Identification of a novel lactose oxidase in Myrmecridium flexuosum NUK-21.

Lactobionic acid (O-ß-galactosyl-(1-4)-gluconic acid) (LBA) is a high-value lactose derivative, produced via oxidation of the reducing terminal of lactose. LBA can be produced by fermentation using certain microorganisms, although subsequent purification is challenging. Therefore, we have attempted to identify an enzyme for possible use in LBA production. Here, we purified a novel lactose oxidase (LOD) to homogeneity from a wheat bran culture of a soil-isolated fungal strain, Myrmecridium flexuosum NUK-21. Maximal activity was observed on the wheat bran solid culture after 3 days of NUK-21 growth, following release from cells at 0.66 unit·mL -1 culture filtrate. This new sugar oxidase was composed of a single polypeptide chain with a molecular mass of 47.2 kDa and was found to contain 2.0 zinc ions per mole of enzyme but no flavin adenine dinucleotide or heme. This enzyme was stable in the pH range 5.5-9.0, with an optimal reaction pH of 7.5. Its optimal reaction temperature was 40 °C, and it was stable up to 50 °C for 1 h at pH 7.5. LOD oxidized disaccharides with reducing-end glucosyl residues linked by an α or ß-1,4 glucosidic bond. The relative activity of LOD toward lactose, cellobiose and maltose was 100 : 83 : 4, respectively. To the best of our knowledge, this is the first report on the discovery of an LOD based on coenzyme moiety and enzyme substrate specificity.

Enhanced hexose-6-phosphate dehydrogenase expression in adipose tissue may contribute to diet-induced visceral adiposity.

BACKGROUND: Visceral fat accumulation increases the risk of developing type 2 diabetes and metabolic syndrome, and is associated with excessive glucocorticoids (GCs). Fat depot-specific GC action is tightly controlled by 11ß-hydroxysteroid dehydrogenase (11ß-HSD1) coupled with the enzyme hexose-6-phosphate dehydrogenase (H6PDH). Mice with inactivation or activation of H6PDH genes show altered adipose 11ß-HSD1 activity and lipid storage. We hypothesized that adipose tissue H6PDH activation is a leading cause for the visceral obesity and insulin resistance. Here, we explored the role and possible mechanism of enhancing adipose H6PDH in the development of visceral adiposity in vivo. METHODS: We investigated the potential contribution of adipose H6PDH activation to the accumulation of visceral fat by characterization of visceral fat obese gene expression profiles, fat distribution, adipocyte metabolic molecules, and abdominal fat-specific GC signaling mechanisms underlying the diet-induced visceral obesity and insulin resistance in H6PDH transgenic mice fed a standard of high-fat diet (HFD). RESULTS: Transgenic H6PDH mice display increased abdominal fat accumulation, which is paralleled by elevated lipid synthesis associated with induction of lipogenic transcriptor C/EBPα and PPARγ mRNA levels within adipose tissue. Transgenic H6PDH mice fed a high-fat diet (HFD) gained more abdominal visceral fat mass coupled with activation of GSK3ß and induction of XBP1/IRE1α, but reduced pThr308 Akt/PKB content and browning gene CD137 and GLUT4 mRNA levels within the visceral adipose tissue than WT controls. HFD-fed H6PDH transgenic mice also had impaired insulin sensitivity and exhibited elevated levels of intra-adipose GCs with induction of adipose 11ß-HSD1. CONCLUSION: These data provide the first in vivo mechanistic evidence for the adverse metabolic effects of adipose H6PDH activation on visceral fat distribution, fat metabolism, and adipocyte function through enhancing 11ß-HSD1-driven intra-adipose GC action.

