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• • 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
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
  • C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01S—DEVICES USING THE PROCESS OF LIGHT AMPLIFICATION BY STIMULATED EMISSION OF RADIATION [LASER] TO AMPLIFY OR GENERATE LIGHT; DEVICES USING STIMULATED EMISSION OF ELECTROMAGNETIC RADIATION IN WAVE RANGES OTHER THAN OPTICAL
  • H01S5/00—Semiconductor lasers
  • H01S5/30—Structure or shape of the active region; Materials used for the active region
  • H01S5/34—Structure or shape of the active region; Materials used for the active region comprising quantum well or superlattice structures, e.g. single quantum well [SQW] lasers, multiple quantum well [MQW] lasers or graded index separate confinement heterostructure [GRINSCH] lasers

Definitions

• New antifungal antibiotic, and new Bacillus sp. microorganism which can produce the above antibiotic and its manufacturing method
• the present invention relates to new antifungal antibiotic which is described the below general formula(l), new Bacillus sp. microorganism which can produce the above antibiotic, and its manufacturing method.
• each R denotes -H, -OH, -NH 2 , alkyl group having 1 to 6 carbon atoms, allyl group having 5 to 20 carbon atoms, or alkylene group having 2 to 6 carbon atoms and, subscript 1, m, or n is an integer less than 3.
• Pyrrolnitrin which was produced by Pseudo ⁇ onas pyrrocinia with exhibiting activity against yeast and gram positive bacteria and showing little virulency, was discovered by Arima et al. Polyoxin discovered by Isono et al. had an activity against fungi causing rice diseases and had little harmful effect against men, beasts, fishes, and crops.
• dapiramicin which was produced by Micromonospora and had a superb effect on Pellicularia filamentosa was developed as well as albopeptin A, fengycin, and octacosamicin.
• kasugamycin, validamycin, and polyoxin have been generally used as agricultural antibiotics, but production cost of these antibiotics is too high resulting in an economic problem.
• the synthetic organic compounds have caused the side effects such as agricultural poisoning and inflow of the residual chemical agents into soil.
• most of the residual chemical agents of synthetic organic compounds in soil have caused an agricultural pesticide poisoning.
• destruction of the ecological system by means of pesticide toxicity has arisen many serious social problems including the environmental pollution. Therefore, various kinds of synthetic organic chemical compounds have been prohibited to use as pesticide because of carcinogenic possibility and the residual toxicity.
• Another object of the present invention is to provide new Bacillus sp. microorganism producing the above antibiotic and manufacturing method of antibiotic.
• Another object of the present invention is to provide composite which contains the above antifungal antibiotic as an effective ingredient.
• new Bacillus sp. microorganism which has a high antibiotic effect on plant fungi, especially Pyricularia oryzae and Pellicularia filamentosa, is isolated from the soil and identified.
• This microorganism is cultured in YS medium and centrifuged. The supernatant is extracted with ethyl acetate. Organic layer is collected and evaporated.
• the active fraction is dissolved in methanol, followed by running silica gel column chromatography using methanol. Methanol is removed and then the residue is dissolved in dichloromethane:methanol (1:5) mixture and put on Sephadex LH-20 column chromatography. Solvent is removed and then the active fraction is collected and dissolved in water and lyophilized. Finally powdery antibiotic which has an excellent antifungal activity is obtained.
• the present invention has succeeded in providing a very simple and economical manufacturing method of new antibiotic.
• Fig.1 is an electron microscope photograph of Bacillus sp. SY-414
• Fig.2 is an electron microscope photograph of spore formed by the invented Bacillus sp. SY-414
• Fig.3 is the graph representing the cell growth and the antibiotic production according to culture time
• Fig.4 is the graph representing the antibiotic production according to culture temperature
• Fig.5 is the graph representing the antibiotic production according to initial pH
• Fig.6 is the graph representing the cell growth and the antibiotic activity against Pyricularia oryzae in NB and YS media
• Fig.7 is the graph representing the cell growth and the antibiotic activity against Pellicularia filamentosa in NB and YS media
• Fig.8 is the photograph of inhibition zone against Pyricularia oryzae
• Fig.9 is the photograph of inhibition zone against Pellicularia filamentosa
• Fig.10 is the photomicrograph of the crystallized antibiotic obtained in example 4
• Fig.11 is the UV spectrum of the antibiotic obtained in example 4
• Trihexocin represents the below general formula(l).
