A microorganism that produces glucose isomerase was isolated from soil and identified as a strain of Streptomyces flavogriseus. The organism produced a large quantity of glucose isomerase when grown on straw hemicellulose, xylan, xylose, and H2SO4 hydrolysate of ryegrass straw. The organism produced glucose isomerase both intra-and extra-cellularly. The highest level of intracellular glucose isomerase (3.5 U/ml) was obtained in about 36 h by a culture grown on straw hemicellulose; the extracellular enzyme (1.5 U/ml) appeared in cultures grown for about 72 h. About equal levels of enzyme were produced in cultures grown on straw hemicellulose, xylan, xylose, and H2SO4 hydrolysate of straw, but production of the enzyme was drastically reduced when the organism was grown on other carbon sources. As a nitrogen source, corn steep liquor produced the best results. Soy flour extract, yeast extract, and various peptones also were adequate substrates for g!ucose isomerase production. Addition of Mg2+, Mn2+, or Fe2" to the growth medium significantly enhanced enzyme production. The organism, however, did not require Co2+, which is commonly required by microorganisms used in the production of glucose isomerase.

Glucose isomerase (EC 5.3.1.5) produced from Streptomyces flavogriseus was purified by fractionation with (NH4)2SO4 and chromatography on diethylaminoethyl (DEAE)-cellulose and DEAE-Sephadex A-50 columns. The purified enzyme was homogeneous as shown by ultracentrifugation and sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Benzyl DEAE-cellulose, triethylaminoethylcellulose, 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 Km values for glucose and xylose; the soluble enzyme had the lowest values. The optimum temperature for activity of the soluble and immobilized enzymes was 700C; that for whole cells was 750C. The pH optimum for the three enzyme preparations was 7.5. Magnesium ion or C02+ was required for enzyme activity; an addition effect resulted from the presence of both Mg2e and Co2+. The enzyme activity was inhibited by Hg2+, Ag+, or Cu2. 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 700C 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 Mg2+ and C02+, was 75 and 55%, respectively. Elimination of C02+ decreased operational stability.

SummaryThe effects of two grinding methods, hammer milling and defibrizing by disk refining, on the fermentability of ryegrass straw were investigated. Disk refined or defibrized straw produced more sugar than hammer milled straw. Release of sugar was especially pronounced when H,SO, was added to the straw during the defibrizing process. I n v i m rumen digestibility was significantly higher (P < 0.1) for defibrized than for hammer milled straw. With semisolid culture the level of yeast growth was about three times as high on the defibrized as on hammer milled straw.A scanning electron micrograph revealed that defibrizing removed the waxy surface of the straw as well as separating fiber bundles, so that the surface area of the exposed fiber structure was increased.