Public Health Laboratory Service Board

Microbiological Research Authority

Christopher Philip Price

Peter M Hammond

Anthony Atkinson

Michael D Scawen

John R Ramsey

Stewart R Cambell

Graham Mark Brearley

Helen C Morris

Peter Michael Hammond

A method for producing in culture phenylalanine dehydrogenase at yields of 50+ units of enzyme per litre of culture, one unit measured as the amount required to convert one micro-mole of phenylalanine to phenyl pyrovic acid at 25.degree. C. and pH 10.8, in a growth medium comprising a nitrogen source, a buffer, phenylalanine or phenylpyruvic acid or a salt thereof at 20 mM and optionally a vitamin solution. Using phenylalanine at about 100 mM gives yields of phenylalanine dehydrogenase enzyme of 350+ units per liter of culture.

Phenylalanine dehydrogenase production

A method for the estimation of a salicylate in a sample is characterized by enzymatically converting the salicylate to a catechol by the action of a salicylate mono-oxygenase enzyme on the salicylate in the presence of a reduced pyridine nucleotide, reacting the catechol with a compound selected from compounds of formula I or amine or phenolic compounds of formulae II, III or IV ##STR1##

Assay of salicylates or reduced pyridine nucleotides

A process for the production of an aryl acylamidase enzyme involves culturing bacteria of one of the strains Pseudomonas fluorescens ATCC 39005 (or suitable mutants or variants thereof) or Pseudomonas putida ATCC 39004 (or suitable mutants or variants thereof) in a culture medium in which the bacterial strains produce aryl acylamidase and collecting the enzyme containing material, generally the cell material. Preferably the resulting cells are then disrupted, especially by enzymatic treatment, and the aryl acylamidase is separated from the other unwanted substances, generally the other cell constituents. Preferably the culture medium contains N-acylaniline, especially N-acetyl aniline. The N-acylaniline may form part of a complex or defined salts medium.

Production of aryl acylamidases

A method for the estimation of an anilide in which the anilide is first hydrolyzed enzymatically to an aniline and then the quantity of the aniline produced is estimated spectrophotometrically preferably colorimetrically. The hydrolysis of the anilide may be catalyzed by any enzyme of the type, EC 3.5.1.13, known as aryl acylamidases. Preferably enzymes isolated from the cells of Pseudomonas fluorescens ATCC 39005 or Pseudomonas putida ATCC 39004 are employed. The aniline may be analyzed, for example, by conversion to an indamine, an indophenol or an indoaniline, followed by colorimetric analysis of the colored quinone-type compound produced. This conversion may take place in the presence of an oxidizing agent, such as a copper (II) salt, and/or a base, such as ammonia. A diagnostic kit to allow the routine use of the above method is also provided. In one embodiment of the kit, it comprises,

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Stapleford, United Kingdom

Christopher Philip Price