Chiaki Imada

Enzyme inhibitors and other bioactive compounds from marine actinomycetes

Several enzyme-inhibitor-producing actinomycetes were isolated from various samples collected from the marine environment and characterized. Most of them produced novel compounds that are useful in medicine and agriculture. Actinomycete strain no. 18, which produces antibiotics against Gram-positive bacteria only in the presence of seawater, was isolated from sediment sampled from neritic sea water and characterized. The production of antibiotics was observed at seawater concentrations ranging from 60 to 110% (v/v). Thus, the production was seawater-dependent. The production of tetrodotoxin (TTX), known otherwise as puffer fish toxin, was investigated in various actinomycetes collected from the marine environment. Of 10 isolates from various sea areas, 9 produced TTX as judged by their retention times on high-performance liquid chromatography (HPLC). To our knowledge, this is the first report of actinomycetes from the marine environment that produce TTX.

Bacterial Production of the Tunicate-Derived Antitumor Cyclic Depsipeptide Didemnin B

Natural products obtained from marine invertebrates such as sponges and tunicates are attractive sources of drugs. However, a critical obstacle in the development of these compounds is the problem of supply. In most cases, neither chemical synthesis nor mariculture of invertebrates is economically feasible. Due to structural similarities, many marine natural products are suspected to be produced by associated microorganisms. A favorable strategy for the production of such compounds is to use culturable microorganisms. Here we report that didemnin B, a tunicate-derived depsipeptide, has been isolated from a culturable bacterium, Tistrella mobilis YIT 12409.

Development of a d-alanine sensor for the monitoring of a fermentation using the improved selectivity by the combination of d-amino acid oxidase and pyruvate oxidase

A D-alanine (D-Ala) sensor for the monitoring of a fermentation process was developed using flow injection analysis (FIA). The FIA system consisted of a D-amino acid oxidase (D-AAOx) reactor, a Pyruvate oxidase (PyOx) electrode and a contrast electrode in the flow cell, and through the oxidation of D-amino acids in the D-AAOx reactor, pyruvic acid was formed only from D-Ala. The pyruvic acid was further oxidized with PyOx via the D-AAOx reaction. The amount of oxygen consumed in the PyOx reaction was proportional to the amount of D-Ala. It was possible to continuously repeat the assay up to 60 times at pH 6.8 and a flow rate of 0.18-ml min(-1). A linear relationship was obtained in the range of 0.1-1 mM D-Ala with a correlation coefficient of 0.987 and the detection limit was 0.05 mM. The relative standard deviation (R.S.D.) was 4.9% (n=5) for 0.5 mM D-Ala. The D-Ala content in some fish sauces was also determined using the proposed sensor system. The results obtained indicated a linear relationship between the amounts of D-Ala determined by the proposed sensor system and the conventional method. From the results, even if the substrate specificity of the enzyme (D-AAOx) was low, it was evident that the concentration of the original material (D-Ala) could be determined specifically when the first reaction product was changed by the second reaction (PyOx).

Enzyme Inhibitors of Marine Microbial Origin with Pharmaceutical Importance

Several enzyme inhibitors with various industrial uses were isolated from bacteria and actinomycetes living in the marine environment. These inhibitors are useful in medicine and agriculture. All the compounds, except the monoamine oxidase inhibitors, are novel, and their activities have been characterized.

Effect of culture conditions on lactic acid production of Tetragenococcus species

Aims:  To investigate the effects of the salt concentration, incubation temperature and initial pH of the medium on the fermentative ability of the halophilic lactic acid bacteria, Tetragenococcus muriaticus and T. halophilus.

Purification and Characterization of a Novel Extracellular .BETA.-1,3-Glucanase Produced by Bacillus clausii NM-1 Isolated from Ezo Abalone Haliotis discus hannai.

A novel extracellular alkaline stable beta-1,3-glucanase produced by Bacillus clausii NM-1 isolated from the ezo abalone Haliotis discus hannai was purified by ammonium sulfate precipitation, DEAE-Sepharose FF ion exchange chromatography and Sephacryl S-200HR gel filtration. The molecular weight of the purified enzyme was estimated to be 71 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was very stable at pH 5.3 to 11.5 but unstable at pH 4.0 to 4.5. The optimum temperature and thermostability of the enzyme increased in the presence of CaC1, The enzyme hydrolyzed R-1,3-glucan from marine organisms, but did not show activity against any other beta-1,3-glucans. The major hydrolysis products of beta-1,3-glucan from Laminaria digitata and Eisenia bicyclis were laminaritriose and laminaritetraose, respectively. The N-terminal amino acid sequence of the purified enzyme was similar to that of several beta-1,3-glucanases in the glycoside hydrolase family 16.

