CN114369555A - Alcaligenes faecalis T16 and application thereof - Google Patents
Alcaligenes faecalis T16 and application thereof Download PDFInfo
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- CN114369555A CN114369555A CN202210098108.3A CN202210098108A CN114369555A CN 114369555 A CN114369555 A CN 114369555A CN 202210098108 A CN202210098108 A CN 202210098108A CN 114369555 A CN114369555 A CN 114369555A
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- alcaligenes faecalis
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- 241000588813 Alcaligenes faecalis Species 0.000 title claims abstract description 33
- 229940005347 alcaligenes faecalis Drugs 0.000 title claims abstract description 33
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- RUDATBOHQWOJDD-UZVSRGJWSA-N ursodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-UZVSRGJWSA-N 0.000 claims abstract description 25
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- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical compound O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses an alcaligenes faecalis T16 and application thereof. The strain is named Alcaligenes faecalis T16, is deposited in China center for type culture Collection at 11/17/2021, and has a preservation number of CCTCC NO: m20211431. The invention discovers an Alcaligenes faecalis T16 which can convert LCA into UDCA, has certain tolerance to high-concentration LCA and can meet the requirement of industrial application.
Description
Technical Field
The invention belongs to the field of ursodeoxycholic acid synthesis, and particularly relates to an alcaligenes faecalis T16 and application thereof.
Background
Lithocholic acid (LCA) is not directly biosynthesized from cholesterol in the liver, but is a substance produced by chenodeoxycholic acid through bacterial metabolism in the intestine, which is a secondary bile acid present in bile of higher vertebrates. This substance produced in the intestine is mostly excreted together with the feces, and a very small part is absorbed, and a binding reaction occurs in the liver, and it appears in bile in the form of taurolithocholic acid (taurolithocholic acid) or glycolithocholic acid (glycolithocholic acid). LCA is widely present in bile of animals such as pig, cattle and sheep, and can be extracted and separated from the bile of the animals, however, in ton of slaughter wastes, many bile acids such as LCA are still unavailable and are finally discarded. In addition, LCA is a less toxic important secondary bile acid, and its accumulation is a major factor in liver damage during cholestasis, and also a risk factor for colon cancer and inflammation. LCA has stronger toxicity than other bile acids, and a large amount of feed to experimental animals can cause various kinds of trouble of liver and biliary tract systems.
Ursodeoxycholic acid (UDCA) is an effective component of traditional Chinese medicines, is also a medicine approved by the U.S. Food and Drug Administration (FDA) as only one first-line clinical medicine for treating Primary Biliary Cirrhosis (PBC), has very wide clinical application and excellent medicinal value, and can regulate metabolism in the liver. Research results show that the biological functions of ursodeoxycholic acid in vivo mainly include the following aspects: capable of increasing low density lipoprotein binding; can stimulate proliferation of liver cells; can enhance the therapeutic effect of interferon on chronic viral hepatitis; can promote the receptor activation of glucocorticoid and inhibit the tumor initiation and the migration of tumor cells; has good curative effect in the aspects of treating gallstone, bile reflux gastritis, alcoholic liver, biliary cirrhosis, hepatitis induced by medicaments, promoting liver transplantation and the like, and has large market consumption. Ursodeoxycholic acid, originally mainly extracted from natural bear gall, but live bear resources are very limited and violate animal protection laws.
Therefore, it is very interesting to propose a new method for synthesizing UDCA from lithocholic acid.
Disclosure of Invention
The invention adopts LCA as a unique carbon source to screen an Alcaligenes faecalis T16 which is tolerant to LCA and can convert LCA into UDCA, wherein the Alcaligenes faecalis T16 is named as Alcaligenes faecalis T16, is preserved in China center for type culture collection at 11.17.2021, and has a preservation number of CCTCC NO: m20211431. The 16S rDNA gene sequence of the gene has the sequence shown in SEQ ID No: 1 is shown. When the concentration of LCA is 5g/L, the conversion rate is the highest and reaches 73.8%, and the strain can tolerate 10g/L of LCA and meets the requirement of industrial application.
