Deracemization and Stereoinversion of α‐Amino Acids by l‐Amino Acid Deaminase

Abstract

Enantiomerically pure a-amino acids are compounds of primary interest for the fine chemical, pharmaceutical, and agrochemicals ectors.A mino acid oxidases are usedf or resolving d,l-amino acids in biocatalysis. We recently demonstrated that lamino acid deaminase from Proteus myxofaciens (PmaLAAD) showsp eculiar features for biotechnologicala pplications,s uch as ah igh production level as soluble protein in Escherichia coli and as table binding with the flavin cofactor.S ince l-amino acid deaminases are membrane-bound enzymes,p revious applications were mainly based on the use of cellbased methods.N ow,t aking advantage of the broad substrate specificity of PmaLAAD, an umber of natural and synthetic l-amino acids were fully converted by the purified enzyme into the corresponding aketo acids:t he fastest conversion was obtained for 4nitrophenylalanine.A nalogously,s tarting from race-mic solutions,t he full resolution (ee > 99%) was also achieved. Notably, d,l-1-naphthylalanine was resolved either into the d-o rt he l-enantiomer by using PmaLAADo rt he d-aminoa cid oxidase variant having ag lycine at position 213, respectively, and was fully deracemized when the two enzymes were used jointly.M oreover, the complete stereoinversion of l-4-nitrophenylalaninew as achieved using PmaLAADa nd as mall molar excesso fb orane tertbutylamine complex. Taken together, recombinant PmaLAADr epresents an l-specific amino acid deaminase suitablef or producing the pure enantiomers of several natural and synthetic amino acids or the corresponding keto acids,c ompounds of biotechnologicalo rp harmaceutical relevance.

Highly selective synthesis of d-amino acids from readily available l-amino acids by a one-pot biocatalytic stereoinversion cascade

“…36 During the oxidative deamination reaction, PmLAAD catalyzes the deamination of L-Phe to the corresponding a-imino acid with oxygen as the co-substrate, aer which spontaneous hydrolysis produces phenylpyruvic acid (PPA) (Scheme 2). [37][38][39] Because PmLAAD is a membrane-bound protein, 40 different forms of the biocatalyst were prepared in order to investigate their effects on the yield of PPA from L-Phe. As shown in Table S1, † the yield of PPA was higher when the biocatalyst was in whole-cell form compared to other biocatalyst types.…”

Ancestral L-amino acid oxidases for deracemization and stereoinversion of amino acids

“…Proteinogenic amino acids have been produced on a large scale using a fermentation approach, whereas synthesis of non-proteinogenic amino acids, such as enantiopure amino acid derivatives, remains a challenging task 2 . Many attempted approaches to solve this challenge have been reported and all involve synthesizing chiral amino acids [3][4][5][6][7][8][9][10][11] ; in this study, we focused on the deracemization approach utilizing amino acid oxidases and chemical reductants 12,13 . The deracemization was performed by the following steps repeatedly; D-or L-amino acids in racemates were oxidized to α-imino acids by amino acid oxidases, and the resulting imino acids were reduced to racemates by chemical reductants (Fig.…”

“…This low productivity may be due to high toxicity of the enzymes in the host cells 22 . L-amino acid deaminase (LAAD) is utilized as an alternative to LAAO 3,11,23 , and is a FAD-dependent enzyme which catalyzes the deamination of L-amino acids [24][25][26] . LAAD generates little H 2 O 2 during the reaction which is a key factor to improving productivity and the ability to synthesize αketo acids from L-amino acids 24 .…”

Biotransformation and chiral resolution of d,l‐alanine into pyruvate and d‐alanine with a whole‐cell biocatalyst expressing l‐amino acid deaminase

“…In previous research, l-amino acid deaminases (l-AAD, EC 1.4.3.2) were able to catalyze the stereoselective oxidative deamination of l-amino acids into the corresponding αketo acids and ammonia [18][19][20]. Therefore, l-AAD can be used to catalyze racemic mixtures of amino acids into single enantiomer, which is of great economic interest since in general single enantiomers are more economically valuable than the corresponding racemic mixtures of amino acids.…”

“…At present, l-AAD was identified in various microorganisms such as Morganella, Provindecia, and Proteus [21]. The recombinant PmaLAAD derived from Proteus myxofaciens was used to produce the pure enantiomers of several amino acids and the respective α-keto acids [19]. An engineered l-AAD from P. mirabilis KCTC 2566 (Pm1) has been used to produce αketoglutaric acid from glutamic acid [22].…”