L-Alanine dehydrogenase

Several sensors for L-alanine are obtained by immobilizing L-alanine dehydrogenase over an ammonia gas-sensing electrode (288) or over an O2 sensor (280). The enzyme catalyzes the specific deamination of alanine in the presence of the coenzyme NAD ... 

Co-immobilizing a second enzyme, NADH oxidase (280) or lactate dehydrogenase (288), permits the regeneration of NAD+. Measurements may be completed in Tris-HCl or carbonate buffers, but borate and glycine buffers inhibit L-alanine dehydrogenase (289). Highly selective L-histidine electrodes are available (290, 291) to determine histidine in urine (291). 

The application of the same principle to the formation of o-alanine is possible but lacks applicability due to its slowness. In this case, in the presence of ammonia, pyruvate is in equilibrium with its imine. This is reduced at the cathode under formation of racemic alanine. The L-alanine of the racemic mixture is reoxidized by L-alanine dehydrogenase under anodic regeneration of the necessary cofactor NAD to give pyruvate and ammonia, while the o-alanine is not accepted by the enzyme and accumulates in the reaction mixture. The drawback of this reaction is the kinetic control by imine formation, which is very slow, so that a complete inversion of a lOmAf solution of L-alanine would require 140 h [105]. 

When reaction (22) is 50—100 times faster than reaction (21), the oxygen consumption is proportional to the activity of L-alanine dehydrogenase (Figure 8). Since NAD+ is continuously regenerated, the enzyme activity is proportional to time, and does not show the typical progressive inhibition due to the accumulation of reduced pyridine coenzyme. This technique allows many dehydrogenases be studied under simple experimental conditions. Moreover, in the presence of excess pyridine-linked dehydrogenase, experiments can be conducted with very low substrate concentrations (1—5 nmol). 

Alanine racemase is a bacterial enzyme that catalyzes racemization of l- and d-alanine, and requires pyridoxal 5 -phosphate (PLP) as a cofactor. The enzyme plays an important role in the bacterial growth by providing D-alanine, a central molecule in the peptidoglycan assembly and cross-linking, and has been purified from various sources15 161. The enzyme has been used for the production of stereospecifically deuterated NADH and various D-amino acids by combination of L-alanine dehydrogenase (E. C. 1.4.1.1), D-amino acid aminotransferase (E. C. 2.6.1.21), and formate dehydrogenase (E.C. 1.2.1.2)I17, 18. ... 

Figure 17-3. A, Preparation of [4S-2H]-NADH by coupling of alanine racemase and L-alanine dehydrogenase. B, In situ determination of stereospecificity of H-transfer by H-NMR. AlaR, AlaDH, and DH represent alanine racemase, L-alanine dehydrogenase, and dehydrogenase, respectively.

A simple procedure was established for the synthesis of various D-amino adds by means of four types of thermostable enzymes alanine racemase, D-amino acid aminotransferase 49, 501, L-alanine dehydrogenase 51, and formate dehydrogenase (Fig. 17-4) 171. The commercial preparation of formate dehydrogenase from Candida boidinii used by Wichmanri et al. 38 is not sufficiently stable. However, Galkin et al.1521 doned and expressed the gene of thermostable formate dehydrogenase in E. coli. 

Table 17-2. Synthesis of D-amino acids from a-keto acids by combination of four purified enzymes alanine racemase, L-alanine dehydrogenase, formate dehydrogenase, and D-amino acid aminotransferase.

Normal strains of B. subtilis possess an NAD-dependent L-alanine dehydrogenase and are devoid of either NAD-GDH or NADP-GDH (29,236). am+ mutants of this species are known which contain a fairly active NADP-GDH and lack demonstrable alanine dehydrogenase activity (29,236), and it has been suggested that there is conversion of the alanine dehydrogenase to an enzyme with glutamate dehydrogenase activity by mutations induced by nitrous acid (237). 

Another interesting example of a redox neutral cascade has been proposed for the multienzymatic synthesis of (JJ)-3-fluorolactic acid together with the resolution of racemic 3-fluoroalanine (Scheme 11.5b) [13]. Optically enriched (S)-3-fluoroalanine (88% ee) was recovered unreacted after the enantioselective oxidative deamination of the racemic substrate catalyzed by the L-alanine dehydrogenase (i-AlaDH) from Bacillus subtilis. This oxidative reaction, which is thermodynamically unfavorable, was driven by the coupled reduction reaction of the intermediate 3-fluoropyruvate catalyzed by rabbit muscle i-lactate dehydrogenase (L-LDH). Since both enzymes are NADH dependent, this coupled... 

L-lsoleucine is a generally used amino acid and is an essential amino acid in feedstuffs and in infusion solutions, with annual production yielding amoxmts of 400 f [106]. A new way to use reductive amination in whole cells was recenfly invented [53]. A coupled enzyme system consisting of an L-alanine-dependenf fransaminase and an L-alanine dehydrogenase for fhe cofactor regeneration is needed to recycle the amino donor L-alanine as shown in Scheme 29.14. Both enzymes were expressed in E. coli. 

---