The invention has the beneficial effects that: the invention discovers an Alcaligenes faecalis T16 which can convert LCA into UDCA, has certain tolerance to high-concentration LCA and can meet the requirement of industrial application.
Drawings
FIG. 1 shows a colony morphology of Alcaligenes faecalis T16.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments and the accompanying drawings to fully understand the objects, aspects and effects of the present invention.
Example 1:
first, screening and related experiments of Alcaligenes faecalis T16:
(1) screening a culture medium:
plate screening culture medium: CH (CH)3COONH3 5g/L、NaNO3 5g/L、LCA 1g/L、MgSO4·7H2O 0.5g/L、MnSO4·H2O 0.01g/L、Na2HPO4 25g/L、NaH2PO44.4g/L, Tween 801 mL/L and agar 20 g/L.
Transformation medium: CH (CH)3COONH3 5g/L、KCl2 2g/L、K2HPO4 2g/L、LCA 1g/L、MgSO4·7H2O 0.5g/L、CaCl2 0.5g/L、FeSO4·7H2O 0.01g/L、CuSO4·5H2O 0.01g/L、Na2MoO4·2H2O 0.01mg/L、FeSO4·7H2O0.01 mg/L and Tween 801 mL/L.
Fermentation medium: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride, 801 mL/L of tween and 1g/L of LCA.
Carbon source optimization culture medium: different carbon sources are 5g/L, tryptone 10g/L, sodium chloride 10g/L, tween 801 mL/L and LCA 1 g/L. The different carbon sources include lactose, soluble starch, glucose, beef extract, citric acid, sucrose and glycerol.
Carbon source optimization culture medium: 5g/L of different nitrogen sources, 5g/L of optimal carbon source, 10g/L of sodium chloride, 801 mL/L of tween and 1g/L of LCA. The different nitrogen sources include beef extract, peptone, urea, ammonium sulfate, diammonium phosphate, ammonium oxalate and diammonium hydrogen citrate.
(2) The screening method comprises the following steps:
1. weighing 10g of duck pond sludge in the Ruzhou city Linchuan river east countryside home village, putting the duck pond sludge into a triangular flask filled with 90mL of sterile water, putting the triangular flask into a shaking table, oscillating at 150rpm for 60min, taking out the duck pond sludge, and gradually diluting the duck pond sludge into 10-1、10-2、10-3、10-4、10-5、10-6The 6 concentrations of the diluted solution are respectively coated on a flat plate for screening, and the diluted solution is statically cultured for 3-6d at 27 ℃. A single colony on a selected plate is inoculated into a transformation medium culture medium and cultured for 14d at the temperature of 27 ℃ and the rpm of 200.
TCL detection LCA: the method comprises the following steps of 1: 1 volume of ethyl acetate extraction, run TCL for preliminary characterization. The chromatography agent is dichloromethane: acetone: glacial acetic acid 20: 10: 1, color developing agent: 4.5% phosphomolybdic acid.
3. Amplification experiment: all possible strains were inoculated into a tube containing 5mL of fermentation medium at 27 ℃ for 2 days at 200rpm, and the cells were collected by centrifugation (50mL centrifuge tube) and washed with PBS.
Each centrifuge tube (with cells) was filled with 40mL of transformation medium, mixed well, and cultured at 27 ℃ for 14 days at 200 rpm.
Dividing 40mL of liquid into two parts, extracting with ethyl acetate with the same volume, drying, dissolving with chromatographic pure methanol, and detecting with liquid chromatography.
4. Liquid chromatography detection of UDCA: c18 column, methanol: phosphoric acid aqueous solution (pH 2) 80: 20, flow rate: 0.8mL/min, wavelength: 210nm, column temperature: 35 ℃ is carried out.
5. Optimizing fermentation conditions: different carbon sources, nitrogen sources, temperatures (15-40 ℃) and pH values (3-10) are optimized under different fermentation conditions, and the optimized results are detected by liquid chromatography.
6. Optimizing transformation conditions: optimizing transformation conditions in transformation media with different temperatures (20-60 ℃) and different pH values (3-10), and detecting the optimized result by liquid chromatography.
