JPH04169191A - Method for fermentation and purification of novel fungicidal substance krf-001 complex - Google Patents

Abstract

PURPOSE: To obtain the subject new complex useful as a nonpollution antifungal agent in good yield and provide mass-production thereof by culturing a new microorganism belonging to the genus Bacillus under immersion aerobic fermenting conditions.

CONSTITUTION: A microorganism Bacillus subtilis subspecies Krictiensis (ATCC 55079) or Bacillus subtilis subspecies Krictiensis M 18-91 (ATCC 55078) or its active mutant capable of producing an antifungal substance KRF-001 complex is fermented or cultured in a culture medium containing an assimilable carbon source and an assimilable nitrogen source under immersion aerobic fermenting conditions to afford the objective complex. The culture is preferably carried out at 30°C and pH 7.

COPYRIGHT: (C)1992,JPO

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a novel microorganism, Bacillus saftilis subsp.
ssubsp, Kr1ctiensis) This mutant strain was cultivated or fermented to produce a novel antifungal substance, KRF.
The present invention relates to a method for producing a -001 complex and a method for purifying the same.

The inventors of the present invention carried out microbial screening research to search for non-polluting antifungal substances, selected new and useful bacterial strains, and discovered that a series of antifungal substances produced by these strains are novel peptides. (“Novel Bacillus subtilis subspecies and antifungal substance KRF-001 complex produced therefrom” filed on the same day as this application)
(see specification).

The inventors of the present invention named the series of novel peptide antifungal substances KRF-001, and described the fermentation method and the produced KRF-001 for increasing the production yield, which is essential in this industrialization process. The present invention was completed by developing a purification method that can efficiently obtain .

Ultimately, the purpose of the present invention is to use KRF-001 as a novel Bacillus subtilis subsp.
Kr1ctiensis, ATCC55079) and Bacillus subtilis subsp.
8-91 (Bacillus 5ubtilis
5ubsp.

Kr1ctiensis, M 18-91. ATC
To provide a method for maximizing the amount that can be obtained by fermenting KRF-001 from C55078), and a method for separating and purifying the KRF-001 produced therefrom, which can efficiently obtain the maximum amount. be.

[Conventional technology]

In the conventional production process using the G strain that produces antifungal substances, the increase in production yield and the purification of the produced antifungal substances, which are essential in the process of industrialization, have not been effectively carried out.

[Invention or problem to be solved]

Under these circumstances, there has been a need for microbial screening to search for non-polluting antifungal substances and the development of fermentation methods and efficient purification methods to increase the production yield of produced substances.

[Means to solve the problem]

In order to solve the above problems, the inventors of the present invention conducted microbial screening research, selected a new and useful bacterial strain, and confirmed that this strain produces a series of antifungal substances and other novel peptide compounds. (See the specification of "Novel Bacillus subtilis subspecies and antifungal substance KRF-001 complex produced therefrom" filed on the same date as the present application).

The inventors of the present invention named the above-mentioned series of novel peptide antifungal substances KRF-001, and described the fermentation method and the produced KRF-001 for increasing the production yield, which is essential in this industrialization process. The present invention was completed by developing a purification method that can efficiently obtain .

Hereinafter, the present invention will be explained in detail.

The novel Bacillus subtilis strain used in the present invention was isolated from a soil sample collected near Gyeryongsan Gaksa Temple, Chungcheongnam-do, Republic of Korea, and the mutant strain was obtained by a conventional method using ultraviolet rays as detailed in the Examples. , was created. The taxonomic identification of these microorganisms can be found in the literature (Bergey's Manual
of Systematic Bacteriolo
gy.

Vol.2. Williams & Wilkins
Co, (1986)).

The new microorganisms used in the present invention are Bacillus subtilis, which was used as a standard strain.
lis: ATCC 6633), but it is negative for spore location and oxidase reaction, has a slightly low salt tolerance, can utilize lactose, and has a lysocyme concentration of 0.0'01%. The cases of sensitivity were worthy of special mention (see Table 2 below).

