody for quantification of laccase proteins. Denatured extracellular laccases from culture supernatants of the basidiomycetes C. cinerea, H. fasciculare, Lentinula edodes, Mycena sp., Piriformospora indica, Pleurotus cornucopiae, Pleurotus ostreatus, Pycnoporus cinnabarinus, Trametes versicolor and furthermore the ascomycete Verpa conica were detected with apparent molecular masses between 60 and 70 kDa by LccCbr2. The identity of extracellular laccases from C. cinerea, H. fasciculare, P. ostreatus, P. cinnabarinus and T. versicolor were verified by tryptic peptides using nanoLC–ESI–MS/MS.     

糙皮侧耳生长发育过程中漆酶基因家族的表达研究

漆酶参与木质素的降解和食用菌的生长发育。为了明晰漆酶基因在糙皮侧耳生长发育过程中的作用,本文采用real-time PCR检测了11条漆酶基因及Lacc2的小亚基sspoxa3在糙皮侧耳生长发育不同阶段的表达。其中lacc6和sspoxa3在整个生长发育阶段表达量均较高;lacc12随着原基的分化和子实体的形成大量表达,与成菇过程有关。lacc4,lacc7和lacc11在原基分化期高表达,与原基的分化有关。lacc2,lacc3和lacc8在成熟子实体阶段表达量显著上升,与子实体的分化和成熟有关。     

Purification and partial characterization of a thermostable laccase from an unidentified basidiomycete

A thermostable laccase was isolated from an unidentified fungal isolate [Enz. Microb. Technol. 33 (2003) 212], and tentatively named UD4. This work indicates that the enzyme has unique properties other than its thermostability. Investigation into the kinetic parameters of the thermostable laccase yielded an unusually high affinity for ABTS as a substrate (low K_m) when compared with available published data for other laccase isozymes. The specificity constant (k_cat/K_m) was found to be considerably higher than laccase from other sources and is comparable to “white” laccase from Pleurotus ostreatus (POXA1). However, POXA1 isozyme exhibits a large turnover number (k_cat) that contributes to its high specificity constant whereas the high specificity constant for UD4 laccase is achieved by having a high substrate affinity. The UD4 thermostable laccase, like most other laccases, is able to utilize guaiacol as a substrate, whereas POXA1 is unable to oxidize guaiacol, indicating a broader substrate range for the thermostable laccase from UD4. The thermostable laccase is inhibited by sodium azide through non-competitive inhibition, and by thioglycolic acid and hydroxylamine through competitive inhibition. The high specificity constant, substrate affinity and broader substrate range of the thermostable laccase from UD4 indicates that it is a highly favourable candidate enzyme for industrial application.     

Immobilization of wood-rotting fungi laccases on modified cellulose and acrylic carriers

Extracellular laccases produced by three different wood-rotting fungi, Cerrena unicolor, Heterobasidion annosum and Trametes versicolor, were immobilized via covalent bonds formation on DEAE-Granocel 500, CM-Granocel 500, and acrylic carriers. Out of the tested carriers, only the DEAE-Granocel 500, which was activated by divinyl sulphone appeared to be a suitable matrix for the expression of enzymic activity. Only one laccase of all the tested enzymes produced by C. unicolor showed the best binding to the carrier and a satisfactory enzymic activity. The immobilized laccase exhibited the highest enzymic activity at pH 5.2 and it was more resistant to thermal denaturation than the native enzyme. At 90 °C, it retained 75% activity compared to the free enzyme. It was also more stable during storage at 4 °C: after 4 months the immobilized laccase retained 98% of initial activity. Immobilized C. unicolor laccase was active in 10–60% concentration of methanol, acetone, isopropanol or acetonitrile. The best enzymic activity was observed in 20% solution of acetonitrile in buffer.     

Conversion of Japanese red cedar (Cryptomeria japonica) into a feed for ruminants by white-rot basidiomycetes

