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Gaseidnes Sigrid; Synstad Bjornar; Jia Xiaohong; Kjellesvik Hege; Vriend Gert; Eijsink Vincent G.H. 《Protein engineering, design & selection : PEDS》2003,16(11):841-846
This paper describes attempts to increase the kinetic stabilityof chitinase B from Serratia marcescens (ChiB) by the introductionof semi-automatically designed rigidifying mutations of theGlyAla and XxxPro 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. 相似文献
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