7. The effect of varying concentrations of LCA on the conversion of LCA by Alcaligenes faecalis T16 to UDCA.
Second, result in
(1) LCA is used as a unique carbon source to screen a bacterial strain Alcaligenes fa eclalis T16 capable of converting LCA to obtain UDCA, wherein the bacterial colony is circular and light yellow, the surface of the bacterial colony is smooth, wet and slightly glossy, the middle of the bacterial colony is convex, the bacterial colony is opaque, the edge of the bacterial colony is neat, the texture of the bacterial colony is thick, gram staining is negative, and the morphology of the bacterial colony is shown in figure 1.
The strain can not ferment saccharides, has positive reaction of oxidase and catalase, has negative reaction of indole test, M-R test, V-P test, gelatin liquefaction test and amylase test, can utilize nitrate and nitrite, meets the description of the Alcaligenes in Bergey's Manual of bacteria identification, and is preliminarily identified as Alcaligenes. Extracting T16 genome, wherein the 16S rDNA gene sequence of the T16 genome is SEQ ID No: 1, and the following components:
AGTATCCCCCGTGGTAGCGCCCTCCTTACGGTTAGGCTACCTACTTCTGGTGAAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGACATTCTGATCCGCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGGTTTCTGAGATTGGCTCCCCCTCGCGGGTTGGCGACCCTCTGTCCCGACCATTGTATGACGTGTGAAGCCCTACCCATAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCTCTTGCGTAGCAACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTTCCcGGTTCTCtTGCGAGCACGGCCAAATCTCTTCGGCTTTCCAGACATGTCAAGGGTAGGTAAGGTTTTtCGCGTTGCATCGAATTAATcCACATCATCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGTCAACTTCACGCGTTAGCTGCGCTACTAAGGCCTAACGGCCCCAACAGCTAGTTGACATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGTCTGAGCGTCAGTATTATCCCAGGGGGCTGCCTTCGCCATCGGTATTCCTCCACATATCTACGCATTTCACTGCTACACGTGGAATTCTACCCCCCTCTGACATACTCTAGCTCGGCAGTTAAAAATGCAGTTCCAAGGTTGAGCCCTGGGATTTCACATCTTTCTTTCCGAACCGCCTACACACGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATtCTGCAGATACCGTCAGCAGTACCTCGTATTAGGAGGTACCTTTTCTTCTCTGCCAAAaGTACTTTACAACCCGAAGGCcTTCATCATACACGCGGGATGGCTGGATCAGGGTTTCCCCCATTGTCCAAAATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCTGGTCGTCCTCTCAAACCAGCTACGGATCGTTGCCTTGGTGAGCCTTTACCCCACCAACTAGCTAATCCGATATCGGCCGCTCCAATAGTGAGAGGTCTTGCGATCCCCCCCTTTCCCCCGTAGGGCGTATGCGGTATTAGCCACTCTTTCGAGTAGTTATCCCCCGCTACTGGGCACGTTCCGATATATTACTCACCCGTCCGCCACTCGCCGCCAAGAGAGCAAGCTCTCTCGCGCTGCCGTTCGACTGCATGTTAAAGCTGCCCAAGG。
the universal forward primer BSF8(5 '-agaggtttgatcctggcctcag-3') and the universal reverse primer BSR1492(5 '-ggctact tagagactt-3') were PCR amplified, and the BLAST result at NCBI after sequencing was Alcaligenes faecalis, so the name is: alcaligenes faecalis T16.
(2) The effect of different carbon sources on the biomass of Alcaligenes faecalis T16 and the conversion of LCA to UDCA:
the fermentation medium takes peptone 10g/L as a nitrogen source, and the following carbon sources of lactose, starch, glucose, beef extract, citric acid, sucrose and glycerol are respectively added according to the concentration of 5 g/L. The results are shown in Table 1. As can be seen from table 1: the carbon source has obvious influence on the biomass of the thalli and has larger influence on the conversion rate, when glucose and sucrose are used as the carbon source, the thalli grow vigorously, the biomass is higher, the conversion rates respectively reach 73.1 percent and 73.8 percent, and the optimal carbon source is sucrose.