• each R denotes -H, -OH, -NH2, alkyl group of Ci - C ⁇ , allyl group of C5- C 20 , or alkylene group of C2- C ⁇ and, subscript 1, m, or n is an integer less than 3.
• the antibiotic of the present invention showed a needle shape and a low R f value in general solvents, and its melting points was 127-1281C. As the results of its positive color reaction with alkaline permanganate, 10% sulfuric acid and iodine including *H NMR analysis, it was elucidated that new antibiotic contained the sugar moiety. 13 C NMR analysis showed that it did not contain any aromatic compounds. From all the above results, we decided the antibiotic of the present invention as the new material, and named it as "Trihexocin". Trihexocin of the present invention had an activity at the concentration of 50-100 tg/ml against Pyricularia oryzae and Pellicularia filamentosa, but did not against gram positive and negative bacteria, and yeast.
• the antibiotic activity against Pyricularia oryzae was stable at pH 6, 7 and 8, but represented 68* at pH 5 and 70* at pH 10.
• the antibiotic activity against Pellicularia filamentosa was stable at pH 6,7 and 8, but represented 0* at pH 5, 82* at pH 9 and 0* at pH 10.
• the antibiotic activity against Pyricularia oryzae was stable at 301C and 40 X but represented 80* at 501C and 60* at 601.
• the antibiotic activity was stable at 30C and 401C but represented 70* at 501C, and disappeared above 60 TC.
• trihexocin of the present invention showing an antifungal antibiotic is the material having sugar moiety, and its derivatives and salts having the biological activity are easily obtained.
• Trihexocin is produced by Bacillus sp. SY-414 (KCCM-10043) and its manufacturing method will be described in detail as follows.
• Kangwon-do Soil collected from the Chunchon area, Kangwon-do was suspended in distilled water and cultured in plate and then the microorganisms were isolated.
• the isolated strain was inoculated in nutrient broth (NB medium: meat extract 0.3*, peptone 0.5*) and cultured at 30 : for 3 days.
• NB medium meat extract 0.3*, peptone 0.5*
• the culture broth was centrifuged at 12,000xg for 20 minutes and the antibiotic activity of supernatant was detected by paper disc method using Pyricularia oryzae IF0 30517 and Pellicularia filamentosa IF0 8985 as a test microorganism.
• the isolated strain which showed the highest activity against the above test microorganisms was selected and identified.
• the above microrganism was similar to Bacillus licheniformis but confirmed as a new microorganism, thus it was named Bacillus sp. SY-414 and deposited as deposit number KCCM-10043 at Korea Federation of Culture Collections (KFCC) on September 14, 1993.
• Bacillus sp. SY-414 of the present invention was identified by general identification methods that were commonly used and, the results were in Table 1-5.
• Bacillus sp. SY-414 of the present invention was rod and motile, and the cell size was 1.5/M x 3.6 ⁇ as shown in Figure 1.
• the spore of Bacillus sp. SY-414 was ellipsoidal and located in central( Figure 2) and the sporangium was unswellen.
• Bacillus sp. SY-414 was within the range of 15-50TC, the pH range was 3-10, and it was able to grow up to 6* salt concentration.
• Bacillus sp. SY-414 represented catalase positive and oxidase negative, utilized propionate and hydrolyzed starch.
• Bacillus sp. SY-414 utilized most of sugars except cellulose for the growth of microorganism. As the results of sugar fermentation, an acid was produced from some sugars but a gas was not produced from all the sugars.
• Bacillus sp. SY-414 was inoculated in 20ml nutrient broth and cultured at 30"C for 48-96 hours. The seed culture was prepared under the above conditions.
