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Literature summary extracted from

  • Han, S.; Shin, J.
    One-pot preparation of D-amino acids through biocatalytic deracemization using alanine dehydrogenase and omega-transaminase (2018), Catal. Lett., 148, 3678-3684 .
No PubMed abstract available

Application

EC Number Application Comment Organism
2.6.1.B21 synthesis in another reaction for deracemization using enantiocomplementary transaminases, the oxidative deamination step is carried out by alpha-transaminase such as branched-chain transaminase (EC 2.6.1.42) and D-amino acid transaminase (EC 2.6.1.21). It is notable that substitution of alpha-transaminase with alanine dehydrogenase in the deracemization method for production of D-amino acids using L-alanine dehydrogenase (AlaDH), D-selective omega-transaminase (omega-TA) ARTAmut, and NADH oxidase (NOX) eliminates the need for an expensive 2-oxoacid cosubstrate. Feasibility of the stereoinversion reaction is dependent on enzyme activities of AlaDH and onega-TA for L-amino acids and its keto acids, respectively. ARTAmut substrate specificity allows various oxoacids as substrates Arthrobacter sp.

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.4.1.1 gene AlaDH, DNA and amino acid sequence determination and analysis, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) Bacillus subtilis
2.6.1.B21 construction of the pET28a vector carrying a codon-optimized gene of an engineered D-selective omega-transaminase (omega-TA) ARTAmut from Arthrobacter sp. (ARTAmut), recombinant expression of His-tagged omega-TA in Escherichia coli strain BL21(DE3) Arthrobacter sp.

Protein Variants

EC Number Protein Variants Comment Organism
1.4.1.1 additional information biocatalytic deracemization of aliphatic amino acids into D-enantiomers by running cascade reactions: (1) stereoinversion of L-alanine to a D-form by L-alanine dehydrogenase and omega-transaminase and (2) regeneration of NAD+ by NADH oxidase. Under the cascade reaction conditions containing 100 mM isopropylamine and 1 mM NAD+, complete deracemization of 100 mM DL-alanine is achieved after 24 h with 95% reaction yield of D-alanine (over 99% enantiomeric excess, 52% isolation yield). AlaDH produces pyruvate from L-alanine with NAD+, NOX oxidizes NADH to NAD+, and reductive amination of the resulting pyruvate back to the amino acid in an enantiomerically opposite form by D-selective omega-transaminase (omega-TA) using isopropylamine as an amino donor cosubstrate. Method evaluation, overview Bacillus subtilis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.4.1.1 L-alanine + H2O + NAD+ Bacillus subtilis
-
 pyruvate + NH3 + NADH + H+
-
 ?
1.4.1.1 L-alanine + H2O + NAD+ Bacillus subtilis 168
-
 pyruvate + NH3 + NADH + H+
-
 ?
2.6.1.B21 (R)-alpha-methylbenzylamine + pyruvate Arthrobacter sp.
-
 acetophenone + L-alanine
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.4.1.1 Bacillus subtilis Q08352
-
-
1.4.1.1 Bacillus subtilis 168 Q08352
-
-
2.6.1.B21 Arthrobacter sp.
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.4.1.1 recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Bacillus subtilis
2.6.1.B21 recombinant His-tagged ARTA mut from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and desalting gel filtration Arthrobacter sp.

