An Efficient Route to Both Enantiomers of allo‐Threonine by Simultaneous Amino Acid Racemase‐Catalyzed Isomerization of Threonine and Crystallization

Abstract

We present an efficient method for the production of d-and l-allo-threonine (allo-Thr) with very high purity by enzymatic isomerization of l-or d-threonine (Thr) and simultaneous crystallization. Isomerization of Thr to allo-Thr is catalyzed by a purified amino acid racemase (AArac12996) from Pseudomonas putida NBRC12996, which can easily be obtained from a recombinant E. coli strain by secretion into the medium and subsequent anion exchange chromatography. Crystallization of d-and l-allo-Thr was performed in a repetitive batch mode over a period of up to 55 days at 30 8C. Total amounts of 30.8 g d-allo-Thr and 32.4 g l-allo-Thr were obtained with a very good diastereomeric excess of de d-allo > 99.2% and de l-allo > 98.4%, respectively, and in good yields (d-allo-Thr: 83%, l-alloThr: 79%). The enzymes remarkable high stability under process conditions resulted in enzyme specific yields of 2.56 g d-allo-Thr per mg AArac12996 and 1.62 g l-allo-Thr per mg AArac12996. In contrast to chemical multi-step syntheses of allo-Thr, our process consists of only one enzyme-catalyzed reaction step with simultaneous product crystallization. The process is performed under low energy consumption (30 8C, atmospheric pressure) in water and avoids the use and production of any toxic or harmful compounds. Recovery of the enantiomerically pure products is performed by simple filtration which reduces downstream processing significantly compared to chromatographic methods which are usually applied.

Efficient Conversion of Threonine to Allothreonine Using Immobilized Amino Acid Racemase and Temperature Cycles

“…For amino acid compounds, in our previous work, we performed TCID of asparagine monohydrate in the presence of immobilized AArac12996 . Hence, the epimerization of Thr to aThr using immobilized AArac12996 should also be possible as immobilized AArac12996 catalyzes an epimerization at the α-C atom, as already confirmed by Würges et al…”

“…There are several chemical and enzymatic syntheses to produce aThr; however, the direct synthesis of allothreonine via chemical and enzymatic approaches requires multiple steps of synthesis. − Besides direct synthesis of aThr, the epimerization of Thr at α-C or β-C atoms is an alternative way to achieve a mixture of Thr and aThr. After epimerization, the separation of aThr from the mixture is essential. , For instance, Würges et al demonstrated a method for aThr production from Thr by a combination of enzymatic epimerization of Thr to aThr using AArac12996 and simultaneous crystallization in a suspension of Thr.…”

“…After epimerization, the separation of aThr from the mixture is essential. 23,24 For instance, Wurges et al 25 demonstrated a method for aThr production from Thr by a combination of enzymatic epimerization of Thr to aThr using AArac12996 and simultaneous crystallization in a suspension of Thr.…”

Temperature Cycling Induced Deracemization of dl-Asparagine Monohydrate with Immobilized Amino Acid Racemase

“…In addition to chemical racemizing agents, several amino acid racemases are available, which allow for racemization of the amino acid at mild temperatures. Examples include alanine racemase from Schizosaccharomyces pombe, arginine racemase from Pseudomonas graveolens, glutamate racemase from Pseudomonas pentosaceus, serine racemase from rat brain, and amino acid racemases from Pseudomonas putida − (P. putida).…”

The use of enzymes in organic synthesis and the life sciences: perspectives from the Swiss Industrial Biocatalysis Consortium (SIBC)

“…38,39 Another example is the combination of preferential crystallisation with enzymatic in situ racemisation, as exemplified for L-asparagine 40 and allo-threonine. 41 3.6. Ligases (EC 6)…”