Отправить статью по E-mail Rational Design of Practically Important Enzymes
- Авторы: Tishkov V.I.1,2,3, Pometun A.A.2,3, Stepashkina A.V.2,3, Fedorchuk V.V.1,3, Zarubina S.A.1,3, Kargov I.S.1,2,3, Atroshenko D.L.1,2, Parshin P.D.1,2, Shelomov M.D.1,2, Kovalevski R.P.1,2, Boiko K.M.3, Eldarov M.A.3, D’Oronzo E.4, Facheris S.4, Secundo F.4, Savin S.S.1,2
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Учреждения:
- Department of Chemistry
- Innovations and High Technologies MSU Ltd
- Bach Institute of Biochemistry
- Istituto di Chimica del Riconoscimento Molecolare
- Выпуск: Том 73, № 1 (2018)
- Страницы: 1-6
- Раздел: Article
- URL: https://journals.rcsi.science/0027-1314/article/view/163580
- DOI: https://doi.org/10.3103/S0027131418020153
- ID: 163580
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Аннотация
Majority of native enzymes are poorly applicable for practical usage: that is why different methods of enzyme modification are used to obtain the biocatalysts with appropriate characteristics. Development of genome sequencing and various modern approaches in protein engineering allow one to identify protein of interest and to improve the enzyme properties for a particular process. This review describes the results on development of novel biocatalysts based on bioinformatics and rational design. New genes encoding formate dehydrogenase (FDH) from bacterium Staphylococcus aureus, yeasts Ogataea parapolymorpha and Saccharomyces cerevisiae and moss Physcomitrella patens (SauFDH, OpaFDH, SceFDH and PpaFDH, respectively), have been cloned. New FDHs were produced in the active form and characterized. SauFDH was shown to have at least 2-fold higher catalytic constant than other known FDHs. OpaFDH has catalytic parameters as good as those for soy FDH mutant forms, and in addition, is more thermostable. Apo- and holo-forms of SauFDH have been crystallized. Mutation of two Cys residues in Pseudomonas sp.101 enzyme (PseFDH) yields enzyme preparations with improved kinetic parameters and enhanced thermal and chemical stability. New generation of PseFDH preparations with the coenzyme specificity changed from NAD+ to NADP+ have been obtained. The effect of ionic liquids on the catalytic properties and thermal stability of six wild-type recombinant FDHs, and a number of their mutants, have been studied. In case of D-amino acid oxidase (DAAO), single-point mutations have been combined to create multi-point mutants. The introduced amino acid replacements have been shown to exert an additive effect, improving both kinetic parameters and increasing thermal and chemical stability. DAAO genes from Hansenula polymorpha yeast have been cloned. α-Amino acid ester hydrolase (AEH) gene has been cloned and expressed in the active form in E. coli. Structural modeling has been performed and the effectiveness in amino beta-lactams synthesis studied. The structure of a single-chain penicillin acylase from Alcaligenes faecalis (scAfPA) has been modeled and two variants of scAfPA gene was generated by PCR. Both variants have been expressed in E. coli, isolated and characterized. Catalytic properties of scAfPA were slightly better than those of its natural heterodimer.
Об авторах
V. Tishkov
Department of Chemistry; Innovations and High Technologies MSU Ltd; Bach Institute of Biochemistry
Автор, ответственный за переписку.
Email: vitishkov@gmail.com
Россия, Moscow, 119991; Moscow, 109559; Moscow, 119071
A. Pometun
Innovations and High Technologies MSU Ltd; Bach Institute of Biochemistry
Email: vitishkov@gmail.com
Россия, Moscow, 109559; Moscow, 119071
A. Stepashkina
Innovations and High Technologies MSU Ltd; Bach Institute of Biochemistry
Email: vitishkov@gmail.com
Россия, Moscow, 109559; Moscow, 119071
V. Fedorchuk
Department of Chemistry; Bach Institute of Biochemistry
Email: vitishkov@gmail.com
Россия, Moscow, 119991; Moscow, 119071
S. Zarubina
Department of Chemistry; Bach Institute of Biochemistry
Email: vitishkov@gmail.com
Россия, Moscow, 119991; Moscow, 119071
I. Kargov
Department of Chemistry; Innovations and High Technologies MSU Ltd; Bach Institute of Biochemistry
Email: vitishkov@gmail.com
Россия, Moscow, 119991; Moscow, 109559; Moscow, 119071
D. Atroshenko
Department of Chemistry; Innovations and High Technologies MSU Ltd
Email: vitishkov@gmail.com
Россия, Moscow, 119991; Moscow, 109559
P. Parshin
Department of Chemistry; Innovations and High Technologies MSU Ltd
Email: vitishkov@gmail.com
Россия, Moscow, 119991; Moscow, 109559
M. Shelomov
Department of Chemistry; Innovations and High Technologies MSU Ltd
Email: vitishkov@gmail.com
Россия, Moscow, 119991; Moscow, 109559
R. Kovalevski
Department of Chemistry; Innovations and High Technologies MSU Ltd
Email: vitishkov@gmail.com
Россия, Moscow, 119991; Moscow, 109559
K. Boiko
Bach Institute of Biochemistry
Email: vitishkov@gmail.com
Россия, Moscow, 119071
M. Eldarov
Bach Institute of Biochemistry
Email: vitishkov@gmail.com
Россия, Moscow, 119071
E. D’Oronzo
Istituto di Chimica del Riconoscimento Molecolare
Email: vitishkov@gmail.com
Италия, Milan, 20131
S. Facheris
Istituto di Chimica del Riconoscimento Molecolare
Email: vitishkov@gmail.com
Италия, Milan, 20131
F. Secundo
Istituto di Chimica del Riconoscimento Molecolare
Email: vitishkov@gmail.com
Италия, Milan, 20131
S. Savin
Department of Chemistry; Innovations and High Technologies MSU Ltd
Email: vitishkov@gmail.com
Россия, Moscow, 119991; Moscow, 109559
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