For this reason, the novel microorganism used in the present invention is Bacillus subtilis subsp.
s 5ubsp,), and the wild strain was Panrus subtilis subsp.
s 5ubtilis 5ubsp, Kr1cti
The mutant strain was named Panrus subtilis subsp. chrysoccensus Ml 8-91).

These new microorganisms were published on July 26, 1990 by the American Type Cuisine Society.
deposit numbers ATCC55079 and ATCC5, respectively.
It has been deposited as No. 5078.

Tables 1, 2, and 3 below describe the characteristics of the microorganisms used in the present invention.

Table 1 New microorganisms used in the present invention
s5uhsp, Kri:n1ensis) 1: I↓"°"'°"1- Table 2 Comparison of physiological characteristics of the standard strain and the new microorganism used in the present invention −
-I Okintase reaction l --: Hobsoupros □ --1 Kosuo medium
: ] Utilization of propionic acid 1 -
−□ Nidorate reducibility □
10-1, Indole generation
--1 Hydrogen sulfide generation 1
− −: Urase action
1 ′ □ − (Starch degradability −
I casein degradability 1 -
Liquefaction 1
4 -□ 1 Salinity tolerance 1 0 to 109θ
,, 0 to τ011□ 1 Growth at pH57 1 -
, ,.

:1 Arginine decomposition □ −-■ l Tyronolysis Ni-・] Phenyl-alanine amine (N □ Table 3 Comparison of sugar usability between the standard strain and the new microorganism used in the present invention'1-7 Lahinose Ni-'d - Cyrus --・d-g'ji-11" -: d-mannitol 1 + -□: Lactose:, -11 Sarin
1
:□ , i／＞)4 l =
: =□ :d-10s)-1-・-□'d-blacktoss □-10id-trehalose--□1 cellohiose 1-
−: ・Maltose □ −−1 Atonitol 1 − −1 Dalzytl −−■ Sucrose 1 +:
-1 child tuxtorile 1 -
- In the present invention, the novel antifungal substance KRF-001 produced from a novel microorganism etc. refers to a complex of a series of peptide compounds etc. produced by culturing or fermenting the above novel microorganism. do.

That is, KRF-001 has components A, B, and C, respectively.

A complex of a series of peptide compounds named D, E, and F, which is produced by culturing or fermenting the novel microorganism used in the present invention, and then purifying the produced antifungal substance using high-pressure liquid chroma). When analyzed by HPLC, it was separated into the above six types of peptide components. The separated components were subjected to FAB-MS (JEOL product, Model DX
303゜positive 1onization,
argon gas, gun voltage3k
v, emission 30mA)
Molecular weight: 1042.1056.1056.1070 respectively
．． Confirmed as 1070 and 1084.

The Rf value of KRF-001 on thin layer chromatography (TLC) is 075 when only methanol is used as the developing solvent, methanol:acetonitrile (1: ]
), it appears as 0.53, and it can be seen that it is a hydrophilic substance because it dissolves well in water and methanol but does not dissolve well in chloroform and hexane. Furthermore, KRF-00°1 has a pH of 1,0 to 11
．． It is relatively stable at 0, and even after high temperature treatment (100-12 picC)
) It was found to be a stable substance for a considerable period of time (5 hours at 100°C, 15 minutes at 121°C).

KRF-001 showed negative results in the ninhydrin reaction, or was hydrolyzed with 6N hydrochloric acid and the decomposition products were analyzed by GC-M.
As a result of analysis using S, it was confirmed that asparagine, glutamine, proline, serine, and tyrosine were commonly present in each component. Furthermore, according to the elemental analysis results, the above KRF
A in each peptide component from -001 is C+tH2sNO
z, B and C were confirmed to contain the specific amino acids CI 6821 NO□, D and E were C, H22NO, and F was C1□H2sO2.

Each of these was dissolved in 6N HCI and reacted at 116°C for 6 hours using an amino acid automatic analyzer (amin).

acid autoanalyzer, Waters
As a result of analysis, the presence of asparagine, glutamine, serine, proline, tyrosine, etc. was confirmed, and the molar ratio was found to be 3:1:1:1:1.