Growth and enzymatic responses of phytopathogenic fungi to glucose in culture media and soil

The effect of inoculation of Aspergillus flavus, Fusarium verticillioides, and Penicillium sp. in Dystrophic Red Latosol (DRL) and Eutroferric Red Latosol (ERL) soils with or without glucose on the total carbohydrate content and the dehydrogenase and amylase activities was studied. The fungal growth and spore production in culture medium with and without glucose were also evaluated. A completely randomized design with factorial arrangement was used. The addition of glucose in the culture medium increased the growth rate of A. flavus and Penicillium sp. but not of F. verticillioides. The number of spores increased 1.2 for F. verticillioides and 8.2 times for A. flavus in the medium with glucose, but was reduced 3.5 times for Penicillium sp. The total carbohydrates contents reduced significantly according to first and second degree equations. The consumption of total carbohydrates by A. flavus and Penicillium sp. was higher than the control or soil inoculated with F. verticillioides. The addition of glucose to soils benefited the use of carbohydrates, probably due to the stimulation of fungal growth. Dehydrogenase activity increased between 1.5 to 1.8 times (p <0.05) in soils with glucose and inoculated with the fungi (except F. verticillioides), in relation to soil without glucose. Amylase activity increased 1.3 to 1.5 times due to the addition of glucose in the soil. Increased amylase activity was observed in the DRL soil with glucose and inoculated with A. flavus and Penicillium sp. when compared to control.

High-throughput screening for cellobiose dehydrogenases by Prussian Blue in situ formation.

Extracellular fungal flavocytochrome cellobiose dehydrogenase (CDH) is a promising enzyme for both bioelectronics and lignocellulose bioconversion. A selective high-throughput screening assay for CDH in the presence of various fungal oxidoreductases was developed. It is based on Prussian Blue (PB) in situ formation in the presence of cellobiose (<0.25 mM), ferric acetate, and ferricyanide. CDH induces PB formation via both reduction of ferricyanide to ferrocyanide reacting with an excess of Fe³âº (pathway 1) and reduction of ferric ions to Fe²âº reacting with the excess of ferricyanide (pathway 2). Basidiomycetous and ascomycetous CDH formed PB optimally at pH 3.5 and 4.5, respectively. In contrast to the holoenzyme CDH, its FAD-containing dehydrogenase domain lacking the cytochrome domain formed PB only via pathway 1 and was less active than the parent enzyme. The assay can be applied on active growing cultures on agar plates or on fungal culture supernatants in 96-well plates under aerobic conditions. Neither other carbohydrate oxidoreductases (pyranose dehydrogenase, FAD-dependent glucose dehydrogenase, glucose oxidase) nor laccase interfered with CDH activity in this assay. Applicability of the developed assay for the selection of new ascomycetous CDH producers as well as possibility of the controlled synthesis of new PB nanocomposites by CDH are discussed.

A homogeneous, high-throughput-compatible, fluorescence intensity-based assay for UDP-N-acetylenolpyruvylglucosamine reductase (MurB) with nanomolar product detection.

A novel assay for the NADPH-dependent bacterial enzyme UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is described that has nanomolar sensitivity for product formation and is suitable for high-throughput applications. MurB catalyzes an essential cytoplasmic step in the synthesis of peptidoglycan for the bacterial cell wall, reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramic acid (UNAM). Interruption of this biosynthetic pathway leads to cell death, making MurB an attractive target for antibacterial drug discovery. In the new assay, the UNAM product of the MurB reaction is ligated to L-alanine by the next enzyme in the peptidoglycan biosynthesis pathway, MurC, resulting in hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The ADP is detected with nanomolar sensitivity by converting it to oligomeric RNA with polynucleotide phosphorylase and detecting the oligomeric RNA with a fluorescent dye. The product sensitivity of the new assay is 1000-fold greater than that of the standard assay that follows the absorbance decrease resulting from the conversion of NADPH to NADP(+). This sensitivity allows inhibitor screening to be performed at the low substrate concentrations needed to make the assay sensitive to competitive inhibition of MurB.

Effect of diabetes on enzymes involved in rat hepatic corticosterone production.

