Abstract
l-β-Chloroalanine is a useful intermediate for the synthesis of several l-amino acids. Conditions for synthesizing optically pure l-β-chloroalanine from 3-chloropyruvate using alanine dehydrogenase (AlaDH), leucine dehydrogenase and phenylalanine dehydrogenase with a regeneration of NADH by formate dehydrogenase (FDH) were investigated. The enzymatic reaction was carried out at neutral pH because of a chemical instability of 3-chloropyruvate on the alkaline side. Commercially available AlaDH from Bacillus stearothermophilus IFO 12550 showed the highest activity for the production of l-β-chloroalanine at pH 7.5. The K m and V max values for 3-chloropyruvate of AlaDH were calculated to be 300 units/mg and 62.5 mm, respectively. Although 3-chloropyruvate had no inhibitory effect on AlaDH, it acted as a non-competitive inhibitor with FDH. 3-Chloropyruvate was added into the reaction mixture in a stepwise manner to avoid the inhibition. l-β-Chloroalanine was produced with high chemical (>90%) and optical yields (100% enantiometric excess) and at a high concentration (43 g/l).
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Asano Y, Nakazawa A, Endo K, Hibino Y, Ohmori M, Numao N, Kondo K (1987a) Phenylalanine dehydrogenase of Bacillus badius. Eur J Biochem 168:153–159
Asano Y, Nakazawa A, Kondo K (1987b) Novel phenylalanine dehydrogenases from Sporosarcina ureae and Bacillus sphaericus. J Biol Chem 262:10346–10354
Asano Y, Yamada A, Kato Y, Yamaguchi K, Hibino Y, Hirai K, Kondo K (1990) Enantioselective synthesis of (S)-amino acids by phenylalanine dehydrogenase from Bacillus sphaericus: use of natural and recombinant enzymes. J Org Chem 55:5567–5571
Cargoe EJ, Robb CM (1960) Synthesis of 3-hydroxyquinoline. Org Synth 40:54–60
Chenault HK, Dahmer J, Whitesides GM (1989) Kinetic resolution of unnatural and rarely occuring amino acids. J Am Chem Soc 111:6354–6365
Chibata I, Ishikawa T, Yamada S (1957) Studies on the enzymatic resolution; a survey of the acylase in mold. Bull Agric Chem Soc Jpn 21:300–303
Chibata I, Tosa T, Sato T (1986) Decarboxylase to l-alanine. Appl Biochem Biotechnol 13:231–240
Fischer E, Raske E (1907) Verwandlung des l-serins in d-alanin. Chem Ber 40:3717–3724
Hümmel W, Kula M-R (1989) Dehydrogenase for the synthesis of chiral compounds. Eur J Biochem 184:1–13
Inoue O, Murayama A (1986) Optical resolutions of β-chloro-dl-alanine. Japanese patent no. 86-152645
Itoh N, Morikawa R (1983) Crystallization and properties of l-alanine dehydrogenase from Streptomyces phaeochromogenes. Agric Biol Chem 47:2511–2519
Kato N, Kano M, Tani Y, Ogata K (1974) Purification and characterization of formate dehydrogenase in a methanol-utilizing yeast, Kloeckera sp. no. 2201. Agric Biol Chem 38:111–116
Kumagai H, Kiuchi M (1986) A process for producing S-carboxymethyl-l-cysteine. Japanese patent no. 86-1582293
Kuroda S, Tanizawa K, Sakamoto Y, Tanaka H and Soda K (1990) Alanine dehydrogenases from two Bacillus species with distinct thermostabilities: molecular cloning, DNA and protein sequence determination, and structural comparison with other NAD(P)+-dependent dehydrogenases. Biochemistry 29:1009–1015
Nagasawa T, Yamada H (1986) Enzymatic transformations of 3-chloroalanine into useful amino acids. Appl Biochem Biotechnol 13:147–165
Nakayasu K, Furuya O, Inoue C, Moriguchi S (1981) β-Chloroalanine. Deutsches Patent no. 3021566
Ohoka M, Kato T, Mita R, Kawashima N, Higuchi C (1987) A process for producing β-chloroalanine. World patent no. 8204043
Ohshima T, Soda K (1979) Purification and properties of alanine dehydrogenase from Bacillus sphaericus. Eur J Biochem 100:29–39
Ohshima T, Nagata S, Soda K (1985) Purification and characterization of thermostable leucine dehydrogenase from Bacillus stearothermophilus. Arch Microbiol 141:407–411
Ohshima T, Wandrey C, Conrad D (1989) Continuous production of 3-fluoro-l-alanine with alanine dehydrogenase. Biotechnol Bioeng 34:394–397
Ohshima T, Takada H, Yoshimura T, Esaki N, Soda K (1991) Distribution, purification and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes. J Bacteriol 173:3943–3948
Rosazza JP (1982) Microbial transformation of bioactive compounds. CRC Press, Boca Raton, Fl.
Sakamoto Y, Nagata S, Esaki N, Tanaka H, Soda K (1990) Genecloning, purification and characterization of thermostable alanine dehydrogenase of Bacillus stearothermophilus. J Ferm Bioeng 69:154–158
Soda K, Misono H, Mori K, Sakato H (1971) Crystalline l-leucine dehydrogenase. Biochem Biophys Res Commun 44:931–935
Takahashi S, Ohashi T, Kii Y, Kumagai H, Yamada H (1979) Microbial transformation of hydrolysis to N-carbamyl-d-amino acids. J Ferment Technol 57:328–332
Walsh CT, Schonbrunn A, Abeles RH (1971) Studies on the mechanism of action of d-amino acid oxidase. J Biol Chem 246:6855–6866
Yamada H, Shimizu S (1988) Microbial and enzymatic processes for the production of biologically and chemically useful compounds. Angew Chem Int Ed Engl 27:622–642
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kato, Y., Fukumoto, K. & Asano, Y. Enzymatic synthesis of l-β-chloroalanine using amino acid dehydrogenase. Appl Microbiol Biotechnol 39, 301–304 (1993). https://doi.org/10.1007/BF00192082
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00192082