Therefore, KRF-001 can be seen as an oligopeptide compound complex of 3 moles of asparagine, 1 mole of glutamine, 1 mole of serine, 1 mole of proline, 1 mole of tyrosine, and especially 1 mole of amino acids, and is a complex of chymotrypsin proteolytic agents. Since these oligopeptides are not hydrolyzed by enzymes, they have a circular structure as shown in the general formula below.
As a result of interpreting the structure of the unique amino acid whose structure is unknown above using high-resolution H'-NMR, this structure has an integral value at δ1.25 based on the methylene chain and the 3rd carbon (C-3). The absorption that shows that the proton is coupled with the other four protons is δ4,2
It was found that the structure of the above amino acid was β-amino α.

Furthermore, the terminal structure of the β-amino acid confirmed through the signal of the methyl group appearing at 60.9 of the peptide compounds of each component A, B, C, DSE, and F is that peptide A is a normal type (δ0.9.3H1t). , peptide B is ante
iso type (δ0.9.6H, m), peptide C is i
so type (δ0.9.6H, d), peptide D is
o type (δ0.9.6H, o), peptide E is normal type (δ0.9.3H, t), and peptide F is an
It can be seen that it is a teiso type (δ0.9.6H, m).

Therefore, the β-amino acid structure of each peptide constituting the KRF-001 complex as determined by NHR analysis is as shown in the following general formula.

Component A: CH, (CH2)1. cH(NH2)CH
2COOH component B 2CH2CH2CH(CH2) (C
H2), CH(NH2)CH2COOH component C: component, CH(CH3XCF(2), CH(NH2)CH2
COOH component D: component, CH(CH,)(CH2)
,. CH(NH2)CH2COOH component E: component,
(CH2),,CH(NH2)CH2COOl(component F
: CH, CH2Cl(CH2XCH2),, CH(
NH2)CH2COOH The fermentation and purification process according to the invention was generally carried out as follows.

Detection and quantification of KRF-001 Quantification of KRF-001 was performed using Pyricularia oryzae (P.
yricularia oryzae) as a test bacterium on an agar plate.
) and decided. Purely separated KR is used as a reference material.
Using F-001, the normal production volume is the diameter of the blocking ring (mm
) appeared. For example, after inoculating test bacterial spores onto a botate textulose plate agar medium, place a sterile stainless steel pot on the plate and add the fermentation filtrate (0,
Fermentation liquid filtered with a 22% m sterile Millibo filter)
The diameter of the growth inhibition ring that appeared after culturing for 24 hours to 2 days was measured. KR of fermentation broth and partially purified samples
The presence of F-001 can be determined by thin layer chromatography (TLC).
), confirmed by the fluorescent spots observed by ultraviolet irradiation and the reaction of yellow spots by iodine.

This commercially available (TLC) board was manufactured by Merck Co., Ltd.
l) silica gel 60 F2
54, thickness 0.2 mm), and the developing solvents were mainly chloroform, methanol, aqueous ammonia (15%) and isopropanol (4:3:2:1,
volume ratio) was used. KRF-0 under the above TLC conditions
The Rf value of 01 was 0.57, and the bioautograph using Piricularia oryzae as the test bacteria was performed.
am) to confirm the part showing antifungal activity. When only methanol was used as a developing solvent, Rf was 0.75, and when methanol acetonitrile (1.1) was used, Rf was 0.53.

Measurement of the amount of bacterial cells The absorbance at 550 nm (linear interval between 0 and 0.3) and the amount of bacterial cells were expressed as the weight of dry cells with respect to a constant volume of sample. At this time, the bacterial cells were dried at 105° C. overnight, and then the weight of the dried bacterial cells was measured.