BACKGROUND: Numerous studies have explored the etiologic or permissive role of 11ß-hydroxysteroid dehydrogenase (11ß-HSD1) in obesity and Type 2 diabetes, biochemical conditions often with concurrent hyperinsulinism. In contrast, there are limited data on the effect of insulin deficiency (i.e. Type 1 diabetes) on 11ß-HSD1 or endoplasmic reticulum enzymes that generate the reduced pyridine cofactor NADPH. Thus, the aim of the present study was to examine the effect of insulin-deficient, streptozotozin diabetes on key microsomal enzymes involved in rat hepatic corticosterone production. METHODS: After rats had been rendered diabetic with streptozotocin and some had been treated with insulin (2-6 units, s.c., long-acting insulin once daily) for 7 days, hepatic microsomes were isolated. Serum corticosterone and fructosamine were obtained premortem. Intact microsomes were incubated in vitro and 11ß-HSD1, hexose-6-phosphate dehydrogenase (H6PDH), and isocitrate dehydrogenase (IDH) measured. RESULTS: Although diabetes markedly altered body weight gain and serum protein glycosylation (assessed by fructosamine), there was no significant change in hepatic 11ß-HSD1 reductase activity, with or without insulin treatment. However, serum corticosterone levels were significantly correlated with 11ß-HSD1 reductase activity when all groups were analyzed together (P < 0.05). Untreated diabetes modified (P < 0.01) two hepatic microsomal NADPH-generating enzymes, namely H6PDH and IDH, resulting in a 37% decrease and 14% increase in enzyme levels, respectively. CONCLUSIONS: Consistent with most in vivo studies, chronic insulin deficiency with attendant hyperglycemia does not significantly modify hepatic 11ß-HSD1 reductase activity, but does alter the activity of two microsomal enzymes coupled with pyridine cofactors.

[Isolation of wood-decaying fungi and evaluation of their enzymatic activity (Quindío, Colombia)]. / Aislamiento y evaluación de la actividad enzimática de hongos descomponedores de madera (Quindío, Colombia).

White rot fungi (Ascomycota and Basidiomycota) were collected on fallen trunks with different decay stages, in a subandean forest (La Montaña del Ocaso nature reserve), and it was evaluated their ligninolitic activity. They were cultured on malt extract agar. Then it was performed semiquantitative tests for laccase and cellobiose dehydrogenase (CDH) activity using ABTS and DCPIP as enzymatic inducers. Based on the results of these tests, the fungi with higher activities from trunks with different decay stages were selected: Cookeina sulcipes (for stage 1), a fungus from the family Corticiaceae (for stage 2), Xylaria polymorpha (for stage 3) and Earliella sp. (for stage 4). A fermentation was performed at 28 degrees C, during 11 days, in a rotatory shaker at 150 rpm. Biomass, glucose, proteins and enzyme activities measurements were performed daily. The fungi that were in the trunks with decay states from 1 to 3, showed higher laccase activity as the state of decay increased. A higher DCH activity was also associated with a higher. Also, there was a positive relationship between both enzymes' activities. Erliella was the fungus which presented the highest biomass production (1140,19 g/l), laccase activity (157 UL(-1)) and CDH activity (43,50 UL(-1)). This work is the first report of laccase and CDH activity for Cookeina sulcipes and Earliella sp. Moreover, it gives basis for the use of these native fungi in biotechnological applications and the acknowledgment of their function in the wood decay process in native forest.

Probing active-site residues of pyranose 2-oxidase from Trametes multicolor by semi-rational protein design.

D-Tagatose is a sweetener with low caloric and non-glycemic characteristics. It can be produced by an enzymatic oxidation of D-galactose specifically at C2 followed by chemical hydrogenation. Pyranose 2-oxidase (P2Ox) from Trametes multicolor catalyzes the oxidation of many aldopyranoses to their corresponding 2-keto derivatives. Since D-galactose is not the preferred substrate of P2Ox, semi-rational design was employed to improve the catalytic efficiency with this poor substrate. Saturation mutagenesis was applied on all positions in the active site of the enzyme, resulting in a library of mutants, which were screened for improved activity in a 96-well microtiter plate format. Mutants with higher activity than wild-type P2Ox were chosen for further kinetic investigations. Variant V546C was found to show a 2.5-fold increase of k(cat) with both D-glucose and D-galactose when oxygen was used as electron acceptor. Because of weak substrate binding, however, k(cat)/K(M) is lower for both sugar substrates compared to wild-type TmP2Ox. Furthermore, variants at position T169, i.e., T169S and T169N, showed an improvement of the catalytic characteristics of P2Ox with D-galactose. Batch conversion experiments of D-galactose to 2-keto-D-galactose were performed with wild-type TmP2O as well as with variants T169S, T169N, V546C and V546C/T169N to corroborate the kinetic properties determined by Michaelis-Menten kinetics.