The potential of five white-rot fungi to convert Japanese red cedar (Cryptomeria japonica) into feed for ruminants was determined. Pleurotus ostreatus (ATCC 66376), Pholiota nameko (IFO 30373), Dichomitus squalens (CBS 432.34), Lentinula edodes (IFO 6654) and Ceriporiopsis subvermispora (ATCC 90467) were inoculated into chips of cedar sapwood. L. edodes was cultured at 25 °C, and the other fungi at 28 °C, for 4, 8, 12, or 20 weeks. The in vitro organic matter (OM) digestibility (IVOMD) in cedar wood cultured without fungus were between 0.047 and 0.068, while it was elevated to 0.446 by culturing with C. subvermispora and to 0.281 by culturing with L. edodes for 20 weeks. In contrast, the IVOMD were 0.200, or lower, in cedar wood cultured with P. ostreatus, P. nameko, or D. squalens. The in vitro gas production (IVGP) in cedar wood cultured with P. ostreatus, P. nameko, or D. squalens was 37 ml/g OM, or lower, while that in cedar not inoculated with fungus was between 4 and 17 ml/g OM. In contrast, the IVGP in cedar wood cultured with C. subvermispora for 20 weeks increased to 107 ml/g OM, and that in cedar wood cultured with L. edodes increased to 58 ml/g OM. Lignin degradability in cedar wood cultured with C. subvermispora and L. edodes for 20 weeks were 0.578 and 0.288, respectively. These changes in IVOMD and IVGP demonstrate that a selective white-rot fungus, C. subvermispora has the ability to convert cedar wood into feed for ruminants, although further increase is required before the cultured cedar wood would have widespread feed potential.     

卵孢侧耳野生菌株人工栽培及营养成分分析

采自西藏日喀则地区的两份野生蘑菇标本及其分离菌株,通过形态学及基于ITS序列的分子系统发育分析鉴定为卵孢侧耳Pleurotus placentodes。该物种在2016年首次报道于中国。人工驯化栽培试验表明卵孢侧耳可以在人工条件下出菇,生长周期80-95d;初步的营养成分分析表明,卵孢侧耳蛋白质、粗多糖、氨基酸含量比香菇、糙皮侧耳（平菇）、金针菇、柱状田头菇（茶树菇）等都高,尤其含有较高的组氨酸和丰富的矿物质,如铁、钙、锌等。卵孢侧耳可作为新的食用菌进行开发利用。     

Decolorization of dyes using white-rot fungi and radical-generating reactions

Decolorization of synthetic dyes was performed using cultures of white-rot fungi producing ligninolytic enzymes and radical-generating reactions that could be involved in the mechanism of fungal decolorization. Among the white-rot fungi tested, Pleurotus ostreatus exhibited the highest decolorization rates, and also the highest production of laccase and Mn-peroxidase. P. ostreatus strain f6 gave 69% decolorization of Eosin Yellowish, 96% of Evans Blue, 75% of Phenol Red (all at 1 mM) and 88% of Poly B-411 (20 ppm) during a 14-day treatment. Treatment with Cu/succinic acid/H₂O₂ resulted in 96% decolorization of Evans Blue and Poly B-411 within 24 h. However, only 48% and 2% decolorization was achieved with Phenol Red and Eosin Yellowish, respectively. Similar decolorization rates were also obtained when Cu was replaced with Co. The results show that treatment of dye-containing solutions with both fungal cultures and biomimetic catalytic reactions results in decolorization.     

铜离子调控木质纤维素降解和糙皮侧耳形态发育的研究

漆酶是一种含铜的多酚氧化酶,在木质素的降解中起重要作用。铜离子对漆酶的产生和活性有重要影响。通过向秸秆固体培养基中添加铜离子,研究铜离子对糙皮侧耳木质纤维素降解酶、木质纤维素降解和形态发育的影响。结果表明,添加铜离子能在一定程度上提高漆酶的活性,添加3mmol/L铜离子在第7天漆酶活性比对照高出71.2%,28d后添加铜离子的样品木质素降解率稍高出对照,添加铜离子能促进糙皮侧耳原基的分化和子实体的发育。     

磷酸氢二铵对糙皮侧耳菌丝生长及基质降解酶活性的影响

氮源种类和浓度均会对糙皮侧耳Pleurotus ostreatus菌丝长势、生长速率以及代谢产生影响,而氮匮乏与氮过量积累都会对糙皮侧耳的生长产生不利影响.为了研究氮源对糙皮侧耳菌丝生长和基质降解酶活性的影响,本研究选取磷酸氢二铵[(NH4)2HP04]作为唯一氮源,通过在培养基中添加不同浓度的磷酸氢二铵检测糙皮侧耳菌...     

Determination of laccase gene expression during degradation of 2,4,6-trinitrotoluene and its catabolic intermediates in Trametes versicolor

We have cloned a laccase gene fragment isolated from a Trametes versicolor strain in Korea. It showed high similarity in nucleotide sequences when compared with other fungal laccases. TNT (2,4,6-trinitrotoluene), a widely used explosive, was transformed rapidly by T. versicolor. When TNT and its catabolic intermediates were added to the fungal culture, they were transformed during the first few hours and the expression level of the laccase gene was increased during the early stage of cultivation.     

