D-(-)-Fructose → aldehydo-D-Xylose

Reference XI or XI mutein is produced and purified as described in Example I. The purified protein is adjusted to an average activity of 1.8-2.0 U/ml as determined by HPLC assay, and after precooling in a salt and ice bath, is mixed with glucose solution also precooled (400 g/l glucose, 25 mM maleic acid, 10 mM MgSO4 --H2O, 1 mM Fe++ (FeSO4.7H2O, pH 6.5) in a 1:1 ratio at 1° C. and distributed into 100 μl thin-walled glass micropipettes (Fisher Scientific, Pittsburgh, Pa.) which are flamed sealed and incubated in heating baths at different isomerization temperatures for 40 minutes. Temperatures of the heating baths are set at 1° C. intervals over the range at which the enzyme shows the full range of inactivation (85° C. to 100° C. for the reference XI). One bath is set at the reference temperature approximately 10° C. below the temperature of half maximum enzyme activity. For the blank samples, the buffer solution is mixed with the glucose substrate in a 1:1 ratio and distributed into 100 μl thin-walled glass micropipettes which are sealed and incubated in water baths at the same temperatures and incubation time as used for the enzyme test samples. The reaction is terminated by immersing the micropipettes in a salt-ice bath, and 65 μl of isomerase is removed from each micropipette. 2 μl of 1N HCl is added to stop the isomerase reaction. The isomerase is assayed for fructose and glucose by HPLC using a Beckman Liquid Chromatograph as described in Example I.

1. custompurified
2. additionis mixed with glucose solution
3. customat 1° C.
4. temperatureare flamed
5. customsealed
6. temperaturein heating baths at different isomerization temperatures for 40 minutes
7. customare set at 1° C.
8. custom85° C. to 100° C.
9. customis set at the reference temperature approximately 10° C. below the temperature of half maximum enzyme activity
10. additionFor the blank samples, the buffer solution is mixed with the glucose substrate in a 1:1 ratio
11. customare sealed
12. customThe reaction is terminated
13. custom65 μl of isomerase is removed from each micropipette
14. addition2 μl of 1N HCl is added
15. customthe isomerase reaction

ORD record

ord-ec0485fc05df489fb419d9bf7786973b

Source dataset

uspto-grants-1991_08

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US05041378