Abstract
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 MatrexTM 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 2,480 mg 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/mg protein. The affinity of MatrexTM 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 Alcaligenes eutrophus are discussed and compared to large scale purification methods applied for other enzymes.
Published Version
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