Rapid screening and scale-up of transaminase catalysed reactions (original) (raw)
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Non-Canonical Amino Acid-Based Engineering of (R)-Amine Transaminase
Frontiers in Chemistry, 2022
Non-canonical amino acids (ncAAs) have been utilized as an invaluable tool for modulating the active site of the enzymes, probing the complex enzyme mechanisms, improving catalytic activity, and designing new to nature enzymes. Here, we report site-specific incorporation of p-benzoyl phenylalanine (pBpA) to engineer (R)-amine transaminase previously created from d-amino acid aminotransferase scaffold. Replacement of the single Phe88 residue at the active site with pBpA exhibits a significant 15-fold and 8-fold enhancement in activity for 1-phenylpropan-1-amine and benzaldehyde, respectively. Reshaping of the enzyme’s active site afforded an another variant F86A/F88pBpA, with 30% higher thermostability at 55°C without affecting parent enzyme activity. Moreover, various racemic amines were successfully resolved by transaminase variants into (S)-amines with excellent conversions (∼50%) and enantiomeric excess (>99%) using pyruvate as an amino acceptor. Additionally, kinetic resoluti...
Transaminases Applied to the Synthesis of High Added-Value Enantiopure Amines
Organic Process Research & Development, 2014
Critical parameters affecting the stereoselective amination of (hetero)aromatic ketones using transaminases have been studied such as temperature, pH, substrate concentration, cosolvent and source and percentage of amino donor, to further optimize the production of enantiopure amines using both (S)-and (R)-selective biocatalysts from commercial suppliers. Interesting enantiopure amino building blocks have been obtained overcoming some limitations of traditional chemical synthetic methods. Representative processes were scaled-up affording halogenated and heteroaromatic amines in enantiomerically pure form and good isolated yields.
This work aims to establish microscale methods to rapidly explore bioprocess options that might be used to enhance bioconversion reaction yields: either by shifting unfavourable reaction equilibria or by overcoming substrate and/or product inhibition. As a typical and industrially relevant example of the problems faced we have examined the asymmetric synthesis of (2S,3R)-2-amino-1,3,4-butanetriol from L-erythrulose using the x-transaminase from Chromobacterium violaceum DSM30191 (CV2025 x-TAm) and methylbenzylamine as the amino donor. The first process option involves the use of alternative amino donors. The second couples the CV2025 x-TAm with alcohol dehydrogenase and glucose dehydrogenase for removal of the acetophenone (AP) byproduct by in situ conversion to (R)-1-phenylethanol. The final approaches involve physical in-situ product removal methods. Reduced pressure conditions, attained using a 96-well vacuum manifold were used to selectively increase evaporation of the volatile AP while polymeric resins were also utilised for selective adsorption of AP from the bioconversion medium. For the particular reaction studied here the most promising bioprocess options were use of an alternative amino donor, such as isopropylamine, which enabled a 2.8-fold increase in reaction yield, or use of a second enzyme system which achieved a 3.3-fold increase in yield.
Transaminases for the synthesis of enantiopure beta-amino acids
AMB Express, 2012
Optically pure β-amino acids constitute interesting building blocks for peptidomimetics and a great variety of pharmaceutically important compounds. Their efficient synthesis still poses a major challenge. Transaminases (also known as aminotransferases) possess a great potential for the synthesis of optically pure β-amino acids. These pyridoxal 5'-dependent enzymes catalyze the transfer of an amino group from a donor substrate to an acceptor, thus enabling the synthesis of a wide variety of chiral amines and amino acids. Transaminases can be applied either for the kinetic resolution of racemic compounds or the asymmetric synthesis starting from a prochiral substrate. This review gives an overview over microbial transaminases with activity towards β-amino acids and their substrate spectra. It also outlines current strategies for the screening of new biocatalysts. Particular emphasis is placed on activity assays which are applicable to high-throughput screening.
A novel multi-enzymatic high throughput assay for transaminase activity
Tetrahedron, 2012
a b s t r a c t Aminotransferases (ATs) are crucial enzymes for prokaryote and eukaryote metabolism, and have attracted industrial attention for large scale synthesis of amino acids and chiral amines. A high throughput screening procedure for branched chain aminotransferases (BCATs) was developed to identify optimal expression on a large number of expression clones. Escherichia coli BCAT, encoded by the ilvE gene, was expressed in E. coli and Pichia pastoris. This simple colorimetric assay procedure allowed the identification of optimal clones for ilvE expression and enabled the testing of its activity in cell lysates or on whole cell catalysis.
