Glucose isomerase: insights into protein engineering for increased thermostability

Thermostable glucose isomerases are desirable for production of 55% fructos e syrups at > 90 degreesC. Current commercial enzymes operate only at 60 de greesC to produce 45% fructose syrups. Protein engineering to construct mor e stable enzymes has so far been relatively unsuccessful, so this review fo cuses on elucidation of the thermal inactivation pathway as a future guide. The primary and tertiary structures of 11 Class 1 and 20 Class 2 enzymes a re compared. Within each class the structures are almost identical and sequ ence differences are few. Structural differences between Class 1 and Class 2 are less than previously surmised. The thermostabilities of Class 1 enzym es are essentially identical, in contrast to previous reports, but in Class 2 they vary widely. In each class, thermal inactivation proceeds via the t etrameric apoenzyme, so metal ion affinity dominates thermostability. In Cl ass 1 enzymes, subunit dissociation is not involved, but there is an irreve rsible conformational change in the apoenzyme leading to a more thermostabl e inactive tetramer. This may be linked to reversible conformational change s in the apoenzyme at alkaline pH arising from electrostatic repulsions in the active site, which break a buried Arg-30-Asp-299 salt bridge and bring Arg-30 to the surface. There is a different salt bridge in Class 2 enzymes, which might explain their varying thermostability. Previous protein engine ering results are reviewed in light of these insights. (C) 2000 Elsevier Sc ience B.V. All rights reserved.