Rokuro Okamoto

Hyper-production of l-trytophan via fermentation with crystallization

A stable and fast l-tryptophan producer, AGX1757, was isolated from Escherichia coli W3110 trpAE1 trpR tnaA, which carried pSC101-trpI15·14. Cells of AGX1757 did not lose the composite plasmid during fermentation. Whenever a fed-batch culture of AGX1757 attained an l-tryptophan concentration of about 30 g/l, indole began to appear in the broth. The emergence of indole was caused by inhibition of tryptophan synthase due to accumulated l-tryptophan. Hence, the production rate of l-tryptophan sharply decreased. A higher solubility of l-tryptophan in the supernatant of culture broth (about 32 g/l) than that in the initial medium (about 22 g/l) was attributed to some unknown interaction between l-tryptophan and certain macromolecular material(s) coming from the bacterial cells. An addition of non-ionic detergents into the supernatant was effective for decreasing the solubility of l-tryptophan, hence causing crystallization of l-tryptophan. Pluronic L-61 was supplied from outside to an extent of 0.5% in terms of wt% concentration at around 45 h of fermentation when the l-tryptophan accumulated reached about 25 g/l. This addition actually caused crystallization of l-tryptophan and, as a result, the inhibitory effect of tryptophan synthase by l-tryptophan accumulated in the broth could be alleviated. Thus far, further fermentation became possible. l-Tryptophan of more than 50 g/l was finally produced by feeding solutions of both glucose and anthranilic acid.

Application of cyclodextrin to microbial transformation of vitamin D3 to 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3

Abstract Amycolata autotrophica converts vitamin D3(VD3) to 1α,25-dihydroxyvitamin D3(1α,25(OH)2VD3) via 25-hydroxyvitamin D3(25(OH)VD3) by hydroxylation of VD3 at C-25 and C-1. In this microbial hydroxylation, it was found that cyclodextrin (CD) had the ability to enhance the hydroxylation of VD3. Addition of partially-methylated-β-cyclodextrin (PMCD) increased the productivity of 25(OH)VD3 about seven-fold compared to that without CD. Combined use of PMCD and γ-CD increased the production of 1α,25(OH)2VD3 in a tank fermentor about sixteen-fold compared to that without CD.

Glass formation of 4″-O-(4-methoxyphenyl) acetyltylosin and physicochemical stability of the amorphous solid

Glass formation of 4″-O-(4-methoxyphenyl) acetyltylosin (MAT) and the physicochemical stability of amorphous MAT have been investigated. The amorphous form of MAT was prepared by spray drying of an MAT dichloromethane solution. The glassy state was confirmed by DSC and the glass transition temperature was observed to be 102–103°C. It was found that different kinds of the glassy state of MAT could be obtained by changing the inlet temperature of spray drying. Storage experiments on amorphous powders at 40°C and 75% RH revealed that the amorphous powders prepared at a temperature between the glass transition (Tg) and recrystallization (Tc) temperatures were the most stable. A correlation between the stability and the apparent density was observed.

Preferential and high-yield production of a cephamycin C by dissolved oxygen controlled fermentation

Abstract Streptomyces sp. P6621-RS1726, a high cephamycin C producing mutant, usually produces cephamycin C together with its precursor, penicillin N, in flask fermentation. The fermentation conditions for selective and high yield production of cephamycin C were investigated using 20-l jar fermentors. As a result, cephamycin C was preferentially produced without penicillin N when the DO level was maintained at least higher than at 10% during the production phase. However, when the agitation speed was too high at the initial stage of the fermentation, the cell growth was inhibited, resulting in suppression of both cephamycin C and penicillin N production, even if the DO was maintained at a high level. Based on these experiments, a computer control system for preferential and high-yield production of cephamycin C was constructed. By using this computer control system, scale-up from a 20-l jar fermentor to a 1,500-l pilot fermentor was carried out with control of the DO level at 20% of saturation during the cephamycin C production phase. As a result, the profile of cephamycin C production in the former was favorably reproduced in the latter.

A novel process of cyclodextrin production by the use of specific adsorbents: Part II. A new reactor system for selective production of α-cyclodextrin with specific adsorbent

Abstract We have developed a novel process of α-cyclodextrin (α-CD) production by using a new adsorbent that is characterized by its exceedingly powerful selectivity for α-CD compared with other CDs. α-CD production was carried out in a closed reactor system that was composed of a main reactor, wherein liquefied starch was converted to CDs by cyclodextrin glucosyltransferase (CGTase: EC 2.4.1.19), and a column packed with the adsorbent. While the reaction mixture was circulated in the system, α-CD was selectively adsorbed in the column and its concentration in the mixture of the main reactor was kept at a low level. This low concentration of α-CD stimulated the conversion of starch to CDs and as a result, enhanced its yield based on added starch. When 8.3 % (w/v) of liquefied starch was used in the reactor system, the yield of α-CD was 22.2% and α-CD occupied 58.7 % of the reaction mixture of total CDs synthesized. Meanwhile, in a batch system without the adsorbent, the yield of α-CD and its fraction were 10.8% and 45.0%, respectively. After the conversion reaction, and following the preliminary washing with water through the column. α-CD was easily eluted with hot water, resulting in a high purity of about 95%.

