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 has been developed that enables clear-cut separation and isolation of two structurally similar but functionally different aldose reductases (ALRs) in high yield. In the first step, the yeast cell extract is fractionated efficiently by biomimetic chromatography using the dye HE-3B (reactive Red 120) as pseudoaffinity ligand coupled to Sepharose CL-4B. In the second step, optimised high-resolution anion-exchange chromatography using Mono Q yields purified ALR1 and ALR2 in overall yields of 63 and 62%, respectively. ALR1 is strictly specific for NADPH (2.4·105 M−1 s−1) whereas ALR2 utilises NADH and NADPH with similar specificity constants of approximately 2–4·105 M−1 s−1. Both enzymes are dimers with a subunit molecular mass of 36 000 but they differ in pI and the number of titratable sulphydryl groups in the native protein. The chromatographic procedure identifies microheterogeneity in recombinant aldose reductase from Candida tenuis overexpressed in Escherichia coli.