Inoculum culture After storing the inoculum in a liquid state at -80'C, the bacterial cells collected at low temperature in the late logarithmic growth phase were added to fresh LB medium (1% Bacto Tributone, 0.5% Bacto Yeast Extract).
, NaCl 1%, pH 7,0) and further mixed with the same volume of glycerol (final cell concentration A
5. . = 0.01), divide the stocks into 1.0 ml or 0.5 ml sterile vials (Nunc product) and make a stock (stack) of 1°O to 200 at a time, -80 It was used while being stored in a refrigerator at ℃.

The culture medium for the seed culture of the novel microorganism of the present invention to be inoculated into the fermenter is shown in Table 4 below, and the pH value is 7.0 before pressure sterilization.
It was adjusted to

Table 41 Creation of seed culture medium Glucose 10g Bacteau tripton 10g Bacteau yeast extract 5g NaC 110g Distilled water
l 150ml of Riator starter culture medium to 101ml
After sterilizing it in a flask with a baffle at the base of 00O capacity (Bellco product), cool it at room temperature, take out 1 or 2 vials from the refrigerator, thaw it, and add 1 ml of the thawed seed culture. was aseptically inoculated into the above-mentioned seed culture medium and cultured at a temperature of 30° C. for 4 to 6 hours in a rotary shaking incubator (rotation width 2.7 cm, 20 rpm).

The fermenter used for fermentation is an upper rotary fermenter with a total capacity of 14 liters (Marubishi product, MJ-N type).
and a lower rotary fermenter (B) with a total capacity of 18 liters.

Brown, Biostat E type). A typical production medium formulation is as shown in Table 5 below.
Among them, the carbon source, MgSO4, trace elements, etc. were sterilized separately and mixed later. The fermenter was sterilized at 121'C for 40 minutes in the case of the upper rotary fermenter, and the fermenter was sterilized under the same conditions using its own sterilizer in the case of the lower rotary fermenter.

For fermentation, after inoculating 200 ml of inoculum into 10 liters of fermentation liquid, the temperature was 30'C, the pH was 7.0, and the stirring speed was 400.
The conditions were as follows: rpm and air flow rate of 1 vvm. Adjust the pH value during fermentation by pumping 5N HCI and 5N NaOH to within 7±02. If too much foam is generated, use silicone series (silicon 3) as an antifoaming agent.
0%, manufactured by Korean Ginseng Chemical Factory) was diluted 10 times and sterilized under pressure, or rice bran oil was sterilized under pressure and used.

Table 5. Typical KRF-001 Production Medium Sucrose 30, Qg 20.0g Soyton lo, og - yeast extract 5.0g 4
．． 0g(NH4) 2S0.
4. o gK2) (Po,
0.5g 0.5gMg5Oa
0.5g 0.5g Trace element MnCL 4mg 4mg
cact2 5mg 5mg
Fe50.・7H2025mg 25 mg
Distilled water 1 liter liter KRF-001 was produced about 7 to 8 hours after the start of fermentation, and reached its maximum value after about 40 hours, although it differed slightly depending on the medium used. ) In active mutants of Cucillus subtilis subsp. fritiensis, K.
In some cases, RF-001 production reached its highest price. Mostly K
Beneficial conditions for RF-001 production are temperature 30°C and pH 7.
, 0, sufficient oxygen supply, etc., and supply of appropriate types of rare elements. For example, if you add 4■/1 M n C12, the Piricularia oryzae inhibition ring will be 10 to 13.
An increase from 20 to 30 mm was observed.

The production of KRF-001 is greatly influenced by the type of various carbon and nitrogen sources. 1 g of yeast extract, 4 g of (NH4) 2 So as a basal medium to discover a carbon source compatible with the production of KRF-001.

Na2HP0.2g, KH4PO40.3g, Mg5O
= ・7820 0.5g, CaCO52g, Mn
C124mg, Fe5O1'7820 25■, Ca
A medium containing 25 mg of Cl and 10,100 O of distilled water was prepared. After adding 30 g of each carbon source (100 g in the case of molasses), the KRF-001 producing strain was inoculated. The influence of each carbon source on KRF-001 productivity is shown in Table 6 below.
It was described in

Table 6. Production comparison between various carbon sources and KRF-001.
In order to find a nitrogen source suitable for the production of KRF-001, 30 g/l of sucrose was added to the above basal medium, and various nitrogen sources were added to investigate the productivity of KRF-001. It appeared.

