CN1130210A - Method for preparing cyclocyto polypeptide A from Branched layer wall bacterium - Google Patents

Abstract

The invention relates to a process for the preparation of cyclosporin A from Tolypocladium sp comprising in the step of preparing a fermented medium of said Tolypocladium sp. The fungal biomass is extracted of said fermented medium to obtain a methanol extract, which methanol is removed by evaporation to obtain a first residue. An ethyl acetate extract is prepared from the aqueous solution of said first residue, which is decolourized and concentrated to obtain a second residue, the second residue is subjected to a step of purification.

Description

The method for preparing Cyclosporin A from branched layer wall bacterium

The present invention relates to from branched layer wall bacterium as described below (Tolypocladium sp.), prepare the method for Cyclosporin A.

People know usually, Cyclosporin A as prevent in the organ transfer operation organ rejection-kind of immunosuppressor has useful purposes.According to currently known methods, cyclocyto polypeptide can be prepared into mixture by the cultivation to distortion branched layer wall bacterium (Tolypocladium varium) fungal bacterial strain.No. 2227489 Patent publish of Britain a kind of microbial process of production cyclocyto polypeptide mixture or its component (being Cyclosporin A, cyclocyto polypeptide B and cyclocyto polypeptide C).This currently known methods suggestion is cultivated distortion branched layer wall bacterium (Tolypocladium varium) fungal bacterial strain and is produced the cyclocyto polypeptide mixture on a nutritional medium.As used in fermentation broth usually, this nutritional medium contains carbon source, organic and inorganic nitrogen-sourced and inorganic salt.Fermentation is as carrying out under aerobic conditions usually, and temperature is 25～30 ℃.If required, the cyclocyto polypeptide mixture that is generated is separated, and purified and generation purifying mixture.

This currently known methods has been advised a kind of preferred fermention medium, and it contains peptone, ammonium sulfate and Tryptones as nitrogenous source.Preferred carbon source is glucose, maltose and Sorbitol Powder.The culture distortion branched layer wall bacterium Sp nov Cy/93 number of depositing is NCAlM (P) F-001005.Currently known methods is not mentioned and is only produced Cyclosporin A and its purity and be not less than 98% method.

The objective of the invention is to propose a kind of improvement method for preparing Cyclosporin A.

Another object of the present invention is to propose a kind of method for preparing the high yield Cyclosporin A.

A further object of the invention is to propose a kind of preparation to have the method for basic homogeneous purity Cyclosporin A.

The present invention has used a certain bacterial classification of a new fungi branched layer wall bacterium (Tolypocladium), and its preserving number is NRRL 18950.As shown in table 1 with the characteristic of other bacterial classification of branched layer wall bacterium that used bacterial classification is relevant among the present invention.

Table 1

Branched layer wall bacterium different strain characteristic relatively

Characteristic	???T.inflatum	????T.Cylindrosporum	???T.geodus	??Tolypocladium?sp.
					Growth bacterium colony Hypac-size conidiophore bottle stalk load spore stigma	Slowly white, the hairy size of cushion: not clear 1-1.5-2 μ m is short and small, bottle stalk tool squamous basis, side reach, and end has thick swirls not clear thread	Slowly white and cream-coloured, cushion is hairy, the colourless size in the bacterium colony back side: 15-20mm 1-1.5-3 μ m; Cell 2.5-2.7 μ m is long thin, thread with T.inflatum size: 3-5 * 2-2.8 μ m, crooked sometimes size: 1.5-2 * 0.3-0.5 μ m	Slow white, the hairy size of cushion: not clear 1-1.5-2 μ m is not clear thread with T.inflatum	Normal white is extremely cream-coloured, cushion is hairy, and the bacterium colony back side is the beige size: 20-30mm 2-6 μ m; Cell 6-30 μ m length is little, cylindric bottle stalk tool squamous basis, side reach, and end has swirls size: 4-3 * 2-4 μ m thin, thread, sometimes crooked size: 2-4 * 0.6-1.2 μ m

Table 1 (continuing)

