1,721,001 research outputs found
Immobilized hydrolytic enzymes exhibit antibiofilm activity against escherichia coli at sub-lethal concentrations
Full text linkThe effects of two commercially available immobilized enzymes (namely the glycosidase pectinase and the protease subtilisin A) at sub-lethal concentrations were investigated in terms of their influence on biofilm genesis, on the composition of the biofilm matrix, and their antibiotic synergy against Escherichia coli biofilm, used as a model system of bacterial biofilms. The best antibiofilm performance of solid-supported hydrolases was obtained at the surface concentration of 0.022 and 0.095 U/cm2 with a reduction of 1.2 and 2.3 log CFU/biofilm for pectinase and subtilisin, respectively. At these enzyme surface concentrations, the biocatalysts affected the structural composition of the biofilm matrix, impacting biofilm thickness. Finally, the immobilized hydrolases enhanced biofilm sensitivity to a clinically relevant concentration of the antibiotic ampicillin. At the final antibiotic concentration of 0.1 mg/ml, a reduction of 2 and 3.5 log10 units in presence of 0.022 Upectinase/cm2 and 0.095 Usubtilisin/cm2 was obtained, respectively, in comparison the antibiotic alone. Immobilized pectinase and subtilisin at sub-lethal concentrations demonstrated a great potential for antibiofilm applications
Evidence for an Essential Lysyl Residue in Phospholipase D from Streptomyces sp. by Modification with Diethyl Pyrocarbonate and Pyridoxal 5-Phosphate
No full textDiethyl pyrocarbonate inactivated phospholipase D from Streptomyces PMF with second-order rate constants of 0.7 M-1 s-1 at pH 6.1 or 222 M-1 s-1 at pH 8.3 and 25 °C, and modified 5 His residues per enzyme molecule. The His residues, however, were not essential for activity because: (a) the second-order rate constants for reaction of diethyl pyrocarbonate with the His residues of the enzyme, which were 1.4 M-1 s-1 at pH 6.1 or 7.2 M-1 s-1 at pH 8.3 and 25 °C, differed, both at low and high pH values, from the inactivation rates, and (b) the reversal of His modification by hydroxylamine was not accompanied by recovery of activity. As demonstrated by dinitrophenylation experiments carried out on the treated enzyme, diethyl pyrocarbonate also modified up to 20 Lys residues per enzyme molecule. Other amino acid residues and the conformation and hydrodynamic volume of the enzyme were not modified. The involvement of a Lys residue in enzyme activity was confirmed through experiments with pyridoxal 5-phosphate which inactivated phospholipase D, after NaBH4 reduction, with a second-order rate constant of 3.5 M-1 s-1 at pH 8.5 and 15 °C. The inactivation took place with concomitant modification of 4 Lys residues, only one of which was found to be essential using the kinetic method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535−1538). Dicaproyl phosphatidylcholine markedly protected the enzyme against inactivation by DEP or PLP, and this strongly suggests that the essential Lys residue is located in or near the substrate binding site
Nanostructured Gold For Immobilization Of Thioaniline Functionalized Glucose Oxidase And Au Nanoparticles By Electropolymerization
No full textSensors are devices composed of an active sensing material with a
signal transducer. Electrochemical sensors have more advantages
over the others because the electrodes can sense the analyte that is
present in the host without doing any damage to the host system.
The immobilization of a protein on a metallic transducer can be a
crucial step in the development of bionanodevices that find
applications in the field of biomaterials, biocoatings, biofuel cells,
etc. In the present study, we show the immobilization by
electropolymerization of thioaniline functionalized glucose oxidase
and Au nanoparticles on nanostructured gold films prepared by
electrodeposition and compared to sputtered gold. This enzyme is
employed in the preparation of biosensors of biomedical interest.
