Selective Ring‐Opening of Di‐substituted Epoxides Catalysed by Halohydrin Dehalogenases

Authors: Elia Calderini Julia Wessel Philipp Süss Patrick Schrepfer Rainer Wardenga Anett Schallmey

Partners: TUBS, ENZ

Year: 2019

Abstract

Halohydrin dehalogenases (HHDHs) are valuable biocatalysts for the synthesis of β‐substituted alcohols based on their epoxide ring‐opening activity with a number of small anionic nucleophiles. In an attempt to further broaden the scope of substrates accepted by these enzymes, a panel of 22 HHDHs was investigated in the conversion of aliphatic and aromatic vicinally di‐substituted trans‐epoxides using azide as nucleophile. The majority of these HHDHs was able to convert aliphatic methyl‐substituted epoxide substrates to the corresponding azidoalcohols, in some cases even with absolute regioselectivity. HheG from Ilumatobacter coccineus exhibited also high activity towards sterically more demanding di‐substituted epoxides. This further expands the range of β‐substituted alcohols that are accessible by HHDH catalysis.

 

2‐Deoxyribose‐5‐phosphate aldolase from Thermotoga maritima in the synthesis of a statin‐side chain precursor: characterization, modelling and optimization

Authors: Anera Švarc Zvjezdana Findrik Blažević Đurđa Vasić‐Rački Anna Szekrenyi Wolf‐Dieter Fessner Simon J. Charnock Ana Vrsalović Presečki

Partners: TUDA, UZAG

Year: 2019

Abstract

BACKGROUND
The statin side‐chain synthesis by the sequential aldol condensation catalyzed by DERA (EC 4.1.2.4) is the most known and promising route. The advantage of this process is the formation of two stereocenters in a single step starting from inexpensive achiral components, acetaldehyde and chloroacetaldehyde. The limitation for the industrial‐scale application is enzyme inactivation caused by substrates as well by the intermediate produced by single aldol addition.

RESULTS
DERA enzyme from Thermotoga maritima (DERATm) was extensively investigated in this work. The influence of aldehydes on DERATm stability was studied in detail. Based on experimentally determined kinetic parameters of all reactions included in the synthesis of statin side chain, mathematical models in different reactor configurations were developed. Models also include enzyme inactivation by all compounds that have negative impact on its stability. Mathematical model‐based optimization enabled finding the optimal process conditions and choosing the best reactor configuration. The fed‐batch reactor proved to be the most suitable choice. Under optimal conditions product concentration of 78 g/L, productivity of 56 g/(L day) and yield of 95% was achieved.

CONCLUSION
The validated mathematical model and the used methodology can be exploited for further process improvement and for process design of similar systems. 

 

Aldolase‐Catalyzed Asymmetric Synthesis of N‐Heterocycles by Addition of Simple Aliphatic Nucleophiles to Aminoaldehydes

Authors: Raquel Roldan Karel Hernández Jesús Joglar Jordi Bujons Teodor Parella Wolf-Dieter Fessner Pere Clapés

Partners: TUDA, CSIC

Journal: Advanced Synthesis & Catalysis

Year: 2019

Abstract

 Nitrogen heterocycles are structural motifs found in many bioactive natural products and of utmost importance in pharmaceutical drug development. In this work, a stereoselective synthesis of functionalized N heterocycles was accomplished in two steps, comprising the biocatalytic aldol addition of ethanal and simple aliphatic ketones such as propanone, butanone, 3 pentanone, cyclobutanone, and cyclopentanone to N‐Cbz‐protected aminoaldehydes using engineered variants of D‐fructose‐6‐phosphate aldolase from Escherichia coli (FSA) or 2‐deoxy‐D‐ribose‐5‐phosphate aldolase from Thermotoga maritima (DERATma) as catalysts. FSA catalyzed most of the additions of ketones while DERATma was restricted to ethanal and propanone. Subsequent treatment with hydrogen in the presence of palladium over charcoal, yielded low‐level oxygenated N‐heterocyclic derivatives of piperidine, pyrrolidine and N‐bicyclic structures bearing fused cyclobutane and cyclopentane rings, with stereoselectivities of 96–98 ee and 97:3 dr in isolated yields ranging from 35 to79%.

