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%

 

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.

 

Are in vivo selections on the path to extinction?

Authors: José Berenguer, Mario Mencía, Aurelio Hidalgo
Partners: UAM
Journal: Microbial Biotechnology
Year of publication: 2016

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.

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