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Molekular geprägte Polymere (MIPs) sind künstliche Rezeptoren mit selektiven Erkennungsstellen für Zielmoleküle. Eine der vielversprechendsten Strategien für Protein‐MIPs beruht auf der Nutzung kurzer, oberflächenexponierter Proteinfragmente, sogenannter Epitope, als Schablonen für die Prägung von Bindungsstellen in einem Polymergerüst für ein spezifisches (Ziel‐)Protein. Der Mangel an hochauflösenden Strukturdaten von flexiblen oberflächenexponierten Regionen erschwert jedoch die Auswahl geeigneter Epitope. Hier haben wir diesen Nachteil durch die Entwicklung eines MIPs auf Polyscopoletin‐Basis behoben, das rekombinante Proteine über das weit verbreitete Affinitätspeptid Strep‐tag II erkennt. Elektrochemie, oberflächensensitive IR‐Spektroskopie und Molekulardynamiksimulationen wurden eingesetzt, um eine größtmögliche Kontrolle der Strep‐MIP‐Elektrosynthese zu gewährleisten. Die Funktionalität dieser neuartigen Plattform wurde mit zwei Strep‐Tag‐markierten Enzymen überprüft: einer O2‐toleranten [NiFe]‐Hydrogenase und einer alkalischen Phosphatase. Beide Enzyme behielten ihre biokatalytischen Aktivitäten nach mehrfacher Verwendung und bestätigten damit die Effizienz von dem Strep‐MIP als generell biokompatible Plattform zur Bindung rekombinanter Proteine für die Nutzung in der Biotechnologie. [ABSTRACT FROM AUTHOR]

Molecularly imprinted polymers (MIPs) are artificial receptors equipped with selective recognition sites for target molecules. One of the most promising strategies for protein MIPs relies on the exploitation of short surface‐exposed protein fragments, termed epitopes, as templates to imprint binding sites in a polymer scaffold for a desired protein. However, the lack of high‐resolution structural data of flexible surface‐exposed regions challenges the selection of suitable epitopes. Here, we addressed this drawback by developing a polyscopoletin‐based MIP that recognizes recombinant proteins via imprinting of the widely used Strep‐tag II affinity peptide (Strep‐MIP). Electrochemistry, surface‐sensitive IR spectroscopy, and molecular dynamics simulations were employed to ensure an utmost control of the Strep‐MIP electrosynthesis. The functionality of this novel platform was verified with two Strep‐tagged enzymes: an O2‐tolerant [NiFe]‐hydrogenase, and an alkaline phosphatase. The enzymes preserved their biocatalytic activities after multiple utilization confirming the efficiency of Strep‐MIP as a general biocompatible platform to confine recombinant proteins for exploitation in biotechnology. [ABSTRACT FROM AUTHOR]

Sophisticated enzymatic systems have evolved in nature to efficiently couple distinct biochemical reactions in form of cascades. As such, they serve as reference models to understand the indirect interactions of catalytic centers. Herein, we studied, in solution, the coupling of the reactions from membrane‐bound [NiFe] hydrogenase (MBH) from Cupriavidus necator (reversible H2 splitting into H+ and e−) and the molybdenum‐dependent formate dehydrogenase from Rhodobacter capsulatus (reversible formate to CO2 interconversion). To follow their interplay via the characteristic absorptions from the MBH's active site or the respective substrate and product bands of FDH, we utilized in situ IR spectrocopy and GC(‐MS), in the absence and presence of soluble redox mediators. Coarse‐grained molecular dynamics (CGMD) computations revealed the lack of productive enzyme complexes for direct electron transfer (ET). Thus, the observed minor amounts of H2 or formate were produced from transient interactions between the two enzymes. On the contrary, the significantly increased product formation in the presence of methyl viologen can be related to the putative multiple interaction sites of the redox mediator with FDH identified by CGMD. Our study represents a proof‐of‐concept approach that can be used in the future to develop novel coupled biocatalytic systems by identifying potential ET pathways. [ABSTRACT FROM AUTHOR]

For the first time, an enzyme‐based electrochemical biosensor system for determination of trimethylamine N‐oxide (TMAO) is described. It employs an active chimeric variant of TorA in combination with an enzymatically deoxygenating system and a low‐potential mediator for effective regeneration of the enzyme and cathodic current generation. TMAO reductase (TorA) is a molybdoenzyme found in marine and most enterobacteria that specifically catalyzes the reduction of TMAO to trimethylamine (TMA). The chimeric TorA, named TorA‐FDH, corresponds to the apoform of TorA from Escherichia coli reconstituted with the molybdenum cofactor from formate dehydrogenase (FDH). Each enzyme, TorA and TorA‐FDH, was immobilized on the surface of a carbon electrode and protected with a dialysis membrane. The biosensor operates at an applied potential of −0.8 V [vs. Ag/AgCl (1 M KCl)] under ambient air conditions thanks to an additional enzymatic O2‐scavenger system. A comparison between the two enzymatic sensors revealed a much higher sensitivity for the biosensor with immobilized TorA‐FDH. This biosensor exhibits a sensitivity of 14.16 nA/μM TMAO in a useful measuring range of 2–110 μM with a detection limit of LOD=2.96 nM (S/N=3), and was similar for TMAO in buffer and in spiked serum samples. With a response time of 16±2 s, the biosensor is stable over prolonged daily measurements (n=20). This electrochemical biosensor provides suitable applications in detecting TMAO levels in human serum. Chimeras for biosensing: An enzyme‐based electrochemical biosensor for the determination of trimethylamine N‐oxide (TMAO) is presented, where enhanced sensitivity is obtained by utilizing a chimeric TorA, corresponding to the apoform of TorA from Escherichia coli reconstituted with the molybdenum cofactor from the Rhodobacter capsulatus formate dehydrogenase. Structure of Shewanella massilia TorA (PDB: 1tmo). [ABSTRACT FROM AUTHOR]