Characterization of mannitol metabolism in the mangrove red algaCaloglossa leprieurii (Montagne) J.Agardh

A metabolic pathway, known as the mannitol cycle in fungi, has been identified as a new entity in the eulittoral mangrove red algaCaloglossa leprieurii (Montagne) J. Agardh. Three specific enzymes, mannitol-1-phosphate dehydrogenase (Mt1PDH; EC 1.1.1.17), mannitol-1-phosphatase (MtlPase; EC 3.1.3.22), mannitol dehydrogenase (MtDH; EC 1.1.1.67) and one nonspecific hexokinase (HK; EC 2.7.1.1) were determined and biochemically characterized in cell-free extracts. Mannitol-1-phosphate dehydrogenase showed activity maxima at pH 7.0 [fructose-6-phosphate (F6P) reduction] and pH 8.5 [oxidation of mannitol-1-phosphate (Mt1P)], and a very high specificity for both carbohydrate substrates. TheK _m values were 1.4 mM for F6P, 0.09 mM for MOP, 0.020 mM for NADH and 0.023 mM for NAD⁺. For the dephosphorylation of MOP, MtlPase exhibited a pH optimum at 7.2, aK _m value of 1.2 mM and a high requirement of Mg²⁺ for activation. Mannitol dehydrogenase had activity maxima at pH 7.0 (fructose reduction) and pH 9.8 (mannitol oxidation), and was less substrate-specific than Mt1PDH and MtlPase, i.e. it also catalyzed reactions in the oxidative direction with arabitol (64.9%), sorbitol (31%) and xylitol (24.8%). This enzyme showedK _m values of 39 mM for fructose, 7.9 mM for mannitol, 0.14 mM for NADH and 0.075 mM for NAD⁺. For the non-specific HK, only theK _m values for fructose (0.19 mM) and glucose (7.5 mM) were determined. The activities of the anabolic enzymes Mt1PDH and MtlPase were always at least two orders of magnitude higher than those of the degradative enzymes, indicating a net carbon flow towards a high intracellular mannitol pool. The function of mannitol metabolism inC. leprieurii as a biochemical adaptation to the environmental extremes in the mangrove habitat is discussed.Abbreviations F6P fructose-6-phosphate - HK hexokinase - Mt1P mannitol-1-phosphate - Mt1PDH mannitol-1-phosphate dehydrogenase - Mt1Pase mannitol-1-phosphatase - MtDH mannitol dehydrogenase     

The mitochondrial genome of the Basidiomycete fungus Pleurotus ostreatus (oyster mushroom)

In this study, the full mitochondrial genome of a basidiomycete fungus, Pleurotus ostreatus, was sequenced and analyzed. It is a circular DNA molecule of 73 242 bp and contains 44 known genes encoding 18 proteins and 26 RNA genes. The protein-coding genes include 14 common mitochondrial genes, one ribosomal small subunit protein 3 gene, one RNA polymerase gene and two DNA polymerase genes. In addition, one RNA and one DNA polymerase genes were identified in a mitochondrial plasmid. These two genes show relatively low similarities to their homologs in the mitochondrial genome but they are nearly identical to the known mitochondrial plasmid genes from another Pleurotus ostreatus strain. This suggests that the plasmid may mediate the horizontal gene transfer of the DNA and RNA polymerase genes into mitochondrial genome, and such a transfer may be an ancient event. Phylogenetic analysis based on the cox1 ORFs verified the traditional classification of Pleurotus ostreatus among fungi. However, the discordances were observed in the phylogenetic trees based on the six cox1 intronic ORFs of Pleurotus ostreatus and their homologs in other species, suggesting that these intronic ORFs are foreign DNA sequences obtained through HGT. In summary, this analysis provides valuable information towards the understanding of the evolution of fungal mtDNA.     

