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PRACTICAL COURSE

ENZYME ACTIVITY AND INHIBITORS

DR. GILLES TRUAN

TECHNICAL CONSIDERATIONS

Chemical material

The enzyme, substrates and other molecules can be purchased from Sigma-Aldrich.

Name Product # Storage

Ortho-Nitro-Phenyl-Galactoside

MW = 301.3 g/mol N1127 -20°C

Beta-galactosidase from Aspergillus oryzae

MW = 109900 Da G5160 -20°C

Glucose

MW = 180.2 g/mol D9434

Room

Temperature

Lactose

MW = 342.3 g/mol L3625

Room

Temperature

Galactose

MW = 180.2 g/mol G0750

Room

Temperature

Sodium acetate Room

Temperature

Sodium carbonate Room

Temperature

Materials

15 ml test tubes (25)

1.5 ml eppendorf tubes (150)

Floating devices for eppendorf tubes

Test tube racks

Graduated cylinders

Water bath at 37°C

Pipettes (P1000, P200, P10)

Tips for pipettes

Timer

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pH-meter

spectrophotometer

plastic cuvettes

0.1 M sodium acetate (NaAc) buffer, pH 5.0, 500 ml

0.5 M sodium carbonate (Na2CO3), pH 11.0, 500 ml

5 mM (1.5 mg/ml) o-nitrophenyl-beta-galactoside (ONPG) in buffer, 1 ml

2 u/ml (0.25 mg/ml) A. oryzae beta-galactosidase (enzyme) in buffer, 4 ml

10 mM lactose in buffer, 10 ml

10 mM galactose in buffer, 10 ml

10 mM glucose in buffer, 10 ml