	????T.inflatum	??????T.Cylindrosporum	???T.geodus	The Tolypocladium kind
					Conidium	Transparent smooth unicellular	Transparent, smooth, unicellular; Single and 20 or more mostly be group and assemble, usually spherical, closely parallel, cylindric sometimes, the circle slightly crooked size of end sometimes: 4-5.8 * 1.2-1.6 μ m	Transparent smooth unicellular	Transparent, smooth, unicellular sphere is to round shape circle slightly bending mitogenetic separately (2-4 * 1.2 μ m) and side and do not hold the mucus head to assemble (quantity is 2-10, and size is 2-6 * 1-3 μ m) mutually of end sometimes

According to the present invention, a kind of method of preparation Cyclosporin A from following branched layer wall bacterium bacterial classification can be provided, it comprises: the fermentation and obtain a kind of fermention medium in-nutritional medium of above-mentioned branched layer wall bacterium bacterial classification, from above-mentioned fermention medium, extract fungal biomass and obtain a methanol extract liquid with methyl alcohol, from said extracted liquid, remove methyl alcohol by evaporation step and obtain first kind of resistates, from this resistates, prepare the aqueous solution, from this aqueous solution, prepare acetic acid ethyl acetate extract again, this extracting liquid decoloration is obtained second kind of resistates with concentrating, in two steps it being carried out chromatography then purifies, be that the first step is to be solid phase with the silicagel column, and with hexane, chloroform and methanol mixed solvent are mobile phase; Second step was a solid phase with the resin column then, was mobile phase with methyl alcohol.

This method details will talk out subsequently.Comprising two class fermentation techniques, i.e. (i) static fermentation and (ii) solid-state fermenting substrate.

According to the present invention, a small amount of soil is suspended in sterilized water, use the distilled water high dilution then.The sub-fraction of the soil suspension that institute's alkene is released is sprinkling upon on the substratum.

The pH value of substratum can maintain 6.5, in close dextrose, peptone, agar and water.In substratum, add an amount of reagent (Vetstrep) to prevent bacterial growth.

Then, the soil suspension of above-mentioned preparation is sprinkling upon on the solid surface of above-mentioned substratum, gets the suitable containers splendid attire, as Petri dish.

Under about 22-30 ℃ temperature, culture dish is cultivated.Can notice after 7 days that a large amount of fungal colonies generate.When thinking that the growth of fungal colony reaches satisfaction, just end its growth.

Single fungal colony is moved on in the test tube that contains the same medium component on a small quantity, the test tube mouth.Seal and store 25 ℃ of conditions.

Bacterium colony is remained in microscopically to be checked and identifies to the genus level.One of them bacterium colony has the characteristic of branched layer wall bacterium fungi, finds as shown in table 1.This branched layer wall bacterium bacterial classification among the present invention is grown on-nutritional medium, and this substratum contains glucose, peptone, caseinic acid hydrolyzate and sterilized water, and PH remains between the 4-6;-this bacterial classification spore suspension of spoon is inoculated on the agar slant culture-medium, and slant medium contains wort, yeast extract paste and agar, and pH value is 4-6, cultivates agar slant culture-medium and reaches operational degree up to culture.

When growth when enough, culture transferred to carry out freeze-drying in the glass ampoule and handle.

Utilizing solid phase attitude fermentative action to obtain under the situation of Cyclosporin A, the main seed that as above obtains is inoculated into-liquid nutrient medium in.Through after enough growths, culture is transferred in the solid substrate fermentation tray, contain aseptic wheat wheat husband/rice wheat husband in the dish, pH value is controlled between the 4-6.Relative humidity is maintained about 85-90%, and temperature condition is 25-30 ℃ and ventilation, begins to occur fungi growth on solid substrate.Fungal growth is extracted and evaporation process and obtain first kind of resistates with methyl alcohol together with nutritional medium, this resistates is dissolved in the sterilized water and obtains-aqueous solution.The acetic acid ethyl acetate extract of this aqueous solution decolouring and concentrate and obtain second kind of resistates.Second kind of resistates carried out the two-step chromatography method handle, the first step silicagel column, second step was resin column.In the first step with hexane, chloroform and methanol mixed solvent (ratio is 10: 9: 1) as mobile phase.Use methyl alcohol as mobile phase in the second step chromatography.So in this two steps chromatography, obtained highly purified Cyclosporin A.

