Table 2.

Activity of mutant BC621 and wild-type DAPDH for the synthesis of D-amino acids by the reductive amination of the corresponding 2-keto acids with ammonia.

d-Amino acid product	Mutant BC621 (U/mg)[a]	Wild-type (U/mg)[a]	Enhancement over wild-type	Enantiomeric excess
d-Alanine (d-2-aminopropionate)	0.12	0.009	13	77%[e]
d-2-Aminobutyrate	0.25	0.018	14	> 99%
d-Norvaline (d-2-aminopentanoate)	0.69	0.03	23	> 99%
d-Norleucine (d-2-aminohexanoate)	1.41	0.0095	257	> 99%
d-2-Aminoheptanoate	2.15	0.004	538	> 99%
d-2-Aminooctanoate	7.8	0.008	975	95%
d-Valine	0.28	0.005	56	98%
d-tert-Leucine	N/A[b]	N/A	—	N/A
d-Isoleucine	0.22	N/A	∞	96%
d-Leucine	0.62	0.004	155	> 99%
d-Cyclopentylglycine	0.28	0.002	140	> 99%
d-Cyclohexylalanine	2.5	0.004	625	> 99%
d-Methionine	1.0	0.006	153	> 99%
d-Aspartate	N/A	N/A	—	N/A
d-Glutamate	0.025	N/A	∞	N/A
d-Phenylglycine	N/A	N/A	—	N/A
d-Phenylalanine	0.11	N/A	∞	> 99%
d-Tyrosine[d]	0.42	N/D[c]	—	> 99%
d-4-Fluorophenylalanine	0.068	0.002	34	> 99%
d-4-Chlorophenylalanine	0.055	N/A	∞	> 99%
d-Homophenylalanine	N/A	N/A	—	N/A
Control reaction with no 2-keto acid	0.001	0.0004	—

^[a]1 U = 1 μmol NADP⁺/min Activity was normalized to unit weight of lyophilized solids.

^[b]N/A: No measurable activity was observed with this substrate under the assay conditions used.

^[c]N/D: Activity was not determined for this substrate.

^[d]Due to the high absorbance of this substrate at 340 nm, activity towards this compound was assayed via HPLC using 25 mM 2-keto acid, 200 mM NH₄Cl, 5 mM NADPH, 100 mM sodium carbonate buffer, pH 9.0. The HPLC conditions were the following: Column: Synergi 4μ Polar RP (Phenomenex, Torrance, CA), 250 × 4.6 mm, 4 μ particle size; Program: 1 ml/min of 95% aqueous 20 mM potassium phosphate pH 5.7, 5% MeOH, isocratic; product eluted at 4.5 min and was detected via UV absorbance 225 nm.

^[e]Formation of l-alanine due to presence of alanine racemase, see discussion.