Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 95, Issue 2, Pages 389-403Publisher
SPRINGER
DOI: 10.1007/s00253-011-3750-0
Keywords
Carbohydrate oxidase; Flavoenzyme; Thermostable; ThermoFAD; Alditols
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Funding
- Dutch Technology Foundation STW [GBC.7726]
- Ministry of Economic Affairs
- applied science division of NWO
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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.
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