Реакция #2161243

Уравнение реакции

D-glucose

sodium phosphate

→

glucose

fructose

Растворители

trichloroacetic acid

Условия реакции

Обработка

1. 1
   Концентрированиеconcentration) and 5 mM MgCl2 (final
2. 2
   Концентрированиеconcentration)
3. 3
   workup.ADDITIONSixty μl of the substrate solution B was mixed with 40 μl of glucose
4. 4
   Другоеprepared
5. 5
   Другоеthe reaction
6. 6
   ДругоеTen μl of the supernatant, collected by centrifugation at 17,800 g for 15 minutes at 10° C.
7. 7
   workup.ADDITIONwas diluted 100 fold
8. 8
   Другоеequipped with detector ELSD 500

Методика

The measurement was based on Kaneko et. al., (Biosci Biotechnol Biochem. 2000, 64:940-7) with modifications. Substrate solution B stock containing 50%(w/v) D-glucose, 20 mM sodium phosphate buffer (pH 6.5), 250 μM CoCl2 (final concentration) and 5 mM MgCl2 (final concentration) was adjusted to pH 6.5. Sixty μl of the substrate solution B was mixed with 40 μl of glucose isomerase mutant MGI-4 prepared as described in Example 10, incubated at 80° C. for 4 h and 100 μl of 20% trichloroacetic acid was added to stop the reaction. Ten μl of the supernatant, collected by centrifugation at 17,800 g for 15 minutes at 10° C., was diluted 100 fold and applied to high pressure liquid chromatography (HPLC) column μBNONDAPAK NH2 SS COL 3.9×300 (Waters, Calif., USA) equipped with detector ELSD 500. The mobile phase acetonitrile:water (85:15) was run at a flow rate of 0.5 ml/min. The volume of the sample loaded was 10 μl. FIG. 2 shows the results of the conversion of D-glucose to fructose by glucose isomerase mutant MGI-4 at 80° C.

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