 

Enantioselective Synthesis of Pharmaceutically Active γ-Aminobutyric Acids Using a Tailor-Made Artificial Michaelase in One-Pot Cascade Reactions 

Authors: Lieuwe Biewenga, Thangavelu Saravanan, Andreas Kunzendorf, Jan-Ytzen van der Meer, Tjaard Pijning, Pieter G. Teppe, Ronald van Merkerk, Simon J. Charnock, Andy-Mark W. H. Thunnisse, a Gerrit J. Poelarends

PartnersRUGPROZO

Journal: ACS Catalysis

Year: 2019

Abstract

Chiral γ-aminobutyric acid (GABA) analogues represent abundantly prescribed drugs, which are broadly applied as anticonvulsants, as antidepressants, and for the treatment of neuropathic pain. Here we report a one-pot two-step biocatalytic cascade route for synthesis of the pharmaceutically relevant enantiomers of γ-nitrobutyric acids, starting from simple precursors (acetaldehyde and nitroalkenes), using a tailor-made highly enantioselective artificial “Michaelase” (4-oxalocrotonate tautomerase mutant L8Y/M45Y/F50A), an aldehyde dehydrogenase with a broad non-natural substrate scope, and a cofactor recycling system. We also report a three-step chemoenzymatic cascade route for the efficient chemical reduction of enzymatically prepared γ-nitrobutyric acids into GABA analogues in one pot, achieving high enantiopurity (e.r. up to 99:1) and high overall yields (up to 70%). This chemoenzymatic methodology offers a step-economic alternative route to important pharmaceutically active GABA analogues, and highlights the exciting opportunities available for combining chemocatalysts, natural enzymes, and designed artificial biocatalysts in multistep syntheses.

 

Application of Chemical Engineering Methodology in Process Development: A Case Study of MenD-catalyzed Synthesis of 6-Cyano-4-oxohexanoic Acid

Authors: Martina Sudar, Igor Dejanović, Michael Müller, Đurda Vasić-Rački, Zvjezdana F. Blažević

PartnersUZAGUFREI

Journal: Chemical and biochemical engineering quarterly

Year: 2018

Abstract

To speed up evaluation, development and upscaling of new processes, the use of engineering methodology can have a great impact. Here we show the application of an engineering approach to find the reaction conditions allowing the best process metrics. An experimentally validated mathematical model for the MenD-catalyzed synthesis of a commercially unavailable product, 6-cyano-4-oxohexanoic acid, with a potential industrial use as a building block, was used for process optimization. Using the optimized conditions, 62.4 g dm–3 of product, volume productivity of 87.1 g dm–3 d–1, product yield of 96 %, and biocatalyst productivity of 25.8 kgP kg–1 MenD can be achieved. Based on the optimized production procedure, economic analysis was performed to determine minimal product price required for project to be profitable in 8 years economic lifetime. In addition, Monte Carlo analysis (MCA) was used to assess the influence of uncertainties in estimation of input variables on overall economic performance. (This work is licensed under a Creative Commons Attribution 4.0 International License.)

 

Structural and Mutagenesis Studies of the Thiamine‐Dependent, Ketone‐Accepting YerE from Pseudomonas protegens

Authors: Sabrina Hampel, Jan‐Patrick Steitz, Anna Baierl, Patrizia Lehwald, Luzia Wiesli, Michael Richter, Alexander Fries, Martina Pohl, Gunter Schneider, Doreen Dobritzsch, Michael Mülle

Partners: UFREI

Journal: ChemBioChem

Abstract

A wide range of thiamine diphosphate (ThDP)‐dependent enzymes catalyze the benzoin‐type carboligation of pyruvate with aldehydes. A few ThDP‐dependent enzymes, such as YerE from Yersinia pseudotuberculosis (YpYerE), are known to accept ketones as acceptor substrates. Catalysis by YpYerE gives access to chiral tertiary alcohols, a group of products difficult to obtain in an enantioenriched form by other means. Hence, knowledge of the three‐dimensional structure of the enzyme is crucial to identify structure–activity relationships. However, YpYerE has yet to be crystallized, despite several attempts. Herein, we show that a homologue of YpYerE, namely, PpYerE from Pseudomonas protegens (59 % amino acid identity), displays similar catalytic activity: benzaldehyde and its derivatives as well as ketones are converted into chiral 2‐hydroxy ketones by using pyruvate as a donor. To enable comparison of aldehyde‐ and ketone‐accepting enzymes and to guide site‐directed mutagenesis studies, PpYerE was crystallized and its structure was determined to a resolution of 1.55 Å.

