ABSTRACT
The application of enzymes as biocatalysts in industrial processes has great potential due to their outstanding stereo-, regio- and chemoselectivity. Using autodisplay, enzymes can be immobilized on the cell surface of Gram-negative bacteria such as Escherichia coli. In the present study, the surface display of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO) on E. coli was investigated. Displaying these enzymes on the surface of E. coli resulted in whole-cell biocatalysts accessible for substrates without further purification. An apparent maximal reaction velocity VMAX(app) for the oxidation of cyclohexanol with the ADH whole-cell biocatalysts was determined as 59.9 mU ml-1 . For the oxidation of cyclohexanone with the CHMO whole-cell biocatalysts a VMAX(app) of 491 mU ml-1 was obtained. A direct conversion of cyclohexanol to ε-caprolactone, which is a known building block for the valuable biodegradable polymer polycaprolactone, was possible by combining the two whole-cell biocatalysts. Gas chromatography was applied to quantify the yield of ε-caprolactone. 1.12 mM ε-caprolactone was produced using ADH and CHMO displaying whole-cell biocatalysts in a ratio of 1:5 after 4 h in a cell suspension of OD578nm 10. Furthermore, the reaction cascade as applied provided a self-sufficient regeneration of NADPH for CHMO by the ADH whole-cell biocatalyst.
Subject(s)
Alcohol Dehydrogenase , Escherichia coli , Alcohol Dehydrogenase/metabolism , Caproates , Cyclohexanols/metabolism , Escherichia coli/metabolism , Lactones , NADP/metabolism , Oxidation-Reduction , Oxygenases/metabolismABSTRACT
Multi-enzyme cascade reactions capture the essence of nature's efficiency by increasing the productivity of a process. Here we describe one such three-enzyme cascade for the synthesis of 6-hydroxyhexanoic acid. Whole cells of Escherichia coli co-expressing an alcohol dehydrogenase and a Baeyer-Villiger monooxygenase (CHMO) for internal cofactor regeneration were used without the supply of external NADPH or NADP+. The product inhibition caused by the ε-caprolactone formed by the CHMO was overcome by the use of lipase CAL-B for in situ conversion into 6-hydroxyhexanoic acid. A stirred tank reactor under fed-batch mode was chosen for efficient catalysis. By using this setup, a product titre of >20 g L-1 was achieved in a 500 mL scale with an isolated yield of 81% 6-hydroxyhexanoic acid.
Subject(s)
Alcohol Dehydrogenase/genetics , Caproates/chemical synthesis , Escherichia coli Proteins/genetics , Escherichia coli/enzymology , Fungal Proteins/chemistry , Hydroxy Acids/chemical synthesis , Lipase/chemistry , Mixed Function Oxygenases/genetics , Alcohol Dehydrogenase/metabolism , Batch Cell Culture Techniques , Biocatalysis , Bioreactors , Caproates/chemistry , Caproates/metabolism , Coenzymes/biosynthesis , Coenzymes/chemistry , Escherichia coli/genetics , Escherichia coli Proteins/metabolism , Fungal Proteins/metabolism , Gene Expression , Hydroxy Acids/metabolism , Kinetics , Lactones/chemistry , Lactones/metabolism , Lipase/metabolism , Mixed Function Oxygenases/metabolism , NADP/biosynthesis , NADP/chemistryABSTRACT
The introduction of a three-enzyme cascade (comprising a cyclohexanone monooxygenase (CHMO), an alcohol dehydrogenase (ADH) and a lipase (CAL-A)) for the production of oligo-ε-caprolactone provided self-sufficiency with respect to NADPH-cofactor regeneration and reduced inhibiting effects on the central CHMO enzyme. For further optimization of cofactor regeneration, now a co-expression of CHMO and ADH in E. coli using a Duet™ vector was performed. This led to higher conversion values of the substrate cyclohexanol in whole-cell biocatalysis compared to an expression of both enzymes from two separate plasmids. Furthermore, a more advantageous balance of expression levels between the partial cascade enzymes was achieved via engineering of the ribosome binding site. This contributed to an even faster cofactor regeneration rate.