北风菌中的甾体和甾体苷类化合物

从口蘑科真菌北风菌(Pleurotus ostreatus (Jacq.:Fr.) Kummer)的乙酸乙酯部分分离鉴定了12个甾体类化合物,其中包括8个麦角甾醇类甾体及4个甾体苷.利用现代波谱技术及化学方法,鉴定了一个新化合物3-O-β-D-glucopyranosyl-22E, 24R-ergosta-7, 22-diene-5α, 6β, 9α-triol.其余11个已知化合物分别为22E, 24R-ergosta-7, 22-diene-3β, 5α, 6β, 9α-tetraol、 3-O-β-D-glucopyranosyl-22E, 24R-ergosta-7, 22-diene-5α, 6β-diol、 22E, 24R-ergosta-7, 22-diene-3β, 5α, 6β-triol、 22E, 24R-ergosta-5, 7, 22-trien-3β-ol 3-O-β-D-glucopyranoside、 ergosterol、 22E, 24R-ergosta-7, 22-dien-3β-ol 3-O-β-D-glucopyranoside、 22E, 24R-ergosta-7, 22-diene-3β-ol、 22E, 24R-ergosta-4, 6, 8, 22-tetraen-3-one、 22E, 24R-ergosta-7, 22-diene-3β, 5α, 6α-triol、 ergosterol peroxide 和22E, 24R-ergosta-7, 22-diene-3β, 5α-diol-6-one.     

Bioremediation of hexachlorocyclohexane (HCH) in soil using spent mushroom compost of Pleurotus ostreatus

Abstract

Hexachlorocyclohexane (HCH), a highly chlorinated pesticide, was used worldwide in the 1950s and 1960s. HCH toxic residues are still detected in environmental compartments. Thus, effective, viable and eco-friendly strategy is required for its remediation. In this study, degradation of four HCH isomers was evaluated by amending contaminated soil using four treatments of spent mushroom compost (SMC) of Pleurotus ostraetus. The soil was incubated for 5 weeks and was sampled every seven days. Quantitative attenuation in HCH was calculated using gas chromatography–electron capture detector (GC-ECD) and metabolite was identified using gas chromatography–mass selective detector (GC-MSD). Maximum reduction 58%, 26%, 45%, and 64% for α-, β-, γ- and δ-HCH isomers, respectively, using SMC and soil (both unsterilized) showed that this treatment was the best for bioremediation of HCH in soil. However, when one of the factors, either soil or SMC, was sterilized, a significant reduction in HCH degradation was noticed. The second most reduction of isomers was seen during treatment where unsterilized SMC was added in sterilized soil followed by treatment where SMC was sterilized but soil was not. Abiotic control did not remove any significant quantities of HCH. Simple first-order (SFO) kinetic confirmed that SMC reduced the half-live manifolds as compared to biotic control. Only one metabolite δ-PCCH was identified during the course of study.     

平菇种内原生质体分离与融合杂交育种技术的研究

将16个平菇资源品种的菌丝体,在25℃、pH5．8-6．0环境下,分别用1．5％溶菌酶加0．6mol／L KCl稳渗剂溶解其细胞壁2h,原生质体数得量最高。将获得的原生质体两两配对成39个组合,在30℃、pH9、0．6mol／L KCl稳渗剂、不加融合促进剂CaCl2和PEG6000的融合条件下,得到种内杂交融合子5株。其中4株融合子再生出菌丝体和子实体;同皿接种实验显示,新菌株与其双亲具明显的拮抗线。经栽培试验与生产示范,新育的“优生1号”品系表现适应性强,优质、丰产、抗杂、耐热。     

平菇菌粗多糖的抗氧化活性研究

采用深层发酵技术生产平菇粗多糖,时其清除DPPH自由基、羟自由基的能力、铁离子螯合能力以及还原力进行了比较分析。结果表明：菌丝体粗多糖和发酵液粗多糖均具有较强的抗氧化能力,但2种多糖的抗氧化能力存在差异;茵丝体粗多糖清除DPPH自由基的能力较强,其EC。。值为2．20mg／mL;发酵液粗多糖清除羟自由基的能力、铁离子螯合能力以及还原力较强,其EC50值分别为0．72mg／mL、3．32mg／mL和7．91mg／mL。在一定的浓度范围内,多糖的浓度增加,其抗氧化能力也随之增强,呈量效依赖关系。     