NanoDSF as screening tool for enzyme libraries and biotechnology development

Authors: Anders O. Magnusson, Anna Szekrenyi, Henk‐Jan Joosten, James Finnigan, Simon Charnock, Wolf‐Dieter Fessner

PartnersTUDABIOPPROZO

Journal: The FEBS Journal

Year: 2018

Abstract

Enzymes are attractive tools for synthetic applications. To be viable for industrial use, enzymes need sufficient stability towards the desired reaction conditions such as high substrate and cosolvent concentration, non‐neutral pH and elevated temperatures. Thermal stability is an attractive feature not only because it allows for protein purification by thermal treatment and higher process temperatures but also due to the associated higher stability against other destabilising factors. Therefore, high‐throughput screening (HTS) methods are desirable for the identification of thermostable biocatalysts by discovery from nature or by protein engineering but current methods have low throughput and require time‐demanding purification of protein samples. We found that nanoscale differential scanning fluorimetry (nanoDSF) is a valuable tool to rapidly and reliably determine melting points of native proteins. To avoid intrinsic problems posed by crude protein extracts, hypotonic extraction of overexpressed protein from bacterial host cells resulted in higher sample quality and accurate manual determination of several hundred melting temperatures per day. We have probed the use of nanoDSF for HTS of a phylogenetically diverse aldolase library to identify novel thermostable enzymes from metagenomic sources and for the rapid measurements of variants from saturation mutagenesis. The feasibility of nanoDSF for the screening of synthetic reaction conditions was proved by studies of cosolvent tolerance, which showed protein melting temperature to decrease linearly with increasing cosolvent concentration for all combinations of six enzymes and eight water‐miscible cosolvents investigated, and of substrate affinity, which showed stabilisation of hexokinase by sugars in the absence of ATP cofactor.

 

Biocatalytic Aldol Addition of Simple Aliphatic Nucleophiles to Hydroxyaldehydes

Aurthors: Raquel Roldán, Karel Hernandez, Jesús Joglar, Jordi Bujons, Teodor Parella , Israel Sánchez-Moreno, Virgil Hélaine, Marielle Lemaire, Christine Guérard-Hélaine, Wolf-Dieter Fessner, and Pere Clapés 

Partners:  CSICTUDA

Journal: ACS Catalysis

Year: 2018

Abstract

Asymmetric aldol addition of simple aldehydes and ketones to electrophiles is a cornerstone reaction for the synthesis of unusual sugars and chiral building blocks. We investigated d-fructose-6-phosphate aldolase from E. coli (FSA) D6X variants as catalysts for the aldol additions of ethanal and nonfunctionalized linear and cyclic aliphatic ketones as nucleophiles to nonphosphorylated hydroxyaldehydes. Thus, addition of propanone, cyclobutanone, cyclopentanone, or ethanal to 3-hydroxypropanal or (S)- or (R)-3-hydroxybutanal catalyzed by FSA D6H and D6Q variants furnished rare deoxysugars in 8–77% isolated yields with high stereoselectivity (97:3 dr and >95% ee).

