Purification and Characterization of Xylose Isomerase of a Methanol Yeast, Candida boidinii, Which Is Involved in Sorbitol Production from Glucose

Abstract

D-Xylose (xylose) isomerase was extracted from xylose-grown cells of a methanol yeast, Candida boidinii (Kloeckera sp.) No. 2201. The enzyme was purified 70-fold, over the original cellfree extract, with a yield of 2.4% in a homogeneous state, as judged on sodium dodecyl sulfatepolyacrylamide gel electrophoresis and high performance liquid chromatography. The molecular weight of the enzyme was determined to be 130, 000, the enzyme being composed of two subunits of 65, 000. The optimum pH and temperature for activity were 4.5 and 37-45°C, respectively. The enzyme activity was markedly enhanced by Mn²⁺, Mg²⁺ and Co²⁺, and the enzyme isomerized aldopentoses and aldohexoses. The Km values for xylose and D-glucose were 5.6 × 10^-1M and 4.1×10^-1M, and the V_max values were 5.8 × 10² and 3.3 × 10² μmol/min/mg, respectively. NaHAsO₄•7H₂O and NaCN strongly inhibited the activity, but HgCl₂, NaN₃, dithiothreitol, monoiodoacetate and polyols such as D-sorbitol, xylitol and D-mannitol did not inhibit the activity.