Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
  • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
  • C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/44—Preparation of O-glycosides, e.g. glucosides
  • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
  • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin

Definitions

• A83543 which is comprised of individual components A83543A, A83543B, A83543C, A83543D, A83543E, A83543F, A83543G, A83543H, and A83543J, is a useful insecticide produced by a strain of Saccharopolyspora spinosa sp. nov.
• the improved process of this invention comprises:
• an immiscible solvent such as ethyl acetate or dichloromethane
• A83543 is comprised of individual components A83543A, A83543B, A83543C, A83543D, A83543E, A83543F, A83543G, A83543H and A83543J, which have the structures shown in formula .1:
• R is :
• aminosugar in A83543A has been shown to be ⁇ -D-forosamine; and the neutral sugar in A83543A is ⁇ -2,3, -tri-O-methylrhamnose.
• A83543 and the individual A83543 components are useful for the control of insects, particularly epidoptera species such as Southern armyworm, Diptera species such as blow fly, stable fly and mosquito, and Homoptera species such as cotton aphids and leaf- hoppers.
• A83543 is produced by culturing an A83543- producing strain of Saccharopolyspora spinosa sp. nov. under submerged aerobic conditions by methods disclosed in the copending U.S. patent application of Boeck, Chio, Eaton, Godfrey, Michel, Nakatsukasa and Yao, Serial No.
• A83543-producing Saccharopolyspora spinosa cultures NRRL 18395, NRRL 18537, NRRL 18538 or NRRL 18539, have been deposited and made part of the stock culture collection of the Midwest Area Northern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 1815 North University Street, Peoria, Illinois, 61604, from which they are available to the public under the specified NRRL accession numbers.
• Saccharopolyspora spinosa cultures can be any one of a number of media.
• Preferred carbon sources in large-scale fermentation are glucose and maltose, although ribose, xylose, fructose, galactose, mannose, mannitol, soluble starch, potato dextrin, methyl oleate, oils such as soybean oil and the like can also be used.
• Preferred nitrogen sources are cottonseed flour, peptionized milk and digested soybean meal, although fish meal, corn steep liquor, yeast extract, enzyme-hydrolyzed casein, beef extract, and the like can also be used.
• the customary soluble salts capable of yielding zinc, sodium, magne ⁇ sium, calcium, ammonium, chloride, carbonate, sulfate, nitrate and like ions.
• Essential trace elements necessary for the growth and development of the organism should also be included in the culture medium. Such trace elements commonly occur as impurities in other substituents of the medium in amounts sufficient to meet the growth requirements of the organism.
• small amounts i.e., 0.2 ml/L
• an antifoam agent such as polypropylene glycol
• Foaming can be controlled by including soybean oil or pluronic L-101 (BASF) in the medium (1-3%). Additional oil may be added if foaming develops.
• A83543 The ratio of the components in A83543 will vary, depending upon the fermentation conditions used to produce it. In general, A83543 contains about 85-90%* A83543A, about 10-15% A83543D and minor amounts of A83543B, C, E, F, G, H and J. The percentage of a particular A83543 component may be varied by media changes. For example, adding valine or isobutyric or propionic acids increases the percentage of A83543D produced.
• A83543 may be obtained by shake-flask culture.
• the vegetative inoculum is prepared by inoculating a small volume of culture medium with the spore form or myceliall fragments of the organism to obtain a fresh, actively growing culture of the organism. The vegeta- tive inoculum is then transferred to a larger bioreactor.
• the vegetative inoculum medium can be the same as that used for larger fermentations, but other media are also suitable.
• A83543 is produced by the A83543-producing organisms when grown at temperatures between about 24° and about 33°C. Optimum temperatures for A83543 production appear to be about 28-30°C.
• sterile air is blown into the vessel from the bottom while the medium is stirred with conventional turbine impellors.
• the aeration rate and agitation rate should be sufficient to maintain the level of dissolved oxygen at or above 35%, and preferably at or above 50%, of air saturation with an internal vessel pressure of 0.34 atmospheres.
• Production of the A83543 components can be followed during the fermentation by testing extracts of the broth.
• HPLC using a system as described in Example 1, is a useful assay for this purpose.
• This invention relates to an improved process for recovering A83543 from the fermentation medium in which it is produced.
• the A83543 produced during fermen ⁇ tation of the A83543-producing organism occurs in both the mycelial mass (the biomass) and the broth.
• A83543 appears to be lipophilic.
• traditional defoaming agents inhibit A83543 production.
• foaming is a problem, and a substantial amount of oil has to be used to control it.
• separation of the lipophilic A83543 from the medium involved a simultaneous separation of the oil.
• complete recovery of A83543 required additional separations to remove the oil.
