CN112209912A - Method for improving fermentation yield of xenorhabdus nematophilus CB6 strain metabolite Xencouumacin 1 - Google Patents
Method for improving fermentation yield of xenorhabdus nematophilus CB6 strain metabolite Xencouumacin 1 Download PDFInfo
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- CN112209912A CN112209912A CN202011105621.8A CN202011105621A CN112209912A CN 112209912 A CN112209912 A CN 112209912A CN 202011105621 A CN202011105621 A CN 202011105621A CN 112209912 A CN112209912 A CN 112209912A
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Abstract
The invention relates to a method for improving the fermentation yield of a xenorhabdus nematophila CB6 strain metabolite Xencoumacin 1, which adopts an in-situ separation technology based on macroporous adsorption resin to effectively adsorb Xencoumacin 1 generated in the fermentation process. According to the method, the macroporous adsorption resin is added in the fermentation process, Xcn1 can be effectively adsorbed, the degradation of the Xcn1 by the enzyme generated by the CB6 strain is prevented, the concentration of Xcn1 in the fermentation liquid is reduced, the toxicity to the CB6 strain is reduced, and the strain is promoted to generate more Xcn 1. Tests show that the yield of the Xencouumacin 1 fermented by the method is at least over 60.32 mu g/mL. Is much higher than the fermentation yield without adding the macroporous absorption resin. The method is simple and easy to implement, and can effectively reduce the production cost of Xcn1 industrial development.
Description
Technical Field
The invention relates to the technical field of biological fermentation, and particularly relates to a method for improving the fermentation yield of a xenorhabdus nematophilus CB6 strain metabolite Xencoumacin 1.
Background
Xencoumacen 1(Xcn1) is an isocoumarin derivative formed by arginine residue, leucine residue and four acetate units, and has the biological function of antagonizing plant fungal diseases such as potato late blight, tomato late blight, cucumber powdery mildew, pepper phytophthora blight and the like. Xcn1 is the main active substance produced by Xenorhabdus nematophilus CB6, but during the metabolic process of the strain, the excessively accumulated Xcn1 can generate toxicity to the CB6 strain, thereby activating the catabolic pathway of Xcn 1. The yield of Xcn1 obtained by the traditional fermentation production is further reduced along with the advance of time, meanwhile, Xcn1 generated in the fermentation process is converted into derivatives with low activity such as Xcn2, and the yield of Xcn1 is reduced on one hand; on the other hand, various Xcn1 derivatives are generated, which increases the difficulty of the subsequent purification step Xcn 1.
Disclosure of Invention
Based on the technical defects, the technical purpose of the invention is to provide a method for improving the fermentation yield of a metabolite Xencouumacin 1 of a Xenorhabdus nematophilus strain CB 6. The method is based on an in-situ separation technology of macroporous adsorption resin, and is used for effectively adsorbing Xencoumacacin 1 generated in the fermentation process, reducing the concentration of Xcn1 in the fermentation liquor, reducing the toxicity to a CB6 strain and promoting the strain to generate more Xcn 1. Not only improves the fermentation yield of Xcn1, but also reduces the generation of other derivatives, thereby reducing the production cost of Xcn1 industrial development.
The technical scheme of the invention is as follows: a method for improving fermentation yield of xenorhabdus nematophilus CB6 strain metabolite Xencoumacin 1 adopts an in-situ separation technology based on macroporous adsorption resin to effectively adsorb Xencoumacin 1 generated in a fermentation process. Wherein the in-situ separation technology based on the macroporous adsorption resin is to add 2 to 5 weight percent of macroporous separation resin into a CB6 fermentation culture medium.
The process of the present invention, wherein the macroporous adsorbent resin is preferably macroporous adsorbent resin X-5, may also be carried out using other similar types of macroporous adsorbent resins, such as XAD-16.
The method of the invention, wherein the macroporous adsorption resin is activated before being added into the culture medium, and the activation step is as follows: sequentially soaking the macroporous adsorption resin in absolute ethyl alcohol, shaking and rinsing with an alkali solution, and soaking and activating with an alkali solution. The activation step is a conventional step before the macroporous adsorption resin is used, and a person skilled in the art can purposefully select the activation step corresponding to the resin when different macroporous adsorption resins are adopted, and the activation step is not limited to the specific one.
