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Stabilization of cyclohexanone monooxygenase by computational and experimental library design
- Source :
- Biotechnology and Bioengineering, Biotechnology and Bioengineering, 116(9), 2167-2177. Wiley-Blackwell
- Publication Year :
- 2019
- Publisher :
- Wiley-Blackwell, 2019.
-
Abstract
- Enzymes often by far exceed the activity, selectivity, and sustainability achieved with chemical catalysts. One of the main reasons for the lack of biocatalysis in the chemical industry is the poor stability exhibited by many enzymes when exposed to process conditions. This dilemma is exemplified in the usually very temperature‐sensitive enzymes catalyzing the Baeyer–Villiger reaction, which display excellent stereo‐ and regioselectivity and offer a green alternative to the commonly used, explosive peracids. Here we describe a protein engineering approach applied to cyclohexanone monooxygenase from Rhodococcus sp. HI‐31, a substrate‐promiscuous enzyme that efficiently catalyzes the production of the nylon‐6 precursor ε‐caprolactone. We used a framework for rapid enzyme stabilization by computational libraries (FRESCO), which predicts protein‐stabilizing mutations. From 128 screened point mutants, approximately half had a stabilizing effect, albeit mostly to a small degree. To overcome incompatibility effects observed upon combining the best hits, an easy shuffled library design strategy was devised. The most stable and highly active mutant displayed an increase in unfolding temperature of 13°C and an approximately 33x increase in half‐life at 30°C. In contrast to the wild‐type enzyme, this thermostable 8x mutant is an attractive biocatalyst for biotechnological applications.<br />This work describes the stabilization of an e‐caprolactone‐producing cyclohexanone monooxygenase through protein engineering. Applying a computational pipeline for predicting stabilizing point mutations and an innovative shuffled library strategy for their combination, the authors report an increase of 13 °C of the melting temperature of the notoriously unstable enzyme, while the half‐life improved 33‐fold.
- Subjects :
- Biocatalysis, Protein Engineering and Nanobiotechnology
computational design
Cyclohexanone
Bioengineering
ALCOHOL
Baeyer–Villiger monooxygenase
010402 general chemistry
01 natural sciences
Applied Microbiology and Biotechnology
Article
Catalysis
ARTICLES
03 medical and health sciences
chemistry.chemical_compound
Bacterial Proteins
Peptide Library
BAEYER-VILLIGER-MONOOXYGENASES
Enzyme Stability
Rhodococcus
CRYSTAL-STRUCTURE
030304 developmental biology
Phenylacetone monooxygenase
Thermostability
chemistry.chemical_classification
0303 health sciences
CATALYST
STABILITY
Chemistry
MUTATIONS
Computational Biology
Substrate (chemistry)
Regioselectivity
Combinatorial chemistry
thermostability
0104 chemical sciences
stabilization
Enzyme
Biocatalysis
Mutation
Oxygenases
cyclohexanone
ENZYMES
Biotechnology
PHENYLACETONE MONOOXYGENASE
Subjects
Details
- Language :
- English
- ISSN :
- 00063592
- Volume :
- 116
- Issue :
- 9
- Database :
- OpenAIRE
- Journal :
- Biotechnology and Bioengineering
- Accession number :
- edsair.doi.dedup.....27e2158088d9780533c01f84b57c034c