In the static fermentation method, main seed divide the two-stage be inoculated into-liquid nutrient medium in and form the kind bacterium of this bacterial classification.This liquid nutrient medium contains glucose, peptone, caseinic acid hydrolyzate and sterilized water, cultivates 3-4 days, and this is the fs.Cultivated 2-3 days down at 25 ℃, this is a subordinate phase, and resulting kind of bacterium transferred in the static fermentation substratum, and medium component is glucose, glycerine, caseinic acid hydrolyzate, wort, peptone, PL-α aminobutyric acid and sterilized water, pH value is 4～6, and ferments 21 days.Above-mentioned substratum specifically consists of 2～6% glucose, 2～5.5% glycerine, 1.5～5.5% caseinic acid hydrolyzate, 0.5～4.5% wort, 0.25～2.5% peptone, 0.1～3% DL-α aminobutyric acid, and all the other are sterilized water.

For instance, substratum contains 4% glucose, 4% glycerine, 3% caseinic acid hydrolyzate, 2% wort, 1% peptone, 0.5% DL-α aminobutyric acid.

If medium component exceeds above-mentioned scope, the output of Cyclosporin A is just very low.

The Tolypocladium fungal biomass that to grow on above-mentioned static fermentation substratum filters, and the fungal biomass that reclaims is thus extracted in organic solvent (as methyl alcohol).Methyl alcohol is evaporated from this methanol extract liquid and obtain first kind of resistates, this resistates is dissolved in the distilled water and obtains-aqueous solution; This aqueous solution is extracted and get-acetic acid ethyl acetate extract with ethyl acetate.This extracting solution is obtained second kind of resistates with sodium solution flushing and evaporation, this resistates is carried out two step column chromatography for separation recited above.

Two kinds of column chromatographies can both obtain the Cyclosporin A of 98% purity.

In the flow process diagram below, we have explained the canonical process that utilizes the static fermentation step to prepare Cyclosporin A.

Table 2

Utilize the output of Cyclosporin A of the inventive method of static fermentation

Test	Cyclosporin A output (substratum mg/litre)	Purity %
			The pure product form of crude product form
	??1. ??2. ??3. ??4. ??5. ??6.			??1519.6??????1132.0 ??1798.0??????1268.8 ??1889.0??????1226.4 ??1749.8??????1280.0 ??1801.4??????1238.0 ??1887.0??????1394.0	????98.2 ????98.5 ????99.6 ???100.0 ????99.9 ????99.9
		??1774.1??????1256.3	????99.4

It is as follows to utilize solid-state fermenting substrate (SSF) to prepare the method for Cyclosporin A.(10 batches mean values)

The separation source and the cyclocyto polypeptide output thereof of different branched layer wall bacterium kinds

Bacterial classification	The source	Cyclocyto polypeptide (mg/litre)	Bibliography
				Branched layer wall bacterium t bacteria .inflatum UAMH Acc.No. 2,472 2,880 4,002 4,553 4,594 4,740 4,828 4900	The high product fertile soil of the organic granular scrubbed in the native alpine meadow of soil muskec under soil/mulch cover mulch-covering habitat Lu Sheng invertebrate Acarasiales sporangium Glyptostrobus P.contorta soil pupa surface (Mycobbates certain)	Not clear 101 ± 61 34 ± 41 1223 ± 75 32 ± 27 45 ± 38 12 ± 11 60 ± 35 15 ± 8	Gams.Persoonia) 1971) 6:185-191 Isaac et al. Antimicrob.Agents Chemother (1990) 34:121-127. is the same the same the same

(continuing)

Bacterial classification	The source	Cyclocyto polypeptide (mg/litre)	Bibliography
				The used branched layer wall bacterium bacterial classification (Tolypocldium sp.) of static fermentation among 4901 Atcc, 34921 NRRL, 8044 T.Cylindrosporum the present invention	The young mosquito mosquito breeding habitat soil of the not clear Aedes kind of moist organic substance	24 ± 12 130 and 170 101 not clear 2000 ± 100	The same Lee ﹠ Agathos; Aopl.Microbiol; Biotechnol (1991) 34:513-517. Von Wartburg ﹠ Traber Proc.Allercv (1986) 38:28-45. Soares et.al.Proc. Pao.Annu.Conf.Calif. Mosq.Vector Control Assoc. (1979) 47:51-54 and Weiser and Pillai, the unexposed data of Entomophaga (1981) 26:357-361

