Arthrobacter sp. and application thereof in degrading aristolochic acid

Abstract

The present invention belongs to the field of microorganisms and discloses a strain of Arthrobacter and its application in degrading aristolochic acid. The strain of the present invention is Arthrobacter ( Arthrobacter sp.) NyZ610, which has a deposit number of GDMCC No. 61799; the Arthrobacter sp. NyZ610 comprises the gene sequence described in SEQ ID NO. 1. The strain of the present invention can degrade aristolochic acid under aerobic conditions, can reproduce and grow using aristolochic acid as the sole carbon, nitrogen, and energy source, mineralizes aristolochic acid through an oxidative pathway, removes the nitro group, and ultimately decomposes it into carbon dioxide, water, and nitrite, achieving complete detoxification / detoxification of aristolochic acid. Therefore, the strain can be used for in situ remediation of aristolochic acid contamination in the environment, and can also be used for the preparation and detoxification of traditional Chinese medicines containing aristolochic acid, and has the advantages of rapid reproduction rate and complete detoxification.

Description

[0001] This application is a divisional application of the Chinese invention patent with application number 2021113524645. The application date of the original application is August 18, 2021, the application number is 2021113524645, and the name of the invention is "A strain of Arthrobacter and its application in degrading aristolochic acid". Technical Field

[0002] The present invention belongs to the field of microorganisms and relates to an Arthrobacter sp. NyZ610 and its application in degrading aristolochic acid. The Arthrobacter sp. can reproduce and grow using aristolochic acid as the sole carbon, nitrogen and energy raw material, and mineralizes aristolochic acid through an oxidation pathway, during which the nitro group is removed and nitrite is generated. Background Art

[0003] Aristolochic acid (AAs) is a nitrophenanthrene organic acid compound, mainly containing aristolochic acid A, aristolochic acid B, etc., and is naturally present in Aristolochicaceae plants such as Aristolochia and Asarum. These plants have been widely processed and detoxified and used as native medicinal materials, but long-term use of Chinese herbal medicines containing aristolochic acid and their preparations will lead to nephrotoxicity and carcinogenicity. Aristolochic acid has been shown to be related to liver cancer and kidney disease. Aristolochic acid and plants containing aristolochic acid have been included in the list of Class I carcinogens. Currently, many countries have banned the use of medicinal materials containing aristolochic acid. In addition, aristolochic acid secreted by plants is stable in nature and easily accumulates in the environment and enters the human food chain, causing food contamination caused by aristolochic acid.

[0004] Research has also shown that various aristolochic acid compounds can be detected in the soil and nearby water bodies where aristolochic acid-producing plants grow. These compounds can be absorbed by crop roots and accumulate in fruits, seeds, and other parts of the crops. Long-term consumption of foods containing aristolochic acid can cause irreversible kidney damage, and in severe cases, renal failure. Therefore, remediating aristolochic acid contamination in soil and water bodies is of practical significance.

[0005] The preparation and detoxification of traditional Chinese medicines containing aristolochic acid is an important step in the application of such traditional Chinese medicines. Among them, the use of microorganisms for fermentation attenuation or detoxification is a very economical, green and sustainable method. However, due to the stable chemical properties of aristolochic acid, current research on its microbial attenuation includes the use of mold fermentation to convert aristolochic acid I into its demethoxy compound, which achieves partial attenuation, but the most important nitro group that causes its toxicity is not removed. Therefore, it is of great significance to find a microorganism that can achieve complete detoxification of aristolochic acid and aristolochic acid-containing plants. Summary of the Invention

[0006] To address the aforementioned issues in the prior art, the present invention provides a strain of Arthrobacter sp. NyZ610 and its use in degrading aristolochic acid. The strain, isolated from the rhizosphere soil of Aristolochia spp., is capable of growing and reproducing using aristolochic acid A and aristolochic acid B as its sole carbon, nitrogen, and energy sources. The strain mineralizes aristolochic acid via an oxidative pathway, with the removal of nitro groups and the generation of nitrites.

[0007] The technical solutions of the present invention are as follows:

[0008] The Arthrobacter sp. NyZ610 of the present invention was deposited in the Guangdong Microbial Culture Collection Center (GDMCC for short, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510075) on July 21, 2021, with the deposit number GDMCC No: 61799; the Arthrobacter sp. NyZ610 includes the gene sequence described in SEQ ID NO.1.