热带、亚热带典型森林-土壤系统植硅体碳演变规律

分别选取中国亚热带毛竹林、马尾松林、青冈林、杉木林和热带青梅林、芭蕉林、橡胶林、马占相思林8种森林类型,采集其鲜叶、凋落叶以及0～10和10～30 cm土层土壤,通过微波消解法提取其中的植硅体,并采用碱溶法测定植硅体中碳含量.结果表明: 4种亚热带森林类型鲜叶、凋落叶和0～10 cm土层中植硅体碳含量均以马尾松林(230.24、229.17、20.87 g·kg^-1)最高,毛竹林(30.55、37.37、3.38 g·kg^-1)最低,10～30 cm土层则以青冈林(18.54 g·kg^-1)最高,毛竹林(2.90 g·kg^-1)最低.热带森林鲜叶中植硅体碳含量以马占相思林(377.66 g·kg^-1)最高,青梅林(46.83 g·kg^-1)最低,凋落叶中则是橡胶林(218.23 g·kg^-1)最高,芭蕉林(27.66 g·kg^-1)最低,而0～10和10～30 cm土层土壤中均以马占相思林(23.84、24.90 g·kg^-1)最高,芭蕉林(3.89、3.93 g·kg^-1)最低.与0～10 cm表层土相比,杉木林、青冈林、马尾松林、毛竹林、橡胶林、马占相思林、芭蕉林和青梅林鲜叶植硅体碳含量分别下降97.4%、94.9%、90.9%、88.9%、95.9%、93.7%、93.3%和63.7%.青冈林、芭蕉林和马占相思林鲜叶植硅体碳含量显著高于凋落叶,而毛竹林、马尾松林、杉木林、青梅林和橡胶林之间无显著差异.8种森林类型土壤植硅体碳含量均显著低于鲜叶和凋落叶,表明植硅体在通过凋落物释放到土壤的过程中是不稳定的.     

四种水库消落带植物对水淹环境的适应性研究

研究了百花湖消落带四种优势植物双穗雀稗(Paspalum distichum)、空心莲子草(Alternanthera philoxeroides)、牛鞭草(Hemarthria altissima)和狗牙根(Cynodon dactylon)在模拟水淹环境下的生长指标和叶绿素含量,以期了解其对水淹环境的适应特征。结果表明:1) 水淹条件下,双穗雀稗、空心莲子草和牛鞭草生长加剧,狗牙根生长缓慢,四种植物损失了不适应全淹环境的分枝和叶片。2)双穗雀稗、牛鞭草和狗牙根表现为茎的生长,空心莲子草表现为茎和分枝的生长。植株顶端冒出水面后,双穗雀稗、空心莲子草和牛鞭草叶的生长加剧。3)四种植物的叶绿素a、b的含量降低,叶绿素b降低比叶绿素a更显著,叶绿素a/b值显著升高。植株顶端冒出水面后,双穗雀稗、空心莲子草和牛鞭草叶绿素a、b含量显著升高,叶绿素a/b值显著降低。4)经过不断地适应和进化后,四种植物能更好地适应水淹环境。     

Ostreolysin enhances fruiting initiation in the oyster mushroom (Pleurotus ostreatus)

Fruiting initiation in mushrooms can be triggered by a variety of environmental and biochemical stimuli, including substances of natural or synthetic origin. In this work ostreolysin, a cytolytic protein specifically expressed during the formation of primordia and fruit bodies of Pleurotus ostreatus, was applied to nutrient media inoculated with mycelium of P. ostreatus, and its effects on mycelial growth and fructification of the mushroom studied. The addition of ostreolysin slightly inhibited the growth of mycelium, but strongly induced the formation of primordia, which appeared 10 d earlier than in control plates supplemented with bovine serum albumin or with the dissolving buffer alone. Moreover, ostreolysin stimulated the subsequent development of primordia into fruit bodies. However, direct involvement of this protein in the sporulation of the mushroom is unlikely, as it was also detected in large amounts in the non-sporulating strain of P. ostreatus.     

