Ebelactone, an inhibitor of esterase, produced by actinomycetes.

Sir: We have demonstrated that aminopeptidases, phosphatases and esterases are located not only in cells but also on the cellular membrane of various kinds of animal cells1-3) and that inhibitors of these enzymes modified immune responses4,5). Among these inhibitors, bestatin, amastatin and forphenicine enhanced immune responses, but esterastin which inhibited esterase suppressed immune responses6,7). We continued the screening of esterase inhibitors and found another group of inhibitors which we named ebelactone. Ebelactone inhibited also N-formylmethionine aminopeptidase. This inhibitor enhanced immune responses. In this paper, we report on the isolation and characterization of ebelactone. In the screening study, culture filtrates of many strains of various species of soli actinomycetes showed the activity to inhibit esterase and we were successful in the isolation of an inhibitor from the strain MG7-Gl. This strain, closely related to Streptomyces aburaviensis, was isolated from a soil sample collected in Rissho University, Kumagaya City, Saitama Prefecture. In determining the inhibitory activity against esterase, the method used previouslye~ was modified as follows: 1 ml of 0.1 m phosphate buffer containing 0.06%. Triton X-100 (pH 7.0), 0.95 ml of water with or without a test sample and 0.025 ml of esterase solution (from hog liver, Sigma Chemical Co., U.S.A.) were pipetted into a series of test tubes (1.5 x 10 cm) at room temperature; after 3 minutes, 0.025 ml of 40 mgt p-nitrophenyl acetate (PNPA, Sigma Chemical Co., U.S.A.) was added and well mixed. Exactly 15 minutes thereafter, the absorbance of the reaction mixture was read at 400 nm. The concentration of enzyme in the solution was adjusted to yield 50 nmoles of p-nitrophenol. The reaction was also carried out without the enzyme solution to obtain the blank value. Ebelactone was produced by shaken culture and tank fermentation of the strain MG7-G I in media containing various carbon and nitrogen sources. Glycerol, glucose, lactose, maltose and starch are examples of carbon sources and fish meal, N-Z amine, yeast extract and soy-bean meal are examples of nitrogen sources suitable for the production of ebelactone. A typical medium used for production contained 3.0 glycerol, 2.0% fish meal and 0.2 % CaCO3. The maximum production was attained in 2 days in the shaken culture. Ebelactone was extracted from the whole broth with same volume of butyl acetate. pH was not adjusted. The butyl acetate extract was concentrated under reduced pressure and the brownish oily residue thus obtained was chromatographed on a silica gel column using n-hexane chloroform ethyl acetate (5: 5: 1). The active fractions were combined and concentrated under reduced pressure. The concentrate was subjected to reversed phase silica gel column chromatography with methanol water (1: 1). Two active peaks were obtained. The active compound in the first peak was named ebelactone A and that in the second peak was named ebelactone B. The yield of ebelactone A is twice of ebelactone B. The two compounds obtained