Modification of carboxyl groups in Geotrichum candidum lipase

Lipase from Geotrichum (Geo.) candidum was rapidly inactivated by incubation with water-soluble carbodiimide, 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC), or 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl) carbodiimide metho-p-toluenesulfonate (CMC), at pH 4.8. The pH dependence of the rate of inactivation was consistent with the modification of carboxyl groups in the lipase. Reaction of the lipase with EDC in the presence of the nucleophile taurine showed that about 9 carboxyl groups per molecule of enzyme were modified with concomitant total loss of activity. This number was reduced to 4 when CMC was used as a carbodiimide instead of EDC. The modification had no effect on the CD spectrum in the ultraviolet region. Kinetic analysis of the effect of CMC on the lipase indicated that at least 1 CMC molecule bound to the enzyme during inactivation.