枸杞岛海藻场6种大型海藻光合荧光特性比较

海藻光合荧光特性研究对分析藻类光合作用和固碳能力具有重要的作用.利用水下调制荧光仪(DIVING-PAM)测定了枸杞岛后头湾夏季常见6种大型海藻孔石莼、斯氏刚毛藻、舌状蜈蚣藻、鼠尾藻、多管藻和羊栖菜的量子产量、快速光曲线(RLC)相关参数.结果显示: 6种常见海藻孔石莼、斯氏刚毛藻、舌状蜈蚣藻、鼠尾藻、多管藻和羊栖菜最大量子产量(F_v/F_m)分别为0.702、0.704、0.457、0.618、0.421、0.567,各物种的开放PSⅡ反应中心原初光能捕获效率(F_v′/F_m′)的大小依次是斯氏刚毛藻>孔石莼>鼠尾藻>羊栖菜>舌状蜈蚣藻>多管藻,且孔石莼、斯氏刚毛藻、羊栖菜的F_v′/F_m′与其他5种大型海藻的差异均达到显著水平;在快速光响应曲线中,羊栖菜、鼠尾藻和孔石莼的最大相对电子传递速率与初始斜率α较高,证明其具有较强的光合能力和捕光能力;舌状蜈蚣藻较高的RLC初始斜率α和较低的I_k表明其有较强的耐弱光能力.3种门类大型海藻之间光合特性有较为显著的差异,其中褐藻门的羊栖菜、鼠尾藻与绿藻门的孔石莼拥有极高的光合活性和抗强光能力,研究结果可为藻场保护工作和大型海藻固碳能力评估提供理论依据.     

Overlap of laccases/cellobiose dehydrogenase activities during the decolourisation of anthraquinonic dyes with close chemical structures by Pycnoporus strains

Pycnoporus strains were used as model to understand the role of laccases in the in vivo decolourisation of three anthraquinonic dyes. The decolourisation capability of Pycnoporus sanguineus MUCL 41582 (PS7), which produces laccases as the main oxidative enzyme, was assayed and compared with the decolourisation capability of a control strain, Pycnoporus cinnabarinus MUCL 39533 (PC330) described as laccase-deficient strain. In absence of dye, laccase activity was observed during the trophophase and the idiophase with PS7, while no laccase activity was observed with PC330. Acid Blue 62 (ABu62), Acid Blue 281 (ABu281) and Reactive Blue 19 (RBu19) caused an increase in laccase activity and surprisingly laccase activity was detected with PC330. In vitro, oxidation of all three anthraquinones by a laccase preparation was obtained to a lesser extent than the whole cell process; suggesting that other factor(s) could be required for a complete decolourisation. As the time space of laccase production in the tested fungi was not perfectly coincidental with the decolourisation process, the activity of cellobiose dehydrogenase (CDH) was monitored. Present early in the broth during the growth of the fungi, CDH displayed in vitro a synergism with laccases in the decolourisation of ABu62, and an antagonism with laccases in the decolourisation of ABu281 and RBu19.     

刺芹侧耳产漆酶条件优化及对偶氮染料甲基橙的脱色

漆酶是一种含铜的单电子多酚氧化酶,能够催化氧化各种酚类及多种染料,在处理染料废水方面具有巨大的潜力。刺芹侧耳Pleurotus eryngii具有较强的产漆酶能力,但漆酶产量在较大程度上受环境条件限制。本文研究了氮源含量、pH、温度、金属离子等环境条件对刺芹侧耳产漆酶能力的影响,优化了其产漆酶条件,并用其粗酶液对典型偶氮类染料甲基橙进行脱色,结果表明,在氮源0.5%（W/W）、pH 5.5、温度28℃、添加5.0mmol/L Mg ²⁺的培养条件下,刺芹侧耳产漆酶能力最强,培养6d时,漆酶酶活可达78.0U/L。用优化培养的刺芹侧耳粗酶液对偶氮染料甲基橙进行脱色,28h后脱色率可达90%,脱色反应为准一级动力学反应,甲基橙并未完全矿化,而是生成小分子中间产物。     

Yeasts as biological agents to control Botrytis cinerea

Yeasts, isolated from different sources, were identified and tested for inhibition using YMA-MB plates seeded with Botrytis cinerea strains. A total of 42 yeast strains of 20 different species were tested in vitro for antagonism against 18 pathogenic B. cinerea strains. Pichia membranifaciens, P. anomala and Debaryomyces hansenii displayed the most important inhibitory effect against Botrytis strains. In small-scale trials, post-harvest application of P. membranifaciens CYC 1106 to apple wounds inhibited B. cinerea CYC 20010. Purified killer toxin from P. membranifaciens CYC 1106 inhibited B. cinerea CYC 20010. Results indicated that certain yeasts, or their toxins such us P. membranifaciens CYC 1106 killer toxin, might have potential as novel agents to control B. cinerea.