Hidetsugu Nakazawa

Enzymatic preparation of L-tryptophan and 5-hydroxy-L-tryptophan

In equations (5) and (6), RI,, represents -OH, -SH, or phenolyl radicals, and R, phenolyl radicals. In recent studies, we [9] proved that /3-tyrosine catalyze the synthesis of L-tyrosine from pyruvate, ammonia and phenol, apparently by the reversal of a,/5elimination reaction. In appropriate studieb, it has been proved that the crystalline tryptophanase from E. coli [lo] and Proteus rettgeri [ 1 l] also catalyzes the synthesis of L-tryptophan by the reversal of &elmination reaction; at rates similar to the forward reaction. We herein describe an enzymatic method for the preparation of L-tryptophan or S-hydroxy-L-tryptophan from pyruvate, ammonia and indole or 5hydroxyindole, respectively.

Enzymatic preparation of L-tyrosine or 3,4-dihydroxyphenyl-L-alanine from pyruvate, ammonia and phenol or pyrocatechol

Tyrosine phenol lyase is an enzyme which catalyzes the stoichiometric conversion of L-tyrosine to pyruvate, ammonia atid phenol, and requires pyridoxal phosphate as a cofactor [I-S] . Apparently homogeneous preparations of the enzyme were prepared in our laboratory from cells of Escherichiu in termedia and Enviniu herbicola grown in media supplemented with L-tyrosine [4,6] . We reported that the crystal: line preparations of the enzyme catalyze a series of (Y, &elimination [4,6] , P-replacement [ 7, 81 and racemization [9] reactions. The reverse of the a, p-elimination reaction to synthesize L-tyrosine from pyruvate, ammonia and phenol was also catalyzed by crystalline preparations of the enzyme [lo] . In recent studies, we proved that L-tyrosine or 3,4-dihydroxyphenyl-L-alanine (L-dopa) was synthesized from pyruvate, ammonia and phenol or pyrocatechol with intact cells directly as enzyme, in significantly high yields. We herein report an enzymatic method for the preparation of L-tyrosine and L-dopa.

In situ analysis of the microbial fermentation process by natural abundance 13C and 31P NMR spectroscopy. Production of adenosine-5′-triphosphate from adenosine

The application of 13C NMR spectroscopy to in situ analysis of the complex microbial metabolites has been reported by Eakin et al. [l] . They studied the fate of [ 1 -13 C] glucose on being fed to yeast cells, Candida utilis, by monitoring the 13C NMR spectra of metabolites without separating them from the incubation broth. Use of I-13C-enriched substrate, as has been pointed out by Eakin et al. [l] , would allow one to use a substrate concentration lower than several millimolar for its increased NMR sensitivity, although usual microbial fermentations, which are carried out in both laboratories and industry, use much higher substrate concentrations. The r3C enrichment may still be essential to study the transient metabolites which might exist in the fermentation process only for a short period of time compared to the duration to obtain the r3C NMR spectra of metabolites at natural 13C abundance of 1 .l%, or to search for the metabolites which might exist at very low concentration, either in intraor extracellular medium. Despite the merit of higher NMR sensitivity described above use of the r3C-enriched substrates in analyzing microbial reactions has obvious drawbacks. As only the enriched portion of the carbons in the metabolites can be detected, unexpected metabolites in the broth may hardly be characterized. The metabolites, by the same reason, which bear no carbons from the enriched part of the substrate may be overlooked.

Cultural Conditions for the Microbial Production of β-Tyrosinase and Tryptophanase

β-Tyrosinase (tyrosine phenol-lyase: EC 4.1.99.2) and tryptophanse (tryptophan indole-lyase: EC 4.1.99.1) are enzymes which respectively catalyze the degradation of L-tyrosine and L-tryptophan, and require pyridoxal 5’-phosphate (PLP) as a cofactor. Crystalline preparations of these enzymes were prepared in our laboratories from Escherichia intermedia and Proteus rettgeri, and their properties were established in some detail. The crystalline enzymes were shown to catalyze a variety of α,β-elimination (Eq. 1), β-replacement (Eq. 2), and the reverse of α,β-elimination reactions (Eq. 3) (1–3).