Antonie van Leeuwenhoek (2005) 87:5963 DOI 10.

1007/s10482-004-6544-x

Springer 2005

-1

Enzyme inhibitors and other bioactive compounds from marine actinomycetes

Chiaki Imada
Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan (e-mail: imada@s.kaiyodai.ac.jp)
Received 1 June 2004; accepted in revised form 5 August 2004

Key words: Actinomycete, Enzyme inhibitor, Marine

Abstract 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 puer sh 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 highperformance liquid chromatography (HPLC). To our knowledge, this is the rst report of actinomycetes from the marine environment that produce TTX. Introduction Actinomycetes are representative of terrestrial microorganisms and usually are isolated from soils. Compared to terrestrial actinomycetes, however, very little work has been conducted on marine actinomycetes. As marine environmental conditions are extremely dierent from terrestrial ones, it is surmised that marine actinomycetes have characteristics dierent from those of terrestrial actinomycetes and therefore may produce dierent types of bioactive compounds (Okami et al. 1976). Actinomycetes in marine and estuarine sediments have not been investigated extensively although their ubiquitous presence in marine sediments has been well documented (Takizawa et al. 1993; Moran et al. 1995). They were believed to be of terrestrial origin, transported to rivers by rain or irrigation water, and nally to the marine environment where they are exposed to water with salt concentrations and temperatures that dier from those of the terrestrial environment. As a result, some metabolic changes may occur in the organisms (Okazaki and Okami 1975). This view is now changing with the discovery of bona de marine actinomycetes (Jensen et al. 1991, 2004; Bull et al. 2004, this issue). From the viewpoint of antibiotic production, it should be noted that the number of marine microorganisms with antimicrobial activity may be higher than that of terrestrial ones. (Grein and Meyer 1958); and (Okazaki and Okami 1976) found respectively that 50% and 27% of Streptomyces isolated from the marine environment showed antimicrobial activity, and these percentages were increased when tests were conducted in the presence of seawater. As mentioned above, the marine environment is considerably dierent from the terrestrial environment, and therefore, it needs to be explored and exploited for new biological products. So far, microorganisms producing various bioactive

60 compounds have been isolated largely from neritic environments because of the sampling diculty. We describe herein several enzyme inhibitors and other bioactive compounds from marine actinomycetes, as well as their general properties. When activity was detected, large-scale cultivation using a baed Erlenmeyer ask or a jar fermenter was carried out and pure substances were extracted from the culture supernatant.

Assays used in the present study Methods Collection of samples Surface seawater was collected with sterilized bottles whereas deep sea water samples were collected with a Niskin buttery water sampler (General Oceanics, Miami). Sediment samples from shallow sea were collected with a mini sediment sampler (Okami 1972). Deep sea sediment samples were collected with a multiple corer. The isolation of microorganisms was carried out immediately upon retrieval of the samples. In the present study, the inhibitory activity against various enzymes of pharmaceutical importance and the antibiotic activity were tested. The production of tetrodotoxin (TTX), otherwise known as puer sh toxin, was also investigated.

Results and discussion b-Glucosidase inhibitor b-Glucosidases are involved in tumor metastasis and the human immunodeciency virus and therefore have potential for use as drug targets. To date, a number of glucosidase inhibitors have been isolated from terrestrial organisms but not from marine microorganisms. One actinomycete strain that was isolated from deep sea sediment collected at 1500 m depth was found to produce b-glucosidase inhibitors only in the presence of seawater. The taxonomic characteristics of this strain were almost identical to those of a terrestrial actinomycete, Streptomyces galbus, except for the production of b-glucosidase inhibitors, the temperature range for growth, and tolerance to NaCl (Table 1). The extracted and puried inhibitors were identical with D-gluconolactam and D-mannonolactam, which have never been isolated from nature. To our knowledge, this is the rst report of a b-glucosidase-inhibitor-producing microorganism from the marine environment (Imada and Okami 1995).

Isolation of actinomycetes from marine environment In the present study, two media were mainly used for the isolation of actinomycetes from the marine environment. HV agar contains humic acid, inorganic salts and vitamins (Hayakawa and Nonomura 1987). Starch-nitrate agar contains starch and inorganic salts (Ghanem et al. 2000). In order to prevent fungal growth, cycloheximide was added to a nal concentration of 50 lg/ml.

