CN1106817A - Production and seperation of avermectin - Google Patents

Abstract

With the existence of a solvent miscible with water, the streptomyces avermitilis strain fermentation liquid is homogenized, a polyacrylate and phenol polycondensation copolymer resin is adopted to absorb the avermitilis, the water or the solvent miscible with water mixture is used for the gradient elution and an A<1a>, B<1a> and B<1b> avermitilis are isolated from the fermentation liquid. The fermentation liquid can be obtained from a novel streptomyces avermitilis strain of VKM AC-1475D.

Description

Production and seperation of avermectin

The present invention relates to avermectin (avermectines) produces and isolating method.More particularly, the present invention relates to A _1a, B _1aAnd B _1bClass avermectin, especially B _1aThe separation of class avermectin.Avermectin is one group of bacterium metabolite, and as described in the German Patent 2717040, they are to be obtained by the strain fermentation that produces these compounds.Also relate to have the parasitocidal activity ability, the macrolide of (I) (as shown in Figure 1) that meet structure, and these different separately compositions are:

Avermectin A _1aR ¹=C ₂H ₅R ²=OCH ₃

B _1bR ¹=CH ₃R ²=OCH ₃

B _1aR ¹=C ₂H ₅R ²=OH

B _1bR ¹=CH ₃R ²=OH

People know, for the separation of bacterial metabolite, might use the sorbent material of synthetic highly cross-linked resin and so on, described in patent US4399274.According to the technology of using in this patent, handle mycelium extract with cross-linking copolymer.

Finding now, is not from extract, but the plastic resin treatment with two kinds of particular types compares the separate easily avermectin from whole fermented liquid:

A) polyacrylic ester, it is different with the resin that US4399274 describes, and is not multipolymer;

B) phenol-aldehyde condensation polymerization copolymer, it is different with the resin that US4399274 describes, and this resin does not have functional group, and described multipolymer has weakly alkaline official energy.

Also find, when using in above-mentioned two resinoids resinoid, might guide into and preferentially produce A _1aAnd B _1bThe separation method of class avermectin.

Therefore, content of the present invention is the separation method of avermectin, it is characterized in that: with the miscible solvent of water in the presence of, the whole fermentation that the Streptomyces avermitilis nutrient solution that produces avermectin obtains is liquefied, with the homogenizing thing that the plastic resin treatment that is selected from polyacrylic ester and phenol-aldehyde condensation polymerization copolymer obtains like this, utilize the avermectin of water/will be adsorbed on the resin with the miscible solvent mixture gradient of water free.

After fermentation ends, whole fermented liquid is mixed with the organic solvent miscible with water (for example acetone, methyl alcohol, Virahol) of proper volume.Said mixture is poured homogenizer into again so that destroy cell and help extracting avermectin.The material that homogenizes is equipped with polyacrylic ester (as Amberlite without filtering or the direct adverse current of centrifugal treating is packed in the post in the post XAD-8 or Diaion

HP) or phenol-aldehyde condensation polymerization copolymer matrix resin.Its resin water/solvent mixture gradient washing.Adopt water/with the avermectin that the miscible solvent mixture gradient elution of water has adsorbed, this solvent needs not to be the same solvent of using in the absorption.

When using suitable gradient, might separate many avermectins.

Separation method of the present invention can be used for the fermented liquid that the bacterial strain by any generation avermectin obtains.The fermented liquid particularly advantageous that obtains by new microbe streptomyces avermitilis, this new microbe sample is deposited with Russian Academy Of Sciences-microbial biochemistry and the (Puschchino of Physiologic Studies institute, the area, Moscow) culture is forever preserved, and its registration number is VKM AC-1475D.Bacterial strain VKM AC-1475D be the present invention on the other hand.Bacterial strain mixture in this generation has the cell of A Fumai bacterium by fermentation, wherein main ingredient is B _1aAnd A _1aAvermectin, the former percentage ratio are 18-26%, and the latter is 10-15%.

Morphological feature and the culture feature of Streptomyces avermitilis VKM AC-1475D are as follows: spore produces thing and forms the ash-brown bacterium colony with outstanding circular cone centre portions, usually at the topped developing medium in whole surface.Around bacterium colony a white edge is arranged, other bacterium colonies seldom occur.

