CN103160556A - Application of Streptomyces parvus - Google Patents

Abstract

The invention discloses application of Streptomyces parvus CGMCC No. 4027 which is used for preparation of arylomycin A2 and arylomycin A4 through fermentation. The invention provides a novel approach for preparation of arylomycins.

Description

The application of streptomyces parvus

Technical field

The invention belongs to the microbial fermentation field, be specifically related to streptomyces parvus (Streptomyces parvus) CGMCC No.4027 for the preparation of the application of arylomycins.

Background technology

Alexandra

Obtain two groups of lipopeptide compound arylomycins Deng in 2002 from Streptomyces sp.T ü 6075 separation, comprised arylomycins A and arylomycins B (Schimana, Gebhardt et al.2002).They all belong to hexapeptide compounds, contain identical peptide chain (D-Me-Ser-D-Ala-Gly-L-Me-Hpg-L-Ala-L-Tyr), and different is that the Tyr in arylomycins B contains-NO ₂Substituting group.

Applicant of the present invention has obtained a strain streptomyces parvus (Streptomyces parvus) by the method for Natural Selection, on July 20th, 2010 in the formal preservation in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preserving number is: CGMCCNo.4027.At present less to the research of its secondary metabolite.

Summary of the invention

Applicant of the present invention separates having obtained two kinds of compounds in the research process to the meta-bolites of streptomyces parvus (Streptomyces parvus) CGMCC No.4027, be arylomycin A2 and arylomycin A4 through Structural Identification.

Therefore, the object of the invention is to, the application of a kind of streptomyces parvus (Streptomyces parvus) CGMCCNo.4027 is provided, be used for preparing arylomycin A2 and arylomycin A4 by fermentation.

According to a preferred embodiment of the present invention, the seed culture condition of described fermentation is:

Culture temperature is 28～30 ℃, and incubation time is 46～50h;

Seed culture medium forms: glucose 2%, and Zulkovsky starch 8%, soybean cake powder 5%, dipotassium hydrogen phosphate 0.02%, pH 7.0～7.5.

According to a preferred embodiment of the present invention, the conditions of flask fermentation of described fermentation is:

Leavening temperature is 28～30 ℃, and fermentation time is 4～6 days;

Fermention medium forms: soybean cake powder 2.4%, and dextrin 3.2%, glucose 0.6%, dipotassium hydrogen phosphate 0.02%, sodium-chlor 0.15%, pH 7.0～7.5.

According to a preferred embodiment of the present invention, the ferment tank condition of described fermentation is:

Leavening temperature is 28～30 ℃, and fermentation time is 4～6 days;

Fermention medium forms: dextrin 6%, and soybean cake powder 2%, dipotassium hydrogen phosphate 0.02%, sodium-chlor 0.15%, pH 7.0～7.5.

According to a preferred embodiment of the present invention, the preparation of described arylomycin A2 and arylomycin A4 also comprises the step that fermented liquid is separated with preparation HPLC through macroporous resin adsorption.

Streptomyces parvus of the present invention (Streptomyces parvus) CGMCC No.4027 can fermentative production arylomycinA2 and arylomycinA4, for a new way has been opened up in the preparation of arylomycins.

Description of drawings

Fig. 1 is the structural representation of arylomycins.

Fig. 2 is preparative liquid chromatography figure.

Fig. 3 is the high resolution mass spectrum figure of compound 1.

Fig. 4 is the high resolution mass spectrum figure of compound 2.

Fig. 5 is the amino acid analysis figure of compound 1.

Fig. 6 is the amino acid analysis figure of compound 2.

Fig. 7 is the MS/MS spectrogram of compound 1.

Fig. 8 is the MS/MS spectrogram of compound 2.

Fig. 9 is the hydrogen spectrogram of compound 1.

Figure 10 is the carbon spectrogram of compound 1.

Figure 11 is the two-dimensional spectrum spectrogram of compound 1, and wherein (a) is the cosy spectrogram, is (b) the dept spectrogram, is (c) the HMBC spectrogram, is (d) the HMQC spectrogram, (e) is the TOCSY spectrogram.

Figure 12 is the hydrogen spectrogram of compound 2.

Figure 13 is the carbon spectrogram of compound 2.

