l‐Amino Acids from a Racemic Mixture of α‐Hydroxy Acids

Abstract

A racemic mixture of a-hydroxy acids can be transformed to the desired optically active L-amino acid by means of a reaction route that goes through the intermediate formation of the corresponding a-keto acid. The continuous realization of this process is possible in a multienzyme membrane reactor, which has been described earlier as to its technical characteristics.'The feasibility of the process is demonstrated by the transformation of (LD)-hCtate via pyruvate to L-alanine, as first suggested by Mosbach ( See FIG. I). ' In the process, a form of NAD covalently bound to water-soluble polyethylene glycol' (PEG-20000-NAD) together with the three enzymes L-LDH, D-LDH and ALADH is retained by an ultrafiltration membrane. A necessary condition for the steady-state continuous realization of the process is that the concentration of the intermediate (pyruvate) in the reactor has to be held at some level above zero by continuous feeding. If this is not done, the soluble intermediate will be washed out from the reactor and the reaction path will be shifted entirely to the formation of the reduced coenzyme form, NADH, that is, the reaction will be stopped. On the other hand, it is conceivable that large intermediate concentrations will favor the reverse reaction to lactate and theoxidized coenzyme form, NAD'; thus, an optimal feed concentration of the intermediate will be expected.The dependence of the rates of the four individual reaction steps on substrate and product concentrations has been investigated by measuring initial rates and subsequently verified by measuring in the entire range of conversion. The data-fitting has been accomplished by a nonlinear optimization p r~c e d u r e .~The theoretical analysis below is based on the initial rate kinetic data.On the basis of the assumption that the sorption capacities of the enzymatic complexes are practically too low to have any measurable dynamic effect, the following model differential equations can be established for the dynamics of the reaction system under consideration: -d(lac) lac, -lac --dt 7 R , + Rz d(a1a) ala, -ala + R , -R4 --dt T 91(3)

Continuous cofactor regeneration Utilization of polymer bound NAD(H) for the production of optically active acids

“…The parallel produced PEG-NADH would accumulate and the reaction system would stop working after а total conversion of the coenzyme to the reduced form PEG-NADH. This fact proves the necessity of а continous with а small amount of the a-keto acid to compensate the loss of the intermediate across the membrane (20).…”

Multi‐Enzymatic Cascade Reactions: Overview and Perspectives

“…[9] As an alternative, internal cofactor regeneration may be established via enzymatic oxidation of an a-hydroxy acid, thus providing both the substrate and the reduced nicotinamide cofactor for the AADH (Scheme 7). [47,50,51] Recently, this redox-neutral cascade has been extended by employing mandelate racemase in combination with d-mandelate dehydrogenase (d-MDH) and different AADHs for the conversion of racemic mandelic acid to l-phenyl glycine in a novel type of "dynamic kinetic asymmetric transformation" (DYKAT) system. [52] In order to avoid substrate inhibition effects caused by high concentrations of phenyl pyruvate in reductive amination processes using phenylalanine dehydrogenase (PheDH), this reaction has been coupled with in situ generation of the corresponding imino acid from N-acetyl dehydrophenylalanine (acetamidocinnamate) by action of acetamidocinnamate acylase (ACA-acylase) in a cascade process (Scheme 8).…”

Engineering of alanine dehydrogenase from Bacillus subtilis for novel cofactor specificity

“…An early example is the transformation of racemic lactate into l-alanine via the intermediate pyruvate, employing a combination of dand l-lactate DHs with l-AlaDH as biocatalysts [17]. Another reaction cascade involving BasAlaDH was reported for the production of (S)-3-fluoroalanine, a potent antibiotic agent [18], via kinetic resolution of racemic 3-fluoroalanine by stereospecific oxidative deamination [19].…”