 

Nucleophile Promiscuity of Natural and Engineered Aldolases

Authors: Karel Hernández,  Anna Szekrenyi,  Dr. Pere Clapés

Partners: CSICTUDA

Journal: ChemBIoChem

Year: 2018

Abstract

The asymmetric aldol addition reaction mediated by aldolases is recognized as a green and sustainable method for carbon–carbon bond formation. Research in this area has unveiled their unprecedented synthetic potential toward diverse, new chemical structures; novel product families; and even as a technology for industrial manufacturing processes. Despite these advances, aldolases have long been regarded as strictly selective catalysts, particularly for nucleophilic substrates, which limits their broad applicability. In recent years, advances in screening technologies and metagenomics have uncovered novel C−C biocatalysts from superfamilies of widely known lyases. Moreover, protein engineering has revealed the extraordinary malleability of different carboligases to offer a toolbox of biocatalysts active towards a large structural diversity of nucleophile substrates. Herein, the nucleophile ambiguity of native and engineered aldolases is discussed with recent examples to prove this novel concept.

 

Efficient Asymmetric Synthesis of Carbohydrates by Aldolase Nano-Confined in Lipidic Cubic Mesophases

Authors: Tao Zhou, Jijo J.Vallooran,  Salvatore Assenza, Anna Szekrenyi, Pere Clapés, Raffaele Mezzenga

Partners: CSIC, TUDA

Journal: ACS Catalysis

Year: 2018

Abstract

Class-I aldolases are known for efficiently catalyzing stereoselective aldol-addition reactions in bulk aqueous media, and considerable efforts are currently being devoted to engineer the enzyme in order to optimize its activity and stability, primarily by modulating the hydrophobicity of the catalytic active site. Here, we opt for a different strategy based on choosing a nanoconfined environment favorable to the enzyme. We report the observation of enhanced activity and stability of a class-I aldolase, d-fructose-6-phosphate aldolase from E. coli (FSA), when incorporated into lipidic cubic mesophases (LCMs), a class of biomimetic amphiphilic complex fluids employed in several nanotechnology applications. We infer that this improved in-meso performance is achieved by optimal location of the FSA in the LCMs, as a result of the known interaction between the residues of FSA and the glycerol moieties, which serve as the lipid head groups and thus locate along the amphiphilic interface encompassing the whole LCM. This continuous interface ensures increased accessibility of the catalytic reaction center to substrates and high activity in LCM.

 

 

 

Exploiting distant homologues for phasing through the generation of compact fragments, local fold refinement and partial solution combination

Authors: Claudia Millán, Massimo Domenico Sammito, Airlie J. McCoy, Andrey F. Ziem Nascimento , Giovanna Petrillo, Robert D. Oeffner, Teresa Domínguez-Gil, Juan A. Hermoso, Randy J. Read, Isabel Usón

Partners: CSIC, BCZ

Journal: Acta Crystallographica Section D

Year: 2018

Abstract

Macromolecular structures can be solved by molecular replacement provided that suitable search models are available. Models from distant homologues may deviate too much from the target structure to succeed, notwithstanding an overall similar fold or even their featuring areas of very close geometry. Successful methods to make the most of such templates usually rely on the degree of conservation to select and improve search models. ARCIMBOLDO_SHREDDER uses fragments derived from distant homologues in a brute-force approach driven by the experimental data, instead of by sequence similarity. The new algorithms implemented in ARCIMBOLDO_SHREDDER are described in detail, illustrating its characteristic aspects in the solution of new and test structures. In an advance from the previously published algorithm, which was based on omitting or extracting contiguous polypeptide spans, model generation now uses three-dimensional volumes respecting structural units. The optimal fragment size is estimated from the expected log-likelihood gain (LLG) values computed assuming that a substructure can be found with a level of accuracy near that required for successful extension of the structure, typically below 0.6 Å root-mean-square deviation (r.m.s.d.) from the target. Better sampling is attempted through model trimming or decomposition into rigid groups and optimization through Phaser's gyre refinement. Also, after model translation, packing filtering and refinement, models are either disassembled into predetermined rigid groups and refined (gimble refinement) or Phaser's LLG-guided pruning is used to trim the model of residues that are not contributing signal to the LLG at the target r.m.s.d. value. Phase combination among consistent partial solutions is performed in reciprocal space with ALIXE. Finally, density modification and main-chain autotracing in SHELXE serve to expand to the full structure and identify successful solutions. The performance on test data and the solution of new structures are described.

