Rhodococcus abyssinus N106, pantothenic acid synthase produced by rhodococcus

Abstract

The invention relates to rhodococcus abyssinus N106, pantothenic acid synthetase produced by the rhodococcus abyssinus N106 and application of the pantothenic acid synthetase, and belongs to the technical field The strain is preserved in the China Center for Type Culture Collection on December 26, 2024, and the preservation number of the strain is CCTCC M 20242905. The method comprises the following steps: inoculating rhodococcus N106 into a seed culture medium, culturing for 18-24 hours at the constant temperature of 20-35 DEG C and 180 r / min in a shaking table, then inoculating into a fermentation culture medium according to a volume ratio of 5-10%, culturing for 24-48 hours at the constant temperature of 20-35 DEG C in the shaking table, centrifugally collecting thalli, crushing the thalli after resuspending, and centrifuging to obtain supernate, namely crude pantothenic acid synthase. D-calcium pantothenate and D-panthenol can be prepared by using the crude enzyme solution.

Description

Technical Field

[0001] The invention relates to a deep-sea rhodococcus N106 and a pantothenate synthase produced by the same and applications thereof, belonging to the technical field of marine microorganisms. Background Art

[0002] Pantothenic acid, also known as vitamin B5 or pantothenic acid, is a water-soluble vitamin and an important precursor for the biosynthesis of coenzyme A (CoA) and acyl carrier protein (ACP), both of which play an important role in cellular processes such as energy metabolism and fatty acid production. The chemical structure of pantothenic acid shows instability in the face of acidic or alkaline environments, light and high temperatures, so its more stable form, calcium D-pantothenate, is commonly seen on the market. With the gradual deterioration of the environment and global warming, people's living environment is facing serious threats. The country has gradually begun to control the production of products that are harmful to the environment, so the industrial production of D-pantothenic acid will be extremely difficult.

[0003] The chemical synthesis method of panthenol is to react D-pantoic acid lactone with 3-amino-1-propanol in ethanol as a solvent, heat and concentrate under reduced pressure to obtain D-panthenol. This process reaction requires ethanol as a solvent. Since D-panthenol is a viscous liquid, reduced pressure concentration often cannot completely remove the reaction solvent ethanol. At the same time, the product color is yellow and the optical rotation value is low. The above cannot meet the requirements of medicinal D-panthenol, especially injections containing D-panthenol, which have higher quality requirements such as purity.

[0004] The bio-enzymatic method has mild conditions, no excessive by-products, simple product separation, is environmentally friendly, and reduces production costs. It provides research ideas and foundations for the industrial production of D-pantothenic acid and D-panthenol by bio-methods.

[0005] Marine microorganisms have significant advantages as enzyme-producing microbial resources due to their unique living environment and environmental adaptability: pressure resistance, alkali resistance, salt resistance, cold resistance, species diversity, easy regulation of metabolism, high activity at low temperatures, etc., which give marine microbial enzymes a wide range of application prospects. Therefore, screening strains with high production of pantothenate synthase from the marine environment is the key to the future research and development of new pantothenate synthases. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a new deep-sea Rhodococcus N106 capable of producing pantothenate synthase and the pantothenate synthase produced by the present invention and its application in view of the deficiencies in the prior art.

[0007] Another technical problem to be provided by this patent is to provide a method and application of the pantothenate synthase produced by the above-mentioned Rhodococcus N106 from the deep sea.

[0008] The problem to be solved by the present invention is achieved through the following technical solutions:

[0009] A Rhodococcus sp. N106 from the deep sea, whose strain preservation number is CCTCC M20242905.

[0010] The strain N106 involved in the present invention is a Rhodococcus sp. N106 isolated and screened from deep-sea sediments in the Mariana Trench. The full 16S rDNA sequence is shown in SEQ ID No: 1, with a total length of 1513 bp. According to the similarity comparison of its 16SrDNA sequence, the 16SrDNA of N106 has 99% homology with Rhodococcus cerastii, and is identified as a bacterium of the genus Rhodococcus, which is a new strain of Rhodococcus. The strain has been deposited in the China Center for Type Culture Collection on December 26, 2024, and the deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, Tel: (027)-68752319.

