1. Academic Validation
  2. Glucose isomerase: insights into protein engineering for increased thermostability

Glucose isomerase: insights into protein engineering for increased thermostability

  • Biochim Biophys Acta. 2000 Dec 29;1543(2):294-335. doi: 10.1016/s0167-4838(00)00246-6.
B S Hartley 1 N Hanlon R J Jackson M Rangarajan
Affiliations

Affiliation

  • 1 Department of Biochemistry, Imperial College, SW7 2AZ, London, UK. b.hartley@bc.ic.ac.uk
Abstract

Thermostable glucose isomerases are desirable for production of 55% fructose syrups at >90 degrees C. Current commercial Enzymes operate only at 60 degrees C to produce 45% fructose syrups. Protein engineering to construct more stable Enzymes has so far been relatively unsuccessful, so this review focuses 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 are compared. Within each class the structures are almost identical and sequence differences are few. Structural differences between Class 1 and Class 2 are less than previously surmised. The thermostabilities of Class 1 Enzymes are essentially identical, in contrast to previous reports, but in Class 2 they vary widely. In each class, thermal inactivation proceeds via the tetrameric apoenzyme, so metal ion affinity dominates thermostability. In Class 1 Enzymes, subunit dissociation is not involved, but there is an irreversible conformational change in the apoenzyme leading to a more thermostable inactive tetramer. This may be linked to reversible conformational changes 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 engineering results are reviewed in light of these insights.

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