Publication

Hot or not? Discovery and characterization of a thermostable alditol oxidase from Acidothermus cellulolyticus 11B

Winter, R. T., Heuts, D. P. H. M., Rijpkema, E. M. A., van Bloois, E., Wijma, H. J. & Fraaije, M. W., Jul-2012, In : Applied Microbiology and Biotechnology. 95, 2, p. 389-403 15 p.

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DOI

We describe the discovery, isolation and characterization of a highly thermostable alditol oxidase from Acidothermus cellulolyticus 11B. This protein was identified by searching the genomes of known thermophiles for enzymes homologous to Streptomyces coelicolor A3(2) alditol oxidase (AldO). A gene (sharing 48% protein sequence identity to AldO) was identified, cloned and expressed in Escherichia coli. Following 6xHis tag purification, characterization revealed the protein to be a covalent flavoprotein of 47 kDa with a remarkably similar reactivity and substrate specificity to that of AldO. A steady-state kinetic analysis with a number of different polyol substrates revealed lower catalytic rates but slightly altered substrate specificity when compared to AldO. Thermostability measurements revealed that the novel AldO is a highly thermostable enzyme with an unfolding temperature of 84 A degrees C and an activity half-life at 75 A degrees C of 112 min, prompting the name HotAldO. Inspired by earlier studies, we attempted a straightforward, exploratory approach to improve the thermostability of AldO by replacing residues with high B-factors with corresponding residues from HotAldO. None of these mutations resulted in a more thermostable oxidase; a fact that was corroborated by in silico analysis.

Original languageEnglish
Pages (from-to)389-403
Number of pages15
JournalApplied Microbiology and Biotechnology
Volume95
Issue number2
Publication statusPublished - Jul-2012

    Keywords

  • Carbohydrate oxidase, Flavoenzyme, Thermostable, ThermoFAD, Alditols, VANILLYL-ALCOHOL OXIDASE, CHROMOBACTERIUM SP DS-1, STREPTOMYCES-COELICOLOR, ESCHERICHIA-COLI, PENICILLIUM-SIMPLICISSIMUM, PROTEIN THERMOSTABILITY, CHOLESTEROL OXIDASE, CARBONIC-ANHYDRASE, PYRANOSE 2-OXIDASE, THERMOBIFIDA-FUSCA

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