Characterization of a lactate oxidase from a strain of gram negative bacterium from soil (original) (raw)
Related papers
Screening for bacterial strains producing lactate oxidase
Journal of Fermentation and Bioengineering, 1996
Six gram negative bacterial strains producing lactate oxidase were screened from 50 bacterial strains isolated from soil. The strains were capable of producing lactate oxidase both in shaking and static culture. Lactate oxidase activity was highest in strain KY6 and this strain was chosen as the enzyme source. The crude extract of strain KY6 containing 1.29 g of protein per liter converted m-lactate (90 mM) to pyruvate yielding 61.8 % in 12 h, which corresponds to a space-time yield of 86.5 mol l-l d-t g-t of cell-free protein. Morphological and physiological studies according to Bergey's Manual of Systematic Bacteriology revealed that the bacterial strain KY6 belongs to the Edwardsiella tarda Biogroup 1.
Purification and characterisation of the water forming NADH-oxidase from Lactococcus lactis
International Dairy Journal, 2001
The NADH-oxidase (NOX) from Lactococcus lactis was isolated from extracts of aerobically grown cells. It was puri"ed by (NH ) SO fractionation followed by ion exchange and a$nity chromatographic steps. Spectra of the oxidized enzyme showed peaks with maxima at wavelengths of 274, 381 and 446 nm and FAD was identi"ed as cofactor. The enzyme is apparently a dimer composed of two identical subunits of 53 kDa, each containing one molecule of non-covalently bound FAD. NOX showed optimum activity between pH 6.0 and 9.0 and was found to have a pI of 4.8. The lactococcal NOX uses dioxygen as the natural electron acceptor and forms water during the oxidation of NADH. The natural electron donor was -NADH (K "4.1 M and V "83.2 mol min\ mg\) whereas -NADH, -NADPH or -NADPH were not oxidized by the enzyme. Hydrogen peroxide, sulfhydryl reagents and quinine inactivated the enzyme activity. The oxidase described in this paper appears to possess kinetic characteristics potentially useful to redirect, in a food-grade manner, the carbon #ux of lactococci.
Journal of …, 2003
The complexity of the coupled NADH oxidase-NADH peroxidase enzyme system in lactic acid bacteria makes it difficult to simultaneously determine the individual levels of both these enzymes spectrophotometrically. This study describes an improved assay to accurately determine low concentrations of NADH oxidase from enzyme suspensions containing NADH oxidase and NADH peroxidase. For the standardisation of the assay, pure NADH oxidase and NADH peroxidase were mixed in various proportions and the percentage recovery was estimated by both the currently available assay as well as by the improved assay reported in this study. The recovery of NADH oxidase using the currently available assay ranged from as low as À 200% to as high as + 102% as against 90-102% in the improved assay. The recovery percentage of NADH peroxidase ranged from 91% to 112% in both assays. The slopes of NADH oxidation by cell-free extracts of six lactic acid bacteria were also measured by both assays for the estimation of NADH oxidase and NADH peroxidase levels. The improved assay can further distinguish between NADH-H 2 O oxidase and NADH-H 2 O 2 oxidase and was successfully applied to identify the type of NADH oxidase in the lactic acid bacteria tested.
L‐Aspartate Oxidase from Escherichia coli
European Journal of Biochemistry
This paper reports the biochemical characterization of the flavoprotein L-aspartate oxidase from Escherichia coli. Modification of a previously published procedure allowed overexpression of the holoenzyme in an unproteolysed form. L-Aspartate oxidase is a monomer of 60 kDa containing 1 mol of noncovalently bound FAD/mol protein. A polarographic and two spectrophotometric coupled assays have been set up to monitor the enzymatic activity continuously. L-Aspartate oxidase was subjected to product inhibition since iminoaspartate, which results from the oxidation of L-aspartate, binds to the enzyme with a dissociation constant ( K J equal to 1.4 pM. The enzyme binds FAD by a simple second-order process with K, 0.67 pM. Site-directed mutagenesis of the residues E43, G44, S45, F47 and Y48 located in the putative binding site of the isoallossazinic portion of FAD reduces the affinity for the coenzyme.
