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 cell- free extract, with a yield of 2.4% in a homogeneous state, as judged on sodium dodecyl sulfate- polyacrylamide 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 Vmax 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.