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

Remko T. Winter, Dominic P. H. M. Heuts, Egon M. A. Rijpkema, Edwin van Bloois, Hein J. Wijma, Marco W. Fraaije*

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

22 Citaten (Scopus)
52 Downloads (Pure)


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.

Originele taal-2English
Pagina's (van-tot)389-403
Aantal pagina's15
TijdschriftApplied Microbiology and Biotechnology
Nummer van het tijdschrift2
StatusPublished - jul.-2012

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