D-Xylose metabolism by Candida intermedia: isolation and characterisation of two forms of aldose reductase with different coenzyme specificities

To study individual enzyme components responsible for the initial step of D -xylose utilisation by the yeast Candida intermedia, a two-step protocol ha s been developed that enables clear-cut separation and isolation of two str ucturally similar but functionally different aldose reductases (ALRs) in hi gh yield. In the first step, the yeast cell extract is fractionated efficie ntly by biomimetic chromatography using the dye HE-SE (reactive Red 120) as pseudoaffinity ligand coupled to Sepharose CL-4B. In the second step, opti mised high-resolution anion-exchange chromatography using Mono Q yields pur ified ALR1 and ALR2 in overall yields of 63 and 62%, respectively. ALR1 is strictly specific for NADPH (2.4.10(5) M-1 s(-1)) whereas ALR2 utilises NAD H and NADPH with similar specificity constants of approximately 2-4.10(5) M -1 s(-1). Both enzymes are dimers with a subunit molecular mass of 36 000bu t they differ in pi and the number of titratable sulphydryl groups in the n ative protein. The chromatographic procedure identifies microheterogeneity in recombinant aldose reductase from Candida tenuis overexpressed in Escher ichia coli. (C) 2000 Published by Elsevier Science B.V. All rights reserved .