ABSTRACT
Twelve strains of Pseudomonas fluorescens and P. putida were grown in a synthetic medium that contained L-lysine as the only source of carbon and nitrogen, and screened for L-lysine-2-monooxygenase production. Best production was by P. putida BKM B-1458 at 30 IU/g wet wt biomass when grown in a shake-flask but 25 IU/g in a 250-l fermenter.
ABSTRACT
A biosensor to quantify L-proline within 10(-5)-10(-3) mole/L concentration is described. Immobilized Pseudomonas sp. cells grown in a medium containing L-proline as the only source of carbon and nitrogen were used to create the biosensor. The cells oxidized L-proline specifically consuming O2 and did not react with other amino acids and sugars. The change in oxygen concentration was detected with a Clark oxygen membrane electrode. The cells were immobilized by entrapment in polyvinyl alcohol (PVA) cryogel. The resultant biocatalyst had a high mechanical strength and retained its L-proline-oxidizing ability for at least two months.
Subject(s)
Biosensing Techniques , Proline/analysis , Pseudomonas/chemistry , Adenosine Triphosphate/analysis , Bacteriological Techniques/instrumentation , Culture Media , Oxygen Consumption , Pseudomonas/growth & developmentABSTRACT
Isolation and purification of L-lysine-2- monooxygenase from the bacterium Pseudomonas species was carried out. The purification procedure included ammonium sulfate fractionation, acid treatment, gel filtration through Sephadex G-200 and ion-exchange chromatography on DEAE-Sephadex A-50. Such treatment resulted in more than 220-fold purification and 22% yield; the specific activity of the enzyme is 14.6 U/mg. The enzyme spectrum is typical for flavoproteins, with peaks at 275, 386 and 462 nm. At 460 nm excitation, the enzyme fluorescence has an emission maximum at 530 nm, whereas at 360 nm extication--at about 520 nm. The molecular mass of L-lysine-2-monooxygenase as determined by SDS/PAAG electrophoresis is about 268 kD. The KM values for oxygen and lysine are equal to 6.5.10(-4) M and 2.3.10(-4) M, respectively. The curve for the dependence of the reaction rate on lysine concentration is sigmoidal. It was assumed that the electrophoretic behaviour of the enzyme confirms the hypothesis on the nature of allosteric regulation of the enzyme activity by alterations in the regulatory site charge.
