Streptavidin-Enzyme Linked Aggregates for the One-Step Assembly and Purification of Enzyme Cascades

Herein, we report enzyme aggregates assembled around covalently cross-linked streptavidin tetramers. The streptavidin oligomeric matrix (Sav(Matrix)) is produced by using SpyTag/SpyCatch technology and binds tightly to fusion proteins bearing a streptavidin-binding peptide (SBP). Fusing the SBPs to different enzymes leads to precipitation of the streptavidin-enzyme aggregates upon mixing the complementary components. This straightforward strategy can be applied to crude cell-free extracts, allowing the one-step assembly and purification of catalytically active aggregates. Enzyme cascade assemblies can be produced upon adding different SBP-fused enzymes to the Sav(Matrix). The reaction rate for lactate dehydrogenase (LDH) is improved tenfold (compared with the soluble enzyme) upon precipitation with the Sav(Matrix) from crude cell-free extracts. Additionally, the kinetic parameters are improved. A cascade combining a transaminase with LDH for the synthesis of enantiopure amines from prochiral ketones displays nearly threefold rate enhancement for the synthesis of (R)-alpha-methylbenzylamine compared with the free enzymes in solution

An Enantioselective Artificial Suzukiase Based on the Biotin–Streptavidin Technology

Introduction of a biotinylated monophosphine palladium complex within streptavidin affords an enantioselective artificial Suzukiase. Site-directed mutagenesis allowed the optimization of the activity and the enantioselectivity of this artificial metalloenzyme. A variety of atropisomeric biaryls were produced in good yields and up to 90% ee. The hybrid catalyst described herein shows comparable TOF to the previous aqueous-asymmetric Suzuki catalysts, and excellent stability under the reaction conditions to realize higher TON through longer reaction time

Genetic Engineering of an Artificial Metalloenzyme for Transfer Hydrogenation of a Self-Immolative Substrate in Escherichia coli's Periplasm

Artificial metalloenzymes (ArMs), which combine an abiotic metal cofactor with a protein scaffold, catalyze various synthetically useful transformations. To complement the natural enzymes' repertoire, effective optimization protocols to improve ArM's performance are required. Here we report on our efforts to optimize the activity of an artificial transfer hydrogenase (ATHase) using Escherichia coli whole cells. For this purpose, we rely on a self-immolative quinolinium substrate which, upon reduction, releases fluorescent umbelliferone, thus allowing efficient screening. Introduction of a loop in the immediate proximity of the Ir-cofactor afforded an ArM with up to 5-fold increase in transfer hydrogenation activity compared to the wild-type ATHase using purified mutants

Breaking Symmetry: Engineering Single-Chain Dimeric Streptavidin as Host for Artificial Metalloenzymes

The biotin-streptavidin technology has been extensively exploited to engineer artificial metalloenzymes (ArMs) that catalyze a dozen different reactions. Despite its versatility, the homotetrameric nature of streptavidin (Sav) and the noncooperative binding of biotinylated cofactors impose two limitations on the genetic optimization of ArMs: (i) point mutations are reflected in all four subunits of Sav, and (ii) the noncooperative binding of biotinylated cofactors to Sav may lead to an erosion in the catalytic performance, depending on the cofactor:biotin-binding site ratio. To address these challenges, we report on our efforts to engineer a (monovalent) single-chain dimeric streptavidin (scdSav) as scaffold for Sav-based ArMs. The versatility of scdSav as host protein is highlighted for the asymmetric transfer hydrogenation of prochiral imines using [Cp*Ir(biot-p-L)Cl] as cofactor. By capitalizing on a more precise genetic fine-tuning of the biotin-binding vestibule, unrivaled levels of activity and selectivity were achieved for the reduction of challenging prochiral imines. Comparison of the saturation kinetic data and X-ray structures of [Cp*Ir(biot-p-L)Cl]·scdSav with a structurally related [Cp*Ir(biot-p-L)Cl]·monovalent scdSav highlights the advantages of the presence of a single biotinylated cofactor precisely localized within the biotin-binding vestibule of the monovalent scdSav. The practicality of scdSav-based ArMs was illustrated for the reduction of the salsolidine precursor (500 mM) to afford (R)-salsolidine in 90% ee and >17âEuro¯000 TONs. Monovalent scdSav thus provides a versatile scaffold to evolve more efficient ArMs for in vivo catalysis and large-scale applications

Upregulation of an Artificial Zymogen by Proteolysis

Regulation of enzymatic activity is vital to living organisms. Here, we report the development and the genetic optimization of an artificial zymogen requiring the action of a natural protease to upregulate its latent asymmetric transfer hydrogenase activity

