Production, partial characterization, and potential diagnostic use of salicylate hydroxylase from Pseudomonas putida UUC-1. 1994

I M Banat, and A Marchant, and P Nigam, and S J Gaston, and B A Kelly, and R Marchant
Department of Biological and Biomedical Sciences, University of Ulster, Coleraine, N. Ireland, UK.

An unusual strain of Pseudomonas putida UUC-1 capable of growth at high salicylate concentration (10 gl-1) was investigated with the aim of developing an assay and a biosensor system for determining salicylate in body fluids by utilizing the salicylate hydroxylase enzyme. Medium and growth condition optimization were carried out under chemostat conditions. The highest biomass yield was at 4.0 gl-1 salicylate, 25 degrees C, pH 6.5, and 0.2 h-1 dilution rate. Growth occurred at up to 0.45 h-1 dilution rate, producing 236 Ul-1 enzyme activity and an output of 424 U h-1. The activity and productivity were higher than any reported in the literature for this enzyme. It had a Km value of 2.07 +/- 0.32 microM and an M(r) of approximately 43,000. In addition, its specific activity in the crude extract (0.8-0.9 U mg-1 protein) was similar to the commercially available enzyme. No plasmid DNA was detected in this strain, and no salicylate-negative isolates were obtained when curing with mitomycin C. It is therefore proposed that our strain has a chromosomally located inducible salicylate hydroxylase gene that enables it to grow at high salicylate. This strain also offers a means of cheaply producing large quantities of the enzyme through standard fermentation techniques.

UI MeSH Term Description Entries
D010957 Plasmids Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS. Episomes,Episome,Plasmid
D003470 Culture Media Any liquid or solid preparation made specifically for the growth, storage, or transport of microorganisms or other types of cells. The variety of media that exist allow for the culturing of specific microorganisms and cell types, such as differential media, selective media, test media, and defined media. Solid media consist of liquid media that have been solidified with an agent such as AGAR or GELATIN. Media, Culture
D005285 Fermentation Anaerobic degradation of GLUCOSE or other organic nutrients to gain energy in the form of ATP. End products vary depending on organisms, substrates, and enzymatic pathways. Common fermentation products include ETHANOL and LACTIC ACID. Fermentations
D006899 Mixed Function Oxygenases Widely distributed enzymes that carry out oxidation-reduction reactions in which one atom of the oxygen molecule is incorporated into the organic substrate; the other oxygen atom is reduced and combined with hydrogen ions to form water. They are also known as monooxygenases or hydroxylases. These reactions require two substrates as reductants for each of the two oxygen atoms. There are different classes of monooxygenases depending on the type of hydrogen-providing cosubstrate (COENZYMES) required in the mixed-function oxidation. Hydroxylase,Hydroxylases,Mixed Function Oxidase,Mixed Function Oxygenase,Monooxygenase,Monooxygenases,Mixed Function Oxidases,Function Oxidase, Mixed,Function Oxygenase, Mixed,Oxidase, Mixed Function,Oxidases, Mixed Function,Oxygenase, Mixed Function,Oxygenases, Mixed Function
D001709 Biotechnology Body of knowledge related to the use of organisms, cells or cell-derived constituents for the purpose of developing products which are technically, scientifically and clinically useful. Alteration of biologic function at the molecular level (i.e., GENETIC ENGINEERING) is a central focus; laboratory methods used include TRANSFECTION and CLONING technologies, sequence and structure analysis algorithms, computer databases, and gene and protein structure function analysis and prediction. Biotechnologies
D012459 Salicylates The salts or esters of salicylic acids, or salicylate esters of an organic acid. Some of these have analgesic, antipyretic, and anti-inflammatory activities by inhibiting prostaglandin synthesis. Salicylate,Salicylic Acids,Acids, Salicylic
D015374 Biosensing Techniques Any of a variety of procedures which use biomolecular probes to measure the presence or concentration of biological molecules, biological structures, microorganisms, etc., by translating a biochemical interaction at the probe surface into a quantifiable physical signal. Bioprobes,Biosensors,Electrodes, Enzyme,Biosensing Technics,Bioprobe,Biosensing Technic,Biosensing Technique,Biosensor,Electrode, Enzyme,Enzyme Electrode,Enzyme Electrodes,Technic, Biosensing,Technics, Biosensing,Technique, Biosensing,Techniques, Biosensing
D016958 Pseudomonas putida A species of gram-negative, aerobic bacteria isolated from soil and water as well as clinical specimens. Occasionally it is an opportunistic pathogen.
D020156 Salicylic Acid A compound obtained from the bark of the white willow and wintergreen leaves. It has bacteriostatic, fungicidal, and keratolytic actions. 2-Hydroxybenzoic Acid,o-Hydroxybenzoic Acid,ortho-Hydroxybenzoic Acid,2 Hydroxybenzoic Acid,Acid, 2-Hydroxybenzoic,Acid, Salicylic,Acid, o-Hydroxybenzoic,Acid, ortho-Hydroxybenzoic,o Hydroxybenzoic Acid,ortho Hydroxybenzoic Acid

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