Table 7. Comparison of productivity of KRF-001 with various nitrogen sources Yeast extract 30 110 bacterial cell isolation KRF-001 is secreted outside the cells, so once fermentation is complete, cool water (10 to 15°C) is circulated to adjust the temperature of the fermentation solution. The bacterial cells and KRF-001 were separated by centrifugation. Refrigerated centrifuge (Sorvall, RC5
When the bacterial cells were separated by centrifugation using a CG5-3 rotor at a speed of 7000 rpm for 20 minutes at 5°C, the amount of bacterial cells per liter of fermentation liquid was approximately 20 to 60.
g (Achieved a fi wet type weight.

Separation and Concentration of Crude KRF-001 In order to separate and concentrate KRF-001 present in a large amount of supernatant liquid, isoelectric precipitation method was used.
pitatio) and adsorption chromatography methods were used.

1. Isoelectric precipitation method Check the temperature and pH of the supernatant liquid from which the bacterial cells have been removed.
Add HCl or 5NH2S○4 to a specified amount (5 to 20 m1/1) and mix completely by stirring at a speed of 120 to 20 Orpm using an impeller with a diameter of 4 cm, and then adjust the pH. 5°C if it becomes 3.0 to 35
If left alone, the sediment will settle. After 2 to 18 hours have passed, discard the supernatant and place the crotch portion in a centrifuge (Sorval).
l■RC5CG5-3.

(KRF-00) Purity: 3 to 5%).

2. Adsorption chromatography In a column packed with Amberlite XAD7 resin (Rohm & Haas product), KRF in the culture solution is absorbed using hydrophobic action as a binding force.
-001 is adsorbed to resin and KRF-001 is adsorbed.
After washing with distilled water, the fraction was eluted and fractionated with methanol (98%).

The column used at that time had a diameter of 10 cm and a length of 90 mm, was filled with XAD-7 resin to a depth of 45 to 50 ann (approximately 45 liters bedholium) from the bottom, and was operated at room temperature.

Samples were stored refrigerated at 5 to 10°C and used as they were after checking the pH, and adsorption and desorption were performed at a flow rate of 50 ml/
Eluate the sample with methanol by 50 l/l.
After each fraction was divided into 0 ml Erlenmeyer flasks, methanol was evaporated from each fraction, diluted again with distilled water, and freeze-dried (KRF-001 purity: 20 to 30%).

Silica gel chromatography silica gel (US Merck product 7734.300g)
After mixing with chloroform, the inner diameter is 4 cm and the length is 50 an.
Fill the column, and in XAD column chromatography, dissolve the obtained separated and purified sample in methanol and put it in the column, fractionate it while flowing methanol to 1 m every 5 seconds, and collect the part that shows antifungal activity. , dried under reduced pressure, dissolved in distilled water, and freeze-dried to obtain a feather-like substance.

Sephatex LH-20 Chromatography Sephatex L I-I-20 (Pharmacia product,
30g) in methanol to remove air under reduced pressure,
After sufficiently inflating, it was filled into a column with an inner diameter of 2 cm and a length of 60 cm. Dissolve the sample in methanol and Sephatex L
It was applied to an H-20 column and fractionated using an elution solvent.

After confirming the antifungal activity of each of the obtained fractions, they were dried under reduced pressure, dissolved in distilled water, and then freeze-dried to obtain a substance with a pale yellow feather pattern.

High pressure liquid chromatography (HPLC) The active sample obtained in Sephadex LH-20 column chromatography was subjected to high pressure liquid chromatography (HPLC).
It was purified with The column used at this time was Rs i IC
+8 (Altech, lOum, 1.OX250mm
), and the sample was dissolved in as little methanol as possible and injected into the column. The sample was eluted using 80% methanol at a flow rate of 5 ml/min.
Monitored by ultraviolet absorption at nm. HPLC
The latter active fraction could be determined from the absorption peak at 220 nm and the results of biological assay.

