scholarly journals Lignocellulose degradation by Phanerochaete chrysosporium: purification and characterization of the main α-galactosidase

1999 ◽  
Vol 339 (1) ◽  
pp. 43-53 ◽  
Author(s):  
Harry BRUMER ◽  
Paul F. G. SIMS ◽  
Michael L. SINNOTT
Keyword(s):  
Phanerochaete Chrysosporium ◽  
Substrate Inhibition ◽  
Enzyme Reaction ◽  
Lignocellulose Degradation ◽  
K Value ◽  
Softwood Pulp ◽  
Leaving Groups ◽  
Kinetics Of ◽  
Optimal Ph

The main α-galactosidase was purified to homogeneity, in 30% yield, from a solid culture of Phanerochaete chrysosporium on 1 part wheat bran/2 parts thermomechanical softwood pulp. It is a glycosylated tetramer of 50 kDa peptide chains, which gives the N-terminal sequence ADNGLAITPQMG(?W)NT(?W)NHFG(?W)DIS(?W)DTI. It is remarkably stable, with crude extracts losing no activity over 3 h at 80 °C, and the purified enzyme retaining its activity over several months at 4 °C. The kinetics of hydrolysis at 25 °C of various substrates by this retaining enzyme were measured, absolute parameters being obtained by active-site titration with 2´,4´,6´-trinitrophenyl 2-deoxy-2,2-difluoro-α-d-galactopyranoside. The variation of kcat/Km for 1-naphthyl-α-d-galactopyranoside with pH is bell-shaped, with pK1 = 1.91 and pK2 = 5.54. The αD(V/K) value for p-nitrophenyl-α-d-glucopyranoside is 1.031±0.007 at the optimal pH of 3.75 and 1.114±0.006 at pH 7.00, indicating masking of the intrinsic effect at optimal pH. There is no α-2H effect on binding galactose [αD(Ki) = 0.994±0.013]. The enzyme hydrolyses p-nitrophenyl β-l-arabinopyranoside ∼ 510 times slower than the galactoside, but has no detectable activity on the α-d-glucopyranoside or α-d-mannopyranoside. Hydrolysis of α-galactosides with poor leaving groups is Michaelian, but that of substrates with good leaving groups exhibits pronounced apparent substrate inhibition, with Kis values similar to Km values. We attribute this to the binding of the second substrate molecule to a β-galactopyranosyl-enzyme intermediate, forming an E•βGal•αGalX complex which turns over slowly, if at all. 1-Fluoro-α-d-galactopyranosyl fluoride, unlike α-d-galactopyranosyl fluoride, is a Michaelian substrate, indicating that the effect of 1-fluorine substitution is greater on the first than on the second step of the enzyme reaction.

Characterization of naphthalene degradation by Streptomyces sp. QWE-5 isolated from active sludge

2014 ◽  
Vol 70 (6) ◽  
pp. 1129-1134 ◽  
Author(s):  
Peng Xu ◽  
Wencheng Ma ◽  
Hongjun Han ◽  
Baolin Hou ◽  
Shengyong Jia
Keyword(s):  
Degradation Rate ◽  
Substrate Inhibition ◽  
Degradation Pathway ◽  
Active Sludge ◽  
Streptomyces Sp ◽  
Metabolic Intermediates ◽  
Naphthalene Degradation ◽  
Specific Degradation ◽  
Kinetics Of

A bacterial strain, QWE-5, which utilized naphthalene as its sole carbon and energy source, was isolated and identified as Streptomyces sp. It was a Gram-positive, spore-forming bacterium with a flagellum, with whole, smooth, convex and wet colonies. The optimal temperature and pH for QWE-5 were 35 °C and 7.0, respectively. The QWE-5 strain was capable of completely degrading naphthalene at a concentration as high as 100 mg/L. At initial naphthalene concentrations of 10, 20, 50, 80 and 100 mg/L, complete degradation was achieved within 32, 56, 96, 120 and 144 h, respectively. Kinetics of naphthalene degradation was described using the Andrews equation. The kinetic parameters were as follows: qmax (maximum specific degradation rate) = 1.56 h−1, Ks (half-rate constant) = 60.34 mg/L, and KI (substrate-inhibition constant) = 81.76 mg/L. Metabolic intermediates were identified by gas chromatography and mass spectrometry, allowing a new degradation pathway for naphthalene to be proposed. In this pathway, monooxygenation of naphthalene yielded naphthalen-1-ol. Further degradation by Streptomyces sp. QWE-5 produced acetophenone, followed by adipic acid, which was produced as a combination of decarboxylation and hydroxylation processes.

