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

Abstract

Temperature cycling induced deracemization (TCID) is an attractive method to provide a pure enantiomer from a racemic solid phase. In this work, we performed deracemization of the nonessential amino acid asparagine using TCID of DL-asparagine monohydrate crystals (DL-ASN•H 2 O) and an immobilized amino acid racemase (imAAR) as a racemization agent. Experiments were performed with varying initial suspension densities and dosages of imAAR to better understand the process and included measurements of both solid-phase and solution-phase enantiomeric excesses. Furthermore, we demonstrate how to improve the productivity and yield of the TCID process by adding a temperature hold at the end of the process to ensure the maximum possible yield and compare these results to a process using only standard temperature cycles. Finally, we also discuss the effect of both temperature programs on the performances of the TCID process to be a strategy to further improve this process for industrial application.

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

“…Figure 5a also indicates that the rate of evolution of % d.e s of L-aThr increased when the range of temperature used in the cycle increased, and this is because of a higher proportion of the D-Thr crystals dissolved in a cycle, allowing for a higher conversion to L-aThr. This agrees with previous deracemization work, for instance, the rate of deracemization of asparagine was increased when the initial suspension density of D-/L-asparagine monohydrate was reduced under constant temperature difference 40 which also results in a higher proportion of the crystal phase dissolved in each cycle.…”

“…This process leads to deracemization of the crystal phase of the suspension. 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…”

“…For amino acid compounds, in our previous work, we performed TCID of asparagine monohydrate in the presence of immobilized AArac12996. 40 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, 41 as already confirmed by Wurges et al 25 Here, we demonstrate a novel process combining temperature cycling and enzymatic epimerization catalyzed by immobilized AArac12996 in a suspension of Thr to accelerate the solid-state conversion of Thr to aThr. The diastereomeric pair of D-Threonine (D-Thr) and L-allothreonine (L-aThr) was selected as a model system since it is of eutectic type and no formation of co-crystals nor stable solid solutions involving the two species are reported.…”

“…Previous research on the TCID technique has shown that the racemization rate can have a substantial effect on the total time required for deracemization. ,, To investigate this effect in the current process, imAAR dosages were adjusted to investigate the influence of the rate of epimerization. Figure a shows that the time to produce 100 % d.e s was strongly depending on the dosage of imAAR, and particularly, the time required to initially nucleate l -aThr crystals changed significantly according to the imAAR dosage.…”

Characterization of an Immobilized Amino Acid Racemase for Potential Application in Enantioselective Chromatographic Resolution Processes

“…The covalent immobilization occurs by the formation of a Schiff base between the aldehyde and the amino groups of the enzyme. Exploiting this strong binding, the immobilized AAR has already successfully been integrated into a temperature cycling induced deracemization [22].…”