Novel biosynthetic approaches to the production of unnatural amino acids using transaminases

Taylor, Paul; Pantaleone, David P.; Senkpeil, Richard F; Fotheringham, Ian

doi:10.1016/s0167-7799(98)01240-2

Cited by 223 publications

(141 citation statements)

References 31 publications

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“…S3), causing severe departure from linearity in Lineweaver-Burk plots. For three other 2-oxoacids similar inhibition became apparent at lower concentrations, making it impossible to obtain linear plots and good kinetic parameters according to Eqn (1).…”

Section: Substrate Inhibitionmentioning

confidence: 99%

“…Apart from their evident metabolic importance, aminotransferases have biotechnological potential in view of their ability to handle a considerable range of substrates. In practice, they have been increasingly applied to large-scale synthesis of non-natural amino acids, which can be used as the building blocks for peptidomimetic and other single-enantiomer drugs in the pharmaceutical industry [1][2][3][4]. The most widely investigated are the a-transaminases, e.g.…”

Section: Introductionmentioning

confidence: 99%

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The specificity and kinetic mechanism of branched‐chain amino acid aminotransferase from Escherichia coli studied with a new improved coupled assay procedure and the enzyme's potential for biocatalysis

Wang

Engel

2013

The FEBS Journal

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Branched-chain amino acid aminotransferase (BCAT) plays a key role in the biosynthesis of hydrophobic amino acids (such as leucine, isoleucine and valine), and its substrate spectrum has not been fully explored or exploited owing to the inescapable restrictions of previous assays, which were mainly based on following the formation/consumption of the specific branched-chain substrates rather than the common amino group donor/ acceptor. In our study, detailed measurements were made using a novel coupled assay, employing (R)-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans as an auxiliary enzyme, to provide accurate and reliable kinetic constants. We show that Escherichia coli BCAT can be used for asymmetric synthesis of a range of non-natural amino acids such as L-norleucine, L-norvaline and L-neopentylglycine and compare the kinetic results with the results of molecular modelling. A full two-substrate steadystate kinetic study for several substrates yields results consistent with a bibi ping-pong mechanism, and detailed analysis of the kinetic constants indicates that, for good 2-oxoacid substrates, release of 2-oxoglutarate is much slower than release of the product amino acid during the transamination reaction. The latter is in fact rate-limiting under conditions of substrate saturation. DatabaseBranched-chain amino acid aminotransferase EC 2.6.1.42; (R)-2-hydroxyglutarate dehydrogenase EC 1.1.99.2

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Section: Substrate Inhibitionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The specificity and kinetic mechanism of branched‐chain amino acid aminotransferase from Escherichia coli studied with a new improved coupled assay procedure and the enzyme's potential for biocatalysis

Wang

Engel

2013

The FEBS Journal

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“…Biocatalytic processes that have been commercialized include the acylase process operated by Degussa (8) and Tanabe Seiyake (16), the D-hydantoinase process operated by Kanegafuchi (58,59), the aminotransferase process operated by NSC Technologies (1,48), and the L-leucine dehydrogenase process operated by Degussa (10). At DSM, we have implemented several chemoenzymatic processes for the production of enantiopure ␣-hydrogen-and ␣,␣-disubstituted ␣-amino acids, which are based on the stereoselective hydrolysis of racemic amino acid amides (46).…”

mentioning

confidence: 99%

l-Selective Amidase with Extremely Broad Substrate Specificity fromOchrobactrum anthropiNCIMB 40321

