Directed evolution and site-specific mutagenesis of l-isoleucine dioxygenase derived from Bacillus weihenstephanensis

Directed evolution and site-specific mutagenesis of l-isoleucine dioxygenase derived from... 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 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biotechnology Letters Springer Journals

Directed evolution and site-specific mutagenesis of l-isoleucine dioxygenase derived from Bacillus weihenstephanensis

Loading next page...
 
/lp/springer_journal/directed-evolution-and-site-specific-mutagenesis-of-l-isoleucine-14miUSH6BZ
Publisher
Springer Journals
Copyright
Copyright © 2018 by Springer Nature B.V.
Subject
Life Sciences; Microbiology; Biotechnology; Applied Microbiology; Biochemistry, general
ISSN
0141-5492
eISSN
1573-6776
D.O.I.
10.1007/s10529-018-2566-8
Publisher site
See Article on Publisher Site

Abstract

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

Journal

Biotechnology LettersSpringer Journals

Published: Jun 5, 2018

References

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off