A thermostable triple mutant of pyranose 2-oxidase from Trametes multicolor with improved properties for biotechnological applications.

In order to increase the thermal stability and the catalytic properties of pyranose oxidase (P2Ox) from Trametes multicolor toward its poor substrate D-galactose and the alternative electron acceptor 1,4-benzoquinone (1,4-BQ), we designed the triple-mutant T169G/E542K/V546C. Whereas the wild-type enzyme clearly favors D-glucose as its substrate over D-galactose [substrate selectivity (k(cat)/K(M))(Glc)/(k(cat)/K(M))(Gal) = 172], the variant oxidizes both sugars equally well [(k(cat)/K(M))(Glc)/(k(cat)/K(M))(Gal) = 0.69], which is of interest for food biotechnology. Furthermore, the variant showed lower K(M) values and approximately ten-fold higher k(cat) values for 1,4-BQ when D-galactose was used as the saturating sugar substrate, which makes this enzyme particularly attractive for use in biofuel cells and enzyme-based biosensors. In addition to the altered substrate specificity and reactivity, this mutant also shows significantly improved thermal stability. The half life time at 60 degrees C was approximately 10 h, compared to 7.6 min for the wild-type enzyme. We performed successfully small-scale bioreactor pilot conversion experiments of D-glucose/D-galactose mixtures at both 30 and 50 degrees C, showing the usefulness of this P2Ox variant in biocatalysis as well as the enhanced thermal stability of the enzyme. Moreover, we determined the crystal structure of the mutant in its unligated form at 1.55 A resolution. Modeling D-galactose in position for oxidation at C2 into the mutant active site shows that substituting Thr for Gly at position 169 favorably accommodates the axial C4 hydroxyl group that would otherwise clash with Thr169 in the wild-type.

Direct protein-protein interaction of 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the endoplasmic reticulum lumen.

Hexose-6-phosphate dehydrogenase (H6PDH) has been shown to stimulate 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1)-dependent local regeneration of active glucocorticoids. Here, we show that coexpression with H6PDH results in a dramatic shift from 11beta-HSD1 oxidase to reductase activity without affecting the activity of the endoplasmic reticular enzyme 17beta-HSD2. Immunoprecipitation experiments revealed coprecipitation of H6PDH with 11beta-HSD1 but not with the related enzymes 11beta-HSD2 and 17beta-HSD2, suggesting a specific interaction between H6PDH and 11beta-HSD1. The use of the 11beta-HSD1/11beta-HSD2 chimera indicates that the N-terminal 39 residues of 11beta-HSD1 are sufficient for interaction with H6PDH. An important role of the N-terminus was indicated further by the significantly stronger interaction of 11beta-HSD1 mutant Y18-21A with H6PDH compared to wild-type 11beta-HSD1. The protein-protein interaction and the involvement of the N-terminus of 11beta-HSD1 were confirmed by Far-Western blotting. Finally, fluorescence resonance energy transfer (FRET) measurements of HEK-293 cells expressing fluorescently labeled proteins provided evidence for an interaction between 11beta-HSD1 and H6PDH in intact cells. Thus, using three different methods, we provide strong evidence that the functional coupling between 11beta-HSD1 and H6PDH involves a direct physical interaction of the two proteins.

Properties of neutral cellobiose dehydrogenase from the ascomycete Chaetomium sp. INBI 2-26(-) and comparison with basidiomycetous cellobiose dehydrogenases.

