Open Accessd ‐Malic Enzyme of Pseudomonas fluorescens
Author(s) -
KNICHEL Wolfgang,
RADLER Ferdinand
Publication year - 1982
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1982.tb06567.x
Subject(s) - pseudomonas fluorescens , malic acid , pseudomonas putida , malic enzyme , chemistry , enterobacter aerogenes , enzyme , biochemistry , enzyme assay , bacteria , dehydrogenase , chromatography , biology , escherichia coli , citric acid , genetics , gene
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 aeruginosa and Klebsiella 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. fluorescens was 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 K m values were for malate K m = 0.3mM and for NAD K m = 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.