Abstract
ε-Caprolactone is a monomer of poly(ε-caprolactone) which has been widely used in tissue engineering due to its biodegradability and biocompatibility. To meet the massive demand for this monomer, an efficient whole-cell biocatalytic approach was constructed to boost the ε-caprolactone production using cyclohexanol as substrate. Combining an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO) in Escherichia coli, a self-sufficient NADPH-cofactor regeneration system was obtained. Furthermore, some improved variants with the better substrate tolerance and higher catalytic ability to ε-caprolactone production were designed by regulating the ribosome binding sites. The best mutant strain exhibited an ε-caprolactone yield of 0.80 mol/mol using 60 mM cyclohexanol as substrate, while the starting strain only got a conversion of 0.38 mol/mol when 20 mM cyclohexanol was supplemented. The engineered whole-cell biocatalyst was used in four sequential batches to achieve a production of 126 mM ε-caprolactone with a high molar yield of 0.78 mol/mol.
Highlights
As people face the global changes in energy, resources, and the environment, biocatalysis attracts great attentions in chemical, pharmaceutical and energy industries because of its high activity, selectivity, specificity and low energy requirements
Many enzymatic redox applications are limited by the dependence on cofactors as hydrogen source, such as nicotinamide adenine dinucleotide (NADH) and its phosphorylated form
alcohol dehydrogenase (ADH) and cyclohexanone monooxygenase (CHMO) expression and enzyme activity determination To balance the NADP(H) regeneration and intermediate consumption during the ε-caprolactone bi-enzymatic synthesis, the ratio of ADH and CHMO is very important for their different specific activities and expression levels
Summary
As people face the global changes in energy, resources, and the environment, biocatalysis attracts great attentions in chemical, pharmaceutical and energy industries because of its high activity, selectivity, specificity and low energy requirements. The regeneration of cofactor NAD(P)H can be conducted via enzymatic, chemical, photocatalytic and electrochemical approaches. Using inorganic salts with high redox potential or cofactor analogues, the NAD(P)H could be regenerated chemically (Wu et al 2013). It has not been widely used for its inherent issues, such as low transformation efficiency, enzyme deactivation, waste generated, etc. Photocatalytic and electrochemical methods attracted many attentions in recent years, more further studies and investigations are needed for industrial application due to its poor efficiency, bad compatibility and low selectivity
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