[0009] The present invention also provides the use of the Arthrobacter NyZ610 in degrading aristolochic acid.

[0010] In one embodiment of the present invention, the aristolochic acid is one or both of aristolochic acid I and aristolochic acid II.

[0011] The present invention also provides a culture medium for culturing the Arthrobacter NyZ610. The preparation method of the culture medium is as follows: 6 g of tryptone soy broth powder is added to 1000 mL of distilled water, stirred and dissolved, and sterilized at 120-125° C. for 15-20 minutes.

[0012] The present invention also provides a method for degrading aristolochic acid, comprising: mixing and culturing the Arthrobacter NyZ610 with an aristolochic acid solution, and degrading the aristolochic acid under aerobic conditions.

[0013] In one embodiment of the present invention, the method for degrading aristolochic acid further comprises inoculating the seed solution of the Arthrobacter NyZ610 into the above-mentioned culture medium for enrichment culture.

[0014] In one embodiment of the present invention, the method for degrading aristolochic acid, the method for preparing the seed solution is: inoculating the Arthrobacter NyZ610 into a seed solution culture medium, culturing at 25-35° C. for 8-12 hours to obtain a seed solution;

[0015] The preparation method of the seed liquid culture medium is as follows: 6g of tryptone soy broth powder and 15g of agar are added to 1000mL of distilled water, stirred evenly, and sterilized at 121°C for 15-20 minutes.

[0016] In one embodiment of the present invention, the method for degrading aristolochic acid further comprises the following steps:

[0017] (1) The enriched culture solution was centrifuged at 5000-8000 g for 5-10 min at 4°C to collect the bacterial cells, which were then washed three times with sterilized basic inorganic salt liquid medium.

[0018] (2) The collected bacteria were resuspended in basic inorganic salt culture solution and mixed with aristolochic acid solution for culture.

[0019] Degrades aristolochic acid under aerobic conditions.

[0020] In one embodiment of the present invention, in the method for degrading aristolochic acid, the conditions for aerobic degradation in step (2) are: a rotation speed of 150-180 r / min, a temperature of 25-37° C., and a time of 25-50 h.

[0021] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0022] A strain of Arthrobacter sp. NyZ610 of the present invention, this strain can be propagated and grown with aristolochic acid as sole carbon, nitrogen and energy raw material, by oxidation pathway, aristolochic acid is mineralized, nitro is removed and finally decomposed into carbon dioxide, water and nitrite, realizes the complete detoxification / detoxification of aristolochic acid, therefore, this strain can be used for the aristolochic acid pollution in the treatment environment, and can also be used for the aristolochic acid in the treatment of Chinese herbal medicine and food. Moreover, this strain growth rate is fast, and when the inoculum size is 1%, it only needs 6h to reach the stable growth phase in tryptone soy broth liquid culture medium, has remarkable advantages in application. Therefore, this strain can be used for the aristolochic acid pollution in the in-situ repair environment, can also be used for the preparation and detoxification of Chinese medicine containing aristolochic acid, has the advantages such as fast reproduction rate, complete detoxification. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the colony morphology of Arthrobacter sp. NyZ610 in Example 1 of the present invention;

[0024] Figure 2 Schematic diagram of the changes in aristolochic acid I, nitrite and bacterial concentrations during the degradation of aristolochic acid I by Arthrobacter in Example 2 of the present invention, wherein AAI is aristolochic acid I, Nitrite is nitrite, OD 600 for Arthrobacter;

[0025] Figure 3Schematic diagram of the changes in aristolochic acid II, nitrite and bacterial concentrations during the degradation of aristolochic acid II by Arthrobacter in Example 3 of the present invention, wherein AAII is aristolochic acid II, Nitrite is nitrite, and OD 600 for Arthrobacter;

[0026] Figure 4 Schematic diagram of the changes in aristolochic acid I, aristolochic acid II, nitrite and bacterial concentrations during the degradation of aristolochic acid by Arthrobacter in Example 4 of the present invention, wherein AAI is aristolochic acid I, AAII is aristolochic acid II, Nitrite is nitrite, OD 600 For Arthrobacter. DETAILED DESCRIPTION

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0028] The following description sets forth numerous specific details to facilitate a thorough understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar generalizations without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific implementations disclosed below.

[0029] Example 1 Screening of Arthrobacter sp. NyZ610

[0030] (1) Prepare culture medium

[0031] Tryptone soy broth liquid culture medium: 6 g of tryptone soy broth powder, 1000 mL of distilled water, sterilize at 121°C for 21 minutes.

