Ultrastructural and Immunocytochemical Studies on the H2O2-Producing Enzyme Pyranose Oxidase in Phanerochaete chrysosporium Grown under Liquid Culture Conditions

The ultrastructural distribution of the sugar-oxidizing enzyme pyranose 2-oxidase (POD) in hyphae of Phanerochaete chrysosporium K-3 grown under liquid culture conditions optimal for the enzyme's production was studied by transmission electron microscopy immunocytochemistry. Using the 3-dimethylaminobenzoic acid-3-methyl-2-benzothiazolinone hydrazone hydrochloride H₂O₂ peroxidase spectrophotometric assay, POD was detected in mycelial extracts from days 7 to 18, with maximum activity recorded on day 12. Onset of POD activity occurred in the secondary phase of hyphal development at a time of stationary growth, glucose limitation, and pH increase. POD was also detected extracellularly in the culture fluid from days 7 to 18, with maximum activity recorded on day 13. At early stages of development (3 to 4 days), using anti-POD antibodies and immunogold labeling, POD was localized in multivesicular and electron-dense bodies and in cell membrane regions. After 10 to 12 days of growth, at maximum POD activity, POD was concentrated within the periplasmic space where it was associated with membrane-bound vesicles and other membrane structures. At later stages of development (17 to 18 days), when the majority of hyphae were lysed, POD was observed associated with residual intracellular membrane systems and vesicles. Transmission electron microscopy immunocytochemical studies also demonstrated an extracellular distribution of the enzyme at the stationary growth phase, showing its association with fungal extracellular slime. In studies of ligninolytic cultures of the same fungus, POD was found to have a similar intracellular and extracellular distribution in slime as that recorded for cultures grown with cornsteep. POD's peripheral cytoplasmic distribution shows similarities to the cellular distribution of that reported previously for H₂O₂-dependent lignin and manganese peroxidases in P. chrysosporium.     

Pyranose Oxidase, a Major Source of H(2)O(2) during Wood Degradation by Phanerochaete chrysosporium, Trametes versicolor, and Oudemansiella mucida

The production of the H(2)O(2)-generating enzyme pyranose oxidase (POD) (EC 1.1.3.10) (synonym, glucose 2-oxidase), two ligninolytic peroxidases, and laccase in wood decayed by three white rot fungi was investigated by correlated biochemical, immunological, and transmission electron microscopic techniques. Enzyme activities were assayed in extracts from decayed birch wood blocks obtained by a novel extraction procedure. With the coupled peroxidase-chromogen (3-dimethylaminobenzoic acid plus 3-methyl-2-benzothiazolinone hydrazone hydrochloride) spectrophotometric assay, the highest POD activities were detected in wood blocks degraded for 4 months and were for Phanerochaete chrysosporium (149 mU g [dry weight] of decayed wood), Trametes versicolor (45 mU g), and Oudemansiella mucida (1.2 mU g), corresponding to wood dry weight losses of 74, 58, and 13%, respectively. Mn-dependent peroxidase activities in the same extracts were comparable to those of POD, while lignin peroxidase activity was below the detection limit for all fungi with the veratryl alcohol assay. Laccase activity was high with T. versicolor (422 mU g after 4 months), in trace levels with O. mucida, and undetectable in P. chrysosporium extracts. Evidence for C-2 specificity of POD was shown by thin-layer chromatography detection of 2-keto-d-glucose as the reaction product. By transmission electron microscopy-immunocytochemistry, POD was found to be preferentially localized in the hyphal periplasmic space of P. chrysosporium and O. mucida and associated with membranous materials in hyphae growing within the cell lumina or cell walls of partially and highly degraded birch fibers. An extracellular distribution of POD associated with slime coating wood cell walls was also noted. The periplasmic distribution in hyphae and extracellular location of POD are consistent with the reported ultrastructural distribution of H(2)O(2)-dependent Mn-dependent peroxidases. This fact and the dominant presence of POD and Mn-dependent peroxidase in extracts from degraded wood suggest a cooperative role of the two enzymes during white rot decay by the test fungi.     

