Shun-ichi Akiyama

Effective production of a human monoclonal antibody against tetanus toxoid by selection of high productivity clones of a heterohybridoma

A mouse.human-human heterohybridoma, N12-16.63, has been described which produces an anti-tetanus toxoid human monoclonal antibody (MoAb). A clone, N12-16.63.49.19, which produces eight times as much MoAb as that produced by the original cell line, was selected by repeating the recloning and selection twice. Two clones, N12-16.63.49.19.69 and N12-16.63.49.19.127, further selected from this clone produced almost 20 times more than that produced by the original cell line. Though the production of MoAb by these clones gradually decreased with repeating transfers, they still produced a large amount of human MoAb even after 3 months of transfer. Human MoAb (IgM) was isolated from the culture supernatants of the original and high productivity clones and the products were confirmed to be identical. Human MoAb was effectively produced by batch culture on the 20 liter scale or a perfusion culture on the 1 liter scale using these high productivity clones.

Stimulatory effect of cyclodextrins on the production of lankacidin-group antibiotics by Streptomyces species

SummaryThe production of lankacidin-group antibiotics was markedly stimulated by adding β-cyclodextrin (β-CyD) to the fermentation medium. This stimulatory effect was observed for all Streptomyces species known to produce lankacidins. β-CyD had no marked effect on microbial growth, consumption rate of carbon source and pH changes throughout fermentation. β-CyD was not consumed by the microorganism during fermentation, and the lankacidins produced existed as inclusion complexes in the culture filtrate. Comparing α-CyD, β-CyD and γ-CyD, β-CyD was the most effective when it was added at the onset of fermentation. From the results of experiments on the replacement culture and the incorporation of a 14C-labelled precursor to the lankacidins, it was confirmed that the cells grown in the presence of β-CyD had a potent productivity of lankacidins.

Measurement of the overall volumetric coefficient of heat transfer of a shaking flask

Abstract The overall volumetric coefficient of heat transfer ( Ua ) of a shaking flask was measured under various shaking conditions using three types of flask. Ua was significantly affected by flask weight ( W F ), which could be attributed to the thickness of the flask wall, the rotational speed of the shaker ( N ), wind velocity ( V W ), and the liquid volume in the flask ( V L ). The limiting step of heat transfer seemed to be the heat radiation process from the surface of the flask to the surroundings. To predict the value of Ua , the following empirical equations were obtained for each type of flask by the least squares method: 1. (1) for deformed creased flask (M-type flask), Ua =17.4 W F −0.43 V L −0.61 N 0.12 V W 0.36 2. (2) for creased flask (N-type flask), Ua =9.2 W F −0.36 V L −0.57 N 0.17 V W 0.27 3. (3) for smooth flask (S-type flask), Ua =5.3 W F −0.26 V L −0.62 N 0.19 V W 0.25 .

Mechanism of uridine production by Bacillus subtilis mutants

SummaryPyrimidine analogue-resistant mutants of Bacillus subtilis were found to produce a large amount of uridine. One of them accumulated 55 mg/ml of uridine in culture medium. The changes in enzymes involved in the metabolism of uridine 5′-monophosphate (UMP) were examined with this mutant. All six enzymes of de novo UMP biosynthesis were completely free from regulation by uridine compounds, and the activities of these enzymes were 16- to 30-fold higher than those of the enzymes of the parental strain. In the mutant strain, the level of uridine phosphorylase, responsible for converting uridine to uracil, was extremely low, compared with that of the parental strain. No apparent change was observed between the strains in the activity of UMP dephosphorylation or uracil phosphoribosyltransferase. The implication of these findings is discussed in relation to the overproduction of uridine by the mutant.

Isolation and Taxonomie Characterization of Acid Urease-producing Bacteria

About 16,000 isolates from animal feces and intestines were assayed for the production of acid urease, and the 700 strains selected as producers were examined as to their taxonomic properties. Of these 700 strains, 370 belonged to the genus Streptococcus, 312 to the genus Lactobacillus, 9 to the genus Escherichia, 6 to the genus Staphylococcus, 2 to the genus Morganella and 1 to the genus Bifidobacterium. The majority of the streptococci were identified as Streptococcus mitior, the remainder being Streptococcus salivarius, Streptococcus faecalis, Streptococcus faecium, Streptococcus avium and Streptococcus gallinarum. The majority of the lactobacilli were considered to be Lactobacillus reuteri or Lactobacillus fermentum and Lactobacillus animalis or Lactobacillus salivarius, the remainder being Lactobacillus ruminis, Lactobacillus viridescens, Lactobacillus vac- cinostercus and strains considered to be Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus or Lactobacillus gasseri.

