Coupling of electrochemical and optical measurements in a microtiter plate for the fast development of electro enzymatic processes with P450s

Ley, Claudia; Schewe, Hendrik; Ströhle, Frank W.; Ruff, Anne Joelle; Schwaneberg, Ulrich; Schrader, Jens; Holtmann, Dirk

doi:10.1016/j.molcatb.2013.03.019

Cited by 24 publications

(12 citation statements)

References 43 publications

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“…A broad set of cofactor regeneration methods has been successfully developed in the last years. Besides enzymatic cofactor regenerations, electrochemical substituting (Çekiç et al, ; Ley et al, ; Reipa et al, ; Udit et al, ) and chemocatalytic cofactor regenerations (Hollmann and Schmid, ; Kölle and Grützel, ; Ruppert et al, ) have also been reported. In this study the surface displayed P450BM3 were combined with electrochemical, chemical and enzymatic regeneration systems.…”

Section: Resultsmentioning

confidence: 99%

A simplified process design for P450 driven hydroxylation based on surface displayed enzymes

Ströhle

Kranen²,

Schrader

et al. 2015

Biotech & Bioengineering

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New production routes for fine and bulk chemicals are important to establish further sustainable processes in industry. Besides the identification of new biocatalysts and new production routes the optimization of existing processes in regard to an improved utilization of the catalysts are needed. In this paper we describe the successful expression of P450BM3 on the surface of E. coli cells with the Autodisplay system. The successful hydroxylation of palmitic acid by using surface-displayed P450BM3 was shown. Besides optimization of surface protein expression, several cofactor regeneration systems were compared and evaluated. Afterwards, the development of a suitable process for the biocatalytic hydroxylation of fatty acids based on the re-use of the catalysts after a simple centrifugation was investigated. It was shown that the catalyst can be used for several times without any loss in activity. By using surface-displayed P450s in combination with an enzymatic cofactor regeneration system a total turnover number of up to 54,700 could be reached, to the knowledge of the authors the highest value reported for a P450 monooxygenase to date. Further optimizations of the described reaction system can have an enormous impact on the process design for more sustainable bioprocesses. Biotechnol. Bioeng. 2016;113: 1225-1233. © 2015 Wiley Periodicals, Inc.

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Section: Resultsmentioning

confidence: 99%

A simplified process design for P450 driven hydroxylation based on surface displayed enzymes

Ströhle

Kranen²,

Schrader

et al. 2015

Biotech & Bioengineering

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“…70 Also increasing the electron transfer rate between the mediator molecule and the P450 heme-site represents a very promising approach to address the Oxygen Dilemma. [84][85][86][87][88][89][90][91][92] Mechanistically, photochemical reduction is very similar to electrochemical regeneration of P450 monooxygenases. Hence it is not very astonishing that also (in)direct photochemical regeneration of P450 monooxygenases has been investigated.…”

Section: Reductive Regeneration Of Heme-enzymesmentioning

confidence: 99%

Nonconventional regeneration of redox enzymes – a practical approach for organic synthesis?

Zhang

Hollmann

2018

Chem. Commun.

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Oxidoreductases have become useful tools in the hands of chemists to perform selective and mild oxidation and reduction reactions. Instead of mimicking native catalytic cycles, generally involving costly and unstable nicotinamide cofactors, more direct, NAD(P)-independent methodologies are being developed. The promise of these approaches not only lies with simpler and cheaper reaction schemes but also with higher selectivity as compared to whole cell approaches and their mimics.

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“…Another challenge while developing an enzyme for electrobiocatalytic application is a high throughput screening system. Screening using a combination of electromicrotiter plates (eMTPs) (Ley et al, 2013)…”

Section: Discussionmentioning

confidence: 99%

Directed evolution of P450cin for mediated electron transfer

Belsare

Horn

Ruff

et al. 2016

Protein Engineering, Design and Selection

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Directed evolution is a powerful method to optimize enzyme properties for application demands. Interesting targets are P450 monooxygenases which catalyze the stereo- and regiospecific hydroxylation of chemically inert C-H bonds. Synthesis employing P450s under cell-free reaction conditions is limited by low total turnover numbers, enzyme instability, low product yields and the requirement of the expensive co-factor NADPH. Bioelectrocatalysis is an alternative to replace NADPH in cell-free P450-catalyzed reactions. However, natural enzymes are often not suitable for using non-natural electron delivery systems. Here we report the directed evolution of a previously engineered P450 CinA-10aa-CinC fusion protein (named P450cin-ADD-CinC) to use zinc/cobalt(III)sepulchrate as electron delivery system for an increased hydroxylation activity of 1,8-cineole. Two rounds of Sequence Saturation Mutagenesis (SeSaM) each followed by one round of multiple site-saturation mutagenesis of the P450 CinA-10aa-CinC fusion protein generated a variant (Gln385His, Val386Ser, Thr77Asn, Leu88Arg; named KB8) with a 3.8-fold increase in catalytic efficiency (28 µM min) compared to P450cin-ADD-CinC (7 µM min). Furthermore, variant KB8 exhibited a 1.5-fold higher product formation (500 µM µM P450) compared to the equimolar mixture of CinA, CinC and Fpr using NADPH as co-factor (315 µM µM P450). In addition, electrochemical experiments with the electron delivery system platinum/cobalt(III)sepulchrate showed that the KB8 variant had a 4-fold higher product formation rate (0.16 nmol (nmol) P450 min cm) than the P450cin-ADD-CinC (0.04 nmol (nmol) P450 min cm). In summary, the current work shows prospects of using directed evolution to generate P450 enzymes suitable for use with alternative electron delivery systems.

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Coupling of electrochemical and optical measurements in a microtiter plate for the fast development of electro enzymatic processes with P450s

Cited by 24 publications

References 43 publications

A simplified process design for P450 driven hydroxylation based on surface displayed enzymes

A simplified process design for P450 driven hydroxylation based on surface displayed enzymes

Nonconventional regeneration of redox enzymes – a practical approach for organic synthesis?

Directed evolution of P450cin for mediated electron transfer

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