Biocatalysis with Escherichia coli-overexpressing cyclopentanone monooxygenase immobilized in polyvinyl alcohol gel.
This is the first reported study on the immobilization of living recombinant Escherichia coli cells that overexpress cyclopentanone monooxygenase in polyvinyl alcohol gel particles LentiKats®. Immobilized cells overexpressing cyclopentanone monooxygenase have been used as a model of biocatalyst for enantioselective Baeyer-Villiger biooxidation of rac-bicyclo[3.2.0]hept-2-en-6-one into regioisomeric lactones. This process is useful for the syntheses of cytostatic sarkomycin, several prostaglandins and other biologically active compounds. The original technique for qualitative analysis of enzyme expression within free cells and cells entrapped in LentiKats® using SDS-PAGE was developed and used for verification of optimal conditions for the induction of cyclopentanone monooxygenase. Here, we successfully performed six repeated batch Baeyer-Villiger biooxidations utilizing entrapped cells using 40% (w/v) polyvinyl alcohol gel particles in flasks with baffles. The latter conditions have been found to be the most appropriate achieving optimal oxygen transfer within LentiKats®. Moreover, immobilized cells retained their catalytic efficiency over six reaction cycles, while the catalytic efficiency of free cells decreased after three reaction cycles.


CONCLUSIONS
Immobilization in polyvinylalcohol gel particles is desirable technique with presumptive impact on industrial applications of recombinant whole-cell Baeyer-Villiger monooxygenases as biocatalysts for production of bioactive compounds and precursors of potentially new drugs. An original immobilization of cells E. coli with overproduced Baeyer-Villiger monooxygenase improved their stability in repetitive batch biooxidations as compared to free cells. Detected autoinduction of recombinant enzyme in pET22b+ plays significant role in application of immobilized cells as it may increase specific activity of cells in repetitive use under growing reaction conditions. Original technique for qualitative analysis of enzyme expression within immobilized cells was developed.
Institute of Biotechnology and Food Science, Faculty of Chemical and Food Technology, Slovak University of Technology, Bratislava, Slovakia.
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Rebroš
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Lipták
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Rosenberg
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Bučko
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Gemeiner
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry; D055162:Biocatalysis; D019149:Bioreactors; D018914:Cells, Immobilized / Q000201:enzymology; D020585:Comamonas / Q000201:enzymology; D003470:Culture Media; D004926:Escherichia coli / Q000378:metabolism*; D005782:Gels; D007783:Lactones; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D010105:Oxygenases / Q000096:biosynthesis* / Q000737:chemistry; D011142:Polyvinyl Alcohol / Q000737:chemistry*; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D003470:Culture Media; D005782:Gels; D007783:Lactones; D011142:Polyvinyl Alcohol; D010105:Oxygenases; C067215:cyclopentanone monooxygenase; D010100:Oxygen
Baeyer-Villiger biooxidation; E. coli; LentiKats®; cyclopentanone monooxygenase; immobilization