We have investigated the structural stability of cutinase, an industrial lipolytic enzyme, from Fusarium solani pisi. We have the gene of this enzyme cloned in a high expression vector in Eschericha coli, to produce the necessary amounts of pure protein. Cutinase is a single domain 25 kD protein. The activity optimum is found to be at a pH of 8.5. The structure of this protein has been solved to 1.0 Å [1].

Native cutinase displays in DSC experiments a clear non two state unfolding with complex underlying unfolding kinetic behaviour. In the alkaline pH range where the enzyme displays its highest catalytic activity (pH 8,5) the unfolding is irreversible due to additional aggregation effects [2, 3]. The introduction of T45P causes a substantial increase in thermostability, by 5 degrees compared with the native enzyme. But more strikingly this single point mutation also altered the unfolding pathway, such that the thermal denaturation of the mutant follows a two state denaturation as well as a remarkable degree of reversibility, even at alkaline pH values. This allows for a thermodynamic characterisation of the unfolding kinetics of this mutant. The thermo stabilisation seen in DSC experiments, could also be confirmed by temperature scanning steady state fluorescence measurements at different pH values (pH 4.0, 6.0, 8.5 and 10.0). In these experiments the change of environment during the unfolding of the single Tryptophan residue of cutinase, was monitored. Circular dichroism confirmed both of the previous methods.

The change in the unfolding behaviour of T45P from a non two-state unfolding to a two-state unfolding indicating a shift in the unfolding kinetics from a second order unfolding kinetics to a first order unfolding kinetic of the protein. This mutation prevents the aggregtion seen for the native enzyme in the alkaline pH range. X-ray crystallography confirmed that this mutation did not cause any structural differences aside from the exchanged residue (T45P). The overall structure of the enzyme is still the same as in the native enzyme.

The investigation of this mutant leads to new insights in the effect of mutations not only on the thermal stability of enzymes, but also that the kinetic of the unfolding reaction itself affects the overall measured thermostability of an enzyme. We present here the thermodynamic and kinetic characterisation of the T45P mutant.

[1] Longhi, S., Czjzek, M., Lamzin, V., Nicolas, A., Cambillau, C, J.Mol.Biol. 779, 268 pp., 1997

[2] Petersen SB, Jonson PH, Fojan P, Petersen EI, Petersen MTN, Hansen S, Ishak RJ, Hough E, J. Biotechnol. 66(1):11-26, 1998.

[3] Petersen, MTN, Fojan P and Petersen SB, J. Biotechnol.,in print.