 

Complete switch of reaction specificity of an aldolase by directed evolution in vitro: Synthesis of generic aliphatic aldol products

Authors: Wolf-Dieter Fessner, Sebastian Junker, Raquel Roldan, Henk-Jan Joosten, Pere Clapés

Partners: TUDA, BIOP, CSIC

Journal: Angewandte Chemie International Edition

Year: 2018

Abstract

A structure‐guided engineering of fructose‐6‐phosphate aldolase was performed to expand its substrate promiscuity toward aliphatic nucleophiles, i.e., unsubstituted alkanones and alkanals. A "smart" combinatorial library was created targeting residues D6, T26 and N28 that form a binding pocket around the nucleophilic carbon atom. Double‐selectivity screening was executed by high‐performance TLC that allowed simultaneous determination of total activity as well as a preference for acetone versus propanal as competing nucleophiles. While any mutation of N28 resulted in inactivation of the enzyme, D6 turned out to be the key residue that enabled activity with non‐hydroxylated nucleophiles. Altogether 25 single‐ and double‐site variants (D6X and D6X/T26X) were discovered that show useful synthetic activity and a varying preference for ketone or aldehyde as the aldol nucleophiles. Remarkably, all of the novel variants had completely lost their native activity for cleavage of fructose 6‐phosphate.

 

Mathematical model of the MenD-catalyzed 1,4-addition (Stetter reaction) of α-ketoglutaric acid to acrylonitrile

Authors: Martina Sudar, Đurđa Vasić-Rački, Michael Müller, Alexandra Walter, Zvjezdana Findrik Blažević

Partners: UZAG, UFREI

Journal: Journal of Biotechnology

Year: 2018

Abstract

The Stetter reaction, a conjugate umpolung reaction, is well known for cyanide-catalyzed transformations of mostly aromatic aldehydes. Enzymatic Stetter reactions, however, have been largely unexplored, especially with respect to preparative transformations. We have investigated the kinetics of the MenD-catalyzed 1,4-addition of α-ketoglutaric acid to acrylonitrile which has shown that acrylonitrile, while an interesting candidate, is a poor substrate for MenD due to low affinity of the enzyme for this substrate. The kinetic model of the reaction was simplified to double substrate Michaelis–Menten kinetics where the reaction rate linearly depends on acrylonitrile concentration. Experiments at different initial concentrations of acrylonitrile under batch, repetitive batch, and fed-batch reactor conditions were carried out to validate the developed mathematical model. Thiamine diphosphate dependent MenD proved to be quite a robust enzyme; nevertheless, enzyme operational stability decay occurs in the reactor. The spontaneous reactivity of acrylonitrile towards polymerization was also taken into account during mathematical modeling. Almost quantitative conversion of acrylonitrile was achieved in all batch reactor experiments, while the yield of the desired product was dependent on initial acrylonitrile concentration (i.e., the concentration of the stabilizer additive). Using the optimized reactor parameters, it was possible to synthesize the product, 6-cyano-4-oxohexanoic acid, in a concentration of 250 mM. The highest concentration of product was achieved in a repetitive batch reactor experiment. A fed-batch reactor experiment also delivered promising results, especially regarding the short reaction time needed to achieve a 200 mM concentration of product. Hence, the enzymatic Stetter reaction with a highly reactive acceptor substrate can be performed on a preparative scale, which should enable similar transformations with acrylate, methacrylate, and methyl vinyl ketone.

 

Nucleophile Promiscuity of Engineered Class II Pyruvate Aldolase from E. Coli, YfaU

Authors: Pere Clapes, Jesús Joglar, Jordi Bujons, Teodor Parella, Karel Hernánde

Partners: CSIC

Journal: Angewandte Chemie International Edition

Year of publication: 2018

 

