title: Continuous testing system for Baeyer-Villiger biooxidation using recombinant Escherichia coli expressing cyclohexanone monooxygenase encapsulated in polyelectrolyte complex capsules.
abstract: An original strategy for universal laboratory testing of Baeyer-Villiger monooxygenases based on continuous packed-bed minireactor connected with flow calorimeter and integrated with bubble-free oxygenation is reported. Model enantioselective Baeyer-Villiger biooxidations of rac-bicyclo[3.2.0]hept-2-en-6-one to corresponding lactones (1R,5S)-3-oxabicyclo-[3.3.0]oct-6-en-3-one and (1S,5R)-2-oxabicyclo-[3.3.0]oct-6-en-3-one as important chiral synthons for the synthesis of bioactive compounds were performed in the minireactor equipped with a column packed with encapsulated recombinant cells Escherichia coli overexpressing cyclohexanone monooxygenase. The cells were encapsulated in polyelectrolyte complex capsules formed by reaction of oppositely charged polymers utilizing highly reproducible and controlled encapsulation process. Encapsulated cells tested in minireactor exhibited high operational stability with 4 complete substrate conversions to products and 6 conversions above 80% within 14 repeated consecutive biooxidation tests. Moreover, encapsulated cells showed high enzyme stability during 91 days of storage with substrate conversions above 80% up to 60 days of storage. Furthermore, usable thermometric signal of Baeyer-Villiger biooxidation obtained by flow calorimetry using encapsulated cells was utilized for preparatory kinetic study in order to guarantee sub-inhibitory initial substrate concentration for biooxidation tests.
authors: Department of Glycobiotechnology, Institute of Chemistry - Center for Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, SK-845 38 Bratislava, Slovakia. Marek.Bucko@savba.sk
authors: Marek
authors: M
authors: Bučko
authors: Andrea
authors: A
authors: Schenkmayerová
authors: Peter
authors: P
authors: Gemeiner
authors: Alica
authors: A
authors: Vikartovská
authors: Marko D
authors: MD
authors: Mihovilovič
authors: Igor
authors: I
authors: Lacík
mesh_terms: D016954:Acinetobacter calcoaceticus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism*; D019149:Bioreactors; D002151:Calorimetry; D004339:Drug Compounding; D004795:Enzyme Stability; D004867:Equipment Design; D004926:Escherichia coli / Q000201:enzymology*; D007218:Industrial Microbiology / Q000295:instrumentation / Q000379:methods*; D007659:Ketones / Q000378:metabolism; D007700:Kinetics; D007783:Lactones / Q000378:metabolism; D008832:Microchemistry / Q000295:instrumentation; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D007659:Ketones; D007783:Lactones; D011993:Recombinant Fusion Proteins; D010105:Oxygenases; C019961:cyclohexanone oxygenase