Atrazine,desethylatrazine (DEA) and desisopropylatrazine (DIA) degradation by Pleurotus ostreatus INCQS 40310

Abstract

Atrazine is the most common herbicide applied in crops of economic relevance, such as sugar cane, soybean, and corn. Atrazine and its derivatives desethylatrazine (DEA) and desisopropylatrazine (DIA) are toxic to the environment, affecting animal and human health. Thus, this study aimed to evaluate the degradation of atrazine and its derivatives by the fungus Pleurotus ostreatus INCQS 40310, as well as the potential of the enzymes involved in this process. P. ostreatus INCQS 40310 was able to degrade atrazine (82%), DEA (71%), and DIA (56%) over 22?days of fungal cultivation. Proteomic analysis indicated the participation of hydrolases and peroxidases during the degradation process. Additionally, resting cells of the fungus were tested to verify the action of intracellular enzymes in the degradation process, suggesting the participation of cytochrome P450 enzymatic complex. Resting cells experiments promoted the degradation of 50% of atrazine, 36% of DIA, 30% of DEA. So far, this is the first work evaluating the biodegradation of DEA and DIA by fungus.     

Saponin Stimulates Fruiting of the Edible Basidiomycete Pleurotus ostreatus

The addition of saponin from Quillaja to malt extract agar dramatically stimulated fruit body development of Pleurotus ostreatus. Higher concentration of Quillaja saponin added to the medium suppressed the growth of pilei of fruit bodies. The results indicate that the triterpenoid-saponin is a bioactive substance for fruiting of P. ostreatus.     

Ultrastructure of wheat straw cell wall delignified by Pleurotus ostreatus

Abstract Transmission electron microscopy of wheat straw cell wall incubated with Pleurotus ostreatus in the presence of glucose or sunflower oil reveals different degradation patterns. Lower lignin content and increased polyphenol-oxidase activity correspond to peculiar features of the substrate at ultrastructural level. Differentiation between cellulose and lignin degradation by electron microscopy might represent a valuable tool to monitor biological delignification during industrial hydrolysis of ligno-cellulose.     

糙皮侧耳(Pleurotus ostreatus)的AFLP指纹图谱分析

在对糙皮侧耳(Pleurotus ostreatus)的AFLP分析条件进行优化的基础上,利用该技术建立了14株产自不同地区的糙皮侧耳：DNA指纹图谱,并进行了数据分析。结果表明,在合成的14条引物的不同组合中,引物对E-3／M-3可以产生较多的DNA多态片段,E-AGC／M-CAT引物对的扩增效果最好,共获得184条DNA扩增带,其中多态性条带i101条,占54．89％。利用UPGMA法对所获数据进行聚类分析,计算得到糙皮侧耳菌株之间的遗传距离,发现不同品种间遗传距离差异较大,从0．192到0．754,说明糙皮侧耳的遗传多样性比较丰富。绘制的指纹分析树状图表明,14个糙皮侧耳菌株被分为6个组群,其中P17和杂3的相似性系数最高,达到了81．2％,而侧5与其他菌株的亲缘关系相对最远。     

启动子替代构建糙皮侧耳漆酶高产菌株

POXC是糙皮侧耳合成最多的一种漆酶。应用启动子替代技术,用构巢曲霉的甘油醛-3-磷酸脱氢酶基因（gpd）启动子替代POXC基因的启动子,构建了超量表达POXC糙皮侧耳转化子。转化子中POXC基因表达量比出发菌株提高了0.72–3倍。在PDA平板培养、PD摇瓶培养和棉籽壳试管培养条件下,转化子漆酶活力显著提高,比出发菌株提高了1.5倍以上。用棉籽壳栽培,转化子菇产量比出发菌株提高了16.2%,培养料中木质素含量比出发菌株减少21%。结果表明,应用高效启动子替代能够显著提高糙皮侧耳漆酶基因的表达量、漆酶活力及其木质纤维素降解能力。     

