Koji Kubota

Purification and Characterization of Novel Transglutaminase from Bacillus subtilis Spores

Transglutaminase activity was detected in suspensions of purified spores prepared from lysozyme-treated sporulating cells of Bacillus subtilis AJ 1307. The enzyme was easily solubilized from the spores upon incubation at pH 10.5 at 37°C. The transglutaminase activity was separated into two fractions upon purification by hydrophobic interaction chromatography (TG1 and TG2). Each enzyme was purified to electrophoretic homogeneity (about 1,000-fold). Both enzymes had the same molecular weight of 29,000 as estimated by SDS-PAGE, had the same N-terminal 30 amino acid sequence, and also showed the same optimal temperature (60°C) and pH (8.2). The purified enzyme catalyzed formation of cross-linked ε-(γ-glutamyl)lysine isopeptides, resulting in the gel-formation of protein solutions such as αs-casein and BSA.

Fermentative production of L-leucine

Mutant strains of the genera Brevibacterium and Corynebacterium having a resistance to 2-thiazolalanine or having said resistance together with a nutrient requirement, produce L-valine in a culture medium.

Mechanism of Asymmetric Production of d-Amino Acids from the Corresponding Hydantoins by Pseudomonas sp.

The mechanism of asymmetric production of d-amino acids from the corresponding hydantoins by Pseudomonas sp. AJ-11220 was examined by investigating the properties of the enzymes involved in the hydrolysis of dl-5-substituted hydantoins. The enzymatic production of d-amino acids from the corresponding hydantoins by Pseudomonas sp. AJ-11220 involved the following two successive reactions; the d-isomer specific hydrolysis, i.e., the ring opening of d-5-substituted hydantoins to d-form N-carbamyl amino acids by an enzyme, d-hydantoin hydrolase (d-HYD hydrolase), followed by the d-isomer specific hydrolysis, i.e., the cleavage of N-carbamyl-d-amino acids to d-amino acids by an enzyme, N-carbamyl-d-amino acid hydrolase (d-NCA hydrolase).l-5-Substituted hydantoins not hydrolyzed by d-HYD hydrolase were converted to d-form 5- substituted hydantoins through spontaneous racemization under the enzymatic reaction conditions.It was proposed that almost all of the dl-5-substituted hydantoins were stoichiometrically and...

Continuous production of l-serine by immobilized growing Corynebacterium glycinophilum cells

SummaryAuxotrophic mutant cells of Corynebacterium glycinophilum with high l-serine production activity were immobilized by entrapment with various gel materials, such as synthetic prepolymers and natural polysaccharides. The entrapped cells were used for estimation of l-serine productivity in a medium supplemented with glycine as a precursor. Based on the above criteria, including cell growth in gels and cell leakage from gels, calcium alginate was the most suitable gel material. Continuous l-serine fermentation with calcium alginate-entrapped growing cells was successfully achieved in an air-bubbled reactor for at least 13 days.

Mechanism of Asymmetric Production of l-Aromatic Amino Acids from the Corresponding Hydantoins by Flavobacterium sp.

The mechanism of asymmetric production of l-aroniatic amino acids from the corresponding hydantoins by Flavobacterium sp. AJ-3912 was examined by investigating the properties of the enzymes involved in the hydrolysis of 5-substituted hydantoins corresponding to aromatic amino acids (A AH). The enzymatic hydrolysis of AAH by Flavobacterium sp. AJ-3912 consisted of the following two successive reactions; a hydrolytic ring opening reaction of dl-AAH to l- and d-form N-carbamyl aromatic amino acids (NCA), involving an enzyme (hydantoin hydrolase), followed by a hydrolytic cleaving reaction of the l-form NCA to l-aromatic amino acids involving another enzyme (N-carbamyl-l-aromatic amino acid hydrolase, abbreviated as l-NCA hydrolase). The ring opening reaction involving hydantoin hydrolase was not stereospecific, but the NCA cleaving reaction involving l-NCA hydrolase was completely l-specific. The pathway for the conversion of the by-produced d-form NCA to l-aromatic amino acids was as follows; conversion of ...

Production of l-serine from glycine by Corynebacterium glycinophilum and properties of serine hydroxymethyltransferase, a key enzyme in l-serine production

Abstract Fermentative production of l -serine from glycine by Corynebacterium glycinophilum AJ-3413, an auxotrophic mutant of Leu and Met with increased productivity of l -serine using a one liter jar fermentor was carried out and the properties of serine hydroxymethyltransferase (SHMT), a key enzyme in l -serine synthesis, of the parental strain AJ-3170 were investigated. SHMT was effectively induced by the addition of glycine to the medium at an early stage of cultivation. Under optimal conditions, AJ-3413 produced 16.0 g/ l of l -serine from 30 g/ l of glycine with a molar yield of 38%. The partially purified SHMT catalyzed the l - allo -threonine degradation in addition to l -serine degradation, but could not catalyze l -threonine degradation. This enzyme showed an absolute tetrahydrofolic acid requirement for l -serine degradation to glycine and formaldehyde, but not for l - allo -threonine degradation. Pyridoxal 5′-phosphate appeared to be required for enzyme activity. The K m values for glycine and formaldehyde in l -serine synthesis, and for l -serine in l -serine degradation were 1.85, 0.29 and 1.64 mM, respectively.

Enzymatic Production of Antiviral Nucleosides by the Application of Nucleoside Phosphorylase

Various chemical methods and several enzymatic methods have been studied on the synthesis of antiviral nucleosides. However, there remain some practical limitations to these approaches because they are laborious and they give unsatisfactory yields. Recently, we have successfully developed novel processes for the production of antiviral nucleosides from cheap nucleosides by the application of nucleoside phosphorylase. These methods seem to overcome the above problems.

Effects of l-serine dehydratase activity on l-serine production by Corynebacterium glycinophilum and an examination of the properties of the enzyme

Abstract The results with Corynebacterium glycinophilum AJ-3170 and various mutants from AJ-3170 indicated that l -serine production was almost inversely proportional to l -serine degrading activity. The crude extract of the parental strain, AJ-3170, showed l -serine and l -threonine degrading activities. The 2 activities were completely separated from each other by gel-filtration, indicating that each activity comes from a different enzyme. The l -serine degrading enzyme, l -serine dehydratase (SD), was purified 30-fold from AJ-3170. Molecular weight of SD was 130,000. The enzyme was specific for l -serine, activated slightly by FeCl 2 and inhibited by MnCl 2 . The double reciprocal plots of SD rate against substrate concentration gave an upwards-curved line. The value of [S] 0.5 was 35 mM.

Effects various adsorbents on mycelium formation and mycophenolic acid production by Penicillium brevicompactum.

A drug-resistant mutant, No. 4–23–11, which had been derived from Penicillium brevicompactum ATCC 16024, was cultured in a liquid medium for the production of mycophenolic acid (MPA) in the presence and absence of various adsorbents, that is, celites, zeolites, aluminas, talc, silica, charcoals, carbon blacks, natural graphite and carbonaceous mesophase spheres. In the absence of an adsorbent, MPA production (0.2 ~4.8 g/1) and the size of mycelium pellets (5~0.5mm) markedly depended on the concentration of spores inoculated (104~ 107/ml), whereas in the presence of one of these adsorbents, small pellets (smaller than 1 mm in diameter) were formed and the high production of MPA (4.5 — 5.4 g/1) was observed, independently of the spore concentration between approximately 104~ 107/ml. Microscopic observation of the pellets revealed that numerous particles of the adsorbents adhered to the surface of the hyphae. These results suggest that the adsorbents might interfere with the adhesion of hyphae to each other ...