Purification and Enzymatic Properties of Glucose Isomerase from Streptomyces griseofuscus, S-41

Abstract

Glucose isomerase was extracted in good yield from cells of Streptomyces griseofuscus, S–41 by keeping the cell suspension at 4°C for a month. The enzyme was purified 4.3-fold, and obtained in crystalline form by adding ammonium sulfate. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis and ultracentrifugation. The optimum pH for activity was 8.5 and optimum temperature 85°C. The enzyme was quite inactive in the absence of metal ions, but was remarkably activated by the addition of magnesium or cobalt ion. The enzyme catalyzed the isomerization of d-xylose, d-glucose and d-ribose. The Km values for d-glucose and d-xylose were 2.2 × 10⁻¹ M and 5.4 × 10⁻² M, respectively, while V_max values were 17.6 μmol/min/mg and 44.2 μmol/min/mg, respectively. The ratio of d-fructose content-to-d-glucose content was approximately 1.0 at reaction equilibrium. The enzyme activity was inhibited by sugars, sugar alcohols and tris(hydroxymethyl)aminomethane in a competitive manner. The Ki values were as follows: d-xylitol, 1.2 × 10⁻³ M; d-sorbitol, 1.1 × 10⁻² M; d-mannitol, 8.3 × 10⁻² M; d-mannose, 3.4 × 10⁻¹ M; d-galactose, 3.2 x 10⁻¹ M; l-arabinose, 2.3 × 10⁻¹ M and tris(hydroxymethyl)aminomethane, 6.2 × 10⁻² M. ρ-Chloromercuribenzoate, monoiodoacetic acid, sodium azide, potassium cyanide, sodium fluoride and 2-mercaptoethanol had no inhibitory effect on the activity of glucose isomerase, while EDTA caused a significant loss of activity.