墨兰菌根的结构及酸性磷酸酶定位研究

利用光学显微镜、电子显微镜及细胞化学方法,对墨兰菌根的结构和酸性磷酸酶定位进行了初步研究。结果表明墨兰具有典型的兰科植物根结构,发现该兰花的根的外皮层不具薄壁通道细胞,菌根真菌通过破坏部分根被和外皮层细胞而侵入根的皮层细胞并在细胞内形成菌丝结,侵入的菌丝被染菌皮层细胞质膜和电子透明物质包围,进一步被消化并聚集成衰败菌丝团块。酸性磷酸酶在染菌皮层细胞及包围菌丝的皮层细胞质膜和衰败菌丝细胞壁上有强烈的酶反应,衰败菌丝周围分布有许多单层膜的含酶小泡,它们可相互愈合形成大的含酶泡或与包围菌丝的质膜融合,类似于兰科植物共生原球茎中观察到的现象。说明皮层细胞可主动释放水解酶参与对菌丝的消化     

Study on Structure and Localization of Acid Phosphatase of Mycorrhizal Root of Cymbidium sinense (Orchidaceae)

The microstructure and ultrastructure of mycorrhizal root of Cymbidum sinenese (Andr.) Wild were studied. The results showed that this species possesses the typical root structure of orchids. There is no passage cells in the exodermis of root. Mycorrhizal fungi invade into the cortex by destroying the velamen and exodermal cells, and form pelotons in cortical cells. The hyphae colonizing cortical cells were separated from the cortical cells by electron- lucent material and cortical cell plasma membrane and digested. They often gathered to form clumps. Localization of acid phosphatase revealed that this enzyme possessed higher activity in the cortical cells containing hyphae. Many products of it also occurred on cortical cell plasma membrane surrounding hyphae and degenerated hyphae cell wall. Higher acid phosphatase activity was observed in many vesicles in the cortical cells infected by fimgi. These enzyme vesicles gathered around the invaded hy-phae and often fused with each other, or with cortical cell plasma membrane surrounding hyphae to digest these hyphae. It means cortical cells were able to release hydrolytic enzyme to digest the invaded hyphae.     

Shedding of membrane vesicles mediates fibroblast growth factor-2 release from cells

Fibroblast growth factor-2 (FGF-2), a polypeptide with regulatory activity on cell growth and differentiation, lacks a conventional secretory signal sequence, and its mechanism of release from cells remains unclear. We characterized the role of extracellular vesicle shedding in FGF-2 release. Viable cells released membrane vesicles in the presence of serum. However, in serum-free medium vesicle shedding was dramatically down-regulated, and the cells did not release FGF-2 activity into their conditioned medium. Addition of serum to serum-starved cells rapidly induced intracellular FGF-2 clustering under the plasma membrane and into granules that colocalized with patches of the cell membrane with typical features of shed vesicle membranes. Shed vesicles carried three FGF-2 isoforms (18, 22, 24 kDa). Addition of vesicles to endothelial cells stimulated chemotaxis and urokinase plasminogen activator production, which were blocked by anti-FGF-2 antibodies. Treatment of intact vesicles with 2.0 m NaCl or heparinase, which release FGF-2 from membrane-bound proteoglycans, did not abolish their stimulatory effect on endothelial cells, indicating that FGF-2 is carried inside vesicles. The comparison of the stimulatory effects of shed vesicles and vesicle-free conditioned medium showed that vesicles represent a major reservoir of FGF-2. Thus, FGF-2 can be released from cells through vesicle shedding.     

pH影响Ca~(2 )和EGTA对水霉(Saprolegnia ferax)菌丝顶端生长的作用效应

水霉(Saprolegia ferax)菌丝在pH6.0-8.0的OM液体培养基中生长良好,在pH5.0时生长速率有所下降,在pH3.0—4.0时停止生长。短时间(30min)作用研究表明,低浓度的CaCl_2促进pH5.0(1—5mmol/L)和pH6.0(1mmol/L)条件下的菌丝顶端生长,抑制pH7.0—8.0条件下的菌丝生长。1mmol/L以上的EGTA则抑制pH5.0条件下菌丝顶端生长,促进pH6.0—8.0条件下的菌丝顶端生长。但CaCl_2和EGTA都不能使pH3.0—4.0条件下的菌丝恢复生长。长时间(8h)作用跟踪观察表明,2mmol/L EGTA(pH6.8)短时间作用可促进菌丝生长,但随着培养时间延长,则产生抑制作用,并诱导原生质从菌丝最顶端喷出。说明细胞壁Ca~(2 )起着提供胞外Ca~(2 )源和细胞壁修饰成分的双重作用。Ca~(2 )通道阻断剂verapamil对菌丝顶端生长的抑制作用也说明顶端生长所需的Ca~(2 )来自胞外。     