Development of Enzymes for Biocatalytic Applications: Protein Engineering, Immobilization and Reactor Concepts

Within this thesis the protein engineering, immobilization and application of enzymes in organic synthesis were studied in order to enhance the productivity of diverse biotransformations. Article I is a review about Baeyer-Villiger monooxygenases (BVMO) and provides a detailed overview of the most recent advantages in the application of that enzyme class in biocatalysis. Protein engineering of a former uncharacterized polyol-dehydrogenase (PDH) identified in the mesothermophilic bacterium Deinococcus geothermalis 11300 is described in Article II. Article III covers the combination of one PDH mutant with a BVMO in a closed-loop cascade reaction, thus enabling direct oxidation of cyclohexanol to ε-caprolactone with an internal cofactor recycling of NADP(H). Article IV and Article V report a process optimization for transamination reactions due to a newly developed immobilization protocol for five (S)- and (R)-selective aminotransferases (ATA) on chitosan support. Furthermore, the immobilized ATAs were applied in asymmetric amine synthesis. In Article VI, an ATA immobilized on chitosan, an encapsulated BVMO whole cell catalyst and a commercially available immobilized lipase were applied in a traditional fixed-bed (FBR) or stirred-tank reactor (STR), and were compared to a novel reactor design (SpinChem, SCR) for heterogeneous biocatalysis.Innerhalb dieser Arbeit wurde das Proteindesign, die Immobilisierung und der Einsatz von Enzymen in der organischen Synthese untersucht, um die Produktivität von Biotransformationen zu verbessern (der biokatalytische Zyklus). Artikel I ist ein Zusammenfassungsartikel über Baeyer-Villiger Monooxygenasen (BVMO) und gibt einen Überblick über die neuesten Erkenntnisse zu diesem Thema. Weiterhin wurde ein Proteindesign einer vorher nicht charakterisierten Polyol-Dehydrogenase (PDH) durchgeführt, welche aus dem mesothermophilen Organismus Deinnococcus geothermalis 11300 kloniert wurde (Artikel II). Eine der PDH Mutanten konnte daraufhin mit einer BVMO kombiniert werden, um eine direkte Doppeloxidation von Cyclohexanol zu epsilon-Caprolacton in einer Kaskadenreaktion zu ermöglichen (mit gleichzeitiger, integrierter Wiedergewinnung des NADP(H) Kofaktors; Artikel III). Artikel IV und V beschreiben die Prozessoptimierung von Transaminierungsreaktionen mittels der Entwicklung eines neues Immobilisierungsprotokolls auf Chitosan Trägermaterial für fünf (R)- und (S)- selektive Transaminasen (ATA). Die erhaltenen Präparate wurden dann in der asymmetrischen Aminsynthese eingesetzt. In Artikel VI wird erstmalig die Etablierung eines neuen Reaktorkonzeptes für immobilisierte Biokatalysatoren getestet (immobilisierte ATA und BVMO, eingeschlossene ganze E.coli Zellen). Hierfür wurde das neue Reaktordesign mit etablierten, traditionellen Reaktorkonzepten verglichen

Library design and screening protocol for artificial metalloenzymes based on the biotin-streptavidin technology

Artificial metalloenzymes (ArMs) based on the incorporation of a biotinylated metal cofactor within streptavidin (Sav) combine attractive features of both homogeneous and enzymatic catalysts. To speed up their optimization, we present a streamlined protocol for the design, expression, partial purification and screening of Sav libraries. Twenty-eight positions have been subjected to mutagenesis to yield 335 Sav isoforms, which can be expressed in 24-deep-well plates using autoinduction medium. The resulting cell-free extracts (CFEs) typically contain >1 mg of soluble Sav. Two straightforward alternatives are presented, which allow the screening of ArMs using CFEs containing Sav. To produce an artificial transfer hydrogenase, Sav is coupled to a biotinylated three-legged iridium pianostool complex Cp*Ir(Biot-p-L)Cl (the cofactor). To screen Sav variants for this application, you would determine the number of free binding sites, treat them with diamide, incubate them with the cofactor and then perform the reaction with your test compound (the example used in this protocol is 1-phenyl-3,4-dihydroisoquinoline). This process takes 20 d. If you want to perform metathesis reactions, Sav is coupled to a biotinylated second-generation Grubbs-Hoveyda catalyst. In this application, it is best to first immobilize Sav on Sepharose-iminobiotin beads and then perform washing steps. Elution from the beads is achieved in an acidic reaction buffer before incubation with the cofactor. Catalysis using your test compound (in this protocol, 2-(4-(N,N-diallylsulfamoyl)phenyl)-N,N,N-trimethylethan-1-aminium iodide) is performed using the formed metalloenzyme. Screening using this approach takes 19 d