Cultivation of actinomycetes and screening of bioactive compounds To screen for bioactive compound producers, four liquid media (CS, OA, I, and Z) were used (Imada and Okami 1995). In order to screen eciently for a large number of microbes within a short period of time, a minishaker (Marubishi Co., Ltd., type MBSS-500) and a 24-well disposable microplate (Sumitomo Bakelite Co., Ltd.) as the cultivation vessel were employed. Test strains were inoculated into various culture media in the microplate and cultivated on the shaker. The shaker can accommodate more than 500 strains at one time. After cultivation, the activities of the culture supernatant were determined with various assay methods.

N-Acetyl-b-D-glucosaminidase inhibitors N-Acetyl-glucosaminidase is an enzyme that catalyzes the hydrolysis of glycosidic linkages as an exoglycosidase and releases N-acetyl-glucosamine from glycoprotein. The activity of this enzyme is increased markedly in patients with diabetes, leukemia or cancer. Therefore, identication of

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Table 1. b-Glucosidase inhibitor: comparison of strain no. 62 with S. galbus ISP-5089 Strain No. 62 Production of inhibitor maximum growth temperature Tolerance to NaCl Positive 31 C 6% S. galbus ISP-5089 Negative 43 C 3%

inhibitors of this enzyme might lead to the elucidation of the causative agent of those diseases. An attempt was made to isolate a novel enzymeinhibitor-producing microbial strain from the marine environment. A strain that was tested positive for the enzyme inhibitor was isolated from marine sediment collected at a depth of approximately 100 m from Otsuchi Bay in Iwate Prefecture, and was classied under the genus Streptomyces. As shown in Figure 1, this strain produces two novel compounds, pyrostatins A and B, in the presence of seawater (Aoyama et al. 1995). These compounds showed specic inhibitory activity against N-acetyl-glucosaminidase but have no signicant antimicrobial activity at 100 lg/ml and show no toxic eects in mice at 100 mg/ml.

lated from a marine sediment collected from the coast of Shanghai City, China, and was identied as a member of the genus Streptomyces. A novel inhibitor, pyrizinostatin, was isolated from this strain (Figure 2). Pyrizinostatin exhibited noncompetitive inhibition of pyroglutamyl peptidase, its IC50 value being 1.8 lg/ml. Pyrizinostatin at this dose exhibited no signicant antimicrobial activity and no toxic eects after iv injection in mice. Pyrizinostatin, therefore, may be used for elucidating the mechanisms of various diseases caused by the enzyme (Aoyagi et al. 1992).

a-Amylase inhibitors Amylase inhibitors have received considerable attention in recent years because they are helpful tools for the determination of amylase isozyme activities, the purication of amylases, and the control of such carbohydrate-dependent diseases as diabetes, obesity or hyperlipemia. To date, most amylase inhibitors of microbial origin have been isolated from terrestrial actinomycetes. As far as we know, there are no reports of amylase-inhibitor-producing marine microorganisms. Of the nearly 5000 isolates from various marine habitats, only strain no. 178, which was isolated from sediment collected from Aburatsubo Inlet in Kanagawa Prefecture at a depth of 5 m, was found to produce an inhibitor. After adding amylase and iodine solution, a purple inhibitory zone was formed around the colony cultured on starch agar. The strain showed taxonomical features that were almost identical to those of Streptomyces corchorusii except for inhibitor production, optimal temperature for growth, tolerance to NaCl,

Pyroglutamyl peptidase inhibitors Pyroglutamyl peptidase acts by catalyzing the release of pyroglutamyl residues that block terminal amino acids in many proteins and peptides. This enzyme is widely distributed in animals, plants and microorganisms. However, its physiological role in the living body has not yet been established. In order to elucidate the relationship between various disease processes and this enzyme, screening for an enzyme inhibitor was performed. A strain that tested positive for the enzyme inhibitor was iso-

Figure 1. Chemical structures of pyrostatins A and B.

Figure 2. Chemical structure of pyrizinostatin.