The extracellular cellobiose dehydrogenase (CDH) obtained from Chaetomium sp. INBI 2-26(-) has a molecular mass of 95 kDa and an isoelectric point of 5. This novel CDH is highly specific for the oxidation of cellobiose (K(m,app) 4.5 microM) and lactose (K(m,app) 56 microM). With 2,6-dichloroindophenol (DCIP) and cytochrome c(3+) (cyt c(3+)) as electron acceptors, CDH was most active at pH 6. The turnover number of the enzyme for cellobiose, lactose, DCIP and cyt c(3+) was in the range of 9-14s(-1) at 20 degrees C and pH 6. The UV-visible spectrum revealed the flavohemoprotein nature of the enzyme. The cytochrome b domain of the enzyme was reduced by ascorbate, dithionite, as well as specifically by cellobiose in a wide range of pH. The apparent first order rate constants of the spontaneous re-oxidation of the reduced heme domain were estimated as 0.01 and 0.00039 s(-1) at pH 4.5 and 6.5, respectively. The half-inactivation time of CDH at pH 6 and 55 degrees C was ca. 100 min; the stability at pH 8 and, particularly, pH 4 was remarkably lower. Cellobiose stabilized the enzyme against thermal inactivation, whereas DCIP in turn sensitized the enzyme. The new enzyme revealed low affinity for crystalline cellulose, but was capable of binding onto H(3)PO(4)-swollen filter paper. The results show significant differences to already known CDHs and perspectives for several biotechnological applications, where CDH with maximal activity at neutral pH and high affinity for cellobiose and lactose night have some advantages.

Cooperativity between 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase is based on a common pyridine nucleotide pool in the lumen of the endoplasmic reticulum.

11Beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) is a NADP(H)-dependent oxidoreductase of the ER lumen, which may have an important role in the pathogenesis of metabolic syndrome. Here, the functional coupling of 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase (H6PDH) was investigated in rat liver microsomal vesicles. The results demonstrate the existence of a separate intraluminal pyridine nucleotide pool in the hepatic endoplasmic reticulum and a close cooperation between 11betaHSD1 and H6PDH based on their co-localization and the mutual generation of cofactors for each other.

Inactivation of GDP-mannose dehydrogenase from Pseudomonas aeruginosa by penicillic acid identifies a critical active site loop.

The pathogenic bacterium Pseudomonas aeruginosa synthesizes alginate as one of a group of virulence factors that are produced during infections. The enzyme GDP-mannose dehydrogenase catalyzes the committed step in alginate biosynthesis. We show here that penicillic acid is an irreversible inactivator of GDP-mannose dehydrogenase. Inactivation occurs with a rate constant of 0.39+/-0.01 mM(-1) min(-1) at pH 8.0, and does not exhibit saturation behavior. Partial protection from inactivation is afforded by GDP-mannose, but not by the other substrate, NAD+. GMP and NAD+ together provide complete protection against inactivation. Analysis by mass spectrometry confirmed that the enzyme is alkylated at multiple cysteine residues by penicillic acid, including Cys 213, Cys 246, and the active site cysteine, Cys 268. However, the pH dependence of the inactivation rate suggested that alkylation of a single cysteine residue is sufficient to inactivate the enzyme. The C268A mutant protein was also susceptible to inactivation by penicillic acid. The presence of NAD+ and GMP provided partial protection of Cys 246 and Cys 268, and almost complete protection of Cys 213. Cys 213 is located on a helix that forms part of the binding pocket for GDP-mannose, and forms a hydrogen bond with Asn 252. Asn 252 is located on a loop that surrounds GDP-mannose. The C213A mutant enzyme exhibits a Vmax that is 1.8-fold greater than the wild-type enzyme, suggesting that the interaction between Cys 213 and Asn 252 helps to hold the loop in place during catalysis, and that opening the loop to release product is partially rate-limiting. Cys 246 is adjacent to the GDP-mannose binding loop, and its alkylation may also interfere with loop movement.

Interference of laccase in determination of cellobiose dehydrogenase activity of Pleurotus ostreatus (Florida) using dichlorophenol indophenol as the electron acceptor.