Peptidase activities during morphogenesis of Mucor racemosus

Multiple peptidase activities are expressed in the dimorphic fungus Mucor racemosus. Peptide hydrolysis was measured using an enzyme-coupled colorimetric assay. Aminopeptidase as well as carboxypeptidase activities increased during spore, swollen spore, and budding yeast-to-hyphae conversions, and activities achieved a maximum level prior to the period of rapid germ tube formation. These increases in peptidase activity were prevented by cycloheximide. Three distinct aminopeptidases (AP) and three distinct carboxypeptidases (CP) were partially purified by gel filtration column chromatography. AP1 (235 kDa), AP2 (112 kDa), and AP3 (70 kDa) were all expressed in spore, yeast, and hyphae. The activity levels of AP2 and AP3 decreased in hyphae entering stationary growth. CP1 (250 kDa) and CP3 (50 kDa) activities were expressed exclusively in hyphae, whereas CP2 (77 kDa) was expressed in spore, yeast, and hyphal forms. CP1 activity was most pronounced in hyphae entering stationary growth. We concluded that M. racemosus expresses a multiplicity of peptidases and that CP1 and CP3 are morphology-specific carboxypeptidases.     

Nongenomic regulation of protein kinase C isoforms by the vitamin D metabolites 1α,25-(OH)2D3 and 24R,25-(OH)2D3

Prior studies have shown that vitamin D regulation of protein kinase C activity (PKC) in the cell layer of chondrocyte cultures is cell maturation-dependent. In the present study, we examined the membrane distribution of PKC and whether 1α,25-(OH)₂D₃ and 24R,25-(OH)₂D₃ can directly regulate enzyme activity in isolated plasma membranes and extracellular matrix vesicles. Matrix vesicle PKC was activated by bryostatin-1 and inhibited by a PKC-specific pseudosubstrate inhibitor peptide. Depletion of membrane PKC activity using isoform-specific anti-PKC antibodies suggested that PKCα is the major isoform in cell layer lysates as well as in plasma membranes isolated from both cell types; PKCζ is the predominant form in matrix vesicles. This was confirmed in Western blots of immunoprecipitates as well as in studies using control peptides to block binding of the isoform specific antibody to the enzyme and using a PKCζ-specific pseudosubstrate inhibitor peptide. The presence of PKCζ in matrix vesicles was further verified by immunoelectron microscopy. Enzyme activity in the matrix vesicle was insensitive to exogenous lipid, whereas that in the plasma membrane required lipid for full activity. 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ inhibited matrix vesicle PKC, but stimulated plasma membrane PKC when added directly to the isolated membrane fractions. PKC activity in the matrix vesicle was calcium-independent, whereas that in the plasma membrane required calcium. Moreover, the vitamin D-sensitive PKC in matrix vesicles was not dependent on calcium, whereas the vitamin D-sensitive enzyme in plasma membranes was calcium-dependent. It is concluded that PKC isoforms are differentially distributed between matrix vesicles and plasma membranes and that enzyme activity is regulated in a membrane-specific manner. This suggests the existence of a nongenomic mechanism whereby the effects of 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ may be mediated via PKC. Further, PKCζ may be important in nongenomic, autocrine signal transduction at sites distal from the cell. © 1996 Wiley-Liss, Inc.     

Extracellular protease and amylase activities in ligninase-producing liquid culture of Phanerochaete chrysosporium

The production of extracellular proteases and -amylase by ligninase-producing liquid culture of the white-rot fungus Phanerochaete chrysosporium NCIM 1197 has been investigated in stationary culture conditions. Acid, neutral, and alkaline proteases were all identified with maximum activities on the 9th day of incubation. Peak production of ligninase and -amylase occured on day 6 and day 3 respectively. The time courses of the production of proteases and those of ligninase and amylase were negatively correlated.

