Stabilization of a chitinase from Serratia marcescens by Gly->Ala and Xxx->Pro mutations |
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Authors: | Gaseidnes Sigrid; Synstad Bjornar; Jia Xiaohong; Kjellesvik Hege; Vriend Gert; Eijsink Vincent GH |
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Affiliation: | 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 |
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Abstract: | This paper describes attempts to increase the kinetic stabilityof chitinase B from Serratia marcescens (ChiB) by the introductionof semi-automatically designed rigidifying mutations of theGly Ala and Xxx Pro type. Of 15 single mutants, several displayedsignificant increases in thermal stability, whereas most mutantsshowed minor effects. All mutations with non-marginal effectson stability clustered in a limited, surface-exposed regionof the enzyme, indicating that this region is involved in apartial unfolding process that triggers irreversible thermalinactivation (aggregation). A double mutant containing two stabilizingmutations in this region (G188A, A234P) displayed a 10-foldincrease in half-life at 57°C and a 4.2°C increase inapparent Tm. These results show that entropic stabilizationworks well for ChiB and they pinpoint a region whose unfoldingmay 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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