On organic agar, Pridham-Gottlieb agar, glycerine-nitrate agar, inorganic agar, glucose-l-asparagine agar, oatmeal agar, glucose-nitrate agar, trophicity agar, observed the spore generation.

Organic agar

(*) SM: the vegetal culture of brown-black (*) SM=

AM: a small amount of grey AM=aerial mycelium

SP: but brown-black SP=lysochrome

Pridham-Sottlieb agar

(*) AM: brownish grey

Glycerine-nitrate agar

(*) AM: few, white

SM: ochre is orange

SP: insignificant, or pale yellow

Inorganic agar

(*) AM: few, white

SM: colourless or by pale yellow to tawny

SP: insignificant.

Glucose-l-asparagine agar

(*) AM: few, white

SM: few, colourless

SP: do not have (insignificant)

Oatmeal agar

(*) AM: grey, adularescent spot sometimes

SM: lark, taupe gray

SP: do not have

Glucose-nitrate agar

(*) AM: taupe gray

Trophicity agar

(*) AM: be mixed with the taupe gray of white

SM: chocolate

SP: brown

Observe behind 28 ℃ of fortnights, all medium pH are near neutral.

Aforesaid colour-change and description that known bacterial strain is done are carefully relatively demonstrated tangible difference, and this shows that this microorganism should classify as Streptomyces avermitilis novel bacterial.

Following embodiment has illustrated recovery avermectin A _1a, B _1a, B _2aNovel method.These embodiment are illustrative, rather than invention is limited.

Preparation

Streptomyces avermitilis VKM AC-1475D fermented liquid.

Use Streptomyces avermitilis VKM AC-1475D to obtain avermectin.Bacterium is kept on glucose-potato agar slant, in+4 ℃ of test tubes in case the test VITAMIN.At least once should inoculate on fresh culture (terrain) with same composition in every month, culture to be preserved should be cultivated 7 days at 28 ℃.

1. bacterium is placed agar to cultivate and cultivates on the inclined-plane and preserve with following composition:

Glucose 10g/L(grams per liter)

Agar 15g/L

Murphy juice is up to 1L

In order to prepare murphy juice, the 300g potato is placed on boiled in the 700ml water 30 minutes.With its solution centrifugal (6000rpm(rev/min), 20 minutes) so that preparation precipitation juice.In order to prepare described substratum, glucose and agar are added in the murphy juice, adding murphy juice again after volume reaches 1 liter, then in Sterilizers in 110 ℃, 4.9 * 10 ^-2Millibar sterilization 30 minutes.Under the suitable occasion of sterilization, substratum is forwarded in the test tube with the test VITAMIN, test tube is 15ml, and its test tube is remained on obliquity, cools down fully up to the agar compost.In order to obtain inoculum, bacterial strain VKM AC-1475D is inoculated new glucose-potato agar slant (loudspeaker mouth shape agar).Its culture should be in 28 ℃ of cultivations 5 days in test tube, with the test VITAMIN (volume of test tube: 35ml).

2. in flask, stir the preparation inoculum

In flask, cultivate inoculum 2.1 utilize the nutritional medium of following composition:

Lactose 20g/L

Soyflour 15g/L

Yeast autolysate 5g/L

The maximum 1L of distilled water

With 10%NaOH pH is transferred to 7.0-7.2.

In order to prepare substratum soyflour and yeast autolysate are put in the distilled water.Firmly mix its mixture, pH is transferred to 7.0-7.2 and is transferred to 1 liter with distilled water with NaON solution.Afterwards liquid nutrient medium is divided in the amount that reaches 100ml in the flask, again in 120 ℃ of Sterilizerss in 9.8 * 10 ^-2Millibar is 1 hour (flask volume 750ml) of sterilization down.

