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Productivity of Bioactive Compounds in Streptomyces Species Isolated from Nagasaki Marine Environments

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Productivity of Bioactive Compounds in Streptomyces Species Isolated from Nagasaki Marine Environments Actinomycetologica (2009) 23:16–20 Copyright Ó 2009 The Society for Actinomycetes Japan VOL. 23, NO. 1 NOTE Productivity of Bioactive Compounds in Streptomyces Species Isolated from Nagasaki Marine Environments. Takuji Nakashima1;2;4Ã, Kozue Anzai1, Rieko Suzuki1, Natsumi Kuwahara1, Satoshi Takeshita3, Akihiko Kanamoto4 and Katsuhiko Ando1 1NITE Biotechnology Development Center (NBDC), National Institute of Technology and Evaluation (NITE), 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan 2Kitasato Institute for Life Scieneces, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan 3Joint Research Center, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan 4OP Bio Factory Co., Ltd., 3-2-1, Tonoshiro, Ishigaki, Okinawa 901-2102, Japan (Received Nov. 25, 2008 / Accepted Feb. 3, 2009 / Published Mar. 31, 2009) Based on a Blast search of 16S rRNA sequences of Streptomyces from marine environments of Nagasaki, Japan, 64 isolates showed the highest similarity scores with NBRC strains. Only 5 out of these 64 strains showed exactly the same biological profiles as the approximately 900 strains preserved at NBRC strains, while the remaining isolates showed different biological profiles. This suggests that the genus Streptomyces has the ability to produce a wide variety of unknown bioactive metabolites. Actinomycetes that grow extensively in soils containing About 800 actinomycete strains were isolated from rich organic matter are good sources of natural products. marine environments around Nagasaki Prefecture, Japan, In fact, it has been estimated that approximately two- including the areas of Omura-Bay, Iki island and the thirds of naturally occurring antibiotics are originated from Shimabara peninsula (Anzai et al., 2008a). Humic acid- actinomycetes (Okami & Hotta, 1988). In particular, the vitamin agar (Hayakawa & Nonomura, 1987), ISP-4 and genus Streptomyces is an important group of actinomycetes Actinomyces isolation agar (Becton Dickinson and Co., because of its ability to produce many types of secondary Franklin Lakes, NJ, USA) were used as isolation media, metabolites. In recent years, identification of microbial containing 50% artificial seawater (Yashima Pure Chem- metabolites has increased, along with the probability of icals Co., Ltd., Osaka, Japan). Each strain was incubated on rediscovering already identified compounds, which were an isolation plate at 28C for 14 to 30 days, and then its 16S produced by Streptomyces strains from different terrestrial rRNA was isolated and sequenced as described previously soil environments. This progress has led to a declining (Anzai et al., 2008a, b). Template DNA was prepared using trend in the discovery of unknown natural products derived Prepman Ultra (Applied Biosystems, Foster City, CA, from microorganisms (Watve et al., 2001), despite identi- USA) and amplified using a pair of universal primers (9F fication of only an estimated 1–3% of the existing and 1541R) (Tamura et al., 1998). The amplified 16S rRNA compounds produced by Streptomyces species (Watve genes were sequenced directly using a BigDye Terminator et al., 2001; Balts, 2006). v3.1 cycle sequencing kit and an ABI PRISM 3100 genetic Actinomycetes populations are known to be distributed analyzer (Applied Biosystems). The 16S rRNA gene in various ecological habitats, including beach sand (Suzuki sequences of all isolates were retrieved using the Blast et al., 1994) and seawater (Takizawa et al., 1993). Thus, program (Karlin & Aitschul, 1990) from the National expanding the isolation sources beyond just terrestrial soils Center for Biotechnology Information. Of all the isolates, may be one approach to discover novel natural products 64 strains (approximately 800 total strains) showed more from Streptomyces species. In previous studies, we have than 97% sequence similarity with 36 Streptomyces-NBRC reported on the diversity in taxonomy and biological species (approximately 900 total strains). To determine the activity between actinomycetes from the National Institute biological activities of their metabolites, these 64 isolates of Technology and Evaluation (NITE) Biological Research and 36 NBRC strains were cultured at 28C for 3 days in Center (NBRC) (Anzai et al., 2008b) and those isolated 18-mm test tubes using two production media, as described from marine environments around Nagasaki Prefecture, previously (Anzai et al., 2008a, b); the production medium Japan (Anzai et al., 2008a). The aim of the present study contained 50% artificial seawater, supplemented with either was to determine the ability of Streptomyces species from (1) 2.5% starch, 1.5% soy bean meal, 0.2% dry yeast, and marine environments to produce bioactive compounds and 0.4% CaCO3 or (2) 2% glycerin, 1% molasses, 0.5% to compare them with the ability of NBRC species with casein, 0.1% polypepton, and 0.4% CaCO3. To each 3-ml high 16S rRNA sequence similarity. culture, 6 ml acetone was added and vigorously mixed, ÃCorresponding author. E-mail: [email protected], Phone & Fax: +81-3-5791-6133 16 ACTINOMYCETOLOGICA VOL. 23, NO. 1 Fig. 1. Phylogenetic tree of Streptomyces-NBRC strains based on 16S rRNA gene sequences and the bioactivities of their metabolites. AK, antibacterial activity against Kocuria rhizophila NBRC 12708; AE, antibacterial activity against Escherichia coli NBRC 102203; AC, antifungal activity against Candida albicans NBRC 1385; AA, antifungal activity against Aspergillus oryzae NBRC 6215; AO, antioxidant activity; TI, anti-tyrosinase activity; HL, hemolytic activity. þ, positive; À, negative activity. after which the mixture was centrifuged at 5;000  g for We screened the metabolites produced by the Strepto- 15 min. The supernatant was then prepared for measuring myces strains for four types of biological activities: the biological activity and stored at À40C until use. antimicrobial, antioxidative, anti-tyrosinase, and hemolytic, 17 ACTINOMYCETOLOGICA VOL. 23, NO. 1 Table 1. Biological profiles of Streptomyces isolates from marine environments around Nagasaki Prefecture, Japan Biological activitiesà Identical strains Similarity (%) No. of (NBRC no.) strains AK AE AC AA AO TI HL 13841 98.9–100 3 0 0 0 0 0 0 0 14887 99.33 1 0 0 0 0 0 0 0 13845 99.10 2 0 0 0 1 0 0 0 14278 99.87 1 0 0 1 1 0 0 0 15440 100 1 0 0 1 1 0 0 0 13043 100 3 0 0 3 3 0 0 0 13672 97.57–97.98 5 0 0 2 3 0 0 0 100773 100 1 1 0 0 0 0 0 0 13044 99.51 1 0 0 0 0 0 0 0 15899 98.56, 99.52 3 0 0 1 1 0 0 0 13976 100 2 2 0 1 1 1 0 0 101004 99.76–100 4 2 0 2 2 0 0 0 12805 100 1 0 0 0 0 0 0 0 13016 100 2 0 0 0 0 1 0 0 13683 100 2 2 0 0 0 0 2 0 13816 99.41 1 0 0 0 0 0 0 0 13847 99.69–99.74 2 0 0 0 1 0 0 0 13768 99.69–100 3 0 0 2 2 0 0 0 12737 99.36 2 2 0 1 1 0 0 0 13676 98.72 1 1 0 0 0 0 0 0 3561 99.53 1 1 0 0 0 0 0 0 13056 100 1 0 0 1 1 0 0 0 13794 100 1 1 0 1 1 0 0 0 12208 100 1 1 0 1 1 0 0 0 15405 100 2 2 0 2 2 0 0 0 13350 99.93, 100 2 2 0 0 1 0 0 0 3199 100 3 3 0 0 0 1 0 0 3404 98.58, 99.65 2 0 0 0 0 0 0 0 13859 98.32 1 1 0 0 0 0 0 0 13818 99.87 1 0 0 0 0 0 0 0 13971 100.00 1 0 0 0 0 0 0 0 16668 99.84–100 2 1 0 0 0 0 0 0 13813 99.87 1 1 0 0 0 0 0 0 12813 99.88 1 1 0 1 1 0 0 0 15390 100 1 1 0 1 0 0 0 0 100913 98.87, 100 2 1 0 1 1 0 0 0 The abbreviations in biological activities are the same in Fig. 1. ÃThe numerical values are numbers of positive strains. as described previously (Anzai et al., 2008a, b). Anti- The 16S rRNA gene sequence data of NBRC strains microbial activity was estimated by the paper disk method were collected from the DNA Data Bank of Japan (DDBJ) on Mueller-Hinton agar (Eiken Chemical Co., Ltd., Tokyo, and NITE. The phylogenetic tree based on the 16S rRNA Japan) for bacteria, and Sabouraud’s dextrose agar (Becton gene sequences and the biological activities of the 36 Dickinson and Co., Franklin Lakes, NJ, USA) for fungi. Streptomyces-NBRC strains are shown in Fig. 1. The Antioxidative activity was estimated by the 1,1-diphenyl- relative, positive biological activities of Streptomyces- 2-picrylhydrazyl (DPPH)-scavenging method. Anti-tyrosi- NBRC strains were 31% for anti-Kocuria rhizophila,6% nase activity was estimated by measuring a reduction in the for anti-Escherichia coli, 22% for anti-Candida albicans, production of 3,4-dihydroxyphenyl alanine (DOPA) qui- 31% for anti-Aspergillus oryzae, 22% for antioxidative, none from L-DOPA. The hemolytic activity was estimated 11% for anti-tyrosinase, and 14% for hemolytic, while using fresh (within 7 days) rabbit erythrocytes (Nippon those of the Streptomyces isolates from the Nagasaki Bio-Test Laboratories, Tokyo, Japan). marine environment were 40%, 0%, 34%, 39%, 5%, 3%, 18 ACTINOMYCETOLOGICA VOL. 23, NO. 1 and 0%, respectively (Table 1). There were significant Bentley et al., 2002) in hopes to discover novel bioactive differences in the positive rates of antioxidative and compounds from microorganisms. The genus Streptomyces hemolytic activities between NBRC strains and isolates may be a key source of bioactive compounds and our (Fisher’s exact probability test, p < 0:01).
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