• Figure 3 represented the growth of Bacillus sp. SY-414 and the antibiotic production according to the culture time in nutrient broth.
• the growth of Bacillus sp. SY-414 reached the stationary phase after 4-days cultivation and the production of the antibiotic was the maximum at the same culture time and decreased after 4-days of cultivation.
• Potato dextrose agar (PDA) plate was prepared with the test microorganism which was layered in PDA soft agar plate.
• the cup (innner diameter 6mm, outer diameter 8mm, height 10mm) was put on the above plate and the culture supernatant was added into the cup.
• the plate was incubated at 30O for 48 -72 hours, and then inhibition zone was measured.
• the activity was the maximum at the initial pK 8.
• organic nitrogen sources including beef extract was fairly good for the production of the antibiotic.
• the antibiotic production was increased remarkably when yeast extract was added and 0.8* (w/v) yeast extract was the optimal concentration.
• all inorganic nitrogen sources had no effect on the production of the antibiotic.
• KCl was the best mineral source for the production of the antibiotic.
• the antibiotic production was not considerably affected with MgS0 4 « 7H2 ⁇ , CaCl 2 , NaCl, or K2HPO.1. The others had no effect on the production of the antibiotic.
• the optimum concentration of KCl was 0.2* (w/v).
• the antibiotic production in YS medium was twice in NB medium and the range of pH was within 1.0.
• Figure 8 and 9 represented the size of inhibition zone against
• the activity of the antibiotic of the present invention showed 24mm inhibition zone against Pyricularia oryzae and 21mm inhibition zone against
• Bacillus sp. SY-414 (KCCM-10043) was cultured at 301C for 96 hours in YS medium and then centrifuged at
• the organic layer was collected and removed by rotary evaporator under the reduced pressure.
• the condensed active fraction was collected and dissolved in water and lyophilized.
• the above lyophilized powder was dissolved in methanol and loaded in silica gel column, and eluted with methanol.
• the active fraction was concentrated in vaccuo and dissolved in water and lyophilized.
• the active fraction was dissolved in water and lyophilized.
• soluble starch, mannitol, xylose and glucose could be used as carbon sources and beef extract, yeast extract, peptone and soybean meal could be used as nitrogen sources.
• NaCl, KCl, MgS0 4 and KH2PO4 could be used as mineral sources.
• medium containing 3* soluble starch, 0.8* yeast extract and 0.2* KCl was the optimum for the antibiotic production. The optimal condition was 25-361C for 48-96 hours with shaking.
• Soil collected from Chunchon area was suspended in distilled water, streaked onto nutrient plate (beef extract 0.3*. peptone 0. ⁇ *. agar 1.2*), and incubated at 301C for 1-2 days.
• the above isolated microorganism was inoculated in 10ml nutrient broth and cultured at 30 * C for 76 hours, and then centrifuged at 12,000xg for 20 minutes. The activity of supernatant was measured. With Pyricularia oryzae IFO 30617 and Pellicularia filamentosa IFO 8986 as a test microorganism, Bacillus sp. SY-414 represented the highest activity against the two test microorganisms.
• the isolated microorganism was cultured in 20ml nutrient broth at 30 1C for 72 hours.
• the cell at the stationary phase was used as the seed of the present invention.
• Example 3 Fermentation 3.0* soluble starch, 0.8* yeast extract, and 0.2* KCl. Fermentation was carried out at 30 . for 72 hours with shaking. The culture broth was centrifuged at 12.000xg for 20 minutes. The supernatant was used for the purification.
• the lyophilized powder was dissolved in methanol and silica gel column chromatography was carried out.
• the active fraction was eluted with methanol, and then concentrated in vacuo.
• the concentate was dissolved in water and lyophilized.
• the lyophilized powder was dissolved in dichloromethane : methanol (1:6) mixture and loaded in Sephadex LH-20 column. The solvent was removed and dissolved in water and lyophilized. The lyophilized powder was dissolved in methanol and stored at AX_ . lOOmg of the purified trihexocin was obtained from 201 culture. Figure 10 represented the photograph of the purified crystalline trihexocin.