To summarize the fermentation conditions for KRF-001, the temperature is 30℃p.
H7.0, dissolved oxygen concentration 1O to 50%, the production strain reached its highest value KRF-001 within 20 to 35 hours after the start of fermentation.
The production yield was expressed (in the case of the wild strain, the production yield was 0.2 g/l). If there was too much phosphate in the fermentation medium, the production yield would drop sharply, and although the supply of complex carbon and nitrogen sources was effective for cell growth, it was not very helpful for KRF-001 production.

Simple separation methods include isoelectric precipitation and adsorption chromatography as mentioned above.
It was more effective to pass it through AD-7 chromatography, and after that, it was possible to separate and purify it using silica gel chromatography, Sephatex LM-20 chromatography, HPLC, etc.

The present invention will be explained in more detail below with reference to Examples.

〔Example〕

Example 1 Culture and fermentation of Bacillus subtilis subsp. fritchiensis Bacillus subtilis subsp.
s 5ubtilis 5ubsp, Kr1cti
ensis.

ATCC55079) After dissolving one vial (1 ml) and inoculating it into 100 ml of Glucos LB medium, at 30°
Shaking culture was performed at C1 pH 7.0. Glucos LB medium contains 10 g of Glucos, 10 g of packed triptone, 5 g of packed yeast extract, 10 g of salt, and 1000 g of distilled water.
1+11, and the pH was adjusted to 7.0.

When the growth of the above-mentioned inoculum reached As5o 0.4 0.5, the whole culture solution was aseptically transferred to a 14 liter fermenter containing 8 liters of production medium.

The temperature, air communication speed, and stirring speed in the fermentation tank were each 30°C 0. Fermentation was carried out while maintaining 5VVM and 400rprn, and after 2 days, the fermentation solution was centrifuged to remove microbial cells, the supernatant was passed through an XAD-7 column, and the adsorbed KRF-001 was eluted with methanol. 12 g of yellowish brown crude KRF-001 (purity 25%) was obtained. The production medium is 30g of sucrose and 10g of soyton;
Yeast extract 5 g, K2 HP 040.5 g,
MgSO4”7H200.5g, MnClx 4mg,
CaCL5■, Fe5CL.7H2025+ng were dissolved in distilled water at 10,100O, the pH was adjusted to 7, and the mixture was sterilized at 121"C for 40 minutes before preparation.

Example 2 Bacillus subtilis subsp.
llus 5ubtilis 5ubsp, Kr1ct
19 at 30°C and pH 7.0 after inoculating the mutant derivative M18-91 into sterile Glucose LB medium.
Shaking culture was performed for 22 hours. The growth of the inoculum is A3
50=1.5 to 2. It was 0. Inoculum culture solution approx. 20
00 ml was transferred using aseptic techniques to a 100 liter fermenter containing 70 liters of production medium. Fermentation temperature is 30
C., an air flow rate of 1 to 2 VVM, and a stirring rate of 200 to 25 Orpm.The pH was not adjusted, and if foaming occurred, an antifoaming agent was added. Production medium is sucrose 3
0g, soybean or soy bean mill 10g, yeast extract 5g 1 to 2 HP Oa O, 5g SMg S 04
・7H100.5g, MnCL 4mg, CaCI
z5mg, Fe50.・7H2025mg, distilled water 1
It was prepared at a ratio of 0.0100O. Furthermore, an experiment was also conducted in which the soyton and soy bean mill in the above production medium was replaced with 20 g/l of Pamamidia.

The production strain M1B-91 used in the 100 liter fermentor had the characteristic of producing KRF-ooi within a short period of time as one of the mutant derivatives. Since the fermentation progressed much faster than the production strain used in Example 1, about 60 g of KRF-001 (purity 25%) was obtained after culturing for about 17 hours.

When Pamamidia was used as a nitrogen source instead of soyton, the production amount of KRF-001 was further increased, and 150 g of crude KRF-001 (purity 25%) was obtained after culturing for about 17 hours.

