Microbial and Enzymatic Processes for Anino Acid Production

-Tyrosinase, tryptophanase and cysteine desulfhydrase were shown to catalyze a variety of a,—elimination, s—replacement and the reverse of the ct,—elimination reactions of amino acids. The mechanism f or these reactions was studied using the general mechanism for pyridoxal— dependent reactions. The enzyme—bound n—amino acrylate is the key intermediate. Enzymatic methods for preparing L—tyrosine, L—tryptophan, L—cysteine and related amino acids were developed using bacterial cells with high enzyme activities. These processes are simple and economical for the production of these amino acids. INTRODUCTION Two processes have been established in Japan for the biological production of amino acids. They are: I. Fermentation (a) Methods employing wild strains (producing L—glutamic acid, L— or DL— alanine, L—valine, etc.). (b) Methods employing mutant strains (producing L—lysine, L—ornithine, L—citrulline, L—arginine, L—homoserine, L—proline, L—histidine, etc.). (c) Methods adding precursors to media (producing L—isoleucine, L—serine, L—tryptophan, etc.). II. Enzymation (a) Methods employing degradative enzymes (producing L—phenylalanine, L—tryptophan, L—alanine, etc.). (b) Methods employing synthesizing enzymes (producing L—aspartic acid). In recently developed enzymatic processes, L—lysine production from DL—n—amino--c—caprolactam (1—3), L—cysteine production from DL—2—amino—thiazoline—4—carboxylate (4), and the D— forms of phenyl— and hydroxyphenylglycines from the corresponding hydantoins (5) have been reported. During investigation of the biological and enzymatic production of amino acids, we have developed new enzymatic processes to produce L—tyrosine, L—tryptophan, L—cysteine and related amino acids, using microbial multifunctional pyridoxal enzymes. CATALYTIC PROPERTIES OF —TYROSINASE, TRYPTOPHANASE AND CYSTEINE DESULFHYDRASE —Tyrosinase (tyrosine phenol—lyase [deaminating]: EC 4.1.99.2), tryptophanase (tryptophan indole—lyase [deaminating]: EC 4.1.99.1) and cysteine desulfhydrase (cysteine hydrogen sulfide—lyase [deaminating]: EC 4.4.1.1) are the enzymes which respectively catalyze the degradation of L—tyrosine, L—tryptophan and L—cysteine, and require pyridoxal 5'—phosphate (PLP) as a cofactor. Crystalline preparations of these enzymes have been prepared in our laboratory from Escherichia interinedia (6), Proteus rettgeri (7,8) and Aerohacter aerogenes (9) and their properties have been established in some detail (6—15). With the crystalline enzymes we found that these enzymes catalyze a variety of a,—elimina— tion (eq.l), s—replacement (eq.2) and the reverse of the a,—elimination (eq.3) reactions (10,12,14,15). L-RCH2CHNH2COOH + H20 " Eli + CH3COCOOH + NH3 (1) L-RCH2CHNH2COOH + R'H ' L-R'CH2CHNH2COOH + RH (2) R'H + CH3COCOOH + NH3 ' L-R'CH2CHNH2COOH + H2O (3) —tyrosinase; R = phenolyl, —OH, —SH, —Cl, R' = phenolyl tryptophanase; R = indolyl, —OH, —SH, —Cl, R' = indolyl cysteine desulfhydrase; R = —SH, —OH, —Cl, R' = —SH, mercaptan radicals 1118 HIDEAKI YAMADA and HIDEHIKO KUNAGAI The catalytic property of —tyrosinase, as an example, will be described in some detail (10). Table 1 shows comparative substrate affinities and activities of —tyrosinase in the catalysis of n,—elimination and s—replacement reactions. In these reactions, L—tyrosine, L—serine, TABLE 1. Comparative substrate affinities and activities of —tyrosinase in catalysis of different reactions Compound Role Product measured Km (mM) Ki (mM) Vmax . (pmole/min/mg) nj-Elimination reactions Substrate Pyruvate 0.23 1.9 L—Tyrosine L—Serine Substrate Pyruvate 34 0.35 S—Methyl—L—cysteine Substrate Pyruvate 1.8 1.2 —Chloro—L—alanine Substrate Pyruvate 4.5 18.2 L—Alanine Inhibitor Pyruvate 6.5 L—Phenylalanine Inhibitor Pyruvate 2.0 Phenol Inhibitor Pyruvate 0.04 Pyrocatechol Inhibitor Pyruvate 0.46 Resorcinol Inhibitor Pyruvate 0.16 a—Replacement reactions Cosubstrate L—Tyrosine 35 0.33 L—Serine S—Methyl—L—cysteine Cosubstrate L—Tyrosine 1.8 0.82 —Chloro—L—alanine Cosubstrate L—Tyrosine 4.5 1.4 Phenol Cosubstrate L—Tyrosine 1.2 S—methyl—L—cysteine and specifically an unnatural amino acid, —ch1oro—L—alanine, are substrates: L—alanine and L—phenylalanine are competitive inhibitors. In the —rep1acement reaction, phenol is the second substrate to synthesize L—tyrosine. When pyrocatechol, resorcinol, pyrogallol and hydroxyhydroquinone were added to the reaction mixture, in place of phenol, 3, 4—dihydroxyphenyl—L—alanine (L—dopa), 2 ,4—dihydroxyphenyl—L—alanine (2 ,4—L—dopa), 2,3,4—trihydroxyphenyl—L—alanine (2,3,4—L—topa) and 2,4,5—trihydroxyphenyl—L—alanine (2,4,5— L—topa), respectively, were synthesized (Table 2). TABLE 2. Relative velocity of synthesis of L—tyrosine related amino acids from —ch1oro—L—alanine and phenol derivatives by —tyrosinase Phenol derivative L—Amino acida) synthesized Relative velocity of synthesis