• A83543 was isolated by: 1) filtering the whole broth to separate the bio ⁇ mass;
• This invention relates to the discovery that the troublesome oil could be removed by extracting the A83543 and oil with an immiscible solvent such as ethyl acetate and then back extracting the A83543 into an aqueous phase by using a dilute solution of an acid, such as tartaric acid. After concentrating the acidic solution to remove the miscible portion of the organic solvent, A83543 could be precipitated as the base, by adding a base such as sodium hydroxide to a pH of about 10-11. This approach enabled recovery of purified A83543 without the multiple columns required in step 8. Furthermore, another aspect of the invention was the discovery that this approach could be used on the whole, unfiltered broth, thereby avoiding the product losses involved with steps 2-7.
• the improved process of this invention comprises:
• Step 2 is preferably accomplished by filtration, using a filter aid.
• Preferable solvents for step 3 are ethyl acetate or dichloromethane.
• step 5 the aqueous phase is best separated by decanting it.
• a preferred acid for step 6 is tartaric acid.
• step 7(i) separation (a) is most readily accomplished under vacuum.
• a preferred base for step 7(i)(c) or 7(ii) is sodium hydroxide.
• This process is much more practical because it can be completed in a substantially shorter time, e.g. in about three days, whereas the prior process took approximately three weeks. Furthermore, it provides higher yields of A83543, results in a material which is easy to precipitate and gives a product of higher purity.
• the A83543 thus recovered can be separated into individual components A83543A, A83543B, A83543C, A83543D, A83543E, A83543F, A83543G, A83543H, and A83543J by chromatographic procedures.
• a preferred separation procedure involves reverse-phase silica-gel (C 18 or C 8 ) chromatograph .
• A83543 HPLC Assay Method The following analytical HPLC method is useful for monitoring the fermentation for production of A83543: Centrifuge a sample of the whole broth, decant and remove the supernatant. Add enough methanol to the biomass to return the sample to the original volume, mix, and allow the mixture to stand a minimum of fifteen minutes. Centrifuge and filter the supernatant through a 0.45 ⁇ filter.
• the whole broth can be extracted with acetonitrile (1:4 broth:solvent) or acetone.
• composition A or B medium B is preferred for large scale production
• Slants or plates can be prepared by adding 2.5% agar to vegetative seed medium A or B.
• the inoculated slant is incubated at 30°C. for from about 10 to 14 days.
• the mature slant culture is scraped with a sterile tool to loosen the spores and remove and macerate the myceliall mat.
• About one-fourth of the loosened spores and culture growth thus obtained is used to inoculate 50 mL of a first-stage vegetative seed medium.
• the first-stage medium may be inoculated from a liquid nitrogen ampoule.
• ampoules are prepared using equal volumes of vegetative culture (48-72 hr incubation, 30°C) and suspending medium.
• the suspending medium contains lactose (100 g), glycerol (200 mL) and deionized water (q.s. to 1 L) .
• a liquid nitrogen ampoule is used to inoculate 100 mL of vegetative medium in 500-mL Erlenmeyer flasks (or 50 mL medium in 250-mL flasks). The cultures are incubated at 30°C for 48 hours on a shaker orbiting in a two-inch (5.08 cm) circle at 250 rpm.
• the incubated culture (5% v/v inoculum) is used to inoculate 100 mL of a production medium having the following composition:
• Cottonseed flour** CaC0 3 (reagent or technical grade)
• the inoculated production medium is incubated in 500-mL Erlenmeyer flasks at 28-30°C. for 6 to 8 days on a shaker orbiting in a two-inch circle at 250 rpm.
• incubated first-stage medium prepared as described in Section A
• 10 mL of incubated first-stage medium is used to inoculate 400 mL of a second-stage vegetative medium having the same composition as that of the first-stage vegetative medium.
• This second-stage medium is incubated in a 2-L wide-mouth Erlenmeyer flask for about 48 hours at 30°C. on a shaker orbiting in a two-inch circle at 250 rpm.
• Incubated second-stage vegetative medium (2 L) thus prepared is used to inoculate 80 to 115 liters of sterile production medium, prepared as described in Section A. Additional soybean oil is added to control foaming, if needed.
• the inoculated production medium is allowed to ferment in a 165-L stirred bioreactor for 5 to 8 days at a temperature of 28°C.
• the airflow and agitator speed in the stirred vessel are computer controlled to maintain a dissolved oxygen level at or above 50% of air saturation.
• Fermentation broth (10 L) was prepared as described in Example 2 Sect. A, except that 1) 200 mL of production medium was used in 1-L flasks; 2) soybean oil was omitted from the production medium; and 3) incubation was at 30° for 4-6 days. The broth was filtered. The filtrate, containing 4 meg of A83543A/ mL and no detectable quantities of A83543B, C, or D/mL, was discarded.
• the biomass was washed with water and extracted for one hour with methanol.
• the extract (7 L) contained 72 meg of A83543A/mL and 7 meg of A83543D/mL.
• the methanol extract was concentrated to a volume of 5 L, and added to HP-20 resin (150 mL, Mitsubishi Chemical Industries, Ltd., Japan) in water (2 L). This mixture was stirred for one hour.
• the HP-20 resin mixture was then placed in a glass column.
• the initial effluent and the eluate using methanol:water (1:1, 1 L) were not active.
