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Open AccessArticle

Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion

1
Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia
2
Innovations and High Technologies MSU Ltd., 109559 Moscow, Russia
3
Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences, 119071 Moscow, Russia
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(18), 4412; https://doi.org/10.3390/ijms20184412
Received: 15 July 2019 / Revised: 29 August 2019 / Accepted: 4 September 2019 / Published: 7 September 2019
(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)
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 C with a two enzymes system. DAAO from the yeast Trigonopsis variabilis (TvDAAO) shows the best catalytic parameters with cephalosporin C among all known DAAOs. We prepared and characterized multipoint TvDAAO mutants to improve their activity towards cephalosporin C 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 C 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 C reported by others. View Full-Text
Keywords: d-amino acid oxidase; Trigonopsis variabilis; cephalosporin C; multipoint mutants; oxidative stability; thermal stability d-amino acid oxidase; Trigonopsis variabilis; cephalosporin C; multipoint mutants; oxidative stability; thermal stability
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Atroshenko, D.L.; Shelomov, M.D.; Zarubina, S.A.; Negru, N.Y.; Golubev, I.V.; Savin, S.S.; Tishkov, V.I. Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion. Int. J. Mol. Sci. 2019, 20, 4412.

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