scholarly journals Biodegradation of phenanthrene by Pseudomonas sp. strain PPD: purification and characterization of 1-hydroxy-2-naphthoic acid dioxygenase

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 3083-3091 ◽  
Author(s):  
Jaigeeth Deveryshetty ◽  
Prashant S. Phale
Keyword(s):  
Molecular Mass ◽  
Substrate Inhibition ◽  
Enzyme Reaction ◽  
Sole Source ◽  
Purification And Characterization ◽  
Spectral Changes ◽  
Native Molecular Mass ◽  
Naphthoic Acid ◽  
Key Enzyme

Pseudomonas sp. strain PPD can metabolize phenanthrene as the sole source of carbon and energy via the ‘phthalic acid’ route. The key enzyme, 1-hydroxy-2-naphthoic acid dioxygenase (1-HNDO, EC 1.13.11.38), was purified to homogeneity using a 3-hydroxy-2-naphthoic acid (3-H2NA)-affinity matrix. The enzyme was a homotetramer with a native molecular mass of 160 kDa and subunit molecular mass of ∼39 kDa. It required Fe(II) as the cofactor and was specific for 1-hydroxy-2-naphthoic acid (1-H2NA), with K m 13.5 μM and V max 114 μmol min−1 mg−1. 1-HNDO failed to show activity with gentisic acid, salicylic acid and other hydroxynaphthoic acids tested. Interestingly, the enzyme showed substrate inhibition with a K i of 116 μM. 1-HNDO was found to be competitively inhibited by 3-H2NA with a K i of 24 μM. Based on the pH-dependent spectral changes, the enzyme reaction product was identified as 2-carboxybenzalpyruvic acid. Under anaerobic conditions, the enzyme failed to convert 1-H2NA to 2-carboxybenzalpyruvic acid. Stoichiometric studies showed the incorporation of 1 mol O2 into the substrate to yield 1 mol product. These results suggest that 1-HNDO from Pseudomonas sp. strain PPD is an extradiol-type ring-cleaving dioxygenase.

scholarly journals Kinetics of inter-domain electron transfer in flavocytochrome cellobiose dehydrogenase from the white-rot fungus Phanerochaete chrysosporium

2002 ◽  
Vol 365 (2) ◽  
pp. 521-526 ◽  
Author(s):  
Kiyohiko IGARASHI ◽  
Ikuo MOMOHARA ◽  
Takeshi NISHINO ◽  
Masahiro SAMEJIMA
Keyword(s):  
Electron Transfer ◽  
Phanerochaete Chrysosporium ◽  
Substrate Concentration ◽  
Substrate Inhibition ◽  
Cellobiose Dehydrogenase ◽  
White Rot ◽  
White Rot Fungus ◽  
Isosbestic Point ◽  
Second Phase ◽  
Kinetics Of

The pre-steady-state kinetics of inter-domain electron transfer in the extracellular flavocytochrome cellobiose dehydrogenase from Phanerochaete chrysosporium was studied using various values of pH and substrate concentration. Monitoring at the isosbestic point of each prosthetic group indicated that the reductive half-reactions of flavin and haem were biphasic and monophasic respectively. When the observed rates of the flavin and haem reactions were plotted against substrate concentration, the behaviour of the second phase of the flavin reduction was almost identical with that of haem reduction at all substrate concentrations and pH values tested, suggesting that the formation of flavin semiquinone and haem reduction involve the same electron transfer reaction. Although flavin reduction by cellobiose was observed in the range of pH3.0–7.0, the velocity of the next electron transfer step decreased with increase of pH and was almost zero above pH6.0. The second phase of flavin reduction and the haem reduction were inhibited similarly by high concentrations of the substrate, whereas the first phase of flavin reduction showed a hyperbolic relation to the cellobiose concentration. Increase in pH enhanced the substrate inhibition of haem reduction but not the initial flavin reduction. Moreover, the dissociation constant Kd of flavin reduction and the substrate inhibition constant Ki of haem reduction decreased similarly with an increase of pH. From these results, it is evident that binding of cellobiose to the active site inhibits electron transfer from flavin to haem.

