Knichel W, Radler F
Eur J Biochem. 1982 Apr;123(3):547-52. doi: 10.1111/j.1432-1033.1982.tb06567.x.
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 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-Malic enzyme requires divalent cations. The Km values were for malate Km = 0.3 mM and for NAD Km = 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-lactic acid and ATP. Determined by gel filtration and gradient gel electrophoresis, the molecular weight was approximately 175 000.
通过富集培养技术，分离出了14株革兰氏阴性菌和2株酵母菌株，它们以D(+)-苹果酸作为唯一碳源。这些细菌被鉴定为恶臭假单胞菌、荧光假单胞菌、铜绿假单胞菌和产气克雷伯菌。在荧光假单胞菌和恶臭假单胞菌的无细胞提取物中，证实了苹果酸脱氢酶、D-苹果酸酶（依赖NAD）和L-苹果酸酶（依赖NADP）的存在。荧光假单胞菌的D-苹果酸酶被纯化。用50 mM硫酸铵和1 mM EDTA对该酶进行稳定化处理至关重要。制备的D-苹果酸酶在圆盘凝胶电泳中呈现一条带，其比活性为4-5 U/mg。D-苹果酸酶需要二价阳离子。其对苹果酸的Km值为Km = 0.3 mM，对NAD的Km值为Km = 0.08 mM。发现该反应的最适pH范围为8.1至8.8。D-苹果酸酶受到草酰乙酸、内消旋酒石酸、D-乳酸和ATP的部分抑制。通过凝胶过滤和梯度凝胶电泳测定，其分子量约为175000。
