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     

Physiological implication of intracellular trehalose and mannitol changes in response of entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis> to thermal stress

To explore possible role of intracellular trehalose accumulation in fungal tolerance to summer-like thermal stress, 3-day colonies of Beauveria bassiana grown on a glucose-free medium at 25°C were separately exposed to 35, 37.5 and 40°C for 1–18 h, respectively. Trehalose accumulation in stressed mycelia increased from initial 4.2 to 88.3, 74.7 and 65.5 mg g⁻¹ biomass after 6-h stress at 35, 37.5 and 40°C, respectively, while intracellular mannitol level generally declined with higher temperatures and longer stress time. The stress-enhanced trehalose level was significantly correlated to decreased trehalase activity (r ² = 0.73) and mannitol content (r ² = 0.38), which was inversely correlated to the activity of mannitol dehydrogenase (r ² = 0.41) or mannitol 1-phosphate dehydrogenase (r ² = 0.30) under the stresses. All stressed cultures were successfully recovered at 25°C but their vigor depended on stressful temperature, time length and the interaction of both (r ² = 0.98). The highest level of 6-h trehalose accumulation at 35°C was found enhancing the tolerance of the stressed cultures to the greater stress of 48°C. The results suggest that the trehalose accumulation result partially from metabolized mannitol and contribute to the fungal thermotolerance. Trehalase also contributed to the thermotolerance by hydrolyzing accumulated trehalose under the conditions of thermal stress and recovery.     

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.     

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。     

电击法介导的紫孢侧耳原生质体转化

使用基因脉冲导入仪成功地将糙皮侧耳DNA导入紫孢侧耳单核原生质体内,获得了具有\"锁状联合”特征的双核转化菌株T₁,和T₂。转化率为8．2×10^-5,转化比为3．6％。酯酶同I酶分析结果表明,转化菌株除具有受体菌的酶带外,还存在供体菌的酶带,由此证明转化菌株确为紫孢侧耳和糙皮侧耳DNA重组的产物。转化菌株子实体形态也发生了变化。两菌株子实体均不释放孢子;T₁。菌柄中生,T₂成熟子实体菌盖中部易长出菌丝。     

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.     

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。     

Gene cloning and catalysis features of a new mannitol-1-phosphate dehydrogenase (BbMPD) from Beauveria bassiana

A long-chain mannitol-1-phosphate dehydrogenase (MPD) was characterized for the first time from fungal entomopathogen Beauveria bassiana by gene cloning, heterogeneous expression and activity analysis. The cloned gene BbMPD consisted of a 1334-bp open reading frame (ORF) with a 158-bp intron and the 935-bp upstream and 780-bp downstream regions. The ORF-encoded 391-aa protein (42 kDa) showed less than 75% sequence identity to 17 fungal MPDs documented and shared two conserved domains with the fungal MPD family at the N- and C-terminus, respectively. The new enzyme was expressed well in the Luria-Bertani culture of engineered Escherichia coli BL21 by 16-h induction of 0.5 mM isopropyl 1-thio-β-d-galactopyranoside at 20 °C after 5-h growth at 37 °C. The purified BbMPD exhibited a high catalytic efficiency (k_cat/K_m) of 1.31 × 10⁴ mM⁻¹ s⁻¹ in the reduction of the highly specific substrate d-fructose-6-phosphate to d-mannitol-1-phosphate. Its activity was maximal at the reaction regime of 37 °C and pH 7.0 and was much more sensitive to Cu²⁺ and Zn²⁺ than to Li⁺ and Mn²⁺. The results indicate a crucial role of BbMPD in the mannitol biosynthesis of B. bassiana.     

侧耳属食用菌高温胁迫条件优化研究

侧耳属真菌是我国最大规模栽培的食用菌,主要是糙皮侧耳Pleurotus ostreatus和白黄侧耳P. cornucopiae。高温是影响侧耳产量和质量的重要因子之一,确定高温胁迫条件是开展高温胁迫机制研究的方法学基础。以糙皮侧耳和白黄侧耳的11个菌株为材料,以硫代巴比妥酸反应产物（thiobarbituric acid reactive substances,TBARS）含量为主要生理指标,结合菌丝形态和生长等微生物学参数,开展了双核体菌丝高温胁迫条件的研究,包括胁迫的适宜温度、时间和菌龄等。结果表明,所有供试材料,高温胁迫后菌丝体内TBARS含量在28-40℃、0-48h范围内与温度和时间呈正相关,与之后的恢复生长率呈负相关,高温胁迫后菌丝体内TBARS含量和之后的恢复生长能力均与自身菌丝体生长和子实体形成的适宜温度无关。TBARS含量可作为菌丝高温胁迫生理研究条件确定的指标。与植物的最适温度加10℃不同,基于TBARS含量的糙皮侧耳和白黄侧耳高温胁迫研究条件应为Difco^TM Potato Dextrose Agar培养基,菌丝培养3d,最适温度加12℃,高温胁迫处理48h。     

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。     

平菇丰收一号高产特性的研究

通过野生种分离选用的平菇丰收一号,染色体数为９（ｎ＝９）,菌丝生长速度比一般品种快２—３４ｃｍ／ｈ,平均投料０．５ｋｇ（块）产鲜菇０．６～２．５ｋｇ,个别单株重１０～２０ｋｇ左右,生物效率１００～１５０％,高者达２００％以上,是一种高产优质、适应性强的新菌种。     

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.     

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。     

Using spectrophotometry and spectral mapping technique for the study of the production of manganese-dependent and manganese-independent peroxidases by Pleurotus ostreatus

The activity of manganese-dependent and manganese-independent peroxidases produced by Pleurotus ostreatus in culture media composed of agro-residues was measured by visible spectrophotometry. The overall enzyme activity and its selectivity were separated by using spectral mapping technique followed by nonlinear mapping. The relationships between the parameters of enzyme production and the composition of culture media and fermentation time was assessed by stepwise regression analysis. Calculations proved that the addition of extract of straw to the culture media significantly decreased the overall production of both enzymes, whereas the selectivity of enzyme production was influenced by amount of potato extract and the concentration of total sugar in the culture media. Enzyme activity depended quadratically on the fermentation time.     

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。     

An enzymatic colorimetric assay for glucose-6-phosphate

A specific colorimetric assay for the determination of glucose-6-phosphate (G6P) was developed. This assay is based on the oxidation of G6P in the presence of glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADP⁺); the NADPH thereby generated reduces the tetrazolium salt WST-1 [2-(4-indophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt] to water-soluble yellow-colored formazan with 1-methoxy-5-methylphenazium methylsulfate (1-mPMS) as an electron carrier. The assay is optimized for reaction buffer pH, enzyme/dye concentration, and reaction time course. The limit of detection of the assay is 0.15 μM (15 pmol/well). The usefulness of the assay is demonstrated by the accurate measurement of the G6P concentration in fetal bovine serum (FBS).