Molecular Analysis of the Gene Encoding a Novel Chitin-Binding Protease from Alteromonas sp. Strain O-7 and Its Role in the Chitinolytic System

Alteromonas sp. strain O-7 secretes several proteins in response to chitin induction. We have found that one of these proteins, designated AprIV, is a novel chitin-binding protease involved in chitinolytic activity. The gene encoding AprIV (aprIV) was cloned in Escherichia coli. DNA sequencing analysis revealed that the open reading frame of aprIV encoded a protein of 547 amino acids with a calculated molecular mass of 57,104 Da. AprIV is a modular enzyme consisting of five domains: the signal sequence, the N-terminal proregion, the family A subtilase region, the polycystic kidney disease domain (PkdD), and the chitin-binding domain type 3 (ChtBD3). Expression plasmids coding for PkdD or both PkdD and ChtBD (PkdD-ChtBD) were constructed. The PkdD-ChtBD but not PkdD exhibited strong binding to alpha-chitin and beta-chitin. Western and Northern analyses demonstrated that aprIV was induced in the presence of N-acetylglucosamine, N-acetylchitobiose, or chitin. Native AprIV was purified to homogeneity from Alteromonas sp. strain O-7 and characterized. The molecular mass of mature AprIV was estimated to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature of AprIV were pH 11.5 and 35 degrees C, respectively, and even at 10 degrees C the enzyme showed 25% of the maximum activity. Pretreatment of native chitin with AprIV significantly promoted chitinase activity.

Nocapyrones: α- and γ-Pyrones from a Marine-Derived Nocardiopsis sp.

One new α-pyrone (nocapyrone R (1)), and three known γ-pyrones (nocapyrones B, H and L (2–4)) were isolated from the culture extract of a Nocardiopsis strain collected from marine sediment. Structures of these compounds were determined on the basis of spectroscopic data including NMR and MS. γ-Pyrones 2–4 were found to induce adiponectin production in murine ST-13 preadipocyte cells but the α-pyrone 1 had no activity. The absolute configuration of the anteiso-methyl branching in 4 was determined by HPLC comparison of a degraded product of 4 with standard samples as a 2:3 enantiomeric mixture of (R)- and (S)-isomers.

Actinomycetal Community Structures in Seawater and Freshwater Examined by DGGE Analysis of 16S rRNA Gene Fragments

The actinomycetal community structures in marine and freshwater environments (the Pacific Ocean, East China Sea, Tokyo Bay, and Arakawa River) were investigated by a culture-independent molecular method to clarify spatial and seasonal distributions. Deoxyribonucleic acid (DNA) was extracted from environmental water samples, and a community analysis was carried out on polymerase chain reaction-amplified 16S ribosomal DNA. The amplified DNA fragments were analyzed by denaturing gradient gel electrophoresis (DGGE) and nonmetric multidimensional scaling analysis, followed by sequencing analysis. The actinomycetal community structures were different at each station in the Pacific Ocean, the East China Sea, Tokyo Bay, and Arakawa River, and different populations predominated in each area. There were vertical variations in actinomycetal communities in the Pacific Ocean and East China Sea between the surface and 100-m depth, but communities were similar from 200- to 1,000-m depths. There were also distinct seasonal variations in communities in Tokyo Bay. Phylogenetic analysis of DNA fragments recovered from DGGE bands revealed that most of the predominant actinomycetal strains were uncultured and were quite different from well known culturable strains, such as the Streptomyces, Micromonospora, Microbispora, Salinispora, and Actinoplanes groups. These results suggest that the marine environment is an attractive target for discovering new actinomycetal populations producing bioactive compounds and that sampling depth and season are important considerations for isolating various populations effectively.

Hyaluromycin, a New Hyaluronidase Inhibitor of Polyketide Origin from Marine Streptomyces sp.

Hyaluromycin (1), a new member of the rubromycin family of antibiotics, was isolated from the culture extract of a marine-derived Streptomyces sp. as a HAase inhibitor on the basis of HAase activity screening. The structure of 1 was elucidated through the interpretation of NMR data for the compound and its 3″-O-methyl derivative in combination with an incorporation experiment with [1,2-13C2]acetate. The compound’s absolute configuration was determined by the comparison of its circular dichroism (CD) spectrum with those of other rubromycins. Hyaluromycin (1) consists of a γ-rubromycin core structure possessing a 2-amino-3-hydroxycyclopent-2-enone (C5N) unit as an amide substituent of the carboxyl function; both structural units have been reported only from actinomycetes. Hyaluromycin (1) displayed approximately 25-fold more potent hyaluronidase inhibitory activity against hyaluronidase than did glycyrrhizin, a known inhibitor of plant origin.