原生质体再生对糙皮侧耳Pleurotus ostreatus病毒脱除的效果

以糙皮侧耳Pleurotus ostreatus菌株新831和豫6为材料,从这两个菌株的菌丝体中提取了糙皮侧耳病毒基因组,共有3个dsRNA片段,大小分别为8.2kb、2.6kb和1.1kb。采用菌丝尖端分离脱毒、原基组织分离脱毒和原生质体再生脱毒技术对糙皮侧耳菌丝体进行脱毒处理,利用dsRNA技术对脱毒效果进行检测。结果显示,原基组织分离脱毒后3个条带依然存在;菌丝尖端分离脱毒后,8.2kb和1.1kb 2个条带完全脱除,2.6kb的条带亮度有所减弱;原生质体再生脱毒后3个条带完全脱除;对3种脱毒技术得到的菌株进行生理生化指标测定,结果显示,原生质体再生脱毒菌株的菌丝生长速度、生物量、呼吸强度、纤维素酶活等均明显优于出发菌株、原基组织分离脱毒和菌丝尖端分离脱毒菌株;栽培结果表明,原生质体再生脱毒菌株前两茬菇的生物学效率达到96.4%－99.1%,比出发菌株提高19.9%－25.4%,并且菌盖宽度和长度有所增加,表明原生质体再生技术可以有效脱除糙皮侧耳菌株病毒,提高糙皮侧耳栽培产量。     

平菇病毒dsRNA传播途径的探究

通过对平菇孢子dsRNA检测、单双杂交和单单杂交,从垂直传播和水平传播2个方面来研究平菇病毒dsRNA的传播途径,结果表明：病毒dsRNA通过孢子垂直传播的几率很小,通过细胞融合的水平传播几率相对比较高,在实际生产中平菇病毒dsRNA的主要传播途径还有待进一步的研究。     

人小肠三叶因子在糙皮侧耳中的整合、表达及检测

用原生质体法将含双 35S_AMV启动子及人小肠三叶因子 (hITF)cDNA的表达载体导入糙皮侧耳 (Pleurotusostreatus)中 ,原生质体转化子在含有除草剂的培养基上初步筛选 ,获得抗性菌株。通过PCR分析证明hITFcDNA已整合到侧耳基因组中。以hITF为抗原免疫家兔制备了抗血清 ,纯化后的IgG以直接型酶联免疫吸附检测 (ELISA)方法测定效价 ,并建立起hITF的竞争型酶联免疫吸附检测 (ELISA)方法 ,给出了标准工作曲线 ,以应用于大规模表达菌株的筛选确立。检测表明 ,hITF在侧耳新鲜菌丝体中分 3个表达量段 :10 0 0～ 12 5 0ng g、14 80～ 170 0ng g、最高达2 0 0 0～ 2 2 5 0ng g ,约占总可溶性蛋白的 0 7%～ 1 5 %。Western印迹进一步分析证明hITF在侧耳中的表达。     

平菇(Pleurotus ostreatus)对稻草中木质素的生物降解及降解产物分析

变色圈试验证明平菇可以选择性优先降解稻草中的木质素,培养15d后,平菇对稻草中的木质素降解率为17.86%,对综纤维素降解率为2.44%,选择性指数为9.79。生料栽培平菇后,稻草中的木质素被降解50.24%。用气—质色谱(GC/MS)和红外光谱(IR)对木质素降解产物分析结果表明,平菇对稻草中木质素的降解效果十分明显,降解产物中检测出了大量含有苯环的小分子,证明木质素聚合体的降解首先发生在单体的侧链及单体间的连键上,发生Cα-Cβ、β-O-4等断裂,形成了单体。在进一步的降解过程中,平菇表现了其自身特有的降解机制,取代苯环单体上的甲氧基为甲基,而后发生苯环的开裂,这与报道的白腐菌降解过程有所不同。红外光谱分析中,平菇对木质素的降解明显,降解产物中含有很多木质素单体所特有的基团,如紫丁香基、愈创木基等,说明木质素的降解首先发生的是侧链的氧化反应。     

