The Secretary of State for Defence in Her Britannic Majesty's Government

Public Health Laboratory Service, Center for Applied Microbiology &

Michael J Comer

Anthony Atkinson

Christopher J Bruton

Richard J Sharp

A thermostable glycerokinase enzyme useful in the detection and estimation of glycerol derivatives has a half-life in excess of 1 hour at 55.degree. C. at a protein concentration of less than 2 mg/ml and a pH of 7.8 .+-.0.5 in the absence of any substrate. The enzyme is produced by culturing at least one micro-organism which is capable of growth at a temperature of at least 50.degree. C. in a culture medium in which it will produce said enzyme, disrupting the resulting cells of the micro-organism to release the enzyme and separating the enzyme from the cell debris. The medium normally contains at least 0.1% glycerol but certain strains of micro-organism have been found to be capable of producing thermostable glycerokinase enzyme even in the absence of glycerol. The micro-organism is preferably a Bacillus organism, especially of the stearothermophilus species.

Thermostable glycerokinases and process for its production

Glycerol dehydrogenase enzymes having exeptionally good thermal stability are produced by culturing novel strains of Bacillus stearothermophilus. Procedures for deriving and identifying suitable strains are described. The strains are grown in conventional culture media, preferably containing 0.05 to 4.0%, especially 0.1 to 1.0%, by weight of glycerol or a glycerol analogue at 40.degree.-65.degree. C. and pH 5 to 8. The enzyme is isolated by conventional cell disruption and separation techniques, and typically has a molcular weight of 240,000.+-.30,000, composed of four similar sub-units, and a specific activity of greater than 5 Units per mg protein at 30.degree. C. by the modified assay described. They may be stored as aqueous solutions or a freeze dried solids. The enzymes may be used for assay of serum triglycerides by conventional assay methods, but preferably by the nictotinamide adenine dinucleotide spectrophotometric assay at a pH of 7 to 8.8. The pH is preferably controlled by an amine, especially triethanolamine/HCl, buffer.

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Weilheim, Germany

Michael J Comer