[0011] The present invention also discloses a method for producing pantothenate synthase from Rhodococcus sp. N106 from the deep sea as described in the above technical scheme, wherein the method comprises the following steps: inoculating Rhodococcus sp. N106 into a seed culture medium (pH=7.0) that has been sterilized at high temperature, culturing the culture medium at 20-35°C and 180 r / min on a shaking table for 18-24 hours, taking the culture solution at a volume ratio of 5-10% and adding it to a fermentation culture medium, culturing the culture medium at 20-35°C on a shaking table for 24-48 hours, collecting the bacterial bodies by centrifugation, crushing the bacterial bodies after resuspending, and centrifuging to obtain a supernatant, which is a crude enzyme solution of pantothenate synthase.

[0012] Furthermore, the seed culture medium is: 1000 ml of distilled water containing 5 g of yeast extract, 10.0 g of sodium chloride, and 10.0 g of tryptone, with a pH of 6.8-7.2.

[0013] The fermentation medium is: 5.0 g of tryptone, 5.0 g of yeast extract, 5.0 g of sucrose, and 5.0 g of NaCl in 1000 ml of distilled water, pH 6.8-7.2.

[0014] The invention also discloses a pantothenate synthase from Rhodococcus sp. N106 from the deep sea. The optimum reaction temperature of the pantothenate synthase is 35° C. and the optimum reaction pH is 8.0.

[0015] The present invention also discloses an application of using the pantothenate synthetase to prepare D-calcium pantothenate. The reaction system comprises 25mM sodium D-pantothenate, 25mM alanine, 4.5mM adenosine triphosphate (ATP), 10mM MgCl2, 15mM KCl, and 1% by volume of a crude pantothenate synthetase enzyme solution. The reaction system is placed at 30-50°C for a full reaction of 1-5h. At the end of the reaction, 1M HCl is added to terminate the reaction. After the reaction is completed, Ca(OH)2 is added. After centrifugation and filtration, a D-calcium pantothenate reaction solution can be obtained.

[0016] The present invention also discloses an application of using the pantothenate synthase to prepare D-panthenol, wherein the reaction system comprises 25mM sodium D-pantothenate, 25mM 3-amino-1-propanol, 4.5mM ATP, 10mM MgCl2, 15mM KCl, and 1% by volume of a crude pantothenate synthase solution; the reaction system is placed at 30-50°C for sufficient reaction for 1-5h to obtain a D-panthenol reaction solution.

[0017] The beneficial effects of the present invention compared with the prior art are as follows: the present invention provides a new Rhodococcus strain N106 that is screened from Mariana Trench sediment samples and has a high production of pantothenate synthase, and discloses an enzyme production method. The strain is the first reported Rhodococcus microorganism with pantothenate synthase production activity, and the present invention effectively broadens the source of pantothenate synthase. The enzyme produced by the present invention has better enzyme properties, can synthesize both D-panthenol and D-calcium pantothenate, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the optimal reaction temperature diagram of pantothenate synthase;

[0019] Figure 2 This is the optimal reaction pH diagram of pantothenate synthase;

[0020] Figure 3 It is the liquid phase diagram of D-calcium pantothenate reaction solution;

[0021] Figure 4 It is the liquid phase diagram of D-panthenol reaction solution;

[0022] Figure 5 This is the phylogenetic tree of N106 bacteria. DETAILED DESCRIPTION

[0023] The specific technical scheme of the present invention is further described below in conjunction with specific examples, so that those skilled in the art can further understand the present invention, but it does not constitute a limitation on the rights thereof. The culture medium used in the embodiments is:

[0024] Liquid seed culture medium: 1.0% tryptone, 0.5% yeast extract, 1.0% sodium chloride, the above percentages are all mass percentages, and the remaining ingredients are water;

[0025] Liquid fermentation medium: 0.5% tryptone, 0.5% yeast extract, 0.5% sucrose, 0.5% sodium chloride, the above percentages are all mass percentages, and the remaining component is water.

