Prozessentwicklung mit maßgeschneiderten rekombinanten Biokatalysatoren zur Synthese einer Bulk- und einer Feinchemikalie

Development of a biocatalytic process is a challenging task. Thus, herein general molecular biological and engineering concepts were developed to overcome typical limitations in industrial process development exemplified by the production of the bulk-chemical ε-caprolactone and the fine chemical (S)-2,2,2-trifluor-1-phenylethanol.<br /><br /> Based on the literature known proof of concept for the enzymatic double oxidation of cyclohexanol by means of an alcohol dehydrogenase and a cyclohexanone monooxygenase towards ε-caprolactone without isolation of the intermediate cyclohexanone, inhibition of the monooxygenase especially by the product was addressed by solvent engineering. However, increase of productivity was limited since the organic solvents caused biocatalyst inactivation. Separation of the aqueous and organic phase with a polydimethylsiloxane membrane, being permeable for ε-caprolactone but impermeable for the extracting solvent methyl cyclohexane, significantly improved the productivity. Whole-cell catalyst design based on harmonisation of the process windows (activity, half-life and melting temperature) of both enzymes enabled self-sufficient regeneration of host internal cofactors, so that high costs for external addition of cofactors or auxiliaries are saved. Buffer screening and selection further improved process economy but also sustainability. The monooxygenase as the limiting enzyme was subjected to in vivo circularization as a new protein engineering approach in the field of applied biocatalysis. The best circular mutant was superior to the wildtype and previously described mutants in terms of process activity and stability. The concept of in situ extraction was extended for the production of polycaprolactone by direct polymerization of ε-caprolactone in the organic compartment by means of lipase B yielding a polymer fraction having a degree of polymerization comparable to commercially available polycaprolactone. During experimental work, it was observed that the productivity of biotransformations dramatically depends on the enzyme´s storage time, having tremendous consequences for industry and academia. Combined with results from literature a model explaining this phenomen could be evolved.<br /><br /> The asymmetric reduction of 2,2,2-trifluoracetophenone by means of an alcohol dehydrogenase combined with a glucose dehydrogenase for cofactor regeneration was used to search for a general rule for the selection of organic solvents applied for increasing substrate solubility. Herein, no correlation between characeristics of the organic solvent like logPo/w and the activity or stability of the enzymes in presenc of the solvents could be found.<br /><br /> To date measurement of the melting temperature was limited to purified enzymes. Thus scrrening for enzymes and mutants with improved thermostability was time- and cost-intensive process. By means of nano differential scanning fluorimetry a high-throughput protocoll could be devised enabling the determination of the melting temperature of even two simultaneously recombinant, overexpressed enzymes in crude extracts.<br /><br /> Finally, basics for the investigation of organic solvent induced cofactor leaching were layed.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bielefeld

Year

2019

Author dc:creator

• Wedde, Severin

Repository record source_url

https://pub.uni-bielefeld.de/record/2999827

OAI identifier oai:identifier

oai:pub.uni-bielefeld.de:2999827