Pleurotus ostreatus (Florida), ITCC 3308 produces approximately 9.0 U/ml extracellular cellobiose dehydrogenase (CDH) in cellulose medium after 7 days of growth. However, no activity could be detected if the assay was done with cellobiose as the substrate and 2,6-dichlorophenol indophenol (DPIP) as the electron acceptor in absence of any laccase inhibitor. Kinetic study showed that V(max)/K(m) value was very high for rDPIP (reduced 2,6-dichlrophenol indophenol) oxidation by laccase. Oxygen consumption rate of rDPIP oxidation by the enzyme was found to be highest among all the tested substrates. The present study indicated that rDPIP was a good substrate for laccase. Therefore, caution is needed to measure CDH activity by monitoring DPIP reduction in a system where laccase is likely to be present.

Electrochemical investigation of cellobiose dehydrogenase from new fungal sources on Au electrodes.

Following previous electrochemical investigations of cellobiose dehydrogenase (CDH), the present investigation reports on the initial screening of the electrochemistry of three new CDHs, two from the white rot basidiomycetes Trametes villosa and Phanerochaete sordida and one from the soft rot ascomycete Myriococcum thermophilum, for their ability to directly exchange electrons with 10 different alkanethiol-modified Au electrodes. Direct electron transfer (DET) between the enzymes and some of the modified Au electrodes was shown, both, in the presence and in the absence of cellobiose. However, the length and the head functionality of the alkanethiols drastically influenced the efficiency of the DET reaction and also influenced the effect of pH on the biocatalytic/redox currents, suggesting the importance of structural/sequence differences between these CDH enzymes. In this respect, the white rot CDHs exhibit excellent biocatalytic and redox currents, whereas for the soft rot CDH the DET communication is much less efficient. Cyclic voltammograms indicate that the heme domain of the CDHs is the part of the enzymes that most readily exchanges electrons with the electrode. However, for P. sordida CDH on 11-mercaptoundecanol or dithiopropionic acid-modified Au electrodes, a second voltammetric wave was noticed suggesting that for some orientations of the enzyme, DET communication with the FAD cofactor can also be obtained.

[Degradation of the herbicide atrazine by the soil mycelial fungus INBI 2-26(-)--a producer of cellobiose dehydrogenase]. / Razlozhenie gerbitsida atrazina pochvennym mitselial'nym gribom INBI 2-26(-)--produtsentom tsellobiozodegidrogenazy.

Nonsporulating mycelial fungi producing cellobiose dehydrogenase (CDH) and isolated from soils of South Vietnam with high residual content of dioxins are capable of growing on a solid medium in the presence of high atrazine concentrations (to 500 mg/l). At 20 and 50 mg/l atrazine, the area of fungal colonies was 1.5-1.2-fold larger, respectively, compared with control colonies of the same age, whereas development of the colonies at 500 mg/l atrazine was delayed by 5 days, compared with controls grown in the absence of atrazine. Surface cultivation of the fungus on a minimal medium with glucose as a sole source of carbon and energy decreased the initial concentration of atrazine (20 mg/l) 50 times in 40 days; in addition, no pronounced sorption of atrazine by mycelium was detected. This was paralleled by accumulation in the culture medium of extracellular CDH; atrazine increased the synthesis of this enzyme two- to threefold. Accumulation of beta-glucosidase (a mycelium-associated enzyme) and cellulases preceded the formation of CDH.

Anatomy and fiber type composition of human interarytenoid muscle.

Intrinsic laryngeal muscle investigations, especially those of the interarytenoid (IA) muscle, have been primarily teleologically based. We determined IA muscle anatomy and histochemical and immunohistochemical classification of extrafusal and intrafusal (muscle spindle) fibers in 5 patients. Extrafusal fibers were oxidative type I and glycolytic types IIA and IIX. Intrafusal fibers of muscle spindles were identified by the presence of tonic and neonatal myosin. The results demonstrate that the IA muscle has a phenotype similar to that of limb skeletal muscle. Myosin coexpression, the absence of intrafusal fibers, and fiber type grouping were unusual features found previously in the thyroarytenoid and posterior cricoarytenoid muscles, but they were not present in the IA muscle. These findings lead to the conclusion that the IA muscle has functional significance beyond its assumed importance in maintaining vocal fold position during phonation. The presence of spindles demonstrates differences in motor control as compared to the thyroarytenoid and posterior cricoarytenoid muscles. Further, extrafusal fiber characteristics implicate IA muscle involvement in muscle tension dysphonia and adductor spasmodic dysphonia. Given the unique physiologic characteristics of the human IA muscle, further research into the role of the IA muscle in voice disorders is warranted.

