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Protein Engineering vol. 16 no. 11 pp. 841-846, 2003
© 2003 Oxford University Press

Stabilization of a chitinase from Serratia marcescens by Gly->Ala and Xxx->Pro mutations

Sigrid Gåseidnes1, Bjørnar Synstad1, Xiaohong Jia1, Hege Kjellesvik1, Gert Vriend2 and Vincent G.H. Eijsink1,3

1Department of Chemistry and Biotechnology, Agricultural University of Norway, PO Box 5040, 1432 Ås, Norway and 2Center for Molecular and Biomolecular Informatics, University of Nijmegen, PO Box 9010, 6500 GL Nijmegen, The Netherlands

3 To whom correspondence should be addressed. e-mail: vincent.eijsink{at}ikb.nlh.no

This paper describes attempts to increase the kinetic stability of chitinase B from Serratia marcescens (ChiB) by the introduction of semi-automatically designed rigidifying mutations of the Gly->Ala and Xxx->Pro type. Of 15 single mutants, several displayed significant increases in thermal stability, whereas most mutants showed minor effects. All mutations with non-marginal effects on stability clustered in a limited, surface-exposed region of the enzyme, indicating that this region is involved in a partial unfolding process that triggers irreversible thermal inactivation (aggregation). A double mutant containing two stabilizing mutations in this region (G188A, A234P) displayed a 10-fold increase in half-life at 57°C and a 4.2°C increase in apparent Tm. These results show that entropic stabilization works well for ChiB and they pinpoint a region whose unfolding may be crucial for the kinetic stability of this enzyme.

Received June 18, 2003; revised September 3, 2003; accepted September 12, 2003.


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