• Table 9 represented the Rf value of the antibiotic of the present invention.
• the antibiotic of the present invention showed a positive reaction with alkaline potassium permanganate, 10* sulfuric acid and iodine. Thus it was concluded that the antibiotic contained the sugar moiety in structure.
• the antibiotic (lOmg) obtained in example 4 was hydrolyzed at 110 * 0 for 10-12 hours with 0. IN HC1.
• the hydrolysate was analyzed on HPLC to identify the sugar components.
• the antibiotic consisted of fructose only.
• this antibiotic was named as trihexocin.
• the inhibition zones against Pyricularia oryaze varying antibiotic concentration were 9.8mm at ⁇ O ⁇ g/ml, 14.6mm at lOO/zg/ml, 19.5 mm at 200ig/ml, and 23.3mm at 400/ig/ml and, against Pellicularia filamentosa, 12.3mm at lOOjug/ml, 16.2mm at 200ig/ml and 20.2mm at 400i g/ml.
• Antimicrobial spectrum was tested through the following paper disc agar diffusion method against the test microorganisms to observe the antibiotic activity of the present invention.
• test microorganism inoculated in soft agar (agar 0.8*) and overlayed.
• agar 0.8* soft agar
• fungi the spores were suspended homogeneously and 0.1ml suspension was inoculated in PDA (Potato Dextrose Agar) and overlayed.
• the paper disk (lOOjug/ml) was put on plate and incubated at 30 ! for
• Table 11 represented the antimicrobial spectrum of the antibiotic.
• the antibiotic of the present invention did not have an effect on gram positive and negative bacteria, and yeast, but showed an activity against fungi.
• the antibiotic (lOO ⁇ g/ml) was treated with 0.06M sodium citrate-HCl buffer, potassium phosphate buffer, and ammonium hydroxide-ammonium chloride buffer for two hours.
• the antibiotic solution was added into cup and incubated at 30 * 0 for
• the antibiotic was stable at pH 6-8 against Pyricularia ozyzae and
• the antibiotic solution (100/g/ml) was treated for 1 hour at 30 -2 , 401C, ⁇ OlC, 60TC and 70 O.
• the antibiotic solution was added into cup and incubated at 30"C for 48 hours. The size of inhibition zone was measured.
• the activity of the antibiotic against Pyricularia oryzae was stable at 301C-40 ; but represented 80* at 50"C and 60* at 60"C.
• the activity of the antibiotic against Pellicularia filamentosa was stable at 30 * 0 -401C but represented 70* at 50"C.
• the antimicrobial activity of the present invention was observed through the following method.
• Rice plant which had become 3-4 leaves was infected by Pyricularia oryzae or Pellicularia filamentosa, and then treated with the antibiotic. The effect of the antibiotic was observed for 7 and 14 days with comparison to the control.
• the infected rice plant was grown well at the concentration of 50-100 g/ml.
• the antibiotic was prescribed to 5 ICR mice (age: 6-8weeks) for intraperitoneal injection or oral administration. After two weeks, all the tested mice were survived, indicating the survival of 100*. It was proved that this antibiotic had no toxicity.
• the antibiotic of the present invention can be used alone or in a mixture with fillers, conventionally in the field of the present invention.
• the composite as an effective ingredient can be manufactured.

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• 1994
  • 1994-08-03 KR KR1019940019211A patent/KR970010603B1/ko not_active IP Right Cessation
  • 1994-09-15 CN CN94193799A patent/CN1135238A/zh active Pending
  • 1994-09-15 CA CA002171947A patent/CA2171947A1/en not_active Abandoned
  • 1994-09-15 JP JP7509095A patent/JPH09510862A/ja active Pending
  • 1994-09-15 GB GB9605096A patent/GB2296716A/en not_active Withdrawn
  • 1994-09-15 AU AU77090/94A patent/AU7709094A/en not_active Abandoned
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