Screening of basidiomycete fungi for the quinone-dependent sugar C-2/C-3 oxidoreductase, pyranose dehydrogenase, and properties of the enzyme from Macrolepiota rhacodes.

Mycelial cultures of 76 strains of lignocellulose-degrading basidiomycete fungi were screened for the activity of pyranose dehydrogenase, a novel sugar oxidoreductase recently detected in Agaricus bisporus. Of these fungi, 37 strains belonging to seven phylogenetically related genera of mostly litter-decomposing Agaricales were positive for the dehydrogenase, based on activity assays towards D-glucose with 1,4-benzoquinone or ferricenium ion as electron acceptors, and on TLC/HPLC analyses of the reaction products. Lack of activity with O(2) as the oxidant, specificity for C-3 of D-glucose, and active extracellular secretion of the enzyme were used as criteria to differentiate pyranose dehydrogenase from pyranose 2-oxidase (EC 1.1.3.10), known to be produced by numerous wood-rotting fungi. Extracellular pyranose dehydrogenase from Macrolepiota rhacodes was heavily glycosylated. The enzyme was characterized as a 78-kDa flavoprotein under denaturing conditions and a 76-kDa native protein using gel filtration. This enzyme had a maximum extracellular activity of 4.1 U ml(-1) in 39-day liquid cultures. It exhibited broad selectivity for sugar substrates and oxidized D-glucose (K(m)=1.82) exclusively at C-3 to 3-dehydro-D-glucose (D-ribo-hexos-3-ulose), in contrast to pyranose dehydrogenases from Agaricus species, which acted at both C-3 and C-2 of D-glucose. The N-terminal sequence, AVVYRHPDEL, showed significant similarity with that reported for A. bisporus.

Actinic keratosis--a histoenzymological study.

Actinic keratosis or senile keratosis is the most frequent premalign epithelial lesion that occurs in the elderly with a history of prolonged and intense exposure to the UV radiation and with an inborn susceptibility. Although the genetics and risk factors are clear nowadays, the histogenesis of this lesion is still under study. The histoenzymologic tests (such as those that demonstrate the ATP-ase and SDH-ase activity) are very useful for early detection of the structural and biochemical changes within the actinic keratosis lesion.

Localization of cellobiose dehydrogenase in cellulose-grown cultures of Phanerochaete chrysosporium.

To elucidate the function of cellobiose dehydrogenase (CDH) in cellulose degradation by Phanerochaete chrysosporium, production and localization of CDH were investigated and compared with those in shaking and aerated static cultures grown on cellulose. Substantial CDH activity was detected in the medium of the shake cultures after 8 days of incubation, while no CDH activity was detected in the medium of static cultures at any point during the incubation period. Light microscopy clearly showed that many cellulose particles were adsorbed on the surface of the hypha in static cultures, whereas no cellulose particles were absorbed to the hypha is shake cultures. The addition of laminarinase to static cultures was very effective in detaching cellulose particles from the hypha surfaces. Using a potentiometric assay performed with an oxidation-reduction potential electrode, some CDH activity could be detected on the hypha/cellulose complexes in static cultures. Thus, CDH is produced also in static cultures, albeit in lower amounts that in shake cultures, but the enzyme is not released into the medium. It seem likely that the beta-1,3-glucan layer plays an important role in CDH localization and cellulose degradation. Immunocytochemical confocal laser scanning microscopy for the static cultures demonstrated that most CDH was adsorbed on the surface of the cellulose, especially around the cracks, which were formed by the action of cellulases during the course of incubation. From these observations, we conclude a direct participation of CDH in the degradation of cellulose in cooperation with cellulases.