• the second eluate using methanol:water (7:3, 1 L) contained trace quantities of A83543A.
• the following eluate using methanol (1 L) contained the A83543A and A83543D activity.
• the methanol eluate was concentrated and combined with 2 similar fractions from other work-ups and concentrated to dryness.
• the residue was dissolved in 75 mL of methanol:THF (4:1) and precipitated by addition into 10 volumes of acetonitrile. The mixture was filtered, and the filtrate was concentrated to dryness.
• the column was conditioned in methanol: acetonitrile:water (37.5:37.5:25). After sample application, the column was developed using a 180-min linear gradient of the following solvents:
• Fractions containing A83543D were combined with D-containing fractions from 6 similar separations and were concentrated and chromatographed as described supra, using the same column but different solvents.
• the column was conditioned in methanol:acetonitrile: water(40:40:20) .
• the solvent systems used to develop the column in a 180-min linear gradient operation were: Solvent Systems
• Liquid nitrogen ampoules of the culture are prepared as described in Example 2, using the general procedures of Sect. B.
• One ampoule is used to inoculate a first-stage vegetative culture (50 mL of medium C in 250-mL flasks), which is incubated for about 48-72 hours.
• Incubated first-stage culture is used to inoculate (10-mL inoculum) a second-stage culture (400 mL of medium C in 2-L flasks), which is incubated for about 48 hrs.
• the incubated second-stage culture (5 L) is used to inoculate a production medium (115 L) having the following composition:
• the inoculated production medium is allowed to ferment in a 165-L stirred bioreactor for about 8 to 10 days, or longer, at a temperature of 30°C.
• Dissolved oxygen (DO) levels are regulated by computerized systems set to maintain the DO level above 50% of air saturation as described in Example 2, Section B.
• Liquid nitrogen ampoules of the culture are prepared as described in Example 2, using the general procedures of Sect. B.
• One ampoule is used to inoculate a first-stage vegetative culture (50 mL of medium B in 250-mL flasks), which is incubated for 48 to 72 hours.
• Incubated first-stage culture is used to inoculate (10-mL inoculum) a second-stage culture (400 mL of medium B in 2-L flasks), which is incubated for about 48 hr.
• the incubated second-stage culture (2 L) is used to inoculate a production medium (115 L) having one of the following compositions: PRODUCTION MEDIUM III
• the inoculated production medium is allowed to ferment in a 165-L stirred bioreactor for about 8 to 10 days, or longer, at a temperature of 30°C. DO levels are regulated as described in Example 4.
• the culture Saccharopolyspora spinosa NRRL 18538 is cultivated but using vegetative medium B and production medium III.
• A83543 Separation of A83543 by the New Process Fermentation broth is prepared as described in Example 6. A83543 is separated from the broth as follows:
• A83543 A and D from Fermentation Broth by the New Process Whole fermentation broth is prepared by a procedure similar to that described in Example 2.
• the A83543-producing culture used was a progeny of the NRRL 18395 culture, but an ancestor of the NRRL 18538 and 18539 strains.
• the strain used produces approximately six-fold lower antibiotic titers than the NRRL 18538 and 18539 strains produce.
• Vegative Medium B was used, and the production medium was as follows:
• the production medium was incubated at 30°C for about eight days. After incubation, the broth, contained 141 mcg/mL A83543A and 13 mcg/mL A83543D (total of 16.94 grams of A83543A and A83543D). An equal volume of acetone (110 liters) was added. The broth/acetone was filtered through a plate and frame press to remove the. mycelial solids.
• the recovered filtrate (200 L. ) was adjusted from pH 7.6 to pH 10 with 5 N sodium hydroxide. Then, ethyl acetate (60 L) was added to extract the activity. After stirring, the phases were separated, and the lower phase (100 L. ) was discarded. The upper phase (>50% ethyl acetate) was concentrated from 160 L. to 80 L. to remove acetone.
• the aqueous highly colored phase (20 L. ) was then removed by decantation, and the enriched, concentrated ethyl acetate phase (60 L. ) was back- extracted, using an equal volume of 0.1N tartaric acid in water.
• the enriched aqueous tartaric acid phase (70 L. ) was recovered, concentrated under vacuum to remove miscible ethyl acetate and then concentrated to a volume of 4 L. (20 fold), using a Millipore ProLab reverse osmosis unit.
• the concentrated aqueous tartaric acid solution (4 L. ) was adjusted from pH 3 to pH 10 with sodium hydroxide, slowly allowing a precipitate to form.
• the precipitate was filtered, washed with water and dried under vacuum at 40°C to give 13.4 grams of dry precipitate with a potency of 815 mcg/mg of A83543A and 67 mcg/mg of A83543D (by HPLC).
• These yields represent an overall recovery of 11.8 grams A83543A and A83543D which is a 69.6% yield from whole broth.

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• 1990
  • 1990-10-26 AU AU66414/90A patent/AU631693B2/en not_active Ceased
  • 1990-10-26 JP JP2515669A patent/JPH05504469A/ja active Pending
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