Embodiment

Below by specific embodiment, the present invention is described in further details.Should be understood that following examples are only for explanation the present invention but not for limiting scope of the present invention.

The present invention's bacterial classification used was streptomyces parvus (Streptomyces parvus), had been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preserving number CGMCC No.4027 on July 20th, 2010.

(w/v) is as follows for the formula of the substratum that uses in following examples:

Slant medium: Zulkovsky starch 2%, saltpetre 0.1%, dipotassium hydrogen phosphate 0.05%, sodium-chlor 0.05%, agar 2%, pH 7.2～7.4.

Seed culture medium: glucose 2%, Zulkovsky starch 8%, soybean cake powder 5%, dipotassium hydrogen phosphate 0.02%, pH 7.0～7.5.

The shaking flask fermention medium: soybean cake powder 2.4%, dextrin 3.2%, glucose 0.6%, dipotassium hydrogen phosphate 0.02%, sodium-chlor 0.15%, pH 7.0～7.5.

The fermentor tank fermention medium: dextrin 6%, soybean cake powder 2%, dipotassium hydrogen phosphate 0.02%, sodium-chlor 0.15%, pH 7.0～7.5.

Above substratum uses after 121 ℃ of saturation steam sterilization 30min all with the distilled water preparation.

The preparation of embodiment 1, compound

1.1 fermentation

Streptomyces parvus (Streptomyces parvus) CGMCC No.4027 bacterial classification is inoculated on slant medium from the freeze-drying pipe, in 28 ℃～30 ℃ cultivate four days after, change in the triangular flask that seed culture medium is housed, be placed in shaking table and carry out seed culture, wherein, shaking speed is 200～230rpm, and temperature is 28～30 ℃, and incubation time is 46～50h.Seed culture changes in the triangular flask that fermention medium is housed by 4% transferred species amount after finishing, and carries out shake flask fermentation and cultivates, and shaking speed is 150～300rpm, and 28～30 ℃ of temperature are cultivated after 6 days fermentation ends.

Also can change in the 5L glass fermentation tank that fermention medium is housed by 4% transferred species amount after seed culture, ferment, mixing speed is 300～500rpm, and temperature is controlled at 28～30 ℃, and air flow is 7～9L/min, cultivates and finishes afterwards fermentation in 4～6 days.

1.2 sample separation purifying

The fermented liquid that obtains in 1.1 is placed in 4 ℃ of refrigerator overnight, protein precipitation.Afterwards with fermented liquid in 4 ℃, the centrifugal 30min of 4000rpm.Abandon lower sediment, the supernatant liquor absorption with macroporous adsorbent resin, macroporous adsorbent resin can adopt XAD-1600, SP850, or JD-1, in static or dynamically adsorb all can, adsorption time is no less than 3 hours.The rear pure water, 10%, 30%, 50%, 75% with the twice column volume of absorption, 100% ethanol is wash-out successively, and crude product concentrates in 75% ethanol eluate.Crude product liquid in 40 ℃ of concentrating under reduced pressure, then adopts the medium pressure chromatography system to carry out rough with Rotary Evaporators.Condition is as follows: the crude product concentrated solution is sample introduction sample with 10 times of 45% dilution in acetonitrile, the centrifugal 10min of 12000rpm, supernatant.Applied sample amount is 15～25ml, and compression leg in employing (model: Biotage Si 25+M 1603-2, USA) separates, the column temperature nature, and moving phase is 45% acetonitrile 500ml, isocratic elution.Flow velocity is 3～5ml/min approximately, and sample concentrates in the elutriant of 300～500ml.Elutriant adopts DIONEXP680 double base pump to prepare liquid phase systems after 40 ℃ of concentrating under reduced pressure and is prepared with Rotary Evaporators, the preparation liquid of collecting revolves inspissation in 40 ℃ and is reduced to the dried target compound sterling that obtains.

The liquid phase preparation condition is as follows:

Preparative column: YMC CombiPreP ODS-A 5 μ m 20 * 250mm

Column temperature: natural detector: DAD (210nm, 223nm, 254nm, 280nm)

Flow velocity: 8ml/min sample size: 60～700 μ l

The elutriant formula is as follows: the Glacial acetic acid that the ammonium acetate of 0.2% (w/v) adds 0.12% (v/v) is made into the damping fluid of pH 4.69, then is made into the moving phase of 50% (v/v) acetonitrile, isocratic elution with damping fluid and acetonitrile.