TABLE 1
(3) The effect of different nitrogen sources on the biomass of Alcaligenes faecalis T16 and the conversion of LCA to UDCA:
the enzyme production medium takes 5g/L glucose as a carbon source, and the following nitrogen sources are respectively added according to 10 g/L: beef extract, peptone, urea, ammonium sulfate, diammonium hydrogen phosphate, ammonium oxalate and diammonium hydrogen citrate. The cells cultured at 27 ℃ and 220rpm/min for 2 days were subjected to transformation experiments. The results are shown in Table 2. As can be seen from Table 2, the nitrogen source has a large influence on both biomass and conversion rate of the cells, wherein the cells grow vigorously when peptone and ammonium sulfate are used as carbon sources, the biomass is relatively high, the conversion rates reach 71.9% and 73.2%, respectively, and the optimal nitrogen source is peptone.
TABLE 2
(4) The effect of different culture temperatures on the biomass of Alcaligenes faecalis T16 and the conversion of LCA to UDCA:
temperature is an important factor influencing cell growth and enzyme production by fermentation, influences of different fermentation temperatures on the yield and the conversion rate of thalli of the Alcaligenes faecalalis T16 strain are examined, and as a result, the temperature has certain influences on the biomass and the conversion rate, when the culture temperature of thalli is 30 ℃, the biomass and the conversion rate are the highest, the conversion rate reaches 73.5%, so that 30 ℃ is taken as the optimal fermentation temperature, and the table 3 shows that the biomass and the conversion rate are optimal.
TABLE 3
(5) Effect of initial medium pH on Alcaligenes faecalis T16 biomass and conversion of LCA to UDCA:
the growth of the cells and the activity of various enzymes of the cells are regulated by the initial pH of the medium. To examine the effect of initial pH on biomass of Alcaligenes faecalis T16 and conversion of LCA to UDCA. The fermentation medium was adjusted to different pH values, and the cells obtained by the culture were subjected to LCA transformation to obtain UDCA. As a result, all the microbial cells grew in the medium at pH4.0 to 10.0, the biomass and the conversion rate were highest at pH7, and the conversion rate reached 73.8%, as shown in Table 4.
TABLE 4
(6) Influence of transformation temperature on the biomass of Alcaligenes faecalis T16 and the conversion of LCA to UDCA:
the results of the effect of the conversion temperature on the conversion of LCA to UDCA by Alcaligenes faecalis T16 are shown in Table 5, 30 ℃ being the optimum conversion temperature, and the conversion decreasing with increasing temperature above 30 ℃.
TABLE 5
(7) Effect of transformation medium pH on Alcaligenes faecalis T16 Biomass and transformation of LCA to UDCA:
the results of the effect of pH on the conversion of LCA to UDCA by Alcaligenes faecalis T16 are shown in Table 6. As can be seen from Table 6, the optimum pH of the transformation medium was 8, the catalytic effect was the best under slightly alkaline conditions, and too high or too low pH was detrimental to transformation.
TABLE 6
(8) The effect of different concentrations of LCA on the biomass of Alcaligenes faecalis T16 and the conversion of LCA to UDCA:
the results of the effect of varying concentrations of LCA on the conversion of LCA by Alcaligenes faecalis T16 to UDCA are shown in Table 7. As can be seen from Table 7, the highest conversion reached 73.8% at an LCA concentration of 5g/L, and the conversion decreased gradually with increasing LCA concentration when the LCA concentration exceeded 10g/L, but 10g/L of LCA was tolerated.
TABLE 7
The above description is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiment, and the present invention shall fall within the protection scope of the present invention as long as the technical effects of the present invention are achieved by the same means. The invention is capable of other modifications and variations in its technical solution and/or its implementation, within the scope of protection of the invention.
