Purification, characterization and evaluation of extracellular peroxidase from two Coprinus species for aqueous phenol treatment.

Non-ligninolytic fungal peroxidases produced by Coprinus cinereus UAMH 4103 and Coprinus sp. UAMH 10067 were purified, characterized and evaluated as cost-effective alternatives to horseradish peroxidase for aqueous phenol treatment. Purified Coprinus peroxidases exhibited a molecular weight of 36 kDa on matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Although the catalytic properties of the two Coprinus peroxidases were nearly identical in both crude and purified forms, the stabilities were substantially different. The peroxidase from Coprinus sp. UAMH 10067 was more stable at 50 degrees C and under basic conditions (up to pH 10) than the enzyme from C. cinereus UAMH 4103. The former enzyme also performed better at pH 9 than the latter one in aqueous phenol treatment. The phenol removal efficiency of the Coprinus peroxidase was comparable to those of previously studied plant peroxidases. The broader working pH and higher thermal and alkaline stability of the peroxidase from Coprinus sp. UAMH 10067 may be advantageous for its application to industrial wastewater treatment.

Glycated albumin and risk of death and hospitalizations in diabetic dialysis patients.

BACKGROUND AND OBJECTIVES: Relative to hemoglobin (Hb) A(1c), glycated albumin (GA) more accurately reflects glycemic control in patients with diabetes mellitus and ESRD. We determined the association between GA, HbA(1c), and glucose levels with survival and hospitalizations in diabetic dialysis patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Quarterly GA levels were measured for up to 2.33 years in 444 prevalent patients with diabetes and ESRD. Proportional hazard time-dependent covariate models were computed with adjustment for demographic characteristics, comorbidities, and laboratory variables. Similar analyses were performed for available HbA(1c) and monthly random serum glucose determinations. RESULTS: The participants were 53% male, 54% African American, 43% Caucasian, 90% on hemodialysis, with a mean (SD) age of 62 (12) years and median follow-up duration of 2.25 years. GA and HbA(1c) mean ± SD 21.5% ± 6.0%, median 20.4% and mean ± SD 6.9% ± 6.6%, median 1.6%, respectively. There were 156 deaths during the observation period. In best-fit models, predictors of death included increasing GA, increasing age, presence of peripheral vascular disease, decreasing serum albumin, and decreasing hemoglobin concentrations. HbA(1c) and random serum glucose concentrations were not predictive of survival. Increasing GA levels were associated with hospitalization in the 17 days after measurement, whereas HbA(1c) was not. CONCLUSIONS: In contrast to the HbA(1c) and random serum glucose values, GA accurately predicts the risk of death and hospitalizations in patients with diabetes mellitus and ESRD. The GA assay should be considered by clinicians who care for patients with diabetes on dialysis.

Fungal enzymes for environmental purposes, a molecular biology challenge.

In their capacity to transform xenobiotics and polluting compounds, fungal peroxidases and their use in the environmental field have a recognized and important potential. However, both fundamental and practical issues, such as enzyme stability and availability, have delayed the development of industrial applications. Three main protein engineering challenges have been identified: (1) Enhancement of operational stability, specifically hydrogen peroxide stability in the case of fungal peroxidases. (2) Increase of the enzyme redox potential in order to widen the substrate range. (3) Development of heterologous expression and industrial production. The bottlenecks, advances and strategies that have been proven successful are discussed.

Purification, characterization, and chemical modification of manganese peroxidase from Bjerkandera adusta UAMH 8258.

Ten strains of Bjerkandera adusta from the University of Alberta Microfungus Collection and Herbarium (UAMH) were compared for manganese peroxidase production. The enzyme from B. adusta UAMH 8258 was chosen for further study. After purification the enzyme showed a molecular weight of 43 kDa on 15% SDS-PAGE, 36.6 kDa on matrix-assisted laser desorption ionization-time of flight mass spectrometry, and an isoelectric point of 3.55. The N-terminal amino acid sequence was determined to be VAXPDGVNTATNAAXXALFA, and the amino acid composition showed no tyrosine residues in the enzyme. Manganese peroxidase exhibited both Mn(II)-dependent (optimum pH 5) and Mn(II)-independent activity (optimum pH 3). The purified enzyme was chemically modified with cyanuric chloride-activated methoxypolyethylene glycol to enhance its surface hydrophobicity. The modified and native enzymes showed similar catalytic properties in the oxidation of Mn(II) and other substrates such as 2,6-dimethoxylphenol, veratryl alcohol, guaiacol, and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate). However, the modified enzyme showed greater resistance to denaturation by hydrogen peroxide and stability to organic solvents such as acetonitrile, N,N-dimethylformamide, tetrahydrofuran, methanol, and ethanol. The PEG-modified enzyme also showed greater stability to higher temperatures and lower pH than the native enzyme. Thus, chemical modification of manganese peroxidase from B. adusta increases its potential usefulness for applied studies.

