Production of Galactose Oxidase Inside the Fusarium fujikuroi Species Complex and Recombinant Expression and Characterization of the Galactose Oxidase GaoA Protein from Fusarium subglutinans

Autor: Camila Agnes Lumi Abe, Carla Bertechini Faria, Damaris Batistão Martim, Marco A. S. Oliveira, Fausto Fernandes de Castro, Kelly Valério Prates, Ione Parra Barbosa-Tessmann
Rok vydání: 2019
Předmět:
Models
Molecular
Protein Conformation
alpha-Helical
0106 biological sciences
Fusarium
Genetic Vectors
Gene Expression
Bioengineering
Galactose Oxidase
01 natural sciences
Applied Microbiology and Biotechnology
Biochemistry
Substrate Specificity
03 medical and health sciences
chemistry.chemical_compound
Raffinose
010608 biotechnology
Escherichia coli
Glycerol
Amino Acid Sequence
Enzyme kinetics
Cloning
Molecular
Hydrogen peroxide
Molecular Biology
Melibiose
030304 developmental biology
chemistry.chemical_classification
0303 health sciences
Sequence Homology
Amino Acid
biology
Temperature
Galactose
Hydrogen-Ion Concentration
Methylgalactosides
biology.organism_classification
Recombinant Proteins
Fusarium subglutinans
Enzyme
chemistry
Galactose oxidase
Protein Conformation
beta-Strand
Heterologous expression
Oxidation-Reduction
Sequence Alignment
Biotechnology
Zdroj: Molecular Biotechnology. 61:633-649
ISSN: 1559-0305
1073-6085
DOI: 10.1007/s12033-019-00190-6
Popis: Galactose oxidase catalyzes a two-electron oxidation, mainly from the C6 hydroxyl group of d-galactose, with the concomitant reduction of water to hydrogen peroxide. This enzyme is secreted by Fusarium species and has several biotechnological applications. In this study, a screening of galactose oxidase production among species of the Fusarium fujikuroi species complex demonstrated Fusarium subglutinans to be the main producer. The truncated F. subglutinans gaoA gene coding for the mature galactose oxidase was expressed from the prokaryotic vector pTrcHis2B in the E. coli Rosetta™ (DE3) strain. The purified recombinant enzyme presented temperature and pH optima of 30 °C and 7.0, respectively, KM of 132.6 ± 18.18 mM, Vmax of 3.2 ± 0.18 µmol of H2O2/min, kcat of 12,243 s−1, and a catalytic efficiency (kcat/KM) of 9.2 × 104 M−1 s−1. In the presence of 50% glycerol, the enzyme showed a T50 of 59.77 °C and was stable for several hours at pH 8.0 and 4 °C. Besides d-(+)-galactose, the purified enzyme also acted against d-(+)-raffinose, α-d-(+)-melibiose, and methyl-α-d-galactopyranoside, and was strongly inhibited by SDS. Although the F. subglutinans gaoA gene was successfully expressed in E. coli, its endogenous transcription was not confirmed by RT-PCR.
Databáze: OpenAIRE
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