title: Cold adaptation of a psychrophilic chitinase: a mutagenesis study.
abstract: The gene encoding chitinase ArChiB from the Antarctic Arthrobacter sp. TAD20 has been expressed in Escherichia coli and the recombinant enzyme purified to homogeneity. In an effort to engineer cold-adapted biocatalysts through rational redesign to operate at elevated temperatures, we performed several mutations aiming to increase the rigidity of the molecular edifice of the selected psychrophilic chitinase. The mutations were designed on the basis of a homology-based three-dimensional model of the enzyme, and included an attempt to introduce a salt bridge (mutant N198K) and replacements of selected Gly residues by either Pro (mutants G93P, G254P) or Gln (G406Q). Mutant N198K resulted in a more stable protein (DeltaTm = 0.6 degrees C). Mutant G93P exhibited a DeltaTm of 1.2 degrees C, while mutants G254P and G406Q exhibited decreased stability. We conclude that the effect of mutating Gly residues on enzyme stability is rather complex and can only be understood in the context of the structural environment. Kinetic and spectroscopic analysis of these enzyme variants revealed that the kinetic parameters kcat and Km have been significantly modified.
authors.affiliation: Enzyme Technology Division, Institute of Molecular Biology and Biotechnology, PO Box 1515, 71110 Heraklion, Crete, Greece.
authors.forename: K
authors.initials: K
authors.lastname: Mavromatis
authors.forename: G
authors.initials: G
authors.lastname: Feller
authors.forename: M
authors.initials: M
authors.lastname: Kokkinidis
authors.forename: V
authors.initials: V
authors.lastname: Bouriotis
mesh_terms: D000220:Adaptation, Biological / Q000235:genetics; D019943:Amino Acid Substitution; D001173:Arthrobacter / Q000502:physiology*; D002688:Chitinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003080:Cold Temperature; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D009154:Mutation; D011489:Protein Denaturation
chemical_list: D002688:Chitinases