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Two novel compounds, stachyflin and acetylstachyflin, have been isolated by solid-state fermentation of Stachybotrys sp. RF-7260. The structures of both metabolites, determined by detailed NMR analyses and X-ray crystallographic analysis, are novel with a pentacyclic moiety including cis-fused decalin. The absolute stereochemistry of stachyflins was determined by circular dichroism analysis. Stachyflin showed antiviral activity against influenza A virus (H1N1) in vitro with an IC50 value of 0.003 microM. Acetylstachyflin was about 77-fold less active than stachyflin.

Stachybotrys sp. RF-7260 was found to produce stachyflins, novel anti-influenza virus agents, under solid-state fermentation conditions. Feeding DL-lysine to a culture of Stachybotrys sp. RF-7260 induced the formation of the novel compounds, SQ-02-S-L2 and -L1, and feeding DL-valine the formation of SQ-02-S-VI and -V2. The structures of these metabolites were determined by detailed 2D NMR analyses in comparison with acetylstachyflin. SQ-02-S-L2 and -L1 have the lysine moiety and SQ-02-S-V1 has the valine moiety. SQ-02-S-V2 has an amidine moiety instead of the lactam moiety in acetylstachyflin. SQ-02-S-L2, -L1 and -V1, substituted on the lactam amide hydrogen, displayed only a low level of the antiviral activity. However, deacetyl SQ-02-S-V2 showed potent antiviral activity similar to stachyflin.

In the course of screening for yeast squalene synthase inhibitors, bisabosqual A was isolated from the culture broth of Stachybotrys sp. RF-7260. The related compounds bisabosquals B, C and D were also isolated from Stachybotrys ruwenzoriensis RF-6853. Bisabosquals inhibited squalene synthases. IC50 values of bisabosqual A against the microsomal squalene synthases from Saccharomyces cerevisiae, Candida albicans, HepG2 cell and rat liver were 0.43, 0.25, 0.95 and 2.5 microg/ml, respectively. Bisabosqual C exhibited inhibitory activities similar to bisabosqual A. Bisabosqual A showed broad spectrum antifungal activity in vitro.

The squalene synthase inhibitor bisabosqual A was isolated from the culture broth of Stachybotrys sp. RF-7260, and its structure was determined on the basis of spectroscopic methods including detailed 2D NMR analyses. The structures of bisabosquals B, C and D isolated from Stachybotrys ruwenzoriensis RF-6853 were determined by spectroscopic methods and chemical reactions. The absolute stereochemistry of bisabosquals A, B and D was determined by X-ray crystallographic analysis. They have novel cis-fused tetracyclic structures with a bisabolane-type sesquiterpene and phenol moieties.

Stachybotrys sp. RF-7260 was found to produce stachyflins, novel anti-influenza virus agents, under solid-state fermentation conditions. Feeding DL-lysine to a culture of Stachybotrys sp. RF-7260 induced the formation of the novel compounds, SQ-02-S-L2 and -L1, and feeding DL-valine the formation of SQ-02-S-VI and -V2. The structures of these metabolites were determined by detailed 2D NMR analyses in comparison with acetylstachyflin. SQ-02-S-L2 and -L1 have the lysine moiety and SQ-02-S-V1 has the valine moiety. SQ-02-S-V2 has an amidine moiety instead of the lactam moiety in acetylstachyflin. SQ-02-S-L2, -L1 and -V1, substituted on the lactam amide hydrogen, displayed only a low level of the antiviral activity. However, deacetyl SQ-02-S-V2 showed potent antiviral activity similar to stachyflin.

Bacillus subtilis SHS0133 cephalosporin-C deacetylase (CAH) overexpressed in Escherichia coli was immobilized on an anion-exchange resin, KA-890, using glutaraldehyde. The activity yield of immobilized enzyme was approximately 55% of the free enzyme. The pH range for stability of the immobilized enzyme (pH 5-10) was broader than that for free enzyme. The K(m)(app) value of immobilized enzyme for 7-aminocephalosporanic acid (7-ACA) was similar to that of the free enzyme. This immobilized enzyme obeyed Michaelis-Menten kinetics similar to those of the free enzyme. A batch-type reactor with a water jacket was employed for deacetylation of 7-ACA using CAH immobilized on KA-890. Ten kilograms of 7-ACA were completely converted to deacetyl 7-ACA at pH 8.0 within 90 min. The reaction kinetics agreed well with a computer simulation model. Moreover, the immobilized enzyme exhibited only a slight loss of the initial activity even after repeated use (52 times ) over a period of 70 days. This reaction will thus be useful for the production of cephalosporin-type antibiotics.

非ニュートン流体を示す発酵液中における酸素吸収速度が低下することの原因に対する基礎的解明を目的とした。非ニュートン流体のモデル溶液としてはカルボキシメチルセルロース溶液の見掛けの粘度を変えたものを採用した。この溶液中に単一ノズルより気泡を間歇的または連続的に発生せしめ,気泡径を写真撮影により求め,気泡の滞留時間,液側総括吸収係数と粘性との関係につき検討した。気泡径の分布は水中では対数正規分布するのに対して粘性液中では対数正規分布し難いことを認めた。粘度上昇にしたがって,平均気泡径は大となり,気泡滞留時間は長くなった。液側総括吸収係数は気泡発生後時間が経過するにつれて低下し,粘度の上昇とともに低下する。以上の結果より粘性発酵液の酸素移動容量係数の低下に対しては気泡径の変化が最大の原因であると推察した。

A practical method for the production of calcium 2-keto- l -gulonate (an intermediate in the Reichstein synthesis of l -ascorbic acid) from d -glucose has been established by using a two-stage fermentation system. d -Glucose was first converted to calcium 2,5-diketo- d -gluconate by a mutant strain of Erwinia sp. in a medium containing d -glucose, corn steep liquor, (NH 4 ) 2 HPO 4 , and CaCO 3 . After a 26-h cultivation, 328.6 mg of calcium 2,5-diketo- d -gluconate per ml was obtained, with a 94.5% yield from d -glucose. This broth was used directly for the next conversion without removal of cells by treatment with sodium dodecyl sulfate. The stereospecific reduction of calcium 2,5-diketo- d -gluconate to calcium 2-keto- l -gulonate was performed with a mutant strain of Corynebacterium sp. When the cell growth reached a maximum (about 16 h) in a medium containing d -glucose, corn steep liquor, NaNO 3 , KH 2 PO 4 , and trace elements, NaNO 3 was added to the culture, and then the calcium 2,5-diketo- d -gluconate broth was fed over a period of about 50 h. Since the mutant strain requires a hydrogen donor for reduction, the calcium 2,5-diketo- d -gluconate broth was mixed with d -glucose before being fed. The results of four two-stage fermentations in 10-m 3 conventional fermentors showed that an average of 106.3 mg of calcium 2-keto- l -gulonate per ml was obtained, with a 84.6% yield from d -glucose, the starting material of calcium 2,5-diketo- d -gluconate production. Calcium 2-keto- l -gulonate was stable in the broth. Neither 2-keto- d -gluconic acid nor 5-keto- d -gluconic acid was detected in the final broth.