Analytical Chemistry, 2010
Amine-transaminases (ATAs, ω-transaminases, ω-TA) are PLP-dependent enzymes that catalyze amino group transfer reactions. In contrast to the widespread and wellknown amino acid-transaminases, ATAs are able to convert substrates lacking an r-carboxylic functional group. They have gained increased attention because of their potential for the asymmetric synthesis of optically active amines, which are frequently used as building blocks for the preparation of numerous pharmaceuticals. Having already introduced a fast kinetic assay based on the conversion of the model substrate r-methylbenzylamine for the characterization of the amino acceptor specificity, we now report on a kinetic conductivity assay for investigating the amino donor specificity of a given ATA. The course of an ATA-catalyzed reaction can be followed conductometrically since the conducting substrates, a positively charged amine and a negatively charged keto acid, are converted to nonconducting products, a noncharged ketone and a zwitterionic amino acid. The decrease of conductivity for the investigated reaction systems were determined to be 33-52 µS mM-1. In contrast to other ATA-assays previously described, with this approach all transamination reactions between any amine and any keto acid can be monitored without the need for an additional enzyme or staining solutions. The assay was used for the characterization of a ATA from Rhodobacter sphaeroides, and the data obtained were in excellent agreement with gas chromatography analysis.
Organic & biomolecular chemistry, 2016
Application of amine transaminases (ATAs) for stereoselective amination of prochiral ketones represents an environmentally benign and economically attractive alternative to transition metal catalyzed asymmetric synthesis. However, the restrictive substrate scope has limited the conversion typically to non-sterically demanding scaffolds. Recently, we reported on the identification and design of fold class I ATAs that effect a highly selective asymmetric synthesis of a set of chiral aromatic bulky amines from the corresponding ketone precursors in high yield. However, for the specific amine synthetic approach extension targeted here, the selective formation of an exo- vs. endo-isomer, these biocatalysts required additional refinement. The chosen substrate (exo-3-amino-8-aza-bicyclo[3.2.1]oct-8-yl-phenyl-methanone), apart from its pharmacological relevance, is a demanding target for ATAs as the bridged bicyclic ring provides substantial steric challenges. Protein engineering combining ...
Asymmetric Synthesis of Optically Pure Pharmacologically Relevant Amines Employing ?-Transaminases
Adv Synth Catal, 2008
Various w-transaminases were tested for the synthesis of enantiomerically pure amines from the corresponding ketones employing d-or l-alanine as amino donor and lactate dehydrogenase to remove the side-product pyruvate to shift the unfavourable reaction equilibrium to the product side. Both enantiomers, (R)-and (S)-amines, could be prepared with up to 99% ee and > 99% conversions within 24 h at 50 mM substrate concentration. The activity and stereoselectivity of the amination reaction depended on the w-transaminase and substrate employed; furthermore the co-solvent significantly influenced both the stereoselectivity and activity of the transaminases. Best results were obtained by employing ATA-117 to obtain the (R)-enantiomer and ATA-113 or ATA-103 to access the (S)-enantiomer with 15% v v À1 DMSO.
Electronic Journal of Biotechnology, 2013
Background: New enzymes for biotransformations can be obtained by different approaches including directed mutagenesis and in vitro evolution. These mutants have to be efficiently produced for laboratory research on bioreactions as well as for process development. In the framework of a European ERA-IB project, two different types of enzymes (ammonia lyases and aminotransferases) have been selected as biocatalysts for the synthesis of industrially relevant amines. New mutant enzymes have been obtained: a) aspartases able to recognize β-amino acids; b) ω-transaminases with improved activity. The objectives are to find out a common operational strategy applicable to different mutants expressed in E. coli with the same initial genetic background, the development of an integrated process for production and the preparation of stable useful biocatalysts. Results: Mutant enzymes were expressed in E. coli BL21 under the control of isopropylthiogalactoside (IPTG) inducible promoter. The microorganisms were grown in a formulated defined medium and a high-cell density culture process was set up. Fed-batch operation at constant specific growth rate, employing an exponential addition profile allowed high biomass concentrations. The same operational strategy was applied for different mutants of both aspartase and transaminase enzymes, and the results have shown a common area of satisfactory operation for maximum production at low inducer concentration, around 2 μmol IPTG/g DCW. The operational strategy was validated with new mutants and high-cell density cultures were performed for efficient production. Suitable biocatalysts were prepared after recovery of the enzymes. The obtained aspartase was immobilized by covalent attachment on MANA-agarose, while ω-transaminase biocatalysts were prepared by entrapping whole cells and partially purified enzyme onto Lentikats (polyvinyl alcohol gel lens-shaped particles). Conclusions: The possibility of expressing different mutant enzymes under similar operation conditions has been demonstrated. The process was standardized for production of new aspartases with β-amino acid selectivity and new ω-transaminases with improved substrate acceptance. A whole process including production, cell disruption and partial purification was set up. The partially purified enzymes were immobilized and employed as stable biocatalysts in the synthesis of chiral amines.