Protease inhibition by the addition of phenylmethyl sulphonyl flouride (days 1 and 4) resulted in enhanced activities of ligninase and amylase beyond the 9th day, suggesting an effect of protease on ligninase and amylase activity.     

Homologous expression of recombinant cellobiose dehydrogenase in Phanerochaete chrysosporium

Cellobiose dehydrogenase (CDH) is a novel extracellular hemoflavoenzyme from Phanerochaete chrysosporium and is produced only in cultures supplemented with cellulose. In this report, CDH from P. chrysosporium has been homologously expressed in cultures supplemented with glucose as the sole carbon source when no endogenous CDH is expressed. This was achieved by placing the cdh-1 gene under the control of the D-glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter (1.1 kb) fused upstream of the ATG start codon of cdh-1. The gpd promoter-chd-1 construct was inserted into the multiple cloning site of the expression vector pOGI18, which contained the Schizophyllum commune ade5 as a selectable marker. The P. chrysosporium ade1 auxotrophic strain OGC107-1 was transformed with the pAGC1 construct, and the prototrophic transformants were assayed for CDH activity. Approximately 50% of the Ade(+) transformants exhibited CDH activity in the extracellular medium of stationary cultures. At least one of the transformants produced high levels (500-600 U/liter) of recombinant CDH (rCDH). Purification by ammonium sulfate precipitation, Sephacryl S-200 chromatography, and FPLC using a Mono-Q 5/5 column yielded homogeneous rCDH. Physical, spectral, and kinetic characteristics of purified homologously expressed rCDH were similar to those of wild-type CDH. This expression system will enable site-directed mutagenesis studies to be carried out on CDH.     

Development of a serial bioreactor system for direct ethanol production from starch using<Emphasis Type="Italic">Aspergillus niger</Emphasis> and<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Aspergillus niger hyphae were found to grow with unliquefied potato starch under aerobic conditions, but did not grow under anaerobic conditions. The raw culture ofA. niger catalyzed saccharification of potato starch to glucose, producing approximately 12 g glucose/L/day/ The extracellular enzyme activity was decreased in proportion to incubation time, and approximately 64% of initial activity was maintained after 3 days. At 50°C,A. niger hyphae growth stopped, while the extracellular enzyme activity peaked. On the basis of theA. niger growth property and enzyme activity, we designed a serial bioreactor system composed of four different reactors. Fungal hyphae were cultivated in reactor I at 30°C, uniquefied starch was saccharified to glycose by a fungal hyphae culture in reactors II and III at 50°C, and glucose was fermented to ethanol bySaccharomyces cerevisiae in reactor IV. The total glucose produced by fungal hyphae in reactor I and saccharification in reactor II was about 42 g/L/day. Ethanol production in reactor IV was approximately 22 g/L/day, which corresponds to about 79% of the theoretical maximum produced from 55 g starch/L/day.     

Cell growth and cellulase production inTrichoderma viride on microcrystalline cellulose

The production of CM and FP cellulases was studied during the growth of a wild strain ofTrichoderma viride on microcrystalline cellulose. Part of the enzymes was found to be released into the medium while another part remained bound to the cell. Bound cellulases are released into the medium at the stage of cell lysis which takes place in the post-stationary phase. In this period extracellular CM and FP cellulases attain maximum activities. When the hyphae are subjected to a cold shock, maximum cellulase activity is detected already at the beginning of the stationary phase. An indirect method of dry cell mass determination showed that during exponential growth of cells on microcrystalline cellulose the μmax was 0.23 and the yield coefficient was 41 %.     

Effect of cadmium on the ligninolytic activity ofStereum hirsutum andPhanerochœte chrysosporium

An inhibitory effect of cadmium on the growth and ligninolytic activity of the wood-rotting basidiomycetesPhanerochœte chrysosporium, Pleurotus ostreatus, Pycnoporus cinnabarinus andStereum hirsutum was observed. Delayed decolorization of the polymeric dye Poly R-478 was observed in samples with 0.10 mmol/L Cd. Addition of 0.25 mmol/L Cd to the cultivation medium strongly reduced the activity of both Mn-dependent and Mn-independent peroxidases ofStereum hirsutum, while the activity of laccase was not affected to a similar extent. The maximum of MnP activity in these samples was found during the exponential phase of growth whereas control samples showed the highest activity after the onset of the stationary phase (days 15–21). Cadmium at concentrations higher than 0.50 mmol/L significantly inhibited the activity of all enzymes tested in bothS. hirsutum andP. chrysosporium.     

