Ikuo Kira

Galacto‐oligosaccharide production from lactose by Sirobasidium magnum CBS6803

N. ONISHI, I. KIRA AND K. YOKOZEKI. 1996. Galacto‐oligosaccharide (Gal‐OS) was produced from lactose by a yeast, Sirobasidium magnum CBS6803. With toluene‐treated resting cells, 136 mg ml−1 of Gal‐OS was produced from 360 mg ml−1 of lactose at 50°C for 42 h. Then, the yield of Gal‐OS was increased by a culture method in which cell growth followed the enzymatic reaction : 224 mg ml−1 of Gal‐OS was produced at 30°C for 60 h. Finally, combination of the toluene‐treated resting cells and glucose oxidase plus catalase was applied to improve productivity by the removal of a by‐product, glucose, which inhibits the Gal‐OS production, from the reaction mixture. In this case, 242 mg ml−1 4‐galactosyl‐lactose. of Gal‐OS was produced at 50°C for 42 h without cell growth. The structure of the major product ws identified as 4‐galactosyl‐laetos.

Purification and Characterization of a (R)-1-Phenyl-1,3-propanediol-producing Enzyme from Trichosporon fermentans AJ-5152 and Enzymatic (R)-1-Phenyl-1,3-propanediol Production

An (R)-1-phenyl-1,3-propanediol-producing enzyme was purified from Trichosporon fermentans AJ-5152. It was NADPH-dependent and converted 3-hydroxy-1-phenylpropane-1-one (HPPO) to (R)-1-phenyl-1,3-propanediol [(R)-PPD] with anti-Prelog’s specificity. It showed maximum activity at pH 7.0 and 40 °C. Its K m and V max values toward HPPO were 20.1 mM and 3.4 μmol min−1 mg protein−1 respectively. The relative molecular weight of the enzyme was estimated to be 68,000 on gel filtration and 32,000 on SDS-polyacrylamide gel electrophoresis. An (R)-PPD-producing reaction using the (R)-PPD-producing enzyme and an NADPH recycling system was carried out by successive feeding of HPPO. A total (R)-PPD yield of 8.9 g/l was produced in 16 h. The molar yield was 76%, and the optical purity of the (R)-PPD produced was over 99% e.e.

Enzymatic Production of L -Alanyl- L -glutamine by Recombinant E. coli Expressing α-Amino Acid Ester Acyltransferase from Sphingobacterium siyangensis

An enzymatic production method for synthesizing L-alanyl-L-glutamine (Ala-Gln) from L-alanine methyl ester hydrochloride (AlaOMe) and L-glutamine (Gln) was developed in this study. The cultivation conditions for an Escherichia coli strain overexpressing α-amino acid ester acyltransferase from Sphingobacterium siyangensis AJ 2458 (SAET) and reaction conditions for Ala-Gln production were optimized. A high cell density culture broth prepared by fed-batch cultivation showed 440 units/mL of Ala-Gln-producing activity. In addition, an Ala-Gln-producing reaction using intact E. coli cells overexpressing SAET under optimum conditions was conducted. A total Ala-Gln yield of 69.7 g/L was produced in 40 min. The molar yield was 67% against both AlaOMe and Gln.

Screening, purification, and identification of the enzyme producing N-(l-α-l-aspartyl)-l-phenylalanine methyl ester from l-isoasparagine and l-phenylalanine methyl ester

Screening was carried out for microorganisms able to produce N-(l-alpha-l-aspartyl)-l-phenylalanine methyl ester [APM] from l-isoasparagine and l-phenylalanine methyl ester hydrochloride. Of the 422 strains examined, 44 strains belonging to the family Enterobacteriaceae were found to produce APM. The enzyme catalyzing APM production was purified and identified as dipeptidase E.