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Coupled reactions by coupled enzymes
- Source :
- Applied Microbiology and Biotechnology, Applied Microbiology and Biotechnology, 20(101), 7557-7565. SPRINGER
- Publication Year :
- 2017
- Publisher :
- SPRINGER, 2017.
-
Abstract
- The combination of redox enzymes for redox-neutral cascade reactions has received increasing appreciation. An example is the combination of an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO). The ADH can use NADP+ to oxidize cyclohexanol to form cyclohexanone and NADPH. Both products are then used by CHMO to produce ε-caprolactone. In this study, these two redox-complementary enzymes were fused, to create a self-sufficient bifunctional enzyme that can convert alcohols to esters or lactones. Three different ADH genes were fused to a gene coding for a thermostable CHMO, in both orientations (ADH-CHMO and CHMO-ADH). All six fusion enzymes could be produced and purified. For two of the three ADHs, we found a clear difference between the two orientations: one that showed the expected ADH activity, and one that showed low to no activity. The ADH activity of each fusion enzyme correlated with its oligomerization state. All fusions retained CHMO activity, and stability was hardly affected. The TbADH-TmCHMO fusion was selected to perform a cascade reaction, producing ε-caprolactone from cyclohexanol. By circumventing substrate and product inhibition, a > 99% conversion of 200 mM cyclohexanol could be achieved in 24 h, with > 13,000 turnovers per fusion enzyme molecule. Electronic supplementary material The online version of this article (10.1007/s00253-017-8501-4) contains supplementary material, which is available to authorized users.
- Subjects :
- Stereochemistry
Recombinant Fusion Proteins
Cyclohexanol
Cyclohexanone
010402 general chemistry
01 natural sciences
Applied Microbiology and Biotechnology
Lactones
chemistry.chemical_compound
Cascade reaction
DESIGN
Enzyme fusion
Phosphofructokinase 2
STRATEGY
Biotechnologically Relevant Enzymes and Proteins
Caproates
Alcohol dehydrogenase
chemistry.chemical_classification
EPSILON-CAPROLACTONE
BIOCATALYSIS
biology
CHALLENGES
Cyclohexanones
010405 organic chemistry
General Medicine
Cyclohexanols
0104 chemical sciences
Enzyme
chemistry
Biochemistry
Product inhibition
Biocatalysis
Alcohols
Oxygenases
biology.protein
Cascade
Cyclohexanone monooxygenase
Oxidation-Reduction
NADP
hormones, hormone substitutes, and hormone antagonists
SYSTEM
Biotechnology
Subjects
Details
- Language :
- English
- ISSN :
- 01757598
- Volume :
- 20
- Issue :
- 101
- Database :
- OpenAIRE
- Journal :
- Applied Microbiology and Biotechnology
- Accession number :
- edsair.doi.dedup.....7e1acf274181a468ff15973d565e1fa1