Optimization of Conditions for Conversion of Tylosin to a Novel Antibiotic, Acetyl-Isovaleryl Tylosin (AIV), by Streptomyces thermotolerans and Scale-Up to 200-Liter Pilot-Scale Fermentor

Abstract The rate of substrate (tylosin) supply, pH, dissolved oxygen concentration, and temperature for bioconversion of tylosin to acetyl-isovaleryl tylosin (AIV), were optimized in a flask and a 3- l jar fermentor. An AIV concentration 9.3 g/ l was achieved when the pH was controlled at 7.0 and the tylosin supply rate was maintained at 200 mg/ l /h during the period of bioconversion of tylosin to AIV after the cell growth phase. Based on the data obtained for the flask and jar-fermentor cultures, the bioconversion process was successfully scaled up to a 200- l fermentor. In this pilot-scale fermentor, 13.8 g/ l of AIV was obtained at a yield of 83% based on the amount of tylosin supplied.

Coriolus versicolor Laccase Catalyzes the Decarboxylation of 2-(4-Hydroxyphenyl)-glycine and 4-Hydroxymandelic Acid

Laccasc (benzenediol: oxygen oxidoreductase, EC 1.1O.3.2) has been found in some fungal strains belonging to various classes.i) The enzyme generally catalyzes the removal of hydrogen from phenolic hydroxyl groups or aromatic amino groups using molecular oxygcn as a primary electron acceptor to give radicals, which undergo free-radical additions and related reactions to give various products.2-s) During the investigation n a laccase from the fungus Coriolus versieotor, we found that the enzyme catalyzed the conversion of 2-(4-hydroxyphenyl)glycine (HPG) or 4hydroxyrnandelic acid (HMA) to 4-hydroxybenzaldehyde (HBA), which was a single product from either substrate and was not converted further. Laccase is known to remove

Thin-layer chromatographic analysis of carbapenem antibiotics in fermentation broths

Abstract A one-dimensional silica gel thin-layer chromatographic method has been devised for qualitative and quantitative analysis of carbapenem antibiotics in fermentation broths. The antibiotics were extracted from the broth filtrate with a solvent mixture of 1-butanol and chloroform (1:1) and developed on a silica gel thin-layer plate with a mobile phase of acetonitrile—0.75% acetic acid (9:2). Carbapenem compounds on the thin-layer plate were visualized as reddish pink spots with the Ehrlich reagent and quantitated by densitometry.

Preferential production of a carbapenem antibiotic, PS-5 by dissolved oxygen controlled fermentation

Abstract As Streptomyces fulvoviridis A933-17M9 usually produces several carbapenem analogs including PS-5, the submerged fermentation conditions were optimized for preferential production of PS-5. The scale of PS-5 fermentation was expanded stepwise from a 2.5- l mini-jar fermentor to a 1500- l pilot-scale fermentor. For production of PS-5 as a major product among related carbapenem analogs, the concentration of dissolved oxygen during fermentation was controlled at levels where microbial conversion of PS-5 to epithienamycins A and C and further to MM17880 was largely prevented without deleterious influences on the carbapenem-biosynthesizing activity of mycelia.

Enhanced Production of Antibiotics by Pecilomyces lilacinus under Alkaline Conditions Associated with Morphological Change

The production of the group of peptide antibiotics called No. 1907 by Paecilomyces lilacinus was stabilized and enhanced by mono-spore isolation followed by strain improvement, and the production seemed to be related to a morphological change of the strain. Unstable and a low productivity (below 10 micrograms/ml) of the antibiotics No. 1907 by P. lilacinus was greatly improved up to 140 micrograms/ml by successive mono-spore isolation along with mutagenization. Alkaline conditions caused by adding Na2CO3, K2CO3, NaHCO3 or KHCO3 were essential to obtain high production of the antibiotics following simultaneous morphological change of the cells from filamentous mycelia to round arthroconidia. Resting cell experiments showed that a significant amount of antibiotics (213 micrograms/ml) was synthesized by the filamentous cells along with their morphological change to arthroconidia in the presence of glucose under the alkaline conditions.