Glucose is dissolved in distilled water, in the collector of packing into, its volume is transferred to 7 liters, with this content in 110 ℃ of Sterilizerss in 4.9 * 10 ^-2Millibar sterilization down reaches 30 minutes.Maize extract is placed another container, in 110 ℃ of Sterilizerss in 4.9 * 10 ^-2Millibar sterilization down reaches 30 minutes.Soyflour is dissolved in the cold water, pours fermentor tank into, when agitator moves, be warmed up to 80 ℃, reach 30 minutes 80 ℃ of maintenances, allowing it be cooled to temperature then is 30 ℃.After other component of packing into, except that glucose and maize extract, cumulative volume is transferred to 53 liters, extract sample, measure pH and also put into the pH determinator, with 10%NaOH solution the pH of liquid nutrient medium is transferred to 7.0-7.2.At 120 ℃ of pressure 9.8 * 10 of temperature ^-2Make this medium sterilization reach 1 hour under the millibar, the substratum in the fermentor tank is cooled to after temperature 29-30 ℃, the sterilized solution with glucose and corn extracting solution under aseptic pumps in the substratum, extracts the substratum sample and does biochemistry and microbiological research.25% lactose distilled water solution in addition in 110 ℃ of Sterilizerss in 4.9 * 10 ^-2Millibar was sterilized 30 minutes down.Before implementing inoculation, under aseptic condition, the 8ml lactose solution is added in each flask that liquid nutrient medium has been housed.In order to inoculate the substratum in the flask, get the nutrient agar that sub-fraction (small pieces) has nutrient solution, it be with test tube take out with the test VITAMIN, this operates under aseptic condition.

2.2 inoculum is grown in the flask that stirs

Streptomyces avermitilis VKM AC-1475D inoculum was grown 20-24 hour in 28 ℃ in the 190-200rpm shaking table.The inoculum of cultivating in shaking bottle should meet the following conditions:

1) free of contamination microorganism;

2) inoculum should be stable liquid, and is dark chestnut look;

3) pH should be 6.9-7.2;

4) microscopical appearance: seldom the actinomycetes mycelia with protuberance assembles.

3. the cultivation of Streptomyces avermitilis VKM AC-1475D in fermentation container

3.1 the preparation of fermentation container

Before filling material, fermentation container should strict clean, and with the suds scrutiny at pressure 14.7 * 10 ^-2Resistance to air loss under the millibar.Ready fermentation container to be used is equipped with pH, O ₂Surveying instrument with temperature.

3.2 the nutritional medium of preparation sterilization in fermentation container

For the cultivation of Streptomyces avermitilis VKM AC-1475D, use nutritive medium with following composition

The charging of 60 premium on currency ⁽

⁾

Title base substance content gross weight base substance volume juice

The % of g of %＜(g/L), g (gram) L

(gram) (liter)

Soyflour 12 720 720

Maize extract 50 3 180 90

Glucose 90 65 3,900 3510

Dry yeast 6 360

CaCO ₃98 6 360 352.8

Propargyl alcohol B-400 0.003

Distilled water reaches 60 most

(*) cumulative volume is 60 liters, inoculum volume 50ml

Following biochemical characteristic should be sterilized and have to substratum:

pH＝7.0-7.2

Glucose content=5.0-7.9%

Nutritional medium is inoculated with the inoculum of cultivating in the flask in the fermentation container.

3.3Streptomyces producing being incubated in the fermentation container of avermectin, the cultivation of avermitilis VKM AC-1475D undertaken by following condition:

Temperature: 28 ° ± 1 ℃

Begin to stir: 200rpm(rev/min)

The beginning air flow: the 0L(liter)/minute

Work as pO ₂Value is reduced to 60% o'clock when saturated, should make revolution be increased to 400-450rpm and makes air be increased to the 12-15L/ branch for amount, and promptly per unit volume substratum per minute is a 0.2-0.25 unit volume air, to guarantee its pO ₂Value is in described level.The pH value descends naturally in preceding 24 hours that cultivate, and it is elevated to again and is up to 7.2 afterwards, no longer changes later on, until fermentation ends.The foam that generates during cultivation can be removed with propargyl alcohol B-400 aq suspension.The foam that generates when observing 18-24 hour is maximum, and this moment, biomass sharply increased, and therefrom emitted CO ₂The antifoamer average consumption of 38 ℃ of emulsions is to be 3 liters at every turn.In the training period, the actinomycetes mycelia is attached to the matrix granule surface, and causes the generation aggregate.

