By the enrichment culture technique 14 gram-negative bacteria and two yeast strains were isolated that used u(+)-malic acid as sole carbon source. The bacteria were identified as Pseudomonas putidu, Pseudomonus ,fluerescen.T, Pseudonaonus aeruginosa and K1eb.siella aerogenes. In cell-free extracts of P. fluorescens and P. putidu the presence of malate dehydrogenase, D-malic enzyme (NAD-dependent) and L-malic enzyme (NA DP-dependent) was demonstrated. D-Malic enzyme from P.. f2uort'sct'ns was purified. Stabilization of the enzyme by 50 m M ammonium sulphate an 1 mM 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 -M a k enzyme requires divalent cations. The K, values were for malate K , = 0.3 mM and for NAD K, = 0.08 mM. 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 -k t i c acid and ATP. Determined by gel filtration and gradient gel electrophoresis, the molecular weight was approximately 175000. It was a purpose of the work described in this paper to isolate microorganisms that are able to grow with u-malate as sole carbon source. After an investigation of the metabolism of malic acid, D-malic enzyme could be purified. Because very little information is available on D-malic enzyme, its main characteristics are described. MATERIALS A N D METHODS MicroorganismsThe bacteria and yeasts, isolated by the enrichment technique, were cultured on Standard I nutrient agar or yeast extract/peptone agar. The strains were kept at 4°C and were transferred to fresh medium at six-week intervals. __ -~Ahhrrvintion. TEAE-cellulose. triethylaminoethyl-cellulose. The precultures were grown in 5 ml DL-malic acid medium for one or two days and transferred to 200 ml of the same medium. After incubation for one day this was used to inoculate 2 1 DL-malic acid medium in a 5-1 conical flask. This main culture was incubated on a circular shaker (150 rev./ min) for 36 h at 25 'C. The cells (approx. 6 g wet weight) were spun down, washed twice with 0.05 M phosphate buffer, pH 7.2, and homogenized for 2 min at 4000 rev./min in a homogenizer (Braun, Melsungen). 5 ml buffer and 10 g glass beads (diameter 0.1 1-0.12 mm)/g bacteria (wet weight) were used. Insoluble material was removed from the crude cell-free extracts by centrifugation (40 min at 15000 rev./min). Pur$ication of u-Malic by Method APrec@itation with Protarnine Sulphute. Nucleic acids were removed from about 25 ml cell-free extracts (10 mg protein/ml) with a suspension of protamine sulphate (1 x) of which 1 ml/ 100 mg protein was used After qtirring for 15 min the sediment was spun down and discarded.

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