Biotechnol Lett https://doi.org/10.1007/s10529-018-2566-8 ORIGINAL RESEARCH PAPER Directed evolution and site-speciﬁc mutagenesis of L-isoleucine dioxygenase derived from Bacillus weihenstephanensis Sen Huang Feng Shi Received: 22 March 2018 / Accepted: 14 May 2018 Springer Nature B.V. 2018 N126H Abstract round of EP-PCR, with Ido exhibiting a 2.8-fold Objectives L-isoleucine dioxygenase (IDO) speciﬁ- increase in activity. Two improved mutants were cally transforms L-isoleucine (Ile) to 4-hydroxy- obtained through site-speciﬁc mutagenesis, with T130K isoleucine (4-HIL), and 4-HIL is a promising drug Ido showing a 170% increase in activity. for diabetes. To enhance the activity and catalytic Although the activity of the combined mutant N126H/T130K efﬁciency of IDO, we used directed evolution and site- Ido (0.95 ± 0.08 U/mg) was slightly speciﬁc mutagenesis. higher than that of the wild-type Ido, its catalytic Results The IDO gene (ido) derived from Bacillus efﬁciency was 2.4-fold and 3.0-fold higher than Ido weihenstephanensis was cloned and expressed in with Ile and a-ketoglutaric acid as substrates. After Escherichia coli. Directed evolution using error prone biotransformation of Ile by E. coli BL21(DE3) N126H/T130K (EP)-PCR and site-speciﬁc mutagenesis were con- expressing Ido and Ido, 66.50 ± 0.99 mM ducted. Two improved mutants were obtained after one and 26.09 ± 1.85 mM
Biotechnology Letters – Springer Journals
Published: Jun 5, 2018
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