scholarly journals Isolation and Purification of Pyranose 2-Oxidase from Phanerochaete chrysosporium and Characterization of Gene Structure and Regulation

2004 ◽  
Vol 70 (10) ◽  
pp. 5794-5800 ◽  
Author(s):  
Theodorus H. de Koker ◽  
Michael D. Mozuch ◽  
Daniel Cullen ◽  
Jill Gaskell ◽  
Philip J. Kersten
Keyword(s):  
Phanerochaete Chrysosporium ◽  
Solid Substrate ◽  
Culture Conditions ◽  
Lignocellulose Degradation ◽  
Supporting Evidence ◽  
Isolation And Purification ◽  
Lignin Peroxidases ◽  
Liquid Chromatography Tandem Mass ◽  
Glyoxal Oxidase

ABSTRACT Pyranose 2-oxidase (POX) was recovered from Phanerochaete chrysosporium BKM-F-1767 solid substrate culture using mild extraction conditions and was purified. 13C-nuclear magnetic resonance confirmed production of d-arabino-hexos-2-ulose (glucosone) from d-glucose with the oxidase. Peptide fingerprints generated by liquid chromatography-tandem mass spectrometry of tryptic digests and analysis of the corresponding cDNA revealed a structurally unusual sequence for the P. chrysosporium POX. Relatively high levels of pox transcript were detected under carbon-starved culture conditions but not under nutrient sufficiency. This regulation pattern is similar to that observed for lignin peroxidases, manganese peroxidases, and glyoxal oxidase of P. chrysosporium, supporting evidence that POX has a role in lignocellulose degradation.

Irradiation behavior of pyrolytic silicon carbide

1983 ◽  
Vol 41 ◽  
pp. 72-73
Author(s):  
R. J. Lauf
Keyword(s):  
Silicon Carbide ◽  
Fission Products ◽  
Thermodynamics And Kinetics ◽  
Neutron Fluences ◽  
Fuel Particles ◽  
Sic Coatings ◽  
Irradiation Behavior ◽  
Kinetics Of ◽  
Htgr Fuel

Fuel particles for the High-Temperature Gas-Cooled Reactor (HTGR) contain a layer of pyrolytic silicon carbide to act as a miniature pressure vessel and primary fission product barrier. Optimization of the SiC with respect to fuel performance involves four areas of study: (a) characterization of as-deposited SiC coatings; (b) thermodynamics and kinetics of chemical reactions between SiC and fission products; (c) irradiation behavior of SiC in the absence of fission products; and (d) combined effects of irradiation and fission products. This paper reports the behavior of SiC deposited on inert microspheres and irradiated to fast neutron fluences typical of HTGR fuel at end-of-life.

Elaboration and Characterization of the Properties of Refractory Cr Base Alloys

2010 ◽  
Vol 72 ◽  
pp. 46-52 ◽  
Author(s):  
Laurent Royer ◽  
Stéphane Mathieu ◽  
Christophe Liebaut ◽  
Pierre Steinmetz
Keyword(s):  
Mechanical Properties ◽  
Melting Point ◽  
Molten Glass ◽  
Energy Production ◽  
Creep Properties ◽  
Refractory Alloys ◽  
Industrial Use ◽  
Kinetics Of ◽  
Selection Of