Mechanism of the stimulatory effect of cyclodextrins on lankacidin-producing Streptomyces

SummaryGel-filtration analysis of a mixture of cyclodextrin (CyD) and lankacidin C showed that β-CyD had strong, γ-CyD weak and α-CyD no affinity for lankacidin C. Lankacidin C production activity, which was assayed by measuring the incorporation of l-[methyl-14C-]methionine into the lankacidin molecule, was the greatest with cells grown in the presence of β-CyD, less with γ-CyD and the least with α-CyD. Lankamycin and T-2636M, which are by-products in lankacidin C fermentation, were not included by β-CyD and their production was not stimulated by β-CyD. It was apparent that the stimulatory effect of CyD was closely related to the formation of an inclusion complex between CyD and the antibiotic. Lankacidin C biosynthesis was repressed by preincubating cells with lankacidin C, while the repressive effect of lankacidin C was abrogated by the inclusion by β-CyD. Thus, abrogation of feed-back repression seems to be a main mechanism of the effect of CyD. However, α-CyD, which had no affinity for lankacidin C, stimulated the production to the least extent and exhibited a complementary effect on the stimulation by β-CyD or γ-CyD. α-CyD also caused a change in cell morphology and cell-surface hydrophobicity. It was assumed that the modification of the cell surface is a secondary mechanism of the effect of CyD.

Strain improvement of a mildiomycin producer,Streptoverticillium rimofaciens B-98891

SummaryAminopterin (10 μg/ml) was found to inhibit the formation of 5-hydroxymethylcytosine (HMC), a constituent of mildiomycin, without affecting the growth ofStreptoverticillium rimofaciens. This was available for selecting high-producing mutants.d-Cycloserine caused its morphological mutations at high frequency. In addition, mildiomycin (MIL) production varied widely among the strains picked up from colonies that developed on agar medium containing cycloserine at the inhibitory concentration to the growth. Consequently, we selected the mutants which were capable of producing MIL on agar medium containing 10 μg/ml of aminopterin, among mutants enriched by cycloserine. A high-producing mutant thus obtained, CR4-257, exhibited higher enzymatic activity of the HMC formation and higher resistance todl-serine hydroxamate than the original strain.l-Canavanine resistant mutants were furthermore selected to enhance the biosynthetic activity of the arginine-like moiety of MIL. Among them, we finally obtained an excellent mutant, CVR-48, with an MIL production 2.6 times that of the original strain,S. rimofaciens B-98891.

Selective Accumulation of Ansamitocins P-2, P-3 and P-4, and Biosynthetic Origins of Their Acyl Moieties

The factors involved in the selective accumulation of ansamitocins P-2, P-3 and P-4 by Actinosynnema pretiosum subsp. pretiosum No. C-15003 (Synonym: Nocardia sp. No. C-15003) were studied. The production of ansamitocin P-2, with a propionyl moiety at the C-3 position of the ansa chain, was stimulated more than 3-fold by the addition of isoleucine, propionate, pro-pionaldehyde and ^-propyl alcohol to the fermentation medium. The production of ansamitocin P-3, with an isobutyryl moiety, was enhanced by the addition of valine, isobutyrate, isobutyraldehyde and isobutyl alcohol, and the proportion of P-3 reached more than 90% of the total ansamitocins produced. The production of P-4, with an isovaleryl moiety, was stimulated by leucine, isovalerate, isovaleraldehyde and isoamyl alcohol. The radioactive compounds, which selectively stimulated the production of each ansamitocin component, were preferentially incorporated into their respective acyl moieties of ansamitocins. Based on these results, we propose th...

Oxygen transfer rate in stirred-tank fermentors under the supply of oxygen-enriched air

The oxygen transfer rate (OTR) in 0.2-m 3 and 2-m 3 stirred-tank fermentors having two sets of standard six-blade turbine impellers was determined by the sulfite oxidation method under the supply of oxygen-enriched air. Since the observed OTR values did not correlate very well with the ones calculated using the known empirical equations, a new empirical equation was derived by the least squares method.

Measurement of the evaporation rate of liquid in a shaking flask

Abstract The evaporation rate ( N H2O ) of liquid in a shaking flask was measured under various shaking conditions: temperature, humidity, flask shape, liquid volume in the flask ( V L ), length of the stopper in the flask neck ( L C ), rotational speed of the shaker ( N ), and wind velocity ( V W ). The rate was significantly affected by these factors, and the existence of a distribution of water vapor pressure was suggested inside and outside the flask. To predict the evaporation rate, the following empirical equation was derived by the least squares method: N H2O =1.26×10 −2 S F 1.18 L F −1.3 ( p s – p ) 1.24 V L 0.11 N 0.05 V W 0.26 L C −0.37 where, S F is the cross-sectional area of flask neck, L F is the length of flask neck, p s is the saturated partial pressure of water vapor at the temperature of the air surrounding the flask, and p is the partial pressure of water vapor in the flask.