阅读说明：本技术 一种发酵生产平阳霉素的方法 (Method for producing pingyangmycin by fermentation ) 是由 袁天阳 陈庆源 张海涛 朱颖 禹学军 于 2019-11-20 设计创作，主要内容包括：本发明公开了一种发酵生产平阳霉素的方法。本发明公开了发酵生产平阳霉素的方法,包括将轮枝链霉菌在发酵培养基中进行发酵,在发酵过程中向所述发酵培养基中添加外源前体物得到含平阳霉素的发酵产物,从所述发酵产物中分离得到平阳霉素；其中,所述外源前体物为物质A和/或物质B,所述物质A为腺苷甲硫氨酸或其同类氨基酸衍生物,所述物质B为亚精胺或亚精胺盐。本发明发酵生产平阳霉素方法简单、有效、能够快速提高平阳霉素发酵生产水平,降低生产成本,无需额外对设备与人力,能够大幅度提高平阳霉素工业发酵水平,适合工业化规模生产。(The invention discloses a method for producing pingyangmycin by fermentation. The invention discloses a method for producing pingyangmycin by fermentation, which comprises the steps of fermenting streptomyces verticillata in a fermentation medium, adding an exogenous precursor into the fermentation medium in the fermentation process to obtain a fermentation product containing pingyangmycin, and separating the pingyangmycin from the fermentation product; the exogenous precursor is a substance A and/or a substance B, the substance A is adenosylmethionine or an amino acid derivative of the same type, and the substance B is spermidine or spermidine salt. The method for producing pingyangmycin by fermentation is simple and effective, can quickly improve the fermentation production level of pingyangmycin, reduces the production cost, does not need additional equipment and manpower, can greatly improve the industrial fermentation level of pingyangmycin, and is suitable for industrial mass production.)

1. A method for producing pingyangmycin by fermentation is characterized by comprising the following steps: fermenting streptomyces verticillata in a fermentation medium, adding an exogenous precursor into the fermentation medium in the fermentation process to obtain a fermentation product containing pingyangmycin, and separating pingyangmycin from the fermentation product;

the exogenous precursor is a substance A and/or a substance B, the substance A is adenosylmethionine or an amino acid derivative of the same type, and the substance B is spermidine or spermidine salt.

2. The method of claim 1, wherein: the exogenous precursor is added when the fermentation lasts for 40-60 h.

3. The method according to claim 1 or 2, characterized in that: the addition amount of the exogenous precursor is that the concentration of the exogenous precursor in a fermentation medium is 0.4 g/L-0.6 g/L;

and/or when the exogenous precursor is a substance A and a substance B, the mass ratio of the substance A to the substance B is 1: 4-1: 6.

4. A method according to any one of claims 1 to 3, wherein:

the fermentation medium is a fermentation medium without a substance A and/or a substance B;

and/or the presence of a gas in the gas,

the fermentation medium comprises the following components in percentage by mass: 3.0 to 3.5 percent of maltose, 0.3 to 0.5 percent of soybean peptone, 2.5 to 3.0 percent of starch, 2.5 to 3.5 percent of corn steep liquor, 2.5 to 3.2 percent of soybean cake powder, 0.01 to 0.02 percent of copper sulfate, 0.3 to 0.35 percent of sodium chloride and the balance of water.

5. The method according to any one of claims 1 to 4, wherein the fermentation conditions are as follows:

fermentation temperature: 28 +/-2 ℃;

fermentation time: 140-170 h;

fermentation pressure: 0.02-0.06 MPa;

air flow rate (v/v): 1: 0.6-1;

stirring speed: 50-400 rpm;

controlling dissolved oxygen: the fermentation time is 0-35h, the dissolved oxygen is more than or equal to 25%, the fermentation time is 36-120h, the dissolved oxygen is more than or equal to 30%, and the fermentation time is more than or equal to 40% after 121 h;

and (3) pH control: the fermentation time is controlled to be below 6.90 hours after 0-72 hours, and is controlled to be below 7.45 hours after 72 hours.

6. A kit for producing pingyangmycin, which is characterized in that: the kit contains Streptomyces verticillatus and the fermentation medium of any one of claims 1 to 5.

7. A kit for producing pingyangmycin, which is characterized in that: the kit comprises Streptomyces verticillatus, a fermentation medium according to any one of claims 1 to 5, and exogenous precursors.

8. Use of the method of any one of claims 1 to 5 or the kit of claim 6 or 7 for the preparation of pingyangmycin.

9. Use of substance a and/or substance B according to any one of claims 1 to 5 for the preparation of pingyangmycin.

10. Use of substance A and/or substance B according to any one of claims 1 to 5 for increasing the production of pingyangmycin.

Technical Field

The invention belongs to the field of fermentation engineering, and particularly relates to a method for producing pingyangmycin by fermentation.

Background

Pingyangmycin is a single-component glycopeptide antibiotic with anti-tumor activity found in streptomyces verticillata fermentation products, and the terminal amine is spermidine. Pingyangmycin is a broad-spectrum, low-toxicity antitumor drug. Has good curative effect on malignant lymphoma, head and neck cancer and breast cancer. Also has satisfactory curative effect on liver cancer without effective medicament treatment. Therefore, the method has very important significance for improving the content of pingyangmycin in fermentation liquor, improving the production yield and meeting the market demand.

At present, the domestic research method for improving pingyangmycin mainly changes the fermentation process around strain breeding and fermentation medium prescription optimization, and although certain performance is achieved, the workload is huge, the yield is very low, the fermentation level of industrialized production of single-component pingyangmycin is generally 50-80U/ml, and the fermentation production level is extremely unstable.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the yield of pingyangmycin.

