Abstract
Glucose isomerase (EC 5.3.1.5) produced from Streptomyces flavogriseus was purified by fractionation with (NH(4))(2)SO(4) and chromatography on diethylaminoethyl (DEAE)-cellulose and DEAE-Sephadex A-50 columns. The purified enzyme was homogeneous as shown by ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Benzyl DEAE-cellulose, triethylaminoethyl-cellulose, and DEAE-cellulose were effective in the immobilization of partially purified glucose isomerase. Several differences in properties were found between purified soluble enzyme, immobilized enzyme (DEAE-cellulose-glucose isomerase), and heat-treated whole cells. Glucose and xylose served as substrate for the enzyme. Whole cells had the highest K(m) values for glucose and xylose; the soluble enzyme had the lowest values. The optimum temperature for activity of the soluble and immobilized enzymes was 70 degrees C; that for whole cells was 75 degrees C. The pH optimum for the three enzyme preparations was 7.5. Magnesium ion or Co was required for enzyme activity; an addition effect resulted from the presence of both Mg and Co. The enzyme activity was inhibited by Hg, Ag, or Cu. The conversion ratio of the enzyme for isomerization was about 50%. The soluble and immobilized enzymes showed a greater heat stability than whole cells. The soluble enzyme was stable over a slightly wider pH (5.0 to 9.0) range than the immobilized enzyme and whole cells (pH 5.5 to 9.0). The molecular weight of the enzyme determined by the sedimentation equilibrium method was 171,000. A tetrameric structure for the enzyme was also indicated. After operating at 70 degrees C for 5 days, the remaining enzyme activity of the immobilized enzyme and whole cells, which were used for the continuous isomerization of glucose in a plug-flow type of column in the presence of Mg and Co, was 75 and 55%, respectively. Elimination of Co decreased operational stability.
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