d‐Malic Enzyme of Pseudomonas fluorescens

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Abstract
By the enrichment culture technique 14 gram‐negative bacteria and two yeast strains were isolated that used d(+)‐malic acid as sole carbon source. The bacteria were identified as Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosaandKlebsiella aerogenes.In cell‐free extracts of P. fluorescens and P. putida the presence of malate dehydrogenase, d‐Malic enzyme (NAD‐dependent) and l‐malic enzyme (NADP‐dependent)was demonstrated. d‐Malic enzyme from P. fluorescenswas purified. Stabilization of the enzyme by 50 mM ammonium sulphate an 1mM EDTA was essential. Preparation of d‐malic enzyme that gave one band with disc gel electrophoresis showed a specific activity of 4–5 U/mg. d‐Malic enzyme requires divalent cations. The Kmvalues were for malate Km= 0.3mM and for NAD Km= 0.08mM. The pH optimum for the reaction was found to be in the range of pH 8.1 to pH 8.8 d‐Malic enzyme is partially inhibited by oxaloacetic acid, meso‐tartaric acid, d‐lactic acid and ATP. Determined by gel filtration and gradient gel electrophoresis, the molecular weight was approximately 175000.