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Activity improvement of a Kluyveromyces lactis aldo-keto reductase KlAKR via rational design

Paper ID Volume ID Publish Year Pages File Format Full-Text
22728 43374 2016 7 PDF Available
Title
Activity improvement of a Kluyveromyces lactis aldo-keto reductase KlAKR via rational design
Abstract

•Significantly enhanced the activity of KlAKR via rational design.•Robust mutant for t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate synthesis was gained.•The mechanism was elucidated by molecular docking.•KlAKR-Y295W/W296L showed improved activity toward the tested carbonyl compounds.

Optically pure t-butyl 6-cyano-(3R, 5R)-dihydroxyhexanoate ((R)-1b) is the key chiral precursor for atorvastatin calcium, the most widely used cholesterol-lowering drug. Wild-type aldo-keto reductase KlAKR from Kluyveromyces lactis has ideal diastereoselectivity toward t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (1a, dep >99.5%) but poor activity. A rational engineering was used to improve the KlAKR activity. Based on homology modeling and molecular docking, two amino acid residues (295 and 296) were selected as mutation sites, and two rounds of site-saturation mutagenesis were performed. Among the mutants, KlAKR-Y295W/W296L exhibited the highest catalytic efficiency (kcat/Km) toward 1a up to 12.37 s−1 mM−1, which was 11.25-fold higher than that of wild-type KlAKR. Moreover, the majority of mutations have no negative impact on stereoselectivity. Using KlAKR-Y295W/W296L coupled with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for cofactor regeneration, (R)-1b was accumulated up to 162.7 mM with dep value above 99.5%. KlAKR-Y295W/W296L represents a robust tool for (R)-1b synthesis.

Keywords
Aldo-keto reductase; Atorvastatin calcium; Rational engineering; Site-saturation mutagenesis; T-butyl 6-cyano-(3R, 5R)-dihydroxyhexanoate
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Activity improvement of a Kluyveromyces lactis aldo-keto reductase KlAKR via rational design
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 224, 20 April 2016, Pages 20–26
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us