The goodness of the Au film for enzyme binding is evaluated by
comparison of the enzyme activity and of the interval of linearity
for the determination of glucose concentration
Immobilized enzyme on a plastic surface : how to weaken a biofilm
No full textBiofilms represent a sessile community where cells are irreversibly attached to a substrate, to an interface or to other cells. In comparison with organisms in the planktonic form, cells that aere included into biofilms present different phenotype, gene transcription and growth rate. These features are responsible of the great resistance and aggression of biofilm. Cells into biofilm are immersed into an extracellular polymeric matrix (EPS) which is self-produced and form of 97% of water and 3% of polysaccharides, proteins, nucleic acid, humic substances, metabolites and cellular residues. EPS plays an important role in the formation of biofilm because it alloes the adhesion, aggregation of bacterial cells, biofilm’s cohesion, water retention, enzyme activity and cellular communication. With the intent of developing methods for the preparation of antifouling surfaces, we have been studying the role of protease (alpha-chymotrypsin) as biochemical agent for preventing the growth of biofilm on polyethylene. This protease cleaves peptide amide bonds where the carboxyl side of the amide bond is a tyrosine, tryptophan or phenylalanine. In our preliminary studies alpha-chymotrysin has been immobilized on polyethylee coupons preventively coated with polyethyleneimine (PEI) by crossing-linking with glutaraldehyde of the enzyme to PEI. Methoxypoly(ethylene glycol) (5 KDa) was also added during the cross-linking reaction as enzyme stabilizing additive.
The effect of immobilized alpha-chymotrypsin on biofilm growth was evaluated comparing the growth of a biofilm of Escherichia coli cells in a CDC reactor for 48 H on polyethylene coupons prepared with or without (control) enzyme. The results show that the growth curve of E. coli is similar in term of biomass between the control and the enzyme-coated coupon, but there is a visible difference in terms of resistance. This preliminary result indicates that the use of enzymes as agents to weak a biofilm can be an approach that deserves to be investigated for developing new materials with antifouling properties
Comparative study of the properties of wild type and recombinant cyclohexanone monooxygenase, an enzyme of synthetic interest
No full textCyclohexanone monooxygenase (CHMO), a flavoenzyme of synthetic interest (it catalyses the NADPH-dependent enantioselective oxidation of ketones and of several heteroatoms such as nitrogen, sulfur, phosphorous and selenium present in organic compounds) previously overexpressed in E. coli (TOP10 pQR239), was purified to homogeneity, as demonstrated by SDS-PAGE and MALDI/TOF analysis, and characterised. The recombinant and the wild type (Acinetobacter) enzymes had identical molecular mass, Km values, pH-activity profile and circular dichroism spectra, but slightly differed for pH- and thermo-stability. The latter findings might be due to a different pattern of proteases contaminating the monooxygenases isolated from the two microorganims
Fatty acid composition and fat content in milk from cows grazing in the Alpine region
Full text linkThe variation in the fat profile of pooled milk from cows grazing in pastures in June and July at 400–700 m and at 1400–2250 m of altitude was evaluated by gas chromatography and compared with that from cows stalled in barns and fed with a diet without fresh grass. The ratios unsaturated/saturated fatty acid in milk samples were 1.33, 1.71 and 1.69 in June and 1.21, 1.69 and 1.84 in July for cows fed with prepared diet, grazing at 400–700 m or grazing at 1400–2250 m, respectively. Analogously, the ratios (oleic plus stearic acid)/palmitic acid were, for the same group of cows, 0.59, 0.72 and 0.78 in June and 0.56, 0.73 and 0.81 in July. In milk from pastured cows, the percentage of oleic, vaccenic, rumenic and α-linolenic acids increased as a function of the altitude; instead, that of linoleic acid and of cis-12-octadecenoic acid decreased. The yield of fat was always highest in milk from 1400 to 2250 m of altitude (up to 3.6 g per 100 mL). For the milk collected in July at 1400–2250 m of altitude, it was observed a decrease in the percentage of decanoic (capric) and dodecanoic acids and an increase in pentadecanoic, stearic, arachidic and docosanoic (behenic) acids. Possible reasons for the differences observed in the milk samples were discussed
Asymmetric oxidation of sulfides by cyclohexanone Monooxygenase
No full textCyclohexanonemonooxygenase catalyzes the asymmetric oxidazion of numerous alkyl aryl sulfides with the alkyl chain functionalyzed with Cl, CN, vinyl or hydroxy groups. Sulfoxides with enantiomeric excesses up to 99% were obtained. The structure of the sulfide markedly influenced enzyme enantioselectivity
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