For energy production and also for the glass industry, finding new refractory alloys which could permit to increase the process temperatures to 1200°C or more is a permanent challenge. Chromium base alloys can be good candidates, considering the melting point of Cr itself, and also its low corrosion rate in molten glass. Two families of alloys have been studied for this purpose, Cr-Mo-W and Cr-Ta-X alloys (X= Mo, Si..). A finer selection of compositions has been done, to optimize their chemical and mechanical properties. Kinetics of HT oxidation by air, of corrosion by molten glass and also creep properties of several alloys have been measured up to 1250°C. The results obtained with the best alloys (Cr-Ta base) give positive indications as regards the possibility of their industrial use.

scholarly journals New Trimethoprim-Like Molecules: Bacteriological Evaluation and Insights into Their Action

Antibiotics ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 709
Author(s):  
Marta Jorba ◽  
Marina Pedrola ◽  
Ouldouz Ghashghaei ◽  
Rocío Herráez ◽  
Lluis Campos-Vicens ◽  
...  
Keyword(s):  
Inhibitory Concentration ◽  
Efflux Pump ◽  
Synergistic Effects ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Detailed Characterization ◽  
Low Concentrations ◽  
Pump Activity ◽  
Bacteriological Evaluation ◽  
Kinetics Of

This work reports a detailed characterization of the antimicrobial profile of two trimethoprim-like molecules (compounds 1a and 1b) identified in previous studies. Both molecules displayed remarkable antimicrobial activity, particularly when combined with sulfamethoxazole. In disk diffusion assays on Petri dishes, compounds 1a and 1b showed synergistic effects with colistin. Specifically, in combinations with low concentrations of colistin, very large increases in the activities of compounds 1a and 1b were determined, as demonstrated by alterations in the kinetics of bacterial growth despite only slight changes in the fractional inhibitory concentration index. The effect of colistin may be to increase the rate of antibiotic entry while reducing efflux pump activity. Compounds 1a and 1b were susceptible to extrusion by efflux pumps, whereas the inhibitor phenylalanine arginyl β-naphthylamide (PAβN) exerted effects similar to those of colistin. The interactions between the target enzyme (dihydrofolate reductase), the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH), and the studied molecules were explored using enzymology tools and computational chemistry. A model based on docking results is reported.

scholarly journals Simultaneous Kinetics of Selenite Oxidation and Sorption on δ-MnO2 in Stirred-Flow Reactors

2021 ◽  
Vol 18 (6) ◽  
pp. 2902
Author(s):  
Zheyong Li ◽  
Yajun Yuan ◽  
Lin Ma ◽  
Yihui Zhang ◽  
Hongwei Jiang ◽  
...  
Keyword(s):  
Rate Constant ◽  
Photoelectron Spectroscopy ◽  
Oxide Surfaces ◽  
Kinetic Rate Constant ◽  
Flow Reactors ◽  
Mn Oxide ◽  
Remediation Strategies ◽  
Process Of Se ◽  
Kinetics Of

Selenium (Se) is an essential and crucial micronutrient for humans and animals, but excessive Se brings negativity and toxicity. The adsorption and oxidation of Se(IV) on Mn-oxide surfaces are important processes for understanding the geochemical fate of Se and developing engineered remediation strategies. In this study, the characterization of simultaneous adsorption, oxidation, and desorption of Se(IV) on δ-MnO2 mineral was carried out using stirred-flow reactors. About 9.5% to 25.3% of Se(IV) was oxidized to Se(VI) in the stirred-flow system in a continuous and slow process, with the kinetic rate constant k of 0.032 h−1, which was significantly higher than the apparent rate constant of 0.0014 h−1 obtained by the quasi-level kinetic fit of the batch method. The oxidation reaction was driven by proton concentration, and its rate also depended on the Se(IV) influent concentration, flow rate, and δ-MnO2 dosage. During the reaction of Se(IV) and δ-MnO2, Mn(II) was produced and adsorbed strongly on Mn oxide surfaces, which was evidenced by the total reflectance Fourier transform infrared (ATR-FTIR) results. The X-ray photoelectron spectroscopy (XPS) data indicated that the reaction of Se(VI) on δ-MnO2 produced Mn(III) as the main product. These results contribute to a deeper understanding of the interface chemical process of Se(IV) with δ-MnO2 in the environment.

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