Example 3 Purification of KRF-001 Using 16 liters of the fermentation liquid obtained in Example 1, KRF-001 was purified.
An attempt was made to purify RF-001. First, add about 4 liters of enzyme solution to 4 liters of Amberly 1-
ite) XAD-7 column (Rohm & H, USA)
ASS product, 9X90cm) 40m/min after adsorption
Distilled water (8 liters) twice the bed volume at a flow rate of 1
After washing impurities with
KRF-001 was eluted. The eluted fractions were treated with Viricularia oryzae (Pyric).
After confirming the active substance using M. ularia oryzae as a test bacterium, the active substance was concentrated under reduced pressure and freeze-dried to obtain a yellow-brown active substance.

The above method was repeated four times to obtain about 15 g of yellowish brown crude KRF-001 from 16 liters of fermentation liquid. The next step of purification was performed by silica gel chromatography.

300g of silica gel (MerCk product 7734, USA)
) was filled in a column (4×50 cm) using chloroform, and then the crude KRF-001 was dissolved and adsorbed in a minimum amount of methanol-chloroform (3:7) solution. KRF-001 was eluted using methanol-chloroform (
3: 7) 750ml and (1: 1) 2100ml
I washed it away. The portion containing the active ingredient was freeze-dried to obtain approximately 7 g of crude KRF-001.

The next step of purification was attempted using a separation TLC plate. The developing solvent was butanol-ethyl acetate-water (6
: 1 : 1) was used to recover Rfo, 36 substances, resulting in the recovery of 5 g of active substance. The next step is to apply Sephatex (Sephatex) to 5g of the above active substance over the second layer.
adex) L H-20 column (0.8x90an)
and purified. A sample was dissolved in a small amount of methanol and adsorbed onto the column, and methanol was used as an elution solvent to flow at a flow rate of 0.2 ml per minute. As a result of freeze-drying the active substance in the obtained fraction, 1 g of white active substance was obtained.

As a final purification step, each component of KRF-001 was separated using HPLC (Perkin-Elmer Co, Motel). As an HPLC column, R51l C (10 μl OX
Using a 10.times.250 tube, 1 g of sample was dissolved in 35% acetonitrile and injected at a concentration of 10.times./[111.

Sample elution was performed with 35% acetonitrile for the first 30 minutes and then with 50% acetonitrile for up to 50 minutes, with a flow rate of 4 ml per minute and monitoring by absorption at UV 225 nm. Biological activity search results for absorption peaks, etc. Rt22.8'33.8', 38.7', 45.5
The peaks at ', 46.5', and 49°8' indicate activity, and these are associated with components A, B, and B of KRF-001, respectively.
They were named CSD, E and F. The components of pure KRF-001 finally obtained are component A210, component 83,
0mg, component C 90mg, component D 30■, component E
30■ and component F10■. KR wants to do it
It can be seen that F-001 is a complex of at least six or more antifungal components.

〔Effect of the invention〕

As described above, it has become possible to mass-produce a novel KRF-001 complex having antifungal activity based on the fermentation method of the present invention, and furthermore, the produced KRF-00
1 can now be effectively refined.

Claims (1)

[Scope of Claims] (1) In a culture medium containing an assimilable carbon source and a nitrogen source under immersion aerobic fermentation conditions, the antifungal substance KR
The microorganism that produces the F-001 complex, Bacillus subtilis subsp.
llis subsp. Krictiensis. A.T.
CC55079) or Bacillus subtilis subsp.
ilis subsp. Krictiensis M1
8-91, ATCC 55078) or their active mutant strains are fermented and cultured until a recoverable amount of the above antibiotic is obtained. (2) In paragraph 1, the method of culturing without adjusting the acidity (pH) of the culture solution during culturing. (3) The method according to item 1, wherein the culture medium contains MnCl_2. (4) Directly X the fermentation solution of KRF-001 based on paragraph 1
A method for purifying a KRF-001 complex, which comprises passing it through an AD-7 or activated carbon column and eluting it with an organic solvent.