Sonke¹,

Ernste²,

Tandler³

et al. 2005

Appl Environ Microbiol

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An industrially attractive L-specific amidase was purified to homogeneity from Ochrobactrum anthropi NCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60°C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn 2؉ (to 80%), Mn 2؉ (to 400%), and Mg 2؉ (to 560%). Serine and cysteine protease inhibitors did not influence the purified amidase. This enzyme displayed activity toward a broad range of substrates consisting of ␣-hydrogen-and (bulky) ␣,␣-disubstituted ␣-amino acid amides, ␣-hydroxy acid amides, and ␣-N-hydroxyamino acid amides. In all cases, only the L-enantiomer was hydrolyzed, resulting in E values of more than 150. Simple aliphatic amides, ␤-amino and ␤-hydroxy acid amides, and dipeptides were not converted. The gene encoding this L-amidase was cloned via reverse genetics. It encodes a polypeptide of 314 amino acids with a calculated molecular weight of 33,870. Since the native enzyme has a molecular mass of about 66 kDa, it most likely has a homodimeric structure. The deduced amino acid sequence showed homology to a few other stereoselective amidases and the acetamidase/ formamidase family of proteins (Pfam FmdA_AmdA). Subcloning of the gene in expression vector pTrc99A enabled efficient heterologous expression in Escherichia coli. Altogether, this amidase has a unique set of properties for application in the fine-chemicals industry.Enantiomerically pure ␣-amino acids, both natural and unnatural, form an important class of chiral building blocks for the pharmaceutical and agrochemical industries. Examples of important ␣-hydrogen-␣-amino acids are D-phenylglycine (DPhg) and D-p-hydroxyphenylglycine, which are produced as side chains for the manufacture of semisynthetic ␤-lactam antibiotics such as ampicillin, amoxicillin, and cephalexin (14, 53), and D-valine, an intermediate for the pyrethroid insecticide fluvalinate (28). Another frequently used ␣-hydrogen-␣-amino acid is L-tert-leucine, which is used as a building block for a variety of antiviral (e.g., anti-human immunodeficiency virus), antiarthritis, and anticancer drugs under development and as a chiral auxiliary in chemical asymmetric synthesis (5, 10). Besides ␣-hydrogen-␣-amino acids, ␣,␣-disubstituted ␣-amino acids also constitute a group of compounds of increasing importance, as exemplified by the use of L-␣-methyl-3,4-dihydroxyphenylalanine (L-␣-methyldopa) as an antihypertensive drug (34, 42), ␣-methylvaline as an intermediate for the herbicide Arsenal and related herbicides (47, 49), and L-␣-methylphenylglycine as a building block for the new fungicide fenamidone (27).Enantiomerically pure ␣-amino acids can be produced on a commercial scale by a number of different processes, both chemical and enzymatic. Biocatalytic processes that have been commercialized include the acylase process operated by Degussa (8) a...

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“…Enzymes for the synthesis of chiral amino‐alcohols include imine reductases (Leipold, Hussain, Ghislieri, & Turner, 2013), amino acid dehydrogenases (Zhu & Hua, 2009), transaminases, lyases, and monoamine oxidases (Ghislieri & Turner, 2014; Höhne & Bornscheuer, 2009). Although transaminases with high substrate regio‐ and stereoselectivity have been found, their application is often constrained by unfavorable reaction equilibria and substrate and/or product inhibition (Rios‐Solis et al, 2015; Stewart, 2001; Taylor, Pantaleone, Senkpeil, & Fotheringham, 1998; Villegas‐Torres et al, 2015). These constraints require careful consideration in biocatalytic process development.…”

Section: Introductionmentioning

confidence: 99%

Enzymatic synthesis of chiral amino‐alcohols by coupling transketolase and transaminase‐catalyzed reactions in a cascading continuous‐flow microreactor system

Gruber

Carvalho

Marques

et al. 2017

Biotech & Bioengineering

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Rapid biocatalytic process development and intensification continues to be challenging with currently available methods. Chiral amino‐alcohols are of particular interest as they represent key industrial synthons for the production of complex molecules and optically pure pharmaceuticals. (2S,3R)‐2‐amino‐1,3,4‐butanetriol (ABT), a building block for the synthesis of protease inhibitors and detoxifying agents, can be synthesized from simple, non‐chiral starting materials, by coupling a transketolase‐ and a transaminase‐catalyzed reaction. However, until today, full conversion has not been shown and, typically, long reaction times are reported, making process modifications and improvement challenging. In this contribution, we present a novel microreactor‐based approach based on free enzymes, and we report for the first time full conversion of ABT in a coupled enzyme cascade for both batch and continuous‐flow systems. Using the compartmentalization of the reactions afforded by the microreactor cascade, we overcame inhibitory effects, increased the activity per unit volume, and optimized individual reaction conditions. The transketolase‐catalyzed reaction was completed in under 10 min with a volumetric activity of 3.25 U ml−1. Following optimization of the transaminase‐catalyzed reaction, a volumetric activity of 10.8 U ml−1 was attained which led to full conversion of the coupled reaction in 2 hr. The presented approach illustrates how continuous‐flow microreactors can be applied for the design and optimization of biocatalytic processes.

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Novel biosynthetic approaches to the production of unnatural amino acids using transaminases

Cited by 223 publications

References 31 publications

The specificity and kinetic mechanism of branched‐chain amino acid aminotransferase from Escherichia coli studied with a new improved coupled assay procedure and the enzyme's potential for biocatalysis

The specificity and kinetic mechanism of branched‐chain amino acid aminotransferase from Escherichia coli studied with a new improved coupled assay procedure and the enzyme's potential for biocatalysis

l-Selective Amidase with Extremely Broad Substrate Specificity fromOchrobactrum anthropiNCIMB 40321

Enzymatic synthesis of chiral amino‐alcohols by coupling transketolase and transaminase‐catalyzed reactions in a cascading continuous‐flow microreactor system

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