Abstract

A xylose reductase (XR) with high activity and dual coenzyme activity from Candida tropical was purified to homogeneity by Ni²⁺-chelating column, and immobilized on chitosan bead. Studies on free and immobilized XR systems for determination of optimum temperature, optimum pH, thermal stability, pH stability, operational stability, and kinetic parameters were carried out. Free and immobilized XR showed higher activity at 45 and 50°C, respectively. The optimum pH for free and immobilized XR were 4.5, but immobilized XR had higher activity with a broader pH range of 4.0-6.0. Thermal and pH stability of immobilized XR were higher than that of free XR. The residual activity of immobilized XR was about 40% after 7 cycles of batch operation. The Km value of free XR was 30.3 mM, and that of immobilized XR was 20.1 mM, which indicated that the affinity of xylose was increased for immobilized XR. The immobilized XR activity was stimulated by MnSO₄, and inhibited by NaCl, βME,Glu. In addition, catalytic efficiency with NADH as cofactor of immobilized XR was better enhanced than free XR. It is the first report on immobilizing XR with chitosan, with a relative high activity.

Key words:  Xylose reductase, crosslinked chitosan bead, immobilization, catalytic property, NADH, NADPH.

Abbreviation

XR, Xylose reductase; ALR, aldose reductase; AKR, aldo-keto reductase; NADH, nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate; XDH,  xylose dehydrogenase; IPTG, isopropyl-α-D-thiogalactopyranoside; PBS, phosphate buffered saline; BSA, bovine serum albumin; EDTA, ethylenediaminetetraacetic acid.