Mitsugi Iida

Two forms of NAD-dependent D-mandelate dehydrogenase in Enterococcus faecalis IAM 10071.

ABSTRACT Two forms of NAD-dependent d-mandelate dehydrogenase (d-ManDHs) were purified from Enterococcus faecalis IAM 10071. While these two enzymes consistently exhibited high activity toward large 2-ketoacid substrates that were branched at the C3 or C4 position, they gave distinctly different Km and Vmax values for these substrates and had distinct molecular weights by gel electrophoresis and gel filtration.

Some new intermediates in microbial side chain degradation of β-sitosterol

Abstract By preselection of microorganisms which preferentially attack the side chain of β-sitosterol, we isolated a mutant H-45 of Rhodococcus equi K-3 which produces stigmasta-1,4-dien-3-on-26-oic acid from β-sitosterol in a high yield. The mutant produced new compounds such as ergosta-1,4-dien-3-on-26-oic acid, stigmast-4-en-3-on-26-oic acid, and ergost-4-en-3-on-26-oic acid, as well as the known β-sitosta-1,4-dien-3-one, β-sitost-4-en-3-one, 20-carboxy-pregn-4-en-3-one, 20-carboxy-pregna-1,4-dien-3-one, androst-4-en-3,17-dione, androsta-1,4-dien-3,17-dione, propionic acid, and acetic acid. The structures of these products were established by Mass and 1H-NMR spectroscopy. From microbial transformation of the 25-carboxylic acids obtained, we concluded that stigmasta-1,4-dien-3-on-26-oic acid is a hypothetical intermediate in β-sitosterol side chain degradation. We propose a degradation pathway of the β-sitosterol side chain by Rhodococcus equi.

Microbial production of two new dihydroxylated androstenedione derivatives by acremonium strictum

Abstract We found that Acremonium strictum NN106 converted 4-androstene-3, 17-dione (androstenedione) to 12 compounds. Among them, five products were isolated and found to be hydroxylated at the 11α-, 14α-, 7α, 11α-, 6β, 11α- or 6β, 14α-positions of androstenedione. 6β, 11α-Dihydroxy and 6β, 14α-dihydroxy derivatives of androstenedione have been obtained for the first time. From the time course profile of this transformation, sequential hydroxylation at the 6β-position followed by 11α- or 14α-monohydroxylation was observed. The oxidative product of the 6β, 14α-dihydroxy derivative was found to be the most potent inhibitor of human placentral aromatase.

Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerases produced by Agrobacterium sp. K-03

Abstract A poly(3-hydroxybutyrate) (PHB)-degrading bacterium, Agrobacterium sp. strain K-03, isolated from soil, secreted two PHB depolymerases into the culture fluid when it was cultivated on PHB, 3-hydroxybutyrate (3HB), or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comprising 43 mol% 3-hydroxyvalerate (P(3HB-co-43%3HV)) as the sole source of carbon. The two extracellular PHB depolymerases, designated E1 and E2, were purified from the culture fluid using DEAE-Toyopearl 650M column chromatography followed by gel filtration in Sephadex G-75 column chromatography. E1 and E2 are basic proteins having molecular weights of 46 and 44 kDa, and isoelectric points of 9.0 and 8.9, respectively. The optimal pHs of E1 and E2 for degradation of PHB are 8.1 and 7.9, respectively, and both of them have optimal PHB degradation temperatures of 45°C. Their enzymatic activities were considerably inhibited by dithiothreitol, phenylmethanesulfonyl fluoride, and Tween 20. The K m values of E1 and E2 for PHB were 17.8 μg/ml and 70.5 μg/ml, respectively. At pH 8.0 and 30°C, both enzymes completely degraded PHB, and they degraded 94% of P(3HB-co-43%3HV). The methyl ester of 3HB dimer was degraded by each enzyme to form the free dimer and the free monomer of 3HB.

Microbial Polyhydroxylation of Progesterone by Acremonium strictum

Abstract Acremonium strictum NN106, which has been shown to have a unique hydroxylating ability on androstenedione, also transformed progesterone into a mixture of various products. Six products were isolated in sufficient amounts for structural analysis. Three products were found to have two hydroxy moieties on axial and equatorial positions at 7β, 15β-, 6β, 11α- and 11α, 15β-sites. The other three products had an extra 17α-hydroxyl on the above dihydroxy derivatives to give trihydroxy progesterone. This type of microbial polyhydroxylation of a steroid caused by a fungal strain has not been reported so far and 7β, 15β, 17α-trihydroxy-4-pregnene-3,20-dione is a hitherto unknown compound which was obtained for the first time in the course of this experiment. The 6β, 11α-dihydroxylation has been found in the transformation of androstenedione, but the other positions of hydroxyl introduction are different. The hydroxylating events caused by this strain were greatly influenced by the 17β side chain, especially by C20 keto moiety in the steroidal skeleton.

