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.
The gene encoding an extracellular chitinase from marine Alteromonas sp. strain 0-7 was cloned in Escherichia coli JM109 by using pUC18. The chitinase produced was not secreted into the growth medium but accumulated in the periplasmic space. A chitinase-positive clone of E. coli produced two chitinases with different molecular weights from a single chitinase gene. These proteins showed almost the same enzymatic properties as the native chitinase ofAlteromonas sp. strain 0-7. The N-terminal sequences of the two enzymes were identical. The nucleotide sequence of the 3,394-bp SphI-HindIII fragment that included the chitinase gene was determined. A single open reading frame was found to encode a protein consisting of 820 amino acids with a molecular weight of 87,341. A putative ribosome-binding site, promoter, and signal sequence were identified. The deduced amino acid sequence of the cloned chitinase showed sequence homology with chitinases A (33.4%) and B (15.3%) from Serratia marcescens. Regardless of origin, the enzymes of the two bacteria isolated from marine and terrestrial environments had high homology, suggesting that these organisms evolved from a common ancestor.Chitin is a major component of the exoskeletons of insects and crustaceans, and it is abundantly distributed throughout nature. This polysaccharide is also an important nutrient source of both carbon and nitrogen in the marine environment. Yu et al. (38) pointed out that the oceans would be completely depleted of carbon and nitrogen in a relatively short time if chitin could not be returned to the ecosystem in a biologically usable form. However, marine sediment contains relatively little chitin, despite the production of huge amounts of this insoluble polysaccharide by crustaceans. ZoBell and Rittenberg (39) reported that chitinolytic bacteria, which are abundant and widely distributed in the sea, play an important role in converting insoluble chitin into a biologically usable form. Chitinases (EC 3.2.1.14) are enzymes which degrade chitin, and they have been detected in various microorganisms (3,25,28,30), plants (1, 18), insects (5, 13), and crustaceans (19). Alteromonas sp. strain 0-7 is a gram-negative, flagellated, motile, and aerobic rod-shaped bacterium of marine origin. This strain excretes chitinase into the growth medium in the presence of chitin (32). We have already reported the purification, properties, and partial amino acid sequence of the enzyme from this strain (33). Recently, bacterial chitinase genes from terrestrial and marine bacteria such as Serratia marcescens (6, 10), Bacillus circulans (35, 36), Vibrio harveyi (28), and Vibno vulnificus (37) have been cloned and sequenced. However, the mechanism of hydrolysis, the relationship between structure and function, and the regulatory system involved in enzyme induction are still unclear. In this report, we describe the cloning of a gene coding for chitinase and the purification of the gene products. The nucleotide sequence of the gene was determined, and the deduced amino acid s...
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.
Marine bacteria which produced extracellular alkaline protease inhibitor were isolated and identified. A new and simple casein double layer agar method was employed for a quick screening of the inhibitor producers. Out of the 878 isolates from Aburatsubo Inlet, Kanagawa, Japan, only three strains produced the inhibitor whereas no inhibitor producer was found out of the 1,961isolates from other sea areas. Among the three inhibitor producers, strain B-
Marinostatin is a unique protein protease inhibitor containing two ester linkages. We have purified a 12-residue marinostatin [MST(1-12), (1)FATMRYPSDSDE(12)] and determined the residues involved in the formation of the ester linkages and the solution structure by (1)H NMR spectroscopy and restrained molecular dynamics calculation. The two ester linkages of MST(1-12) are formed between hydroxyl and carboxyl groups, Thr(3)-Asp(9) and Ser(8)-Asp(11), indicating that MST(1-12) has two cyclic regions which are fused at the residues of Ser(8) and Asp(9). A strong NOE cross-peak between Tyr(6) H(alpha) and Pro(7) H(alpha) was observed, indicating that the Pro(7) residue takes a cis-conformation. Well-converged structures and hydrogen-deuterium experiments of MST(1-12) showed that the backbone NH proton of the P1'residue, Arg(5), is hydrogen-bonded to the carbonyl oxygen of the ester linkage between Thr(3) and Asp(9). To reveal the significance of the ester linkages, a marinostatin analogue, MST-2SS ((1)FACMRYPCCSCE(12)) with two disulfide bridges of Cys(3)-Cys(9) and Cys(8)-Cys(11), was also synthesized. The inhibitory activity of MST-2SS was as strong as that of MST(1-12), and the Pro(7) residue of MST-2SS also takes a cis-conformation. However, the exchange rate of the Arg(5) NH proton of MST-2SS was about 100 times faster than that of MST(1-12), and the structure calculation of MST-2SS was not converged on account of the small number of NOEs, indicating that MST-2SS takes a more flexible structure. The hydrogen acceptability of the ester linkage formed by the P2 position residue, Thr(3), is crucial for suppressing the fluctuation of the reactive site and sustaining the inhibitory activity, which enables marinostatin to be one of the smallest protease inhibitors in nature.
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