[0026] Example 1: A Rhodococcus sp. N106 obtained by screening deep-sea sediment samples from the Mariana Trench. The screening method is as follows: weigh 3 g of the Mariana Trench deep-sea sediment sample and put it into 50 ml of liquid seed culture medium, and culture it at 25°C and 180 r / min for 1-3 days. Select a suitable dilution of the culture solution and apply it to a solid seed culture medium (liquid seed culture medium with 3% agar), culture it at 25°C for 1-2 days, pick a single colony after the colony grows, inoculate it into a liquid fermentation medium, culture it at 25°C for 1-2 days, collect the bacteria by centrifugation, crush the bacteria after resuspending, and measure the pantothenate synthase activity. Select a strain with higher enzyme activity and name it N106. The genome of N106 was extracted using a bacterial genome extraction kit, and the universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'SEQ ID NO.2 and 1492R: 5'-GGTTACCTTGTTACGACTT-3'SEQ ID NO.3) for amplifying the 16S rDNA sequence of prokaryotes were used for polymerase chain reaction. The PCR reaction solution (25 μL) consisted of: 2×EasyTaq PCR SuperMix (-dye): 1 μL; Primer 1 (27F): 1 μL; Primer 2: (1492R): 1 μL; Sample DNA: 1 μL; ddH2O: 21 μL. Reaction conditions: 94℃ pre-denaturation for 5 min; (94℃, 30 sec; 60℃, 30 sec; 72℃, 90 sec) for 35 cycles; 72℃ for 10 min. The PCR product was purified by agarose electrophoresis and sent to Shanghai Biotechnology for sequencing. The complete 16S rDNA sequence is shown in SEQ ID No: 1. Figure 5 The 16SrDNA of R19 strain has high homology with other strains of the genus Rhodococcus, and it was identified as a bacterium of the genus Rhodococcus, and is a new strain of Rhodococcus. The strain is deposited in the China Center for Type Culture Collection with the deposit number CCTCC M20242905, and the deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China.

[0027] The strain has the following specific characteristics: the strain is a Gram-positive bacillus with convex light yellow colonies; catalase negative; can utilize arabinose, fructose, galactose, glucose, maltose, sucrose and mannitol; cellobiose, mannose and xylose are not utilized. The strain has a growth temperature range of 10-50°C, with an optimum growth temperature of 30°C; a pH growth range of 6-11, with an optimum growth pH of 7.

[0028] Example 2: A method for producing pantothenate synthase using Rhodococcus sp. N106 from the ocean described in Example 1: inoculate Rhodococcus sp. N106 into a liquid seed culture medium with a liquid volume ratio of 20%, and culture at 30°C and 180 r / min for 24 hours to obtain a seed solution; inoculate the seed solution into a liquid fermentation culture medium with an inoculation volume ratio of 8% and a liquid volume ratio of 20%, and culture at 30°C and 180 r / min for 48 hours, collect the cells by centrifugation, crush the cells after resuspending, and centrifuge to obtain a supernatant, which is a crude pantothenate synthase enzyme solution.

[0029] Example 3: Optimal reaction temperature and pH of pantothenate synthase

[0030] Optimum reaction temperature: The reaction system contains 25mM sodium D-pantothenate, 25mM 3-amino-1-propanol, 4.5mM ATP, 10mM MgCl2, 15mM KCl, and 1% pantothenate synthase crude enzyme solution. Reaction conditions: set the reaction at 20℃, 25℃, 30℃, 35℃, 37℃, 40℃, 50℃, and 60℃ for 20min, and set three groups of parallel at each temperature. After the reaction, add 1M HCI to terminate the reaction, centrifuge at 12000rpm for 10min, and measure the relative enzyme activity.

[0031] Optimum reaction pH: The reaction system contains 25mM sodium D-pantothenate, 25mM 3-amino-1-propanol, 4.5mM ATP, 10mM MgCl2, 15mM KCl, and the volume ratio of crude pantothenate synthase is 1%. Reaction conditions: React for 30min at pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and set three groups of parallel for each pH. After the reaction, add 1M HCI to terminate the reaction, centrifuge at 12000rpm for 10min, and measure the relative enzyme activity.