Lignocellulose degradation by Pleurotus ostreatus in the presence of cadmium

Activities of cellulolytic and hemicellulolytic enzymes endo-1,4-beta-glucanase, exo-1,4-beta-glucanase, 1,4-beta-glucosidase, endo-1,4-beta-xylanase, 1,4-beta-xylosidase and 1,4-beta-mannosidase and ligninolytic enzymes Mn-peroxidase and laccase were detected during the growth of the white-rot fungus Pleurotus ostreatus on wheat straw in the presence and absence of cadmium. The loss of substrate dry weight and Mn-peroxidase activity decreased with increasing Cd concentration, whereas the activities of endo-1,4-beta-glucanase, 1,4-beta-glucosidase and laccase were highly increased in the presence of metal. The onset of hemicellulose-degrading enzyme activity was delayed in the presence of cadmium. The degradation of a model synthetic dye Poly B-411 did not correspond to the activities of ligninolytic enzymes. This is the first report about 1,4-beta-mannosidase in P. ostreatus.     

Transformation of the edible mushroom Pleurotus ostreatus by particle bombardment

Uracil auxotroph of Pleurotus ostreatus was transformed to prototrophy by means of particle bombardment. Five transformants were obtained under three conditions differing in the two parameters of target distance and helium pressure. The transformation frequency was one transformant per microg of DNA. In the transformants, plasmid DNAs were integrated into the genomic DNA and stably maintained. This is the first report on transformation of P. ostreatus by particle bombardment.     

平菇转化叶黄素酯研究

目前针对有关天然产物的生物转化研究较少,首次报道了平菇转化叶黄素酯的研究。在对平菇在叶黄素酯培养基生长的观察中,发现对于固体培养基,菌丝体可利用叶黄素酯但不能增生,而孢子悬液不能生长,但在发酵液中两者生长良好,可见只有菌丝体可分泌水解酶,而且谊酶在溶液环境下效价高。同时利用高效液相色谱测定其降解效果发现,平菇首先将叶黄素酯转化为叶黄素,同时也可将叶黄素转化为其它物质,故控制发酵时间非常重要,这为今后的工业化提供了基础。     

Growth characteristics of Pleurotus ostreatus in bioreactors

Pleurotus ostreatus was cultured in a bioreactor equipped with different impeller geometries under non-limiting nutrient conditions. With a Rushton turbine impeller the specific growth rate decreased 30% and pellet diameter was reduced 15% when the aeration rate was increased from 1 to 1.5 vvm. Agitation rate reduced the pellet diameter from 5.1 mm to 2.8 mm using 200 rpm and 400 rpm of agitation, respectively. Specific growth rates of 0.036, 0.020 and 0.041 h–1 were obtained with Roshton (disc turbine), Helical Ribbon and InterMIG impellers, respectively. Impeller geometry is important to control the pellet size and consequently growth rate of P. ostreatus.     

平菇菌丝体与子实体营养成分的分析比较

采用现代化学分析手段对平菇菌丝体和子实体的营养成分进行分析比较。结果表明,用PDA固体培养的菌丝体的灰分、可溶物、总糖和粗蛋白的含量均明显低于子实体,而粗脂肪和粗纤维的含量则显著高于子实体;菌丝体的Fe、Ca、Zn的含量显著高于子实体,而Mg和K的含量明显低于子实体,Na的含量两者比较接近;菌丝体和子实体的氨基酸组成相似,都含有17种氨基酸,但子实体的各氨基酸的含量均高于菌丝体,从各项氨基酸含量的比值来看,菌丝体则更优于子实体。两者都不失是极好的营养食品源。