| Budget Amount *help |
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1992: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 1991: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
To date, glucose isomerase(D-xylose ketol isomerase EC:5.3.1.5)is the enzyme that catalyzes the isomerization of both D-xylose and D-glucose to D-xylulose and D-fructose,respectively. An enzyme that catalyzes only D-glucose into D-fructose is not yet known. For the first time,I succeeded in isolating, purifying and characterizing a novel enzyme from Bifidobacterium adolescentis that is able to catalyze only the isomerization of D-glucose to D-fructose. To produce the enzyme,the bacterium was cultivated anaerobically in the presence of glucose as carbon source at 37ﾟC for 20 hr. Thereafter,cells(harvested by continuous centrifugation at 12,000 rpm)were disrupted by ultra-sonication and centrifuged at 12,000 rpm for 30 min, The resulting supernatant was then used as crude preparation for purification. The enzyme was purified in 5 step,namely ion exchange chromatography on DE-32 column; Gel filtration chromatography on HW-55-S Toyopearl column; ion exchange chromatography using Smart system with MonoQ-PC16/5 column and HPLC gel filtration chromatography on 300SWxl column. The purified enzyme was characterized and the following are some of its characteristics. (1) Molecular weight was found to be 240,000 by gel filtration method. (2) Optimal pH was 7.0 and the enzyme was stable between pH 6 to 8. (3) Optimal temperature of the enzyme activity was found to be at 40-50ﾟC,and the thermal stability for the enzyme was found to be up to 40ﾟC. (4) The effect of metal ion on the enzyme activity indicated that Mg^<2+> and Mn^<2+> were effective. (5) The Km value(D-glucose)of the enzyme was found to be 8mM. This value was about 2 times higher than that exhabited by xylose isomerase. These experimental data prompt me to say that this is a novel enzyme never characterized before.
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