SEQUENCE LISTING
<110> institute of microbiology of academy of sciences of Jiangxi province
<120> Alcaligenes faecalis T16 and application thereof
<130> 2022
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1436
<212> DNA
<213> Alcaligenes faecalis T16
<400> 1
agtatccccc gtggtagcgc cctccttacg gttaggctac ctacttctgg tgaaacccac 60
tcccatggtg tgacgggcgg tgtgtacaag acccgggaac gtattcaccg cgacattctg 120
atccgcgatt actagcgatt ccgacttcac gcagtcgagt tgcagactgc gatccggact 180
acgatcgggt ttctgagatt ggctccccct cgcgggttgg cgaccctctg tcccgaccat 240
tgtatgacgt gtgaagccct acccataagg gccatgagga cttgacgtca tccccacctt 300
cctccggttt gtcaccggca gtctcattag agtgctcttg cgtagcaact aatgacaagg 360
gttgcgctcg ttgcgggact taacccaaca tctcacgaca cgagctgacg acagccatgc 420
agcacctgtg ttcccggttc tcttgcgagc acggccaaat ctcttcggct ttccagacat 480
gtcaagggta ggtaaggttt ttcgcgttgc atcgaattaa tccacatcat ccaccgcttg 540
tgcgggtccc cgtcaattcc tttgagtttt aatcttgcga ccgtactccc caggcggtca 600
acttcacgcg ttagctgcgc tactaaggcc taacggcccc aacagctagt tgacatcgtt 660
tagggcgtgg actaccaggg tatctaatcc tgtttgctcc ccacgctttc gtgtctgagc 720
gtcagtatta tcccaggggg ctgccttcgc catcggtatt cctccacata tctacgcatt 780
tcactgctac acgtggaatt ctacccccct ctgacatact ctagctcggc agttaaaaat 840
gcagttccaa ggttgagccc tgggatttca catctttctt tccgaaccgc ctacacacgc 900
tttacgccca gtaattccga ttaacgcttg caccctacgt attaccgcgg ctgctggcac 960
gtagttagcc ggtgcttatt ctgcagatac cgtcagcagt acctcgtatt aggaggtacc 1020
ttttcttctc tgccaaaagt actttacaac ccgaaggcct tcatcataca cgcgggatgg 1080
ctggatcagg gtttccccca ttgtccaaaa ttccccactg ctgcctcccg taggagtctg 1140
ggccgtgtct cagtcccagt gtggctggtc gtcctctcaa accagctacg gatcgttgcc 1200
ttggtgagcc tttaccccac caactagcta atccgatatc ggccgctcca atagtgagag 1260
gtcttgcgat cccccccttt cccccgtagg gcgtatgcgg tattagccac tctttcgagt 1320
agttatcccc cgctactggg cacgttccga tatattactc acccgtccgc cactcgccgc 1380
caagagagca agctctctcg cgctgccgtt cgactgcatg ttaaagctgc ccaagg 1436
Claims (4)
1. An Alcaligenes faecalis T16, wherein the Alcaligenes faecalis T16 is named as Alcaligenes faecalis T16, and is preserved in China center for type culture Collection (CCTCC NO) at 11.17.2021, with the preservation number of CCTCC NO: m20211431.
2. The alcaligenes faecalis T16 according to claim 1, wherein the 16S rDNA gene sequence is SEQ ID No: 1 is shown.
3. Use of the alcaligenes faecalis T16 according to any of claims 1-2 for converting lithocholic acid to ursodeoxycholic acid.
4. Use according to claim 3, characterized in that the concentration of lithocholic acid is 5 g/L.
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| CN112852652A (en) * | 2021-01-15 | 2021-05-28 | 江南大学 | Recombinant yeast strain for efficiently converting chenodeoxycholic acid to synthesize ursodeoxycholic acid, construction and application |
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| CN110699297A (en) * | 2019-11-12 | 2020-01-17 | 中国石油集团川庆钻探工程有限公司 | Alcaligenes faecalis phenol subspecies and application thereof |
| CN112852652A (en) * | 2021-01-15 | 2021-05-28 | 江南大学 | Recombinant yeast strain for efficiently converting chenodeoxycholic acid to synthesize ursodeoxycholic acid, construction and application |
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| 代同成等: "片烟产果胶酶细菌的鉴定及酶活测定", 微生物学通报, vol. 38, no. 6, pages 816 - 824 * |
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