[0032] Tryptone soy broth solid medium: 6 g of tryptone soy broth powder, 15 g of agar, 1000 mL of distilled water, sterilized at 121°C for 21 min.

[0033] Basic inorganic salt liquid culture medium: 14.3 g disodium hydrogen phosphate dodecahydrate, 3 g potassium dihydrogen phosphate, 0.28 mg manganese sulfate, 0.3 mg ferrous sulfate, 0.06 mg magnesium sulfate, 1 mg calcium chloride, 0.05 mg copper sulfate, 0.05 mg zinc sulfate, 0.05 mg boric acid, 1000 ml distilled water, pH 7.0.

[0034] Basic inorganic salt solid culture medium: 14.3 g disodium hydrogen phosphate dodecahydrate, 3 g potassium dihydrogen phosphate, 0.28 mg manganese sulfate, 0.3 mg ferrous sulfate, 0.06 mg magnesium sulfate, 1 mg calcium chloride, 0.05 mg copper sulfate, 0.05 mg zinc sulfate, 0.05 mg boric acid, 15 g agar, 1000 ml distilled water, pH 7.0.

[0035] (2) Screening of Arthrobacter

[0036] Select the land area where Aristolochia spp. has been planted for many years, collect its rhizosphere soil samples, bag and store at low temperature. Weigh 0.5g of the above soil sample and add it to 250ml basic inorganic salt liquid culture medium, add 400μM aristolochic acid I at the same time, place it in a shaker, 30℃, 180r / min for enrichment culture. Depending on the degradation of aristolochic acid, transfer once every 2-4 weeks, with an inoculation amount of 1%. Dilute the enrichment solution of the fourth transfer and apply it to a tryptone soy broth plate, and culture it at 30℃ for 16h. Pick a single colony from the above plate for growth verification in a basic inorganic salt liquid culture medium (containing aristolochic acid), and preserve the bacteria that can grow for seed. After streaking and purification on the plate for more than 3 times, a single bacterium is obtained and stored for use.

[0037] (3) Strain characteristics and physiological and biochemical identification

[0038] The strain was Gram-positive and had no spores. After being cultured on a solid plate of tryptone soy broth at 30°C for 18 hours, the bacteria turned yellow and translucent. The colonies were round with a convex and smooth surface. Figure 1 .

[0039] The genomic DNA of strain NyZ610 was extracted and the 16S rRNA was amplified and sequenced. The amplification primer was 27F: 5-AGAGTTTGATCCTGGCTCAG-3

[0040] 1492R:5-GGTTACCTTGTTACGACTT-3

[0041] PCR amplification conditions were as follows: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, and 72°C for 90 s, for 30 cycles; and 72°C for 10 min.

[0042] The PCR product was sequenced for the first generation, and the sequencing results are shown in SEQ ID No. 1. The 16S rRNA sequence has been submitted to the NCBI database. Comparison with the NCBI Nucleotide collection (nr / nt) database revealed that NyZ610 belongs to the genus Arthrobacter. We named it Arthrobacter sp. NyZ610 and deposited it with the Guangdong Provincial Center for Microbial Culture Collection on July 21, 2021; the deposit address is Building 59, 100 Xianlie Middle Road, Guangzhou, China, with the deposit number GDMCC No: 61799.

[0043] Example 2 Degradation of aristolochic acid I by Arthrobacter sp. NyZ610

[0044] (1) The aristolochic acid degrading bacteria NyZ610 screened in Example 1 was inoculated into a solid plate of tryptone soy broth and cultured at 30°C for 10 h. A small amount of bacteria was scraped from the plate with an inoculating loop and inoculated into 5 mL of tryptone soy broth liquid culture medium and cultured at 30°C and 180 r / min for 8 h until the OD 600 The bacterial cell suspension was centrifuged at 5000 g for 5 min at 4°C to collect the cells. The cells were washed three times with sterilized basic inorganic salt liquid medium and set aside.

[0045] (2) The collected bacteria were resuspended in 100 μL of basic inorganic salt culture medium and inoculated into 5 mL of basic inorganic salt culture medium containing 200 μM aristolochic acid I. The initial inoculation OD 600 The concentration of aristolochic acid and OD were determined at 30℃ and 1800r / min. 600 values ​​and nitrite concentrations.