Immunogold-cytochemical labelling of β-glucosidase in the white-rot fungus Coriolus versicolor

Summary Immunogold cytochemical labelling of hyphal sections of Coriolus versicolor showed that -glucosidase was localised in the extracellular mucilage, cell wall layers and cell interior in hyphae grown on glucose-rich malt extract medium whereas in hyphae grown with carboxymethylcellulose (CMC) as sole carbon source, most labelling was in the cell wall layers and cell interior. Little mucilage was visible around hyphae from these cultures. Hyphae from beechwood cultures showed gold labelling of -glucosidase in mucilage and fungal cell walls with some intracellular labelling. Biochemical studies of enzyme activity showed that similar amounts of enzyme were detected in the growth medium when cultures were grown on CMC medium, in agitated liquid cultures or in stationary cultures. In agitated cultures grown on glucose-rich malt extract, the activity of -glucosidase in the medium was 100 times less than that detected in stationary cultures on the same medium. However activity in the hyphae of stationary CMC-grown cultures was similar to that in hyphae from stationary glucose-rich cultures. These data confirm the patterns of gold labelling observed in hyphae from stationary cultures on glucose-rich malt extract when -glucosidase was immobilised in the extracellular mucilage layer around the hyphae. In this paper we propose that a primary function of the extracellular mucilage produced by hyphae of C. versicolor in vivo is to serve as a matrix for immobilisation of -glucosidase. Its substrate, cellobiose, which is released as a result of endo-and exoglucanase hydrolysis of cellulose, is absorbed and retained by the gel filtration properties of the mucilage, so encountering the immobilised -glucosidase. Glucose produced by this reaction is retained within the mucilage matrix around the hyphae before intracellular absorption.Offprint requests to: C. S. Evans     

Partial characterization of lysyl oxidase from several human tissues.

The extracellular release of mycobacillin from Bacillus subtilis first occurred in the medium at the onset of stationary phase and continued at a high rate even after 6 days. Mycobacillin synthetase activity appeared earlier than late-exponential phase in the cytosol of producer cells and was not sedimentable even at 105 000 g. The activity then quickly reached the maximum late in the stationary phase. With further increase in the age of the culture, the activity gradually disappeared from the cytosol, to reappear concomitantly in the membrane in an insoluble particulate form, even in absence of protein synthesis. The membrane-bound synthetase activity was sedimentable at 10 000 g and was fairly active even after 5 days.     

Salicylic acid and methyl jasmonate improve chilling tolerance in cold-stored lemon fruit (Citrus limon)

Chilling injury (CI) is associated with the degradation of membrane integrity which can be aligned to phenolic oxidation activated by polyphenol oxidase (PPO) and peroxidase (POD), enzymes responsible for tissue browning. Phenylalanine ammonia-lyase (PAL) is a further enzyme prominent in the phenolic metabolism that is involved in acclimation against chilling stress. It was hypothesized that treatment with methyl jasmonate (MJ) and salicylic acid (SA) may enhance chilling tolerance in lemon fruit by increasing the synthesis of total phenolics and PAL by activating the key enzyme regulating the shikimic acid pathway whilst inhibiting the activity of POD and PPO. Lemon fruit were treated with 10 μM MJ, 2 mM SA or 10 μM MJ plus 2 mM SA, waxed, stored at −0.5, 2 or 4.5 °C for up to 28 days plus 7 days at 23 °C. Membrane integrity was studied by investigating membrane permeability and the degree of membrane lipid peroxidation in lemon flavedo following cold storage. The 10 μM MJ plus 2 mM SA treatment was most effective in enhancing chilling tolerance of lemon fruit, significantly reducing chilling-induced membrane permeability and membrane lipid peroxidation of lemon flavedo tissue. This treatment also increased total phenolics and PAL activity in such tissue while inhibiting POD activity, the latter possibly contributing to the delay of CI manifestation. PPO activity was found to be a poor biochemical marker of CI. Treatment with 10 μM MJ plus 2 mM SA resulted in an alteration of the phenolic metabolism, enhancing chilling tolerance, possibly through increased production of total phenolics and the activation of PAL and inhibition of POD.     

