Directed evolution and site-specific mutagenesis of L-isoleucine dioxygenase derived from Bacillus weihenstephanensis.
Biotechnol Lett
; 40(8): 1227-1235, 2018 Aug.
Article
em En
| MEDLINE
| ID: mdl-29869760
ABSTRACT
OBJECTIVES:
L-isoleucine dioxygenase (IDO) specifically transforms L-isoleucine (Ile) to 4-hydroxyisoleucine (4-HIL), and 4-HIL is a promising drug for diabetes. To enhance the activity and catalytic efficiency of IDO, we used directed evolution and site-specific mutagenesis.RESULTS:
The IDO gene (ido) derived from Bacillus weihenstephanensis was cloned and expressed in Escherichia coli. Directed evolution using error prone (EP)-PCR and site-specific mutagenesis were conducted. Two improved mutants were obtained after one round of EP-PCR, with IdoN126H exhibiting a 2.8-fold increase in activity. Two improved mutants were obtained through site-specific mutagenesis, with IdoT130K showing a 170% increase in activity. Although the activity of the combined mutant IdoN126H/T130K (0.95 ± 0.08 U/mg) was slightly higher than that of the wild-type Ido, its catalytic efficiency was 2.4-fold and 3.0-fold higher than Ido with Ile and α-ketoglutaric acid as substrates. After biotransformation of Ile by E. coli BL21(DE3) expressing IdoN126H/T130K and Ido, 66.50 ± 0.99 mM and 26.09 ± 1.85 mM 4-HIL was synthesized, respectively, in 24 h.CONCLUSION:
IdoN126H/T130K had a higher enzyme activity and catalytic efficiency and can therefore be used as a more suitable candidate for 4-HIL production.Palavras-chave
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Base de dados:
MEDLINE
Assunto principal:
Bacillus
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Mutagênese Sítio-Dirigida
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Evolução Molecular Direcionada
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Dioxigenases
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Isoleucina
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article