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Table 2. a-Amylase inhibitor: comparison of the inhibitor producer (strain no. 178) with S. corchorusii ISP-5340 Strain No. 178 Inhibitor production Optimal temperature for growth Tolerance to NaCl Production of red pigments Positive 45 C 12% Positive S. corchorusii ISP-5340 Negative 50 C 6% Negative

and production of intracellular red pigments in the presence of seawater (Table 2). From the results, the strain was named Streptomyces corchorusii subsp. rhodomarinus susp. nov (Imada and Simidu 1988). The optimal production of the inhibitor was observed in seawater of quarter strength (Imada and Simidu 1992). This is the rst report of an a-amylase-inhibitorproducing microorganism from the marine environment. Further studies aiming at purication of the inhibitor are under way. Antibacterial substances Marine actinomycetes that produced antibiotics against Gram-positive bacteria only in the presence of seawater were isolated and characterized. Of the 100 isolates from Otsuchi Bay, 41 failed to produce any antibiotics in the absence of seawater. However, of the 41 isolates, two were found to produce antibiotics in the presence of seawater. Strain no. 18 exhibited the highest activity and was therefore selected for further studies. Phylogenetic analysis showed high similarity between strain no. 18 and Micromonospora globosa (Shiomi et al. 1990). However, M. globosa did not produce any

antibiotics in the presence of seawater. Therefore, the eect of seawater on the growth of strain no. 18 and its production of antibiotics was investigated. Strain no. 18 grew in the seawater concentration range of 0140%. The optimal concentration ranged from 10 to 30%. However, the production of antibiotics was observed in the concentration range of 60110% despite poor growth in this range (unpublished data). Thus, antibiotic production is seawater-dependent. The purication of the antibiotic is in progress.

Production of TTX Tetrodotoxin (TTX), otherwise known as puer sh toxin, is a potent neurotoxin that blocks the sodium channel in excitable membranes. It has been the belief for many years that only the puer sh contains TTX. However, this toxin has also been found in other marine animals. Thereafter, TTX-producing bacteria have been isolated from other marine organisms. One report has indicated that a considerable number of bacterial strains isolated from sediment samples produced TTX (Simidu et al. 1987). Actinomycetes are generally present in marine sediments, although their numbers are fewer than other bacteria (see Bull et al. 2004, this issue). They are known to produce various antibiotics and bioactive compounds. However, the production of TTX by actinomycetes has not been examined so far. Sediment samples were collected around Japan and actinomycetes were isolated from the neritic environment. Ten strains were isolated and TTX was extracted from them. The production of TTX

Table 3. Tetrodotoxin (TTX) production by actinomycetes isolated from marine environments Sampling station Aburatsubo Inlet Tokyo Bay Pacic Ocean Lat. (N) 3509.0 3546.3 3311.0 Long. (E) 13937.0 13946.3 14117.3 Depth (m) 5 17 3569 Strain Streptomyces sp. Streptomyces sp. Not identied Streptomyces sp. Streptomyces sp. Streptomyces sp. Streptomyces sp. Streptomyces sp. Streptomyces sp. Streptomyces sp. no. 13 no. 21 no. no. no. no. no. no. no. 7 2 5 8 9 3 6 TTX production Positive Positive Positive Doubtful Positive Positive Positive Positive Positive Positive

Pacic Ocean

3413.0

14112.7

5974

63 was measured by HPLC as reported previously (Do et al. 1991). Table 3 summarizes the production of TTX by actinomycetes isolated from various marine sediment samples. Except for one strain, all the strains produced TTX. This is the rst report of actinomycetes that produce TTX from the marine environment. Future studies will focus on the production of TTX by actinomycetes thriving in the terrestrial environment. Studies of novel bioactive compounds from marine actinomycetes have been actively pursued in recent years. The isolation of various compounds from actinomycetes living in the open ocean or in the deep sea will serve as another source of novel substances, as exemplied in the present study.
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Acknowledgements I would like to thank Dr. Y. Okami, Institute of Microbial Chemistry, who gave pertinent advice, suggestions and encouragement throughout the study. Sincere thanks are also extended to Professor A.T. Bull who arranged for the publication of the manuscript in this Special Issue. I am also grateful to the captain, the ocers and the crews of the research vessels in our university for their cooperation in sample collection.

References
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