[0032] The results showed that the optimum reaction temperature was 35°C and the optimum reaction pH was 8.0. Figure 1 , Figure 2 .

[0033] Example 4: A method for preparing D-pantothenate calcium by converting crude pantothenate synthase liquid produced by Rhodococcus N106, the steps are as follows: the reaction system contains 25mM sodium D-pantothenate, 25mM alanine, 4.5mM ATP, 10mM MgCl2, 15mM KCl, and the volume ratio of crude pantothenate synthase liquid is 1%. The reaction system is placed at 30-50°C for full reaction for 60min, and 1M HCI is added to terminate the reaction. After the reaction is completed, 100μL 25mM Ca(OH)2 is added and centrifuged at 12000rpm for 10min. After filtering with a 0.22μm filter, the D-pantothenate calcium reaction liquid can be obtained.

[0034] Example 5: A method for preparing D-panthenol by converting crude pantothenate synthase solution produced by Rhodococcus sp. N106, wherein the reaction system comprises 25 mM sodium D-pantothenate, 25 mM 3-amino-1-propanol, 4.5 mM ATP, 10 mM MgCl2, 15 mM KCl, and the volume ratio of crude pantothenate synthase solution is 1%. The reaction system is placed at 30-50° C. to fully react for 120 min, and 1 M HCI is added to terminate the reaction. After filtering with a 0.22 μm filter, the D-panthenol reaction solution can be obtained.

[0035] Liquid chromatography detection conditions: The ultraviolet detector equipped with Shimadzu high performance liquid chromatograph was set to a wavelength of 210 nm and Hypersil GOLD TM Chromatographic column (4.6×250 mm). The mobile phase is acetonitrile: 20 mM potassium dihydrogen phosphate = 10:90, the flow rate is 1 mL / min. The column temperature is controlled at 25°C, and the injection volume is 10 μL.

[0036] The HPLC spectrum of the D-calcium pantothenate reaction solution is shown in Figure 1. The peak time of the product D-pantothenate calcium is consistent with that of the standard product, both at 5.42, and the peak time of the product D-panthenol is consistent with that of the standard product, both at 5.04. Figure 3 As shown, the HPLC spectrum of D-panthenol reaction solution is as follows Figure 4 As shown in the chromatogram, it can be seen that sodium D-pantothenate, β-alanine and 3-amino-1-propanol produce D-pantothenate calcium and D-panthenol under the action of pantothenate synthase.

[0037] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

[0038] 16S rDNA of Rhodococcus sp. N106 (SEQ ID NO.1):

