Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion

d-amino acid oxidase (DAAO, EC 1.4.3.3) is used in many biotechnological processes. The main industrial application of DAAO is biocatalytic production of 7-aminocephalosporanic acid from cephalosporin with a two enzymes system. DAAO from the yeast (TvDAAO) shows the best catalytic parameters with ce...

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Bibliographic Details
Published in:International journal of molecular sciences 2019-09, Vol.20 (18), p.4412
Main Authors: Atroshenko, Denis L, Shelomov, Mikhail D, Zarubina, Sophia A, Negru, Nikita Y, Golubev, Igor V, Savin, Svyatoslav S, Tishkov, Vladimir I
Format: Article
Language:English
Subjects:
Amino acid oxidase
Amino Acid Substitution
Amino acids
Antibiotics
Biocatalysis
Bioconversion
Cephalosporin C
Cephalosporins - metabolism
D-Amino-acid oxidase
D-Amino-Acid Oxidase - chemistry
D-Amino-Acid Oxidase - genetics
D-Amino-Acid Oxidase - metabolism
Enzyme Stability
Enzymes
Fungal Proteins - chemistry
Fungal Proteins - genetics
Fungal Proteins - metabolism
Hydrogen peroxide
Hydrogen Peroxide - pharmacology
Methods
multipoint mutants
Mutants
Mutation
Oxidation
oxidative stability
Point Mutation
Saccharomycetales - enzymology
Saccharomycetales - genetics
Shelf life
Storage stability
thermal stability
Thermodynamics
Trigonopsis variabilis
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Summary:d-amino acid oxidase (DAAO, EC 1.4.3.3) is used in many biotechnological processes. The main industrial application of DAAO is biocatalytic production of 7-aminocephalosporanic acid from cephalosporin with a two enzymes system. DAAO from the yeast (TvDAAO) shows the best catalytic parameters with cephalosporin among all known DAAOs. We prepared and characterized multipoint TvDAAO mutants to improve their activity towards cephalosporin and increase stability. All TvDAAO mutants showed better properties in comparison with the wild-type enzyme. The best mutant was TvDAAO with amino acid changes E32R/F33D/F54S/C108F/M156L/C298N. Compared to wild-type TvDAAO, the mutant enzyme exhibits a 4 times higher catalytic constant for cephalosporin oxidation and 8- and 20-fold better stability against hydrogen peroxide inactivation and thermal denaturation, respectively. This makes this mutant promising for use in biotechnology. The paper also presents the comparison of TvDAAO catalytic properties with cephalosporin reported by others.
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms20184412