[0046] (3) The results are as follows Figure 2 As shown in Figure 2, within 35 h, aristolochic acid I was completely degraded by NyZ610, and a small amount of nitrite was detected, indicating that the degradation of aristolochic acid I under aerobic conditions was carried out through the oxidation pathway. At the same time, the accumulation of biomass was detected, and OD 600 The value increased from the initial 0.004 to about 0.02. These results indicate that NyZ610 can utilize aristolochic acid I as the sole carbon source, nitrogen source and energy source for growth.

[0047] Example 3 Degradation of Aristolochic Acid II by Arthrobacter sp. NyZ610

[0048] (1) The aristolochic acid degrading bacteria NyZ610 screened in Example 1 was inoculated into a solid plate of tryptone soy broth and cultured at 30°C for 10 h. A small amount of bacteria was scraped from the plate with an inoculating loop and inoculated into 5 mL of tryptone soy broth liquid culture medium and cultured at 30°C and 180 r / min for 8 h until the OD 600 The bacterial cell suspension was centrifuged at 5000 g for 5 min at 4°C to collect the cells. The cells were washed three times with sterilized basic inorganic salt liquid medium and set aside.

[0049] (2) The collected bacteria were resuspended in 100 μL of basic inorganic salt culture medium and inoculated into 5 mL of basic inorganic salt culture medium containing 200 μM aristolochic acid II. The initial inoculation OD 600 The concentration of aristolochic acid and OD were determined at 30℃ and 1800r / min. 600 values ​​and nitrite concentrations.

[0050] (3) The results are as follows Figure 3 As shown in the figure, after 50 h of growth, the degradation rate of aristolochic acid II in NyZ610 cells was 100%; the biomass accumulated significantly, and the OD 600 The value increased to 0.018.

[0051] Example 4 Degradation of aristolochic acid mixture by Arthrobacter sp. NyZ610

[0052] (1) The aristolochic acid degrading bacteria NyZ610 screened in Example 1 was inoculated into a solid plate of tryptone soy broth and cultured at 30°C for 10 h. A small amount of bacteria was scraped from the plate with an inoculating loop and inoculated into 5 mL of tryptone soy broth liquid culture medium and cultured at 30°C and 180 r / min for 8 h until the OD 600 The bacterial cell suspension was centrifuged at 5000 g for 5 min at 4°C to collect the cells. The cells were washed three times with sterilized basic inorganic salt liquid medium and set aside.

[0053] (2) The collected bacteria were resuspended in 100 μL of basic inorganic salt culture medium and inoculated into 5 mL of basic inorganic salt culture medium containing 200 μM aristolochic acid (100 μM aristolochic acid I and 100 μM aristolochic acid II). The initial inoculation OD 600 The concentration of aristolochic acid and OD were determined at 30℃ and 1800r / min. 600 values ​​and nitrite concentrations.

[0054] (3) The results are as follows Figure 4 As shown in the results, both aristolochic acid I and aristolochic acid II were completely degraded within 30 h, and the degradation rate of aristolochic acid I was faster.

[0055] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A strain of Arthrobacter ( Arthrobacter sp.) NyZ610, whose deposit number is GDMCC No: 61799.

2. Use of the Arthrobacter NyZ610 according to claim 1 in degrading aristolochic acid, wherein the aristolochic acid is one or both of aristolochic acid I and aristolochic acid II.

3. A method for degrading aristolochic acid, characterized in that: The Arthrobacter NyZ610 according to claim 1 is mixed and cultured with an aristolochic acid solution to degrade aristolochic acid under aerobic conditions; the aristolochic acid is one or both of aristolochic acid I and aristolochic acid II.

4. The method for degrading aristolochic acid according to claim 3, wherein: The method further comprises inoculating the seed liquid of the Arthrobacter NyZ610 according to claim 1 into a culture medium for enrichment culture; the culture medium is prepared by adding 6 g of tryptone soy broth powder to 1000 mL of distilled water, stirring and dissolving, and sterilizing at 120-125° C. for 15-20 minutes.

5. The method for degrading aristolochic acid according to claim 4, wherein: The method further comprises the steps of: (1) Centrifuge the enriched liquid obtained from the culture of claim 4 at 5000-8000 g for 5-10 min at 4°C to collect the bacterial cells, and wash the cells three times with sterilized basic inorganic salt liquid medium; (2) The collected bacteria were resuspended in basic inorganic salt culture medium, mixed with aristolochic acid solution and cultured to degrade aristolochic acid under aerobic conditions.

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