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Dive into the research topics where Andreas Taglieber is active.

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Featured researches published by Andreas Taglieber.


Applied Microbiology and Biotechnology | 2008

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates

Joaquin Sanchis; Layla Fernández; J. Daniel Carballeira; Jullien Drone; Yosephine Gumulya; Horst Höbenreich; Daniel Kahakeaw; Sabrina Kille; Renate Lohmer; Jérôme J.-P. Peyralans; John Podtetenieff; Shreenath Prasad; Pankaj Soni; Andreas Taglieber; Sheng Wu; Felipe E. Zilly; Manfred T. Reetz

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene’s QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.


Organic Letters | 2013

Mimicking nature: synthetic nicotinamide cofactors for C═C bioreduction using enoate reductases.

Caroline E. Paul; Serena Gargiulo; Diederik J. Opperman; Iván Lavandera; Vicente Gotor-Fernández; Vicente Gotor; Andreas Taglieber; Isabel W. C. E. Arends; Frank Hollmann

A series of synthetic nicotinamide cofactors were synthesized to replace natural nicotinamide cofactors and promote enoate reductase (ER) catalyzed reactions without compromising the activity or stereoselectivity of the bioreduction process. Conversions and enantioselectivities of >99% were obtained for C═C bioreductions, and the process was successfully upscaled. Furthermore, high chemoselectivity was observed when employing these nicotinamide cofactor mimics (mNADs) with crude extracts in ER-catalyzed reactions.


ChemBioChem | 2008

Light-Driven Biocatalytic Oxidation and Reduction Reactions: Scope and Limitations

Andreas Taglieber; Frank Schulz; Frank Hollmann; Monika Rusek; Manfred T. Reetz

The quest for practical regeneration concepts for nicotinamide‐dependent oxidoreductases continues. Recently we proposed the use of visible light to promote the direct reductive regeneration of a flavin‐dependent monooxygenase. With this enzyme (PAMO‐P3) light‐driven enantioselective Baeyer–Villiger oxidations were performed. In spite of the significant reduction in the complexity achieved, catalytic performance of the novel approach did not meet the requirements for an efficient biocatalytic oxygenation system. Driven by this ultimate goal, we further investigated the limiting factors of our particular system. We discovered that oxidative uncoupling of the flavin‐regeneration reaction from enzymatic O2‐activation accounts for the futile consumption of approximately 95 % of the reducing equivalents provided by the sacrificial electron donor, EDTA. Furthermore, it was found that the apparent turnover frequency (TOF) for PAMO‐P3 in the present setup is approximately two orders of magnitude lower than in conventional setups that use NADPH as reductant. This finding was traced to sluggish electron transfer kinetics that arose from an impeded interaction between PAMO‐P3‐bound FAD and the reducing catalyst. The limiting factors and potential approaches for their circumvention are discussed. Furthermore, we broadened the light‐driven regeneration approach to the class of flavin‐dependent reductases. By using the Old Yellow Enzyme homologue YqjM as a model system, a significantly higher catalytic turnover for the enzyme catalyst was achieved, which we assign to a higher accessibility of the prosthetic group as well as to the absence of oxidative uncoupling.


ChemBioChem | 2008

A Robust Protein Host for Anchoring Chelating Ligands and Organocatalysts

Manfred T. Reetz; Martin Rentzsch; Andreas Pletsch; Andreas Taglieber; Frank Hollmann; Régis Mondière; Norbert Dickmann; Birte Höcker; Simona Cerrone; Michaela C. Haeger; Reinhard Sterner

In order to put the previously proposed concept of directed evolution of hybrid catalysts (proteins that harbor synthetic transition‐metal catalysts or organocatalysts) into practice, several prerequisites must be met. The availability of a robust host protein that can be expressed in sufficiently large amounts, and that can be purified in a simple manner is crucial. The thermostable enzyme tHisF from Thermotoga maritima, which constitutes the synthase subunit of a bi‐enzyme complex that is instrumental in the biosynthesis of histidine, fulfills these requirements. In the present study, fermentation has been miniaturized and parallelized, as has purification of the protein by simple heat treatment. Several mutants with strategically placed cysteines for subsequent bioconjugation have been produced. One of the tHisF mutants, Cys9Ala/Asp11Cys, was subjected to bioconjugation by the introduction of a variety of ligands for potential metal ligation, of a ligand/metal moiety, and of several organocatalytic entities that comprise a flavin or thiazolium salts. Characterization by mass spectrometry and tryptic digestion was achieved. As a result of this study, a platform for performing future directed evolution of these hybrid catalysts is now available.


Chemical Communications | 2009

Deazaflavins as mediators in light-driven cytochrome P450 catalyzed hydroxylations

Felipe E. Zilly; Andreas Taglieber; Frank Schulz; Frank Hollmann; Manfred T. Reetz

A light-driven deazaflavin-dependent direct enzyme regeneration system has been developed for a P450-BM3 catalyzed CH-activating hydroxylation, thereby avoiding the need for the expensive NADPH cofactor.


ChemBioChem | 2018

An engineered alcohol oxidase for the oxidation of primary alcohols

Nicholas J. Turner; Rachel S. Heath; William R. Birmingham; Matthew P. Thompson; Andreas Taglieber; Laurent Daviet

Structure‐guided directed evolution of choline oxidase has been carried out by using the oxidation of hexan‐1‐ol to hexanal as the target reaction. A six‐amino‐acid variant was identified with a 20‐fold increased kcat compared to that of the wild‐type enzyme. This variant enabled the oxidation of 10 mm hexanol to hexanal in less than 24 h with 100 % conversion. Furthermore, this variant showed a marked increase in thermostability with a corresponding increase in Tm of 20 °C. Improved solvent tolerance was demonstrated with organic solvents including ethyl acetate, heptane and cyclohexane, thereby enabling improved conversions to the aldehyde by up to 30 % above conversion for the solvent‐free system. Despite the evolution of choline oxidase towards hexan‐1‐ol, this new variant also showed increased specific activities (by up to 100‐fold) for around 50 primary aliphatic, unsaturated, branched, cyclic, benzylic and halogenated alcohols.


Advanced Synthesis & Catalysis | 2009

Directed Evolution of an Enantioselective Enoate‐Reductase: Testing the Utility of Iterative Saturation Mutagenesis

Despina J. Bougioukou; Sabrina Kille; Andreas Taglieber; Manfred T. Reetz


Angewandte Chemie | 2010

An Artificial Metalloenzyme: Creation of a Designed Copper Binding Site in a Thermostable Protein

John Podtetenieff; Andreas Taglieber; Eckhard Bill; Edward J. Reijerse; Manfred T. Reetz


Angewandte Chemie | 2007

A Light‐Driven Stereoselective Biocatalytic Oxidation

Frank Hollmann; Andreas Taglieber; Frank Schulz; Manfred T. Reetz


Tetrahedron | 2007

Directed evolution of enantioselective hybrid catalysts: a novel concept in asymmetric catalysis

Manfred T. Reetz; Martin Rentzsch; Andreas Pletsch; Matthias Maywald; Peter Maiwald; Jérôme J.-P. Peyralans; Andrea Maichele; Yu Fu; Ning Jiao; Frank Hollmann; Régis Mondière; Andreas Taglieber

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