Cloning, nucleotide sequencing, and expression in Escherichia coli of the gene for formate dehydrogenase of Paracoccus sp. 12-A, a formate-assimilating bacterium

The gene for the NAD-dependent formate dehydrogenase (FDH) of Paracoccus sp. 12-A, a formate-assimilating bacterium, was cloned through screening of the genomic library with activity staining. The FDH gene included an open reading frame of 1,200 base pairs, and encoded a protein of 43,757 Da, which had high amino acid sequence identity with known FDHs, in particular, with bacterial enzymes such as those of Moraxella sp. (86.5%) and Pseudomonas sp. 101 (83.5%). The gene was highly expressed in Escherichia coli cells using an expression plasmid with the pUC ori and tac promoter. The recombinant enzyme was somewhat inactive in the stage of the cell-free extract, but its activity markedly increased with purification, in particular, with the step of heat-treatment at 50°C. The purified enzyme showed essentially the same properties as the enzyme from the original Paracoccus cells.

Microbial degradation of 19-hydroxy-sterol side chains

Abstract Experimental evidence is herein presented to show that C 22 acids are key intermediates in the microbial degradation of cholesterol and campesterol (β-sitosterol) side chains. Exposure of 19-hydroxy-sterols to Rhodococcus mutant K-3 gave four new steroid carboxylic acids in addition to that known as estrone (P1); the chemical structures of these metabolites were characterized as 2(3-hydroxy-1,3,5(10)-estratrien-17-yl)-propionic acid (P2), 2-methyl-6(3-hydroxy-1,3,5(10)-estratrien-17-yl)-heptanoic acid (P3), 2,3-dimethyl-6(3-hydroxy-1,3,5(10)-estratrien-17-yl)-heptanoic acid (P4), and 2(3-hydroxy-1,3,5(10), 17-estratetraen-17-yl)-propionic acid (P5). We propose a degradation pathway of 19-hydroxy-cholesterol and campesterol (β-sitosterol) side chains.

Microbial Hydroxylation of (−)-α-Santonin by Aspergillus niger☆

Abstract The microbiological transformation of a sesquiterpene lactone, (−)-α-santonin was carried out by using Aspergillus niger MIL 5024 and MIL 5025. Strain MIL 5024 brings about the transformation of (−)-α-santonin (400 mg) to 11-hydroxy-(−)-α-santonin (P1) (50.4 mg), 3,6,9-trihydroxy-9,10-seco-selina-1,3,5(10)-trien-12-oic acid-12,6-lactone (P2) (22.4 mg), and 3,6-dihydroxy-9,10-seco-selina-1,3,5(10)-trien-9,12-dioic acid-12,6-lactone (P3) (27.1 mg), which were isolated and characterized by UV, IR, mass and 1H-NMR spectroanalyses. All of these products are described for the first time. Results similar to those with MIL 5024 were also obtained from the transformation of (−)-α-santonin by the other isolate, strain MIL 5025.

A new cerebroside of the n-Alkane-assimilating yeast Candida deformans

Abstract A glucocerebroside was isolated from the n-alkane-assimilating yeast Candida deformans MIL 4041 and named “Deformin”. The cerebroside contained only a single sugar, glucose, attached to ceramide in a β-configuration, and only a single fatty acid, 2-hydroxypalmitic acid. The long-chain base was identified as (5E,9Z)-C19-sphinga-5,9-diene. Therefore, the structure of the cerebroside was determined to be (5E,9Z)-N-2′-hydroxypalmitoyl-1-O-β-glucopyranosyl-2-amino-5,9- nonadecadiene-1,3-diol. Deformin showed the fruiting-inducing activity of fungi.

Microbial hydroxylation of 11,13-dehydrosantonin by Aspergillus nigert☆

Abstract The microbial transformation of a sesquiterpene lactone, 11,13-dehydro-(−)-α-santonin (DS), was carried out by 7 fungi. Although 6 of the fungi transformed the substrate into three (one fungus) or fewer (5 fungi) kinds of products, Aspergillus niger MIL 5024 gave five products including all of those produced by the others. The products were identified by UV, IR, mass, and 1 H-NMR spectroanalyses as (−)-α-santonin (P1), 11-hydroxy-(−)-α-santonin (P2), 13-hydroxy-(−)-α-santonin (P3), 3,6,9-trihydroxy-9,10- seco -selina-1,3,5(10)-trien-12-oic acid- 12,6-lactone (P4), and 8- epi -artemisin (P5). P3, a new compound, was also produced by A. niger MIL 5035, but P4 and P5 were produced only by strain MIL 5024. The biosynthetic pathway of these 5 products from DS was estimated.