Abstract

Pyruvate-dependent aldolases exhibit a stringent selectivity for pyruvate, limiting their synthetic potential application, a drawback shared with other existing aldolases. Structure-guided rational protein engineering rendered a 2-keto-3-deoxy-L-rhamnonate aldolase variant, fused with maltose binding protein (MBP-YfaU W23V/L216A), capable to efficiently convert larger pyruvate analogs, e.g. having linear and branched aliphatic chains, in aldol addition reactions. Combination of these nucleophiles with N-Cbz-alaninal and N-Cbz-prolinal electrophiles gave access to chiral building blocks, e.g. derivatives of (2S,3S,4R)-4-amino-3-hydroxy-2-methylpentanoic acid (68%, dr 90:10) and the enantiomer of Dolaproine (33%, dr 94:6) as well as a collection of unprecedented α-amino acid derivatives of the proline and pyrrolizidine type, with conversions varying between 6-93% and diasteromeric ratios from 50:50 to 95:5 depending on the nucleophilic and electrophilic components

 

Fluorogenic Kinetic Assay for High-Throughput Discovery of Stereoselective Ketoreductases Relevant to Pharmaceutical Synthesis

Authors: Yen-Chi Thai, Anna Szekrenyi, Yuyin Qi, Gary W. Black, Simon J. Charnock, Wolf- Dieter Fessner

Partners: TUDA, PROZO

Journal: Bioorganic & Medicinal Chemistry

Year of publication: 2018

ABSTRACT

Enantiomerically pure 1-(6-methoxynaphth-2-yl) and 1-(6-(dimethylamino)naphth-2-yl)  carbinols are fluorogenic substrates for aldo/keto reductase (KRED) enzymes, which allow the highly sensitive and reliable determination of activity and kinetic constants of known and unknown enzymes, as well as an immediate enantioselectivity typing. Because of its simplicity in microtiter plate format, the assay qualifies for the discovery of novel KREDs of yet unknown specificity among this vast enzyme superfamily. The suitability of this approach for enzyme typing is illustrated by an exemplary screening of a large collection of short-chain dehydrogenase/reductase (SDR) enzymes arrayed from a metagenomic approach. We believe that this assay format should match well the pharmaceutical industry’s demand for acetophenone-type substrates and the continuing interest in new enzymes with broad substrate promiscuity for the synthesis of chiral, non-racemic carbinols.

 

2-Keto-3-Deoxy-l-Rhamnonate Aldolase (YfaU) as Catalyst in Aldol Additions of Pyruvate to Amino Aldehyde Derivatives

Authors: Karel Hernandez, Ariadna Gómez, Jesús Joglar, Jordi Bujons, Teodor Parella, Pere Clapés
 
Partners: CSIC
 
Journal: Advanced Synthesis & Catalysis
 
Year of publication: 2017
 

Abstract

4-Hydroxy-2-keto acid derivatives are versatile building blocks for the synthesis of amino acids, hydroxy carboxylic acids and chiral aldehydes. Pyruvate aldolases are privileged catalysts for a straightforward access to this class of keto acid compounds. In this work, a Class II pyruvate aldolase from Escherichia coli K-12, 2-keto-3-deoxy-l-rhamnonate aldolase (YfaU), was evaluated for the synthesis of amino acid derivatives of proline, pipecolic acid, and pyrrolizidine-3-carboxylic acid. The aldol addition of pyruvate to N-protected amino aldehydes was the key enzymatic aldol addition step followed by catalytic intramolecular reductive amination. The corresponding N-Cbz-amino-4-hydroxy-2-keto acid (Cbz=benzyloxycarbonyl) precursors were obtained in 51–95% isolated yields and enantioselectivity ratios from 26:74 to 95:5, with chiral α-substituted N-Cbz-amino aldehydes. (S)-N-Cbz-amino aldehydes gave aldol adducts with preferentially (R)-configuration at the newly formed stereocenter, whereas the contrary is true for (R)-N-Cbz-amino aldehydes. Addition reactions to achiral amino aldehydes rendered racemic aldol adducts. Molecular models of the pre-reaction ternary complexes YfaU-pyruvate enolate-acceptor aldehyde were constructed to explain the observed stereochemical outcome of the reactions. Catalytic reductive amination of the aldol adducts yielded 4-hydroxy-2-pipecolic acid, and unprecedented C-5 substituted 4-hydroxyproline and pyrrolizidine-3-carboxylic acid derivatives.

