Hot or not? Discovery and characterization of a thermostable alditol oxidase from Acidothermus cellulolyticus 11B
| Autor: | Edwin van Bloois, Marco W. Fraaije, Egon M. A. Rijpkema, Hein J. Wijma, Dominic P. H. M. Heuts, Remko T. Winter |
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| Přispěvatelé: | Biotechnology |
| Jazyk: | angličtina |
| Rok vydání: | 2012 |
| Předmět: |
Models
Molecular Hot Temperature THERMOBIFIDA-FUSCA Cholesterol oxidase Protein Conformation Gene Expression Applied Microbiology and Biotechnology Chromatography Affinity Substrate Specificity Sugar Alcohols Protein structure Enzyme Stability STREPTOMYCES-COELICOLOR Cloning Molecular Biotechnologically Relevant Enzymes and Proteins Peptide sequence Thermostable Thermostability Oxidase test Carbohydrate oxidase PYRANOSE 2-OXIDASE biology Streptomyces coelicolor PROTEIN THERMOSTABILITY General Medicine Biochemistry VANILLYL-ALCOHOL OXIDASE ESCHERICHIA-COLI Biotechnology endocrine system Vanillyl-alcohol oxidase Molecular Sequence Data ThermoFAD CARBONIC-ANHYDRASE CHOLESTEROL OXIDASE Actinomycetales Escherichia coli Amino Acid Sequence Sequence Homology Amino Acid Flavoenzyme Thermophile Computational Biology PENICILLIUM-SIMPLICISSIMUM biology.organism_classification Molecular Weight Alcohol Oxidoreductases Kinetics Alditols CHROMOBACTERIUM SP DS-1 |
| Zdroj: | Applied Microbiology and Biotechnology, 95(2), 389-403. SPRINGER Applied Microbiology and Biotechnology |
| ISSN: | 0175-7598 |
| Popis: | 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. Electronic supplementary material The online version of this article (doi:10.1007/s00253-011-3750-0) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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