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Exporling the Evolvability of Old Yellow Enzymes for Organic Synthesis
In the presented thesis, guidelines for the evolution of flavin-dependent ene reductases, an in-dustrially important catalyst class, are reported. In the first part of this thesis it should be tested if mining the existing knowledge of the Old Yellow Enzyme family (OYE), obtained from directed evol...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2017
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| Online Zugang: | PDF-Volltext |
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| Zusammenfassung: | In the presented thesis, guidelines for the evolution of flavin-dependent ene reductases, an in-dustrially important catalyst class, are reported.
In the first part of this thesis it should be tested if mining the existing knowledge of the Old Yellow Enzyme family (OYE), obtained from directed evolution studies, may allow guided traversing through the sequence space and thereby shortcutting biocatalyst development. Iden-tified hotspot positions of YqjM from Bacillus subtilis, i.e. C26D/I69T and C26G for improvement of activity and stereoselectivity, respectively, were transferred to seven OYE scaffolds. The new-ly created variants were tested with three compounds revealing more stereocomplementary OYE pairs with potent turnover frequencies (up to 660 h-1) and excellent stereoselectivities (up to >99%). Although systematic prediction of absolute enantioselectivity still remains for OYE variants, ‘scaffold sampling’ was confirmed as a fast engineering method for this family allow-ing access to new, potent biocatalysts for organic synthesis.
In the second part of this thesis the development and characterisation of an engineered panel of ene reductases (ERs) from Thermus scotoductus SA-01 (TsER) is reported, that combines control over facial selectivity in the reduction of electron deficient carbon-carbon double bonds with thermostability (up to 70 °C), organic solvent tolerance (up to 40% (v/v)) and a broad substrate scope (23 compounds, three of them new). The panel shows excellent enantiomeric excess (ee) and yields during gram scale synthesis (3.8 g). Exquisite turnover frequencies (TOF) up to 40 000 h-1 are achieved, which are comparable to rates in hetero- and homogeneous metal cata-lysed hydrogenations. Efforts to rationalize the stereocomplementarity are reported, using the obtained crystal structure of TsER C25D/I67T and in silico docking studies. Our holistic charac-terisation, together with the preparative scale reactions, shows that these engineered ERs are truly practical catalysts for preparative organic synthesis.
In the third section the aforementioned panel of TsER variants was screened for bulkier sub-strate classes and further mutation sites were identified over semi-rational design for the suc-cessful biotransformation of coumarin-like structures. Thereby chemoselective variants with either hydrogenation or evidence for acid/base catalysis in the active site of TsER have been discovered. In general there is a great interest in using these highly selective trans-hydrogenation catalysts in the late stage synthesis of complex organic molecules. |
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| Umfang: | 305 Seiten |
| DOI: | 10.17192/z2017.0773 |