According to a preferred embodiment of the present invention, the macroporous adsorbent resin is X-5, and the activation step is: soaking macroporous adsorbent resin X-5 in anhydrous ethanol for 24 hr, and washing with distilled water; removing residual distilled water, and then shaking and rinsing for 4 hours by using three times of 1mol/L NaOH solution; pouring out the NaOH solution, and adding a new 1mol/L NaOH solution to soak for 24 h; and finally, washing the macroporous adsorption resin X-5 with distilled water until the pH value is neutral, and removing residual distilled water for later use.
The method of the invention, wherein the CB6 fermentation medium is a CLB liquid medium and comprises the following components: tryptone 1.0 wt%, yeast extract 0.5 wt%, NaCl 0.5 wt%, MgSO 0.01mM4. Other fermentation media can be selected for use as long as the nutrients required for the production and metabolism of the xenorhabdus nematophilus strain CB6 are met.
The method comprises the step of inoculating CB6 strain seed liquid with the weight percentage of 1% to the fermentation culture medium after adding the macroporous adsorption resin for fermentation culture. The CB6 strain seed liquid is obtained by transferring a single colony of the activated CB6 strain into a CLB liquid culture medium and culturing at 30 ℃ and 200rpm overnight.
According to the method, the macroporous adsorption resin is added in the fermentation process, Xcn1 can be effectively adsorbed, the degradation of the Xcn1 by the enzyme generated by the CB6 strain is prevented, the concentration of Xcn1 in the fermentation liquid is reduced, the toxicity to the CB6 strain is reduced, and the strain is promoted to generate more Xcn 1. Tests show that the yield of the Xencouumacin 1 fermented by the method is at least over 60.32 mu g/mL. Is much higher than the fermentation yield without adding the macroporous absorption resin. The method is simple and easy to implement, and can effectively reduce the production cost of Xcn1 industrial development.
Drawings
FIG. 1 shows the effect of HPLC on Xcn1 of X-5 macroporous adsorbent resin treatment on Xenorhabdus nematophilus CB 6.
FIG. 2 shows the effect of macroporous adsorbent resin X-5 treatment on the antibacterial activity of the extracellular extract of Xenorhabdus nematophilus CB 6; wherein (A) the effect of X-5 treatment on Bacillus subtilis is detected by a cup butterfly method; (B) the influence of X-5 treatment on alternaria solani is detected by adopting a growth inhibition experiment; (C) measuring the diameter of an inhibition zone for resisting bacillus subtilis; (D) the colony diameter of alternaria solani was measured.
Detailed Description
To further illustrate the present invention, reference is made to the following examples:
the Xenorhabdus nematophila (Xenorhabdus nematophila) CB6 strain related by the invention is a known strain, is registered and preserved in the China general microbiological culture Collection center (CGMCC), and has the following addresses: west road No. 1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101, the strain number is CGMCC No. 1173.
The embodiment provides a method for improving fermentation yield of xenorhabdus nematophilus CB6 strain metabolite Xencoumacin 1, which is characterized in that macroporous adsorption resin is activated and then added into a fermentation culture medium of CB6 for fermentation culture, and in the culture process, the macroporous adsorption resin can effectively adsorb metabolite Xencoumacin 1 gradually accumulated in fermentation liquor, so that toxicity of excessively accumulated Xcn1 to the CB6 strain is prevented, and a degradation metabolic pathway of Xcn1 is activated, so that fermentation yield of Xcn1 is reduced. The treatment by the method can effectively adsorb Xcn1, prevent the degradation of the Xcn1 by enzyme generated by the CB6 strain, reduce the concentration of Xcn1 in fermentation liquor, reduce the toxicity to the CB6 strain and promote the strain to generate more Xcn 1.