Involvement of a new enzyme, glyoxal oxidase, in extracellular H2O2 production by Phanerochaete chrysosporium.

The importance of extracellular H2O2 in lignin degradation has become increasingly apparent with the recent discovery of H2O2-requiring ligninases produced by white-rot fungi. Here we describe a new H2O2-producing activity of Phanerochaete chrysosporium that involves extracellular oxidases able to use simple aldehyde, alpha-hydroxycarbonyl, or alpha-dicarbonyl compounds as substrates. The activity is expressed during secondary metabolism, when the ligninases are also expressed. Analytical isoelectric focusing of the extracellular proteins, followed by activity staining, indicated that minor proteins with broad substrate specificities are responsible for the oxidase activity. Two of the oxidase substrates, glyoxal and methylglyoxal, were also identified, as their quinoxaline derivatives, in the culture fluid as secondary metabolites. The significance of these findings is discussed with respect to lignin degradation and other proposed systems for H2O2 production in P. chrysosporium.     

Influence of growth and environmental conditions on cell surface hydrophobicity of Pseudomonas fluorescens in non-specific adhesion

The relative cell surface hydrophobicity (CSH) of 18 soil isolates of Pseudomonas fluorescens, determined by phase exclusion, hydrophobic interaction chromatography (HIC), electrostatic interaction chromatography (ESIC), and contact angle, revealed large degrees of variability. Variation in the adhesion efficiency to Macrophomina phaseolina of the hyphae/sclerotia of these isolates was also examined. Two such isolates with maximum (32.8%; isolate 12-94) and minimum (12%; isolate 30-94) CSH were selected for further study. Early- to mid-log exponential cells of these isolates were more hydrophobic than those in stationary phase, and the CSH of these isolates was also influenced by fluctuations in temperatures and pH. Isolate 12-94 exhibited high CSH (32.3%) at 30 degrees C, compared to lower values (28-24%) in the higher temperature range (35-40 degrees C). Increasing concentrations of either Zn2+, Fe3+, K+, and Mg2+ in the growth medium were associated with the increased CSH. Trypsin, pepsin, and proteinase K (75 to 150 micrograms.mL-1) reduced the CSH of isolate 12-94 cells. CSH was reduced, following exposure to DTT, SDS, Triton X-100, or Tween 80. Prolonged exposure of cells to starvation (60 days) also caused a significant decline in CSH. Several protein bands (18, 21, 23, 26 kDa) of the outer cell membrane were absent in 60-day starved cells compared to unstarved cells. In conclusion, our findings demonstrate that CSH of P. fluorescens isolates may contribute to nonspecific attachment/adhesion onto M. phaseolina hyphae/sclerotia, and the efficiency of adhesion is regulated by growth and other environmental conditions.     

Modelling the growth kinetics of Phanerochaete chrysosporium in submerged static culture.

The potential commercial application of Phanerochaete chrysosporium requires methods for quantitatively predicting growth and substrate utilization. The growth kinetics of P. chrysosporium INA-12 (CNCM I-398) were investigated and modelled under nonlimiting nitrogen and carbon conditions in submerged static culture. This strain, unlike other strains, does not require nutrient limitation for induction of lignin peroxidase. Maximum levels of lignin peroxidase activity were reached 7 days after culture initiation, when almost 80% of the initial glycerol and 70% of the initial nitrogen were still present. Lignin peroxidase levels then decreased, while biomass levels increased until about day 14. The ratio of cell dry weight to wet weight was constant until the maximum biomass concentration was achieved, after which there was a decrease in the water content. The change in this ratio reflects cell lysis as it correlated with increased concentrations of nitrogen in the media, arising from cell leakage. The suitability of four growth models to predict growth, and in some cases glycerol consumption, was evaluated. A simple linear model and the Emerson model performed poorly for the early stages of growth, while a modified Williams model and the Monod model predicted substrate and biomass concentrations equally well. All models will predict biomass concentrations during the active growth phase, but they should not be used to predict biomass concentrations after the stationary growth phase, when cell lysis becomes significant.