Koji Mitsugi

A new method for the synthesis of some 9-β-D-arabinofuranosylpurines by a combination of chemical and enzymatic reactions

Abstract An enzymatic transarabinosylation between 2-chlorohypoxanthine and 1-β- D -arabinofuranosyluracil gave 9-β- D -arabinofuranosyl-2-chlorohypoxanthine which was chemically converted to 9-β- D -arabinofuranosylguanine and its derivatives.

Enzymatic Production of l-Tryptophan from l- and dl-5-Indolyl-methylhydantoin by Newly Isolated Bacterium

Bacteria which can hydrolyze dl-5-indolylmethylhydantoin to l-tryptophan were isolated from various soils. The dl-5-indolylmethylhydantoin-hydrolyzing enzymes were found to be inducible and intracellular. With intact cells, 50 mg/ml as wet base, of newly isolated bacterial strain T-523, 10 mg/ml of dl-5-indolylmethylhydantoin dissapeared and 7.4 mg/ml of l-tryptophan in a molar yield of 82% was produced after 35 hr incubation. Tryptophan produced was confirmed to be l-form regardless of the stereoisomer of the substrates used. A mechanism of asymmetric hydrolysis of dl -5-indolyImethylhydantoin was discussed.

Screening of Microorganisms Producing d-p-Hydroxyphenyl-glycine from Dl-5-(-Hydroxyphenyty)hydantoin

Microorganisms that asymmetrically produce d-p-hydroxyphenylglycine (d-HPG) from Dl-5-(p-hydroxyphenyl)hydantoin (dl-HPH), an intermediate in the chemical synthesis of dl-HPG, were screened from stock cultures and soil samples.Of the 430 strains of bacteria, yeasts and actinomycetes obtained from our stock cultures, only two, Achromobacter delicatulus AJ-2446 and Achromobacter dendriticum AJ-2447, were capable of producing d-HPG from dl-HPH, but the yields were rather low. A bacterium with high productivity was isolated from a soil sample. It was classified as a bacterium belonging to the genus, Pseudomonas. In the presence of intact cells of Pseudomonas sp. AJ-11220 isolated from soil, which was selected as the best strain, d-HPG was produced from d-, l- and dl-HPH at a molar yield of more than 90%. This suggests that racemization and asymmetric hydrolysis occurred simultaneously in this reaction system.

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).

Purification of Proteinases by Affinity Chromatography Techniques

During studies on the microbial production of acid or alkaline proteinases and an investigation of the similarities of these proteinases from the view point of enzyme-inhibitor relationships, we were successful in isolating pepsin and alkaline proteinase inhibitors. The pepsin inhibitor we isolated was found to be an acylpentapeptide which was named SPI (pepsin-inhibitor produced by Streptomyces)(1). Alkaline proteinase inhibitor, on the other hand, was found to be a protein and was abbreviated as SSI (subtilisin BPN′-inhibitor produced by Streptomyces)(2). We tried to immobilize these inhibitors and to purify the proteinases by affinity chromatography on both immobilized inhibitors.

Bacterial Synthesis of Nucleotides: Part IX. Phosphorylation of Nucleoside Monophosphates

Inosine-5′, 2′(or 3′)-diphosphate was prepared by incubating 5′-IMP and p-nitrophenyl-phosphate with the bacteria characterized to phosphorylate at C3′ (&2′), or, on the contrary, by incubating 2′-IMP and a donor with the others capable of synthesizing 5′-nucleotide, via their phosphoryl transfer reactions.Formation of the 5′, 2′(or 3′)-diphosphates of guanosine, cytidine, and uridine was also demonstrated to be carried out under the same relationship between nucleotide isomer as an acceptor and specificities of bacterial phosphotransferases, as observed in the phosphorylation of adenylic acid isomers, while 5′-dTMP was phosphorylated by both groups of bacteria.

Bacterial Synthesis of Nucleotides: Part X. Phosphorylation of 5-Amino-4-imidazole Carboxamide RibosidePart XI. Phosphoryl Transfer between Mononucleotides and Nucleosides via A Phosphotransferase Reaction

5′-Phosphoribosyl 5-amino-4-imidazole carboxamide was prepared by incubating 5-amino-4-imidazole carboxamide riboside and a phosphate compound with the bacteria characterized to phosphorylate at C5′ via the phosphoryl transfer reaction. Aromatic phosphate compounds and 5′-nucleotides were able to act as the phosphate donor. This material was isolated chromatographically and its properties were studied. The other bacteria characterized to phosphorylate at C3′ (or 2′) also phosphorylated a little probably at C3′ (or 2′) of 5-amino-4-imidazole carboxamide riboside.The phosphoryl interconversion between nucleotides and nucleosides was studied to be carried out via the phosphoryl transfer reaction observed in bacteria. The phosphotransferase activity of Ps. trifolii mediated reversibly the phosphoryl transfer between 5′-nucleotides and nucleosides, and its optimal pH was at around 8.5, whereas that of Prot. mirabilis did transfer the phosphoryl radical from 2′- and 3′-nucleotide to nucleoside at its optimal pH...