The macroporous adsorption resin adopted in the embodiment is macroporous adsorption resin X-5 which is activated firstly: soaking macroporous adsorbent resin X-5 in anhydrous ethanol for 24 hr, and washing with distilled water; filtering by using a sand plate funnel to remove residual distilled water, and then oscillating and rinsing by using NaOH solution with the volume concentration being three times of that of 1mol/L for 4 hours; the NaOH solution is poured out, and a new 1mol/L NaOH solution is added for soaking for 24 hours. And finally, washing the macroporous adsorption resin X-5 with distilled water until the pH value is neutral, and filtering the macroporous adsorption resin with a sand plate funnel to remove residual distilled water for later use.
Xenorhabdus nematophilus CB6 was inoculated in CLB (1.0% tryptone, 0.5% yeast extract, 0.5% NaCl, 0.01mM MgSO4) The plate was incubated at 30 ℃ for 24 hours. And (3) selecting a single colony, transferring the single colony into a CLB liquid culture medium, and culturing at 30 ℃ overnight at 200rpm to obtain a fermented seed solution.
Transferring the seed liquid into a CLB liquid culture medium containing 2% of macroporous adsorption resin X-5 according to the inoculation amount of 1% to serve as an experimental group; meanwhile, the cells were inoculated into a CLB liquid medium at an inoculum size of 1% and cultured continuously at 30 ℃ and 200rpm for 10 days as a control. Samples were taken on day 1, day 2, day 3, day 4, day 7 and day 10, respectively.
Collecting a sample:
(1) experimental groups: and filtering and removing the fermentation liquor of the experimental group by using a sand plate funnel to obtain the X-5 resin for adsorbing the metabolite.
(2) Control group: the fermentation liquor after fermentation needs to be purified to obtain the metabolite, and the purification treatment is as follows: firstly centrifuging at 12000rpm for 10min, collecting the supernatant of the fermentation liquor, then adding 2% macroporous adsorption resin X-5 into the supernatant, adsorbing at 30 ℃ and 200rpm for 12h, and filtering by using a sand plate funnel to remove the supernatant to obtain the X-5 resin for adsorbing the metabolite.
(3) The X-5 resin obtained above was eluted once with 50mL of methanol and then again with 25mL of methanol, the two eluates were combined, the methanol was evaporated to dryness with a rotary evaporator, and the metabolite was dissolved in an equal volume of ultrapure water. The dissolved metabolites were filtered through a 0.22 μm filter and tested for Xcn1 and its degradation products by HPLC. The column used for HPLC was a C18 column (Agilent) (4.6 x 150mm) and the mobile phase was water containing 0.1% formic acid and acetonitrile in a volume ratio of 70: 30.
As a result: xcn1 reaches the highest level when the control group without X-5 adsorption resin is fermented and cultured to the next day, and the content of Xcn1 is 56.01 mug/mL; the Xcn1 content gradually decreases with the culture time; when the culture is carried out for 3 days, the content of Xcn1 is reduced to 42.45 mu g/mL, which is reduced by 24.21%; and when the culture is carried out for 10 days, the Xcn1 content is reduced to 12.88 mu g/mL, which is reduced by 77.01%. The experimental group added with macroporous adsorption resin X-5 shows that the yield of Xcn1 is steadily increased firstly, the highest level is reached when the culture reaches the third day, and the content of Xcn1 is 73.77 mug/mL; during the subsequent cultivation, Xcn1 level remained relatively stable, and Xcn1 content was maintained at 60.32. mu.g/mL (as shown in FIG. 1) even after 10 days of cultivation. The addition of macroporous adsorbent resin X-5 significantly inhibited the conversion of Xcn1 to Xcn2, Xcn5 and Xcn6, compared to the control, so that the conversion products were maintained at a lower level throughout the fermentation. In addition, through the detection of the cell metabolic activity, the addition of the macroporous adsorption resin X-5 reduces the cell density of the Xenorhabdus nematophilus CB6, but obviously improves the cell activity compared with the control group. The influence of the addition of the macroporous adsorption resin X-5 on the antibacterial activity is detected by using Bacillus subtilis and Alternaria solani as indicator bacteria. Compared with a control group, the diameter of the inhibition zone of the bacillus subtilis of the experimental group of the resin X-5 is increased from 20.66mm to 26.83mm, and the antibacterial activity is improved by 29.86% (FIG. 2A, C); while the addition of resin X-5 reduced the colony diameter of Alternaria solani growing on PDA plates containing dissolved metabolites from 23.27mm to 16.50mm, and the bacteriostatic activity increased by 28.66% (FIG. 2B, D). The results show that the in situ separation technology based on macroporous adsorption resin X-5 of the embodiment improves the content of the target metabolite Xcn1 by improving the metabolic activity of cells and blocking the degradation of the product.
