A NAD-linked L(+)-lactate dehydrogenase (EC 1.1.1.27) was isolated from Alcaligenes eutrophus N9A. During purification advantage was taken of the high affinity of Matrex TM Gel Green A for this enzyme in crude extracts. One ml of this medium adsorbed 2660 U lactate dehydrogenase if 129 ml crude extract containing 2,480mg protein were applied onto the column, as determined by frontal analysis. The enzyme was purified 275-fold by chromatography on this medium. Subsequent chromatography on Cibacron Blue F3G-A Sepharose 6B-CL resulted in a 3-fold purification and in a homogeneous preparation of lactate dehydrogenase. Starting with a crude extract containing 12 g total protein, the overall purification factor was 712.5, corresponding to a recovery of 36.1% activity and a specific activity of 776.6 U/rag protein. The affinity of Matrex TM Gel Green A medium to lactate dehydrogenase from other sources like A. hydrogenophilus, A. eutrophus A7, A. faecalis, Escherichia coli, Lactobacillus lechmannii, and rabbit muscle was investigated. Advantages of this method for large scale purification of lactate dehydrogenase from AlcaIigenes eutrophus are discussed and compared to large scale purification methods applied for other enzymes.
'hysteretic' enzyme. The purification procedure is the first successful application of Matrex TM Gel Green A for purification of lactate dehydrogenases.
Lactate dehydrogenases are important tools in fields of medical diagnostics and scientific analysis.
Furthermore, the enzyme from A. eutrophus may provide a convenient method for the determination of small quantities of oxaloacetate as suggested previously (Steinbfichel and Schlegel 1983b). Therefore, we simplified the previously described enrichment procedure and are now able to offer a two-step method for purifying lactate dehydrogenase 700-fold to homogeneity. This procedure, which includes chromatography of large quantities of crude extract on small quantities of low cost triazine-dye affinity media (Atkinson et al. 1981), provides a quick and easy method for the large scale purification of the lactate dehydrogenase from A. eutrophus.