 

Combining Aldolases and Transaminases for the Synthesis of 2-Amino-4-Hydroxybutanoic acid

Authors: Karel Hernández, Jordi Bujons, Jesús Joglar, Simon J. Charnock, Pablo Dominguez de Maria, Wolf-Dieter Fessner, and Pere Clapés
Partners: CSIC, SUSMOM, PROZO, TUDA
Journal: ACS Catalysis
Year of publication: 2017

Abstract

Amino acids are of paramount importance as chiral building blocks of life, for drug development in modern medicinal chemistry, and for the manufacture of industrial products. In this work, the stereoselective synthesis of (S)- and (R)-2-amino-4-hydroxybutanoic acid was accomplished using a Systems Biocatalysis approach comprising a biocatalytic one-pot cyclic cascade by coupling of an aldol reaction with an ensuing stereoselective transamination. A Class II pyruvate aldolase from E. coli, expressed as a soluble fusion protein, in tandem with either an (S)- or (R)-selective, pyridoxal phosphate-dependent, transaminase were used as catalysts to realize the conversion, with formaldehyde and alanine being the sole starting materials. Interestingly, the Class II pyruvate aldolase was found to tolerate for-maldehyde concentrations of up to 1.4 M. The cascade system was found to reach product concentrations for (S)- or (R)-2-amino-4-hydroxybutanoic acid of at least 0.4 M, rendering yields between 86% and >95%, respectively, productivities of >80 g L–1 d–1, and ee >99%

 

Are in vivo selections on the path to extinction?

Authors:
Partners: UAM
Journal: Microbial biotechnology
Year of publication: 2017

Abstract

Droplet microfluidics will become a disruptive technology in the field of library screening and replace biological selections if the central dogma of biology and other processes are successfully implemented within microdroplets.

 

The generation and exploitation of protein mutability landscapes for enzyme engineering

Authors: Jan-Ytzen van der Meer, Lieuwe Biewenga and Gerrit J. Poelarends
Partners: RUG
Journal: ChemBioChem
Year of publication: 2016

Abstract

The increasing number of enzyme applications in chemical synthesis calls for new engineering methods to develop the biocatalysts of the future. An interesting concept in enzyme engineering is the generation of large-scale mutational data in order to chart protein mutability landscapes. These landscapes allow the important discrimination between beneficial mutations and those that are neutral or detrimental, thus providing detailed insight into sequence–function relationships. As such, mutability landscapes are a powerful tool with which to identify functional hotspots at any place in the amino acid sequence of an enzyme. These hotspots can be used as targets for combinatorial mutagenesis to yield superior enzymes with improved catalytic properties, stability, or even new enzymatic activities. The generation of mutability landscapes for multiple properties of one enzyme provides the exciting opportunity to select mutations that are beneficial either for one or for several of these properties. This review presents an overview of the recent advances in the construction of mutability landscapes and discusses their importance for enzyme engineering.

 

Recent advances on halohydrin dehalogenases—from enzyme identification to novel biocatalytic applications

Authors: Anett Schallmey, Marcus Schallmey

Partners: TUBS
Journal: Applied Microbiology and Biotechnology
Year of publication: 2016

Abstract

Halohydrin dehalogenases are industrially relevant enzymes that catalyze the reversible dehalogenation of vicinal haloalcohols with formation of the corresponding epoxides. In the reverse reaction, also other negatively charged nucleophiles such as azide, cyanide, or nitrite are accepted besides halides to open the epoxide ring. Thus, novel C-N, C-C, or C-O bonds can be formed by halohydrin dehalogenases, which makes them attractive biocatalysts for the production of various β-substituted alcohols. Despite the fact that only five individual halohydrin dehalogenase enzyme sequences have been known until recently enabling their heterologous production, a large number of different biocatalytic applications have been reported using these enzymes. The recent characterization of specific sequence motifs has facilitated the identification of novel halohydrin dehalogenase sequences available in public databases and has largely increased the number of recombinantly available enzymes. These will help to extend the biocatalytic repertoire of this enzyme family and to foster novel biotechnological applications and developments in the future. This review gives a general overview on the halohydrin dehalogenase enzyme family and their biochemical properties and further focuses on recent developments in halohydrin dehalogenase biocatalysis and protein engineering.

 

 
 
 
 
 
 

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