In order to solve the technical problems, the invention provides a method for producing pingyangmycin by fermentation.

The method for producing pingyangning by fermentation comprises the steps of fermenting streptomyces verticillata (streptomyces verticillatus var. pingyangensis) in a fermentation medium, adding an exogenous precursor into the fermentation medium in the fermentation process to obtain a fermentation product containing pingyangming, and separating pingyangming from the fermentation product; the exogenous precursor is a substance A and/or a substance B, the substance A is adenosylmethionine or an amino acid derivative of the same type, and the substance B is spermidine or spermidine salt.

In the method, the exogenous precursor can be added into a fermentation medium in a solid or solution form, and is added when the fermentation is carried out for 40-60h, at the moment, the formation of a dense net of mycelia can be seen through microscopic examination, and the single-component production effect of the pingyangmycin can be influenced by adding the exogenous precursor after the mycelia are subjected to differentiation and autolysis.

In the method, the exogenous precursor is added in an amount such that the concentration of the exogenous precursor in the fermentation medium is 0.4g/L to 0.6 g/L. When the exogenous precursor is a substance A and a substance B, the concentration is the total mass concentration of the substance A and the substance B, wherein the mass ratio of the substance A to the substance B is 1: 4-1: 6.

In the above method, the fermentation medium is a fermentation medium that does not contain the substance a and/or the substance B.

In the method, the fermentation medium can be composed of the following components in percentage by mass: 2.5-3.0% of starch, 2.5-3.5% of corn steep liquor, 2.5-3.2% of soybean cake powder, 0.01-0.02% of copper sulfate, 3.0-3.5% of maltose, 0.3-0.5% of soybean peptone, 0.3-0.35% of sodium chloride and the balance of water.

In the above method, the fermentation conditions are as follows:

fermentation temperature: 28 +/-2 ℃;

fermentation time: 140-170 h;

fermentation pressure: 0.02-0.06 MPa;

air flow rate (v/v): 1: 0.6-1;

stirring speed: 50-400 rpm;

controlling dissolved oxygen: the fermentation time is 0-35h, the dissolved oxygen is more than or equal to 25%, the fermentation time is 36-120h, the dissolved oxygen is more than or equal to 30%, and the fermentation time is more than or equal to 40% after 121 h;

and (3) pH control: the fermentation time is controlled to be below 6.90 hours after 0-72 hours, and is controlled to be below 7.45 hours after 72 hours.

The method also comprises the step of culturing the streptomyces verticillata by adopting a seed culture medium before fermentation to obtain seed liquid of the streptomyces verticillata, and then transplanting the seed liquid into the fermentation culture medium for fermentation.

In the method, the seed culture medium comprises the following components in percentage by mass: 1.0-2.0% of corn steep liquor, 1.0-1.5% of starch, 1-1.5% of glucose, 0.2-0.4% of sodium chloride, 0.01-0.02% of copper sulfate, 1.2-2.0% of soybean cake powder and the balance of water.

In the method, the inoculation amount of the seed liquid is 5-50% of the volume of the fermentation medium.

In the method, the conditions for culturing the streptomyces verticillata by adopting the seed culture medium are that the culture temperature is 28 +/-2 ℃, the air flow (v/v) is 1: 0.8, the pressure is 0.06Mpa, and the rotating speed is 320 +/-20 rpm.

The invention further provides a kit for producing pingyangmycin, which contains the following A1) or A2) or A3) or A4):

A1) streptomyces verticillata and the fermentation medium;

A2) streptomyces verticillatus, the fermentation medium and the seed medium;

A3) streptomyces verticillata, the fermentation medium and the exogenous precursor;

A4) streptomyces verticillatus, the fermentation medium, the exogenous precursor and the seed culture medium.

The invention also provides application of the substance A and/or the substance B in preparation of pingyangmycin.

The invention further provides application of the substance A or the substance B in improving the yield of pingyangmycin.

In the application, the step of increasing the yield of pingyangmycin refers to the step of increasing the yield of pingyangmycin by utilizing a method for producing pingyangmycin by fermenting streptomyces verticillata.

According to the invention, an exogenous precursor (namely, a composite solution of adenosylmethionine and spermidine, wherein the mass ratio of the adenosylmethionine to the spermidine in the composite solution is 1: 4)) is added in the microbial fermentation process, so that the yield of the pingyangmycin in the fermentation liquid can be obviously improved.

The method for producing pingyangmycin by fermentation is simple and effective, can quickly improve the fermentation production level of pingyangmycin, reduces the production cost, does not need additional equipment and manpower, can greatly improve the industrial fermentation level of pingyangmycin, and is suitable for industrial mass production.

Drawings

FIG. 1 shows the results of the liquid phase assays of the fermentation broths of pingyangmycin of the test group and the control group in example 4.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The formula and preparation method of the culture medium used in the following examples of the invention are as follows:

the slant culture medium is a Gao's improved culture medium, and comprises the following components in percentage by mass: 2.0% of soluble starch, 0.01% of sodium chloride, 0.06% of dipotassium hydrogen phosphate, 0.06% of magnesium sulfate, 0.01% of glucose, 2.0% of agar and the balance of purified water, sterilizing at 120 ℃ for 30 minutes, and paving an inclined plane.

Seed culture medium: the weight percentage of each component is as follows: 2.0% of corn steep liquor, 1.5% of starch, 1% of glucose, 0.2% of sodium chloride, 0.02% of copper sulfate, 2.0% of soybean cake powder and the balance of purified water, and sterilizing at 120 ℃ for 30 minutes.