PRODUCTION, PARTIAL CHARACTERIZATION, AND POTENTIAL DIAGNOSTIC USE OF SALICYLATE HYDROXYLASE FROM PSEUDOMONAS-PUTIDA UUC-1

Ibrahim Banat, A Marchant, Poonam Singh - Nee Nigam, SJS Gaston, BA Kelly, R Marchant

Research output: Contribution to journalArticle

Abstract

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 Uh(-1) The activity and productivity were higher than any reported in the literature for this enzyme. It had a K-m value of 2.07 +/- 0.32 mu M 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.
LanguageEnglish
Pages665-670
JournalEnzyme and Microbial Technology
Volume16
Issue number8
Publication statusPublished - Aug 1994

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salicylate 1-monooxygenase
Pseudomonas putida
Salicylates
Enzymes
Growth
Mitomycin
Biosensing Techniques
Body Fluids
Complex Mixtures
Biomass
Fermentation
Plasmids
DNA

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title = "PRODUCTION, PARTIAL CHARACTERIZATION, AND POTENTIAL DIAGNOSTIC USE OF SALICYLATE HYDROXYLASE FROM PSEUDOMONAS-PUTIDA UUC-1",
abstract = "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 Uh(-1) The activity and productivity were higher than any reported in the literature for this enzyme. It had a K-m value of 2.07 +/- 0.32 mu M 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.",
author = "Ibrahim Banat and A Marchant and {Singh - Nee Nigam}, Poonam and SJS Gaston and BA Kelly and R Marchant",
year = "1994",
month = "8",
language = "English",
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pages = "665--670",
journal = "Enzyme and Microbial Technology",
issn = "0141-0229",
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number = "8",

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T1 - PRODUCTION, PARTIAL CHARACTERIZATION, AND POTENTIAL DIAGNOSTIC USE OF SALICYLATE HYDROXYLASE FROM PSEUDOMONAS-PUTIDA UUC-1

AU - Banat, Ibrahim

AU - Marchant, A

AU - Singh - Nee Nigam, Poonam

AU - Gaston, SJS

AU - Kelly, BA

AU - Marchant, R

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N2 - 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 Uh(-1) The activity and productivity were higher than any reported in the literature for this enzyme. It had a K-m value of 2.07 +/- 0.32 mu M 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.

AB - 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 Uh(-1) The activity and productivity were higher than any reported in the literature for this enzyme. It had a K-m value of 2.07 +/- 0.32 mu M 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.

M3 - Article

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JF - Enzyme and Microbial Technology

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