Archives of Microbiology, 1994
A gram-positive, mesophilic bacterium which assimilated L-phenylalanine but which failed to utilise L-tyrosine was isolated from soil. The isolate, identified as a strain of Bacillus carotarum, converted L-phenylalanine to phenylpyruvate with the initial step catalysed by an inducible, intracellular enzyme which possessed L-phenylalanine oxidase activity. Phenylalanine oxidase has not been previously reported in Gram-positive bacteria, although there are a few examples of non-specific L-amino acid oxidases with activity towards L-phenylalanine. The isolate grew abundantly on complex media but failed to synthesise significant amounts of the enzyme in the absence of Lphenylalanine. The highest enzyme levels were achieved in a chemically defined minimal salts medium containing the amino acid at 10 g/1 as the primary carbon and energy source.
Current Science
Lactobacillus plantarum KSK-II was the most potent oxalate decomposer among various isolated lactic acid bacteria. There was no detectable extracellular or even intracellular oxalate decarboxylase (OxDcase) productivity in the absence of oxalate. The highest enzyme productivity was obtained at 72 h in a medium containing 0.1 M oxalate, 0.1% (w/v) D-glucose, 0.05% (w/v) soybean flour and 0.1% (w/v) of the prebiotics fructo-oligosaccharides and arabinogalactan. Enzyme purification increased its specific activity to 19.6-fold with 14.8% recovery and molecular weight of 63 kDa. The optimal reaction temperature, pH and pI values for OxDcase were 35C, 5.0 and 3.5 respectively, and it was stable till 70C and at pH 4.0-7.0 for 1 h. The apparent K m value of the enzyme was 12.70 mM, the turnover number (K cat) was 64.10 s-1 and the catalytic efficiency (K cat /K m) was 5.05 mM-1 s-1. Treatment of oxaluric rats with L. plantarum KSK-II and a prebiotic mixture significantly decreased oxalate levels inside their bodies suggesting a successful synbiobtic system in the prevention of oxalate stones. KSK-II OxDcase may also be clinically significant from the perspective of its thermo-tolerance and activation by triton X-100 and the reducing agents (sodium-L-ascorbate, potassium ferrocyanide and o-PDA). The non-inhibitory activity of chloride and the oxalate specificity are also significant for clinical applications of the enzyme in measuring of oxalate levels in body fluids.
Purification and characterization of the oxidase from the marine bacterium Pseudomonas nautica 617
European Journal of Biochemistry, 1991
The aerobic respiratory system of the hydrocarbonoclastic marine bacterium Pseudomonas nautica 61 7 ends with a single terminal oxidase. It is a heme-containing membranous protein which has been demonstrated only to reduce molecular oxygen to hydrogen peroxide [Denis, M., Arnaud S. & Malatesta, F. (1989) FEBS Lett. 247, 475-4791. The purification of this oxidase was achieved in a single step through by DEAE-Trisacryl chromatography. SDSjPAGE showed the presence of four subunits. The pZ was found to be 4.45 and a M , of 130000 was determined by gel filtration.
Oxygen metabolism of catalase-negative and catalase-positive strains of Lactobacillus plantarum
Journal of bacteriology, 1975
Two catalase-negative strains of Lactobacillus plantarum and a strain producing the atypical, nonheme catalase were studied to determine if the ability to produce the atypical catalase conferred any growth advantage upon the producing strain. Both catalase-negative strains grew more rapidly than the catalase-positive strain under aerobic or anaerobic conditions in a glucose-containing, complex medium. Upon exhaustion of glucose from the medium, all three strains continued growth under aerobic but not under anaerobic conditions. The continued aerobic growth was accompanied by production of acetic acid in addition to the lactic acid produced during growth on glucose. Oxygen was taken up by exponential phase-cell suspensions grown on glucose when glucose or glycerol were used as substrates. Cells harvested from glucose-exhausted medium oxidized glucose, glycerol, and pyruvate. Oxygen utilization by a catalase-negative strain increased as did the specific activity of reduced nicotinamid...
European Journal of Biochemistry, 1996
This paper reports the biochemical characterization of the flavoprotein L-aspartate oxidase from Escherichia coli. Modification of a previously published procedure allowed overexpression of the holoenzyme in an unproteolysed form. L-Aspartate oxidase is a monomer of 60 kDa containing 1 mol of noncovalently bound FAD/mol protein. A polarographic and two spectrophotometric coupled assays have been set up to monitor the enzymatic activity continuously. L-Aspartate oxidase was subjected to product inhibition since iminoaspartate, which results from the oxidation of L-aspartate, binds to the enzyme with a dissociation constant ( K J equal to 1.4 pM. The enzyme binds FAD by a simple second-order process with K, 0.67 pM. Site-directed mutagenesis of the residues E43, G44, S45, F47 and Y48 located in the putative binding site of the isoallossazinic portion of FAD reduces the affinity for the coenzyme.