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.4.1.1 4
-
 recombinant His-tagged enzyme, pH 7.0, 37°C Bacillus subtilis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.4.1.1 L-alanine + H2O + NAD+
-
 Bacillus subtilis pyruvate + NH3 + NADH + H+
-
 ?
1.4.1.1 L-alanine + H2O + NAD+
-
 Bacillus subtilis 168 pyruvate + NH3 + NADH + H+
-
 ?
1.4.1.1 additional information one-pot preparation of D-amino acids through biocatalytic deracemization using alanine dehydrogenase and omega-transaminase. AlaDH produces pyruvate from L-alanine with NAD+, NOX oxidizes NADH to NAD+, and reductive amination of the resulting pyruvate back to the amino acid in an enantiomerically opposite form by D-selective omega-transaminase (omega-TA) using isopropylamine as an amino donor cosubstrate Bacillus subtilis ?
-
-
1.4.1.1 additional information one-pot preparation of D-amino acids through biocatalytic deracemization using alanine dehydrogenase and omega-transaminase. AlaDH produces pyruvate from L-alanine with NAD+, NOX oxidizes NADH to NAD+, and reductive amination of the resulting pyruvate back to the amino acid in an enantiomerically opposite form by D-selective omega-transaminase (omega-TA) using isopropylamine as an amino donor cosubstrate Bacillus subtilis 168 ?
-
-
2.6.1.B21 (R)-alpha-methylbenzylamine + pyruvate
-
 Arthrobacter sp. acetophenone + L-alanine
-
 r
2.6.1.B21 (R)-alpha-methylbenzylamine + pyruvate isopropylamine is one of the preferred amino donors for omega-TA Arthrobacter sp. acetophenone + L-alanine
-
 r
2.6.1.B21 2-oxo-4-hydroxybutyrate + isopropylamine low activity, 6% activity compared to pyruvate Arthrobacter sp. D-homoserine + acetone
-
 r
2.6.1.B21 2-oxo-hexanoate + isopropylamine low activity, 2% activity compared to pyruvate Arthrobacter sp. D-norleucine + acetone
-
 r
2.6.1.B21 2-oxobutyrate + isopropylamine 33% activity compared to pyruvate Arthrobacter sp. D-2-aminobutyrate + acetone
-
 r
2.6.1.B21 2-oxovalerate + isopropylamine low activity, 2% activity with 2b compared to pyruvate Arthrobacter sp. D-2-amino-pentanoate + acetone
-
 r
2.6.1.B21 additional information developed of a deracemization method for production of D-amino acids using L-alanine dehydrogenase (AlaDH), D-selective omega-transaminase (omega-TA) ARTAmut, and NADH oxidase (NOX). Unwanted L-amino acid in the racemic mixture is converted to a D-form after two consecutive reactions catalyzed by AlaDH and ARTA mut, leading to complete deracemization or enantioenrichment depending on the substrate specificity of the two enzymes, reaction cascade and method evaluation, overview Arthrobacter sp. ?
-
-
2.6.1.B21 pyruvate + isopropylamine
-
 Arthrobacter sp. D-alanine + acetone
-
 r

Subunits

EC Number Subunits Comment Organism
1.4.1.1 homohexamer 6 * 39700, recombinant His-tagged enzyme, SDS-PAGE Bacillus subtilis
2.6.1.B21 homodimer
-
 Arthrobacter sp.

Synonyms

EC Number Synonyms Comment Organism
2.6.1.B21 ARTAmut
-
 Arthrobacter sp.
2.6.1.B21 D-selective omega-TA
-
 Arthrobacter sp.
2.6.1.B21 omega-TA
-
 Arthrobacter sp.
2.6.1.B21 omega-transaminase
-
 Arthrobacter sp.

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.4.1.1 37
-
 assay at Bacillus subtilis
2.6.1.B21 37
-
 assay at Arthrobacter sp.

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.4.1.1 7
-
 assay at Bacillus subtilis
2.6.1.B21 7
-
 assay at Arthrobacter sp.

Cofactor

EC Number Cofactor Comment Organism Structure
1.4.1.1 NAD+
-
 Bacillus subtilis
1.4.1.1 NADH
-
 Bacillus subtilis
2.6.1.B21 pyridoxal 5'-phosphate PLP, dependent on Arthrobacter sp.

General Information

EC Number General Information Comment Organism
2.6.1.B21 evolution the enzyme is a PLP fold type IV transaminase. Transaminases of PLP fold type IV are characterized by (R)- or (S)-stereoselective transfer of amino groups, depending on the substrate profile of the enzyme. PLP fold type IV transaminases include branched-chain amino acid transaminases (BCATs), D-amino acid transaminases, and (R)-amine:pyruvate transaminases. A small substrate binding pocket which is a general property for all the omega-TAs known to date Arthrobacter sp.