From the above examples and experimental results, it can be seen that the method of the present invention can effectively adsorb Xcn1 by adding macroporous adsorption resin during fermentation, prevent it from being degraded by the enzyme produced by CB6 strain, reduce the concentration of Xcn1 in the fermentation broth, reduce the toxicity to CB6 strain, and promote the strain to produce more Xcn 1.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.
Claims (10)
1. A method for improving fermentation yield of Xencouumacin 1 which is a metabolite of a xenorhabdus nematophilus CB6 strain is characterized in that the Xencouumacin 1 generated in the fermentation process is effectively adsorbed by an in-situ separation technology based on macroporous adsorption resin.
2. The method as claimed in claim 1, wherein the in situ separation technique based on macroporous adsorption resin is to add 2-5 wt% macroporous separation resin into CB6 fermentation medium.
3. The process according to claim 1 or 2, wherein the macroporous adsorbent resin is macroporous adsorbent resin X-5 or XAD-16.
4. The method according to claim 1, 2 or 3, wherein the macroporous adsorbent resin is activated by: sequentially soaking the macroporous adsorption resin in absolute ethyl alcohol, shaking and rinsing with an alkali solution, and soaking and activating with an alkali solution.
5. The method of claim 4, wherein the macroporous adsorbent resin is macroporous adsorbent resin X-5, and the activating step is: soaking macroporous adsorbent resin X-5 in anhydrous ethanol for 24 hr, and washing with distilled water; removing residual distilled water, and then shaking and rinsing for 4 hours by using three times of 1mol/L NaOH solution; pouring out the NaOH solution, and adding a new 1mol/L NaOH solution to soak for 24 h; and finally, washing the macroporous adsorption resin X-5 with distilled water until the pH value is neutral, and removing residual distilled water for later use.
6. The method according to claim 2, wherein the CB6 fermentation medium is a CLB liquid medium consisting of: tryptone 1.0 wt%, yeast extract 0.5 wt%, NaCl 0.5 wt%, MgSO 0.01mM4。
7. The method according to claim 2, wherein the fermentation medium is inoculated with 1 weight percent of CB6 strain seed liquid for fermentation culture after being added with the macroporous adsorption resin.
8. The method as claimed in claim 7, wherein the CB6 strain seed solution is obtained by transferring a single colony of the activated CB6 strain into a CLB liquid culture medium and culturing at 30 ℃ and 200rpm overnight.
9. The method according to any one of claims 1 to 8, wherein the yield of Xencouumacin 1 produced by fermentation in said method is above 60.32. mu.g/mL.
10. Use of the process according to any one of claims 1 to 9 for the fermentative production of xenocoumacin 1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113881615A (en) * | 2021-09-29 | 2022-01-04 | 中国农业科学院植物保护研究所 | Xenorhabdus nematophila with high yield of Xcn1 and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1986001509A1 (en) * | 1984-09-05 | 1986-03-13 | Biotechnology Australia Pty. Ltd. | Xenocoumacins |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1986001509A1 (en) * | 1984-09-05 | 1986-03-13 | Biotechnology Australia Pty. Ltd. | Xenocoumacins |
Non-Patent Citations (3)
| Title |
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| DONG YIJIE，ET AL.: "Improving the Yield of Xenocoumacin 1 Enabled by In Situ Product Removal", 《ACS OMEGA》 * |
| JASON M. CRAWFORD,ET AL.: "NRPS Substrate Promiscuity Diversifies the Xenematides", 《ORG. LETT.》 * |
| 黄武仁，等: "嗜线虫致病杆菌代谢产物CB6-1的分离、纯化与结构鉴定", 《中国抗生素杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113881615A (en) * | 2021-09-29 | 2022-01-04 | 中国农业科学院植物保护研究所 | Xenorhabdus nematophila with high yield of Xcn1 and application thereof |
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