Claims (17)

1) a kind of method for preparing Cyclosporin A from branched layer wall bacterium certain (Tolypocladium sp.), it comprises: the fermentation and obtaining-fermention medium in-nutritional medium of above-mentioned branched layer wall bacterium, obtain-methanol extract liquid with the fungal biomass in the above-mentioned fermention medium of methanol extraction, from said extracted liquid, remove methyl alcohol by evaporation step and obtain first kind of resistates, the aqueous solution for preparing above-mentioned first kind of resistates, the acetic acid ethyl acetate extract for preparing this aqueous solution, with said extracted liquid decolouring and concentrate and obtain second kind of resistates, divided for two steps it was carried out purification by chromatography then, be that the first step is solid phase with the silicagel column, with hexane, chloroform and methanol mixed solvent are mobile phase, and second step was solid phase and be mobile phase with methyl alcohol with the resin column.

2) according to the process of claim 1 wherein that fermentation step carries out with static fermentation.

3) according to the process of claim 1 wherein that fermentation step carries out with the solid substrate fermentation.

4) according to the process of claim 1 wherein that the above-mentioned branched layer wall bacterium bacterial classification that is inoculated in the nutritional medium is main seed.

5) according to the method for claim 4, contain above-mentioned branched layer wall bacterium bacterial classification suspension spore in the substratum, medium component is glucose, peptone, caseinic acid hydrolyzate and sterilized water, pH value maintains between the 4-6,-spoon this bacterial classification spore suspension be inoculated in the agar slant culture-medium, this slant medium contains wort, yeast extract paste and agar, pH value is 4-6, agar slant culture-medium cultivated in culture, form spore, obtain main seed like this.

6) according to the method for claim 1, the step that it comprises the kind bacterium that forms this bacterial classification has two kinds of stages at least and be inoculated in the fermention medium.

7) according to the method for claim 6, wherein above-mentioned fs step comprises main seed is transferred in the substratum that this substratum contains glucose, peptone, caseinic acid hydrolyzate and sterilized water, and pH value is 4～6, grows 3-4 days.

8) according to the method for claim 6, wherein above-mentioned subordinate phase step comprise from the culture of fs transfer to-substratum in, this substratum contains glucose, peptone, caseinic acid hydrolyzate and sterilized water, pH value is 4-6, grows 2-3 days.

9) according to the method for claim 5, wherein culturing step carried out 10-14 days.

10) according to the method for claim 1 and 2, wherein nutritional medium contains glucose, glycerine, caseinic acid hydrolyzate, wort, peptone and DL-α aminobutyric acid and sterilized water, pH value are 4-6.

11) according to the method for claim 10, wherein above-mentioned substratum contain peptone, the 0.1-3% of wort, the 0.25-2.5% of caseinic acid hydrolyzate, the 0.5-4.5% of glycerine, the 1.5-5.5% of glucose, the 2-5.5% of 2-6% DL-α aminobutyric acid, all the other are sterilized water.

12) wash the step of decolouring with water behind the sodium hydrogen carbonate solution acetic acid ethyl acetate extract earlier according to the process of claim 1 wherein.

13) basis the process of claim 1 wherein that the methanol extract liquid of above-mentioned biomass is to shake biomass with methyl alcohol to make, and the methanol extract liquid that obtains is thus carried out rapid evaporation handle and obtain a pasty state resistates.

14) according to the method for claim 13, wherein the methyl alcohol resistates is dissolved in the distilled water and obtains-aqueous solution, obtain with ethyl acetate extraction then-ethyl acetate extracting section liquid, this is handled with sodium bicarbonate and the water flushing.

15), obtain the enriched material of ethyl acetate part by rapid evaporation according to the method for claim 14.

16) according to the process of claim 1 wherein that above-mentioned mixed solvent is made up of hexane, chloroform and methyl alcohol, its ratio is 10: 9: 1.

17) by Cyclosporin A with the preparation of claim 1 method.