The mycelia that leaves aggregate during logarithm mutually is long and wide.To the culture growth ending, the mycelia aggregate becomes more closely knit.Growing stationary phase, mycelia is lacked or can not see fully.To fermentation ends, see the actinomycetes self-dissolving usually.Examining under a microscope the aggregate of pressing with a slice iron covering slide is amorphous crumb.Fermentation time is 143-187 hour, on average, is generally 168 hours.Judge the fermentation termination based on following parameter:

-in nutritional medium, there is not glucose;

The content of-avermectin B1 is not less than 100mcg/ml.

During fermentation ends, the nutrient solution of fermentor tank is filtered, and obtain being used to separate the fermented liquid of avermectin.

Embodiment 1

During the fermentation ends in according to preparation, mentioned, have the 30(liter of following composition) all fermented liquids in mixing tank, mix with 30L acetone; Piece speed agitator is installed in the mixing tank:

Total avermectin 720mg/L(mg/litre)

Avermectin B _1a+ B _1b150mg/L

Avermectin A _1a86mg/L

Wet throw out 300g/L

Then its mixture is transferred in the high pressure homogenisers so that break cell.With its equal pledge direct inverse stream Amberlite that packs into

In the XAD-8 post, its charging speed is the 110ml/ branch.Wash part mixture in absorption phase as the 1500ml of elutant and contain the product relevant with biomass, and the spawn relevant with avermectin not.50% acetone soln with 5500ml washs so that wash out the fermented liquid that is trapped in the resin at last.At first, be recovered to the many parts of elutants of 750ml with the avermectin of 60% acetone soln with the absorption of 58ml/ component velocity wash-out.Preceding four parts do not contain avermectin, and 5-14 part contains avermectin B _1a, and quite a spot of B is arranged _2aIn 15-21 part, wash out the avermectin B of largest portion _1aAvermectin A _1aBe in last that part that obtains with the pure acetone wash-out.Avermectin B _1aAnd B _1bThe rate of recovery is 85% approximately.

Embodiment 2

As embodiment 1, handle whole fermented liquids and obtain equal pledge, with its centrifugation with equal-volume methyl alcohol.Transparent supernatant liquor is with the 100ml/ component velocity Duolite that packs into On the A7 post (polycondensation phenol-formaldehyde resin).With alcohol-water gradient mixture wash-out, and simultaneously with the pH of basic solution control elutant.13-20 part elutriant merged be evaporated to driedly, resistates is handled with ethyl acetate.After removing by filter undissolved part.Transparent filtrate vaporising under vacuum is to doing.After with methanol crystallization, obtain 4.82g avermectin B _1a, purity is 86%.

Claims (7)

1. The method of 1, separating avermectin, it is characterized in that: with the miscible solvent of water in the presence of, the whole fermented liquids that come from Streptomyces avermitilis nutrient solution are homogenized, with the equal pledge that the plastic resin treatment that is selected from polyacrylic ester and phenol-aldehyde condensation polymerization copolymer as above obtains, utilize the water/avermectin that adsorbs on the resin is discharged with the mixture gradient of the miscible solvent of water.
2. 2, method according to claim 1, it is characterized in that: equal pledge is contained in to be equipped with what pointed out be on the resin column of matrix so that the absorption avermectin with polyacrylic ester and phenol-aldehyde condensation polymerization copolymer, its feature is that also the wash-out of product is to utilize water/carry out with the miscible solvent mixture gradient of water.
3. 3, method according to claim 1 and 2 is characterized in that: all pledge is to adopt the mechanical means that is selected from ball milling, high pressure homogenizer or ultrasonic apparatus to prepare.
4. 4, according to the described method of arbitrary claim in the aforesaid right requirement, it is characterized in that: just adsorb avermectin more than equal pledge direct inverse stream being installed on the post that polyacrylic ester or phenol-aldehyde condensation polymerization copolymer are housed.
5. 5, require according to aforesaid right in the described method of arbitrary claim, it is characterized in that the wash-out of avermectin on the resin is to adopt water and carry out with the miscible solvent mixture gradient of water, its solvent is selected from acetone, methyl alcohol, ethanol and Virahol.
6. 6, according to the described method of arbitrary claim in the aforesaid right requirement, it is characterized in that whole-broth comes from bacterial strain VKM AC-1475D.
7. 7, microorganism Streptomyces avermitilis VKM AC-1475D.

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