Manganese-lignin peroxidase hybrid from Bjerkandera adusta oxidizes polycyclic aromatic hydrocarbons more actively in the absence of manganese.

We studied polycyclic aromatic hydrocarbon (PAH) oxidation using whole cells and purified manganese-lignin peroxidase (MnLiP) from Bjerkandera adusta UAMH 8258. Although the metabolism of PAHs by B. adusta has been previously demonstrated, less than 5% mineralization of 14C-labelled PAHs occurred in this study over a 40-day period. Oxidation of PAHs was examined by a purified MnLiP hybrid isoenzyme in the presence and absence of manganous ions. The rate of PAH oxidation was decreased by the presence of Mn. The substrates were anthracene and its methyl derivatives, pyrene and benzo[a]pyrene, PAHs with ionization potentials of 7.43 eV or lower. The PAH metabolites of the Mn-independent reaction were identified as the corresponding quinones. The pH optimum of the Mn-independent oxidation was generally about 4, while for the Mn-dependent reaction it was 3. The kinetic constants for the Mn-independent oxidation of 2-methylanthracene at pH 4 were determined, and the values we obtained were a kcat of 145/min, KM,app of 23.8 mmol/L for the aromatic substrate, and KM,app of 0.2 mmol/L for hydrogen peroxide. This is the first report of PAH oxidation by a MnLiP hybrid isoenzyme from white rot fungi.

Optimization of extracellular fungal peroxidase production by 2 Coprinus species.

Optimum culture conditions for the batch production of extracellular peroxidase by Coprinus cinereus UAMH 4103 and Coprinus sp. UAMH 10067 were explored using 2 statistical experimental designs, including 2-level, 7-factor fractional factorial design and 2-factor central composite design. Of the 7 factors examined in the screening study, the concentrations of carbon (glucose) and nitrogen (peptone or casitone) sources showed significant effects on the peroxidase production by Coprinus sp. UAMH 10067. The optimum glucose and peptone concentrations were determined as 2.7% and 0.8% for Coprinus sp. UAMH 10067, and 2.9% and 1.4% for C. cinereus UAMH 4103, respectively. Under the optimized culture condition the maximum peroxidase activity achieved in this study was 34.5 U x mL(-1) for Coprinus sp. UAMH 10067 and 68.0 U x mL(-1) for C. cinereus UAMH 4103, more than 2-fold higher than the results of previous studies.

Cross-linked crystals of chloroperoxidase.

Chloroperoxidase from Caldariomyces fumago was crystallized. The crystals were modified with several cross-linkers, but only glurataldehyde was able to produce catalytically active and insoluble crystals. Unlike other immobilized chloroperoxidase preparations, these catalytic crystals are more thermostable than the unmodified soluble enzyme. The enhanced stability is probably due to the structure conservation in the crystalline matrix. In addition, non-cross-linked chloroperoxidase crystals retained more activity than the soluble enzyme after incubation in an organic solvent with low water content. Although the cross-linked crystals were catalytically active, they showed lower specific activity than the soluble enzyme. This low activity may be due to non-specific reactions between the cross-linker and essential residues for catalysis. Alternative cross-linking strategies are discussed.

Dibenzyl sulfide metabolism by white rot fungi.

Microbial metabolism of organosulfur compounds is of interest in the petroleum industry for in-field viscosity reduction and desulfurization. Here, dibenzyl sulfide (DBS) metabolism in white rot fungi was studied. Trametes trogii UAMH 8156, Trametes hirsuta UAMH 8165, Phanerochaete chrysosporium ATCC 24725, Trametes versicolor IFO 30340 (formerly Coriolus sp.), and Tyromyces palustris IFO 30339 all oxidized DBS to dibenzyl sulfoxide prior to oxidation to dibenzyl sulfone. The cytochrome P-450 inhibitor 1-aminobenzotriazole eliminated dibenzyl sulfoxide oxidation. Laccase activity (0.15 U/ml) was detected in the Trametes cultures, and concentrated culture supernatant and pure laccase catalyzed DBS oxidation to dibenzyl sulfoxide more efficiently in the presence of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) than in its absence. These data suggest that the first oxidation step is catalyzed by extracellular enzymes but that subsequent metabolism is cytochrome P-450 mediated.