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

• Published in: Applied microbiology and biotechnology 2012-07, Vol.95 (2), p.389-403
• Main Authors: Winter, Remko T., Heuts, Dominic P. H. M., Rijpkema, Egon M. A., van Bloois, Edwin, Wijma, Hein J., Fraaije, Marco W.
• Format: Article
• Language: English
• Subjects: Actinomycetales - enzymology; Alcohol; Alcohol Oxidoreductases - chemistry; Alcohol Oxidoreductases - isolation & purification; Alcohol Oxidoreductases - metabolism; Amino Acid Sequence; Analysis; Bacteria; Biocatalysts; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Carbohydrates; Chemical properties; Chromatography, Affinity; Cloning; Cloning, Molecular; Computational Biology; E coli; Engineers; Enzyme Stability; Enzymes; Escherichia coli; Escherichia coli - genetics; Gene Expression; Genomes; Genomics; Hot Temperature; Kinetics; Life Sciences; Microbial Genetics and Genomics; Microbiology; Models, Molecular; Molecular Sequence Data; Molecular Weight; Mutation; Oxidases; Oxidation; Polyols; Protein Conformation; Proteins; Sequence Homology, Amino Acid; Streptomyces coelicolor; Streptomyces coelicolor - genetics; Substrate Specificity; Sugar Alcohols - metabolism
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-011-3750-0

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 °C and an activity half-life at 75 °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.