Liquid phase prepares result as shown in Figure 2.The intercepting retention time is the material of 18.3min and 15.4min, and called after compound 1, compound 2, two materials are white powder respectively, are soluble in methyl alcohol, ethanol, water, acetonitrile.

Embodiment2, the Structural Identification of compound

The sample that obtains in embodiment 1 is detected through high resolution mass spectrum, collection of illustrative plates as shown in Figure 3, Figure 4, the accurate molecular weight of compound 1 is 838.4603, the accurate molecular weight of compound 2 is 824.4437.

The reckoning function of molecular formula in Masslynx software, the molecular formula of compound 1 is speculated as C ₄₃H ₆₂N ₆O ₁₁, the molecular formula of compound 2 is speculated as C ₄₂H ₆₀N ₆O ₁₁

Detect (result such as Fig. 7, shown in Figure 8) in conjunction with amino acid analysis (result such as Fig. 5, shown in Figure 6) and MS/MS, compound 1 and compound 2 are for having identical peptide chain portion as can be known, difference only is the methylene radical-CH2 on side chain, preliminary its amino-acid residue of judgement contains Gly, Ala, similar MS/MS collection of illustrative plates show that two compounds are the structure homologue.Comprehensive above information is retrieved in compound library DNP and Scifinder with these two molecular formula, infers that compound 1 and 2 is respectively Arylomycin A4 and Arylomycin A2 material.

Further, the hydrogen of binding compounds 1 spectrum (Fig. 9), carbon spectrum (Figure 10) and two-dimensional spectrum (Figure 11), and nuclear magnetic data (table 1) its structure is analyzed.

The NMR nuclear magnetic data of table 1 compound 1

By resolving, determined the ownership of all carbon atoms and hydrogen atom, the structure of secondary metabolite compound 1 that has obtained streptomyces parvus is as follows:

Because compound 2 is the structure homologue with compound 1, show that through amino acid analysis difference only is the methylene radical-CH on side chain ₂, on the basis of the structure of deterministic compound 1, in conjunction with hydrogen spectrum (Figure 12), carbon spectrum (Figure 13) and nuclear magnetic data (table 2) to compound 2, its structure is analyzed.

The NMR nuclear magnetic data of table 2 compound 2

By resolving, determined the ownership of all carbon atoms and hydrogen atom, the structure of secondary metabolite compound 2 that has obtained streptomyces parvus is as follows:

Above furanone compound 1 and 2 be respectively Arylomycin A4 and Arylomycin A2.

Claims (5)

1. the application of streptomyces parvus (Streptomyces parvus) CGMCC No.4027, is characterized in that, is used for preparing arylomycins by fermentation, and wherein, described arylomycins comprises arylomycin A2 and arylomycin A4.

2. application as claimed in claim 1, is characterized in that, the seed culture condition of described fermentation is:

Culture temperature is 28～30 ℃, and incubation time is 46～50h;

Seed culture medium forms: glucose 2%, and Zulkovsky starch 8%, soybean cake powder 5%, dipotassium hydrogen phosphate 0.02%, pH 7.0～7.5.

3. application as claimed in claim 1, is characterized in that, the conditions of flask fermentation of described fermentation is:

Leavening temperature is 28～30 ℃, and fermentation time is 4～6 days;

Fermention medium forms: soybean cake powder 2.4%, and dextrin 3.2%, glucose 0.6%, dipotassium hydrogen phosphate 0.02%, sodium-chlor 0.15%, pH 7.0～7.5.

4. application as claimed in claim 1, is characterized in that, the ferment tank condition of described fermentation is:

Leavening temperature is 28～30 ℃, and fermentation time is 4～6 days;

Fermention medium forms: dextrin 6%, and soybean cake powder 2%, dipotassium hydrogen phosphate 0.02%, sodium-chlor 0.15%, pH 7.0～7.5.

5. application as described in any one in claim 1～4, is characterized in that, described preparation arylomycins also comprises the step that fermented liquid is separated with preparation HPLC through macroporous resin adsorption.

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