[0039] TTTTACGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTT

[0040] AACACATGCAAGTCGAGCGGTAAGGCCTTTCGGGGTACACGAGCGGCGA

[0041] ACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTCTGGGATAAGCTTG

[0042] GGAAACTGGGTCTAATACCGGATATGACCGCATGCCGCATGGTGTGTGGT

[0043] GGAAAGATTTATCGGTGCAGGATGGGCCCGCGGCCTATCAGCTTGTTGGT

[0044] GGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTG

[0045] ACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAG

[0046] CAGTGGGAATATTGCACAATGGGCGGAAGCCTGATGCAGCGACGCCGCG

[0047] TGAGGGATGAAGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGACGAAGC

[0048] GTGAGTGACGGTACCTGCAGAAGAAGCACCGGCTAACTACGTGCCAGCA

[0049] GCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAA

[0050] AGAGTTCGTAGGCGGTTTGTCGCGTCGTTTGTGAAAACCCGGGCTCAACT

[0051] TCGGGCTTGCAGGCGATACGGGCAGACTTGAGTGTTTCAGGGGAGACTGG

[0052] AATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGT

[0053] GGCGAAGGCGGGTCTCTGGGAAACAACTGACGCTGAGGAACGAAAGCGT

[0054] GGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTG

[0055] GGCGCTAGGTGTGGGTTCCTTCCACGGGATCTGTGCCGTAGCTAACGCAT

[0056] TAAGCGCCCCGCCTGGGGTACGGCCGCAAGGCTAAAACTCAAAGGAATT

[0057] GACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAAC

[0058] GCGAAGAACCTTACCTGGGTTTGACATACACCGGAAAACCGTAGAGATA

[0059] CGGTCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTC

[0060] GTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTT

[0061] ATGTTGCCAGCGCGTTATGGCGGGGACTCGTAAGAGACTGCCGGGGTCAA

[0062] CTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCCAG

[0063] GGCTTCACACATGCTACAATGGCCAGTACAGAGGGCTGCGAGACCGTGA

[0064] GGTGGAGCGAATCCCTTAAAGCTGGTCTCAGTTCGGATCGGGGTCTGCAA

[0065] CTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTG

[0066] CGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAA

[0067] GTCGGTAACACCCGAAGCCGGTGGCCTAACCCCTCGTGGGAGGGAGCCG

[0068] TCGAAGGTGGGATCGGCGATTGGGACGAAGTCGTAACAAGGTAGCCGTA

[0069] CCGGAAGGTGCGGCTGGATCACCTCCT。

Claims

1. A Rhodococcus N106 from the deep sea, characterized in that: The Rhodococcus N106 was deposited in the China Center for Type Culture Collection on December 26, 2024, with the deposit number CCTCC M 20242905.

2. A method for producing pantothenate synthase using the deep-sea Rhodococcus N106 according to claim 1, characterized in that: The method comprises the following steps: inoculating red coccus N106 into a seed culture medium, culturing the culture medium at a constant temperature of 20-35°C and 180 r / min on a shaking table for 18-24 hours, then inoculating the culture medium at a volume ratio of 5-10%, culturing the culture medium at a constant temperature of 20-35°C on a shaking table for 24-48 hours, collecting the bacterial bodies by centrifugation, crushing the bacterial bodies after resuspending them, and centrifuging to obtain a supernatant, which is a crude pantothenate synthase enzyme solution.

3. The method according to claim 2, characterized in that The seed culture medium is: 1000 ml of distilled water containing 5 g of yeast extract, 10.0 g of sodium chloride, and 10.0 g of tryptone, with a pH of 6.8-7.

2.

4. The method according to claim 2, characterized in that: The fermentation medium is: 5.0 g of tryptone, 5.0 g of yeast extract, 5.0 g of sucrose, and 5.0 g of NaCl in 1000 ml of distilled water, with a pH of 6.8-7.

2.

5. A pantothenate synthetase, characterized in that the pantothenate synthetase is derived from the Rhodococcus sp. N106 according to claim 1, and the optimal reaction temperature of the pantothenate synthetase is 35°C and the optimal reaction pH is 8.

0.

6. Use of the pantothenate synthase according to claim 5 in the preparation of calcium D-pantothenate, characterized in that: The reaction system in the application comprises 25 mM sodium D-pantothenate, 25 mM alanine, 4.5 mM adenine nucleoside triphosphate, 10 mM MgCl2, 15 mM KCl, and 1% by volume of the crude enzyme solution of pantothenate synthetase described in claim 5; the reaction system is placed at 30-50°C to fully react for 1-5 hours, 1 M HCl is added at the end of the reaction to terminate the reaction, and Ca(OH)2 is added after the reaction is completed. After centrifugation and filtration, a reaction solution containing calcium D-pantothenate is obtained.

7. Use of the pantothenate synthase according to claim 5 in the preparation of D-panthenol, characterized in that: The reaction system in the application comprises 25 mM sodium D-pantothenate, 25 mM 3-amino-1-propanol, 4.5 mM ATP, 10 mM MgCl2, 15 mM KCl, and 1% by volume of the crude enzyme solution of pantothenate synthase according to claim 5; the reaction system is placed at 30-50° C. for a full reaction of 1-5 hours to obtain a reaction solution containing D-panthenol.

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