Brevibacterium abyssinus R19 capable of producing D-pantolactone hydrolase and application of brevibacterium abyssinus R19
By screening out Brebacterium deep-sea sediments from Mariana Trench and preparing its D-panolactone hydrolase through a dual culture medium method, the problems of high cost, high pollution and difficult to remove impurities in the prior art were solved, and efficient and low-cost D-panolactone production was achieved.
Patent Information
- Application Number
- CN202411943975.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-04-11
AI Technical Summary
The prior art has problems such as high cost of reagent separation, difficulty in separation, and high pollution when producing D-pantothenic acid, and direct fermentation is difficult to remove impurities, which limits industrial applications.
A strain of Brebacterium Deep-sea sediment samples was screened from the Mariana Trench. This strain was able to produce D-panolactone hydrolase. The highly active D-panolactone hydrolase was prepared by dual culture methods of liquid seed medium and liquid fermentation medium.
This method effectively broadens the source of D-panolactone hydrolase, reduces production costs, and improves the purity of the product and the potential for industrial application.
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Figure CN119799562A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a strain of deep-sea brevibacterium R19 producing D-pantolactone hydrolase and application thereof, belonging to the technical field of marine microorganisms. Background Art
[0002] D-pantolactone is an important intermediate for the preparation of D-pantothenic acid and D-panthenol. It is also a direct precursor for the synthesis of coenzyme A in the body. Pantothenic acid, also known as vitamin B5, is a water-soluble vitamin that has been shown to be closely related to energy metabolism, immune regulation, neurotransmitter synthesis, intestinal health, etc. Since pantothenic acid is unstable, it is mainly sold in the form of calcium pantothenate on the market and is widely used in feed, cosmetics, medicine and other industries. At present, the global annual demand for pantothenic acid is about 20,000 tons.
[0003] At present, the synthesis of D-pantothenic acid mainly includes chemical method, direct fermentation method and microbial enzyme method. The earliest chemical method production adopts Stiller method, i.e. isobutyraldehyde-formaldehyde-sodium cyanide method, or adopts glyoxylic acid-isobutyraldehyde method to synthesize pantothenic acid lactone, and then directly condenses calcium β-alanine with pantothenic acid lactone to obtain DL-pantothenic acid calcium. This method has problems such as high reagent separation cost, difficult separation, and large pollution, and there is no report of large-scale production at present. Because the pantothenic acid produced by direct fermentation is mixed, it is difficult to remove impurities by ion exchange, crystallization, etc., and it is also difficult to apply to industrial production. At present, the large-scale production examples are basically produced by microbial enzyme method, and the most important one is the production of precursor substance D-pantothenic acid lactone.
[0004] Due to the extreme living environment, deep-sea microorganisms have significant advantages as enzyme-producing microbial resources: pressure resistance, alkali resistance, salt resistance, cold resistance, and high activity at low temperatures, which give deep-sea microbial enzymes a wide range of application prospects. Therefore, screening strains that can selectively hydrolyze D-pantolactone from the deep-sea environment is the key to the future research and development of new D-pantolactone hydrolases. Summary of the invention
[0005] The technical problem to be solved by the present invention is to provide a deep-sea Brevibacterium R19 which is derived from a deep-sea sediment sample of the Mariana Trench and can produce D-pantolactone hydrolase in view of the deficiencies of the prior art.
[0006] Another technical problem to be provided by the present invention is to provide a method and application of the D-pantoic acid lactone hydrolase produced by the deep-sea brevibacterium.
[0007] The present invention is achieved through the following technical solutions:
[0008] A strain of Brevibacterium sp. R19 producing D-pantolactone hydrolase, whose strain preservation number is CCTCC NO: M20242906.
[0009] The strain R19 involved in the present invention is Brevibacterium sp. R19 isolated and screened from deep-sea sediments in the Mariana Trench. The strain was deposited in the China Center for Type Culture Collection on December 26, 2024. The deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, Tel: (027)-68752319.
[0010] The invention also discloses D-pantoic acid lactone hydrolase produced by Brevibacterium sp. R19.
[0011] The present invention also provides a preparation method of the D-pantoic acid lactone hydrolase, which comprises inoculating Brevibacterium R19 into a liquid seed culture medium, culturing at 30-37°C and a shaking constant temperature for 18-24 hours, taking the culture solution and inoculating it into a liquid fermentation culture medium at a volume ratio of 5-10% of the inoculation amount, culturing at a shaking constant temperature of 25-30°C for 40-50 hours, and centrifuging to obtain a supernatant, which is a crude enzyme solution of the D-pantoic acid lactone hydrolase.
[0012] Further, liquid seed culture medium: 1% tryptone, 0.5% yeast extract, 1% sodium chloride, the above percentages are all mass percentages, and the remaining components are water;
[0013] Liquid fermentation medium: 0.5% soluble starch, 1.0% tryptone, 1.0% potassium chloride, the above percentages are all mass percentages, and the remaining component is water.
[0014] The present invention also provides an application of Brevibacterium sp. R19. The application method is to use D-pantoic acid lactone as a substrate, add fermentation liquid of Brevibacterium sp. R19, and place the reaction system at 35-42° C. for sufficient reaction for 5 h-7 h to obtain an enzymatic hydrolyzate containing D-pantoic acid.
[0015] The beneficial effects of the present invention compared with the prior art are as follows:
[0016] The invention provides a new deep-sea microbial strain Brevibacterium R19 that produces D-pantolactone hydrolase, which is screened from a Mariana Trench sediment sample, and discloses a method for producing the enzyme. The strain is the first reported microorganism of the genus Brevibacterium that has the activity of producing D-pantolactone hydrolase, and the invention effectively broadens the source of D-pantolactone hydrolase. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 Fermentation carbon source optimization diagram;
[0018] Figure 2 Fermentation nitrogen source optimization diagram;
[0019] Figure 3 Fermentation inorganic salt optimization diagram;
[0020] Figure 4 Liquid phase diagram of D-pantolactone standard;
[0021] Figure 5 Liquid phase diagram of D-pantoic acid reaction solution;
[0022] Figure 6 Phylogenetic tree of Brevibacterium R19. 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] Solid screening medium: 1% D-pantolactone, 1% NaCl, 2% agar, the above percentages are all weight percentages, and the remaining components are water;
[0025] 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;
[0026] Liquid fermentation medium: 0.5% soluble starch, 1.0% tryptone, 1.0% potassium chloride, the above percentages are all mass percentages, and the remaining component is water.
[0027] Example 1
[0028] Weigh 2g of Mariana Trench deep-sea sediment sample and put it into 50ml artificial seawater, shake at 200r / min for 1h. Take 100μL of dilution to spread on solid screening medium (3% agar D-pantoic acid lactone screening medium), culture at 30℃ for 3-5d, pick single colony after colony grows, inoculate in liquid fermentation medium, culture at 30℃ for 1-2d, centrifuge to take supernatant, perform esterase activity determination, select strain with higher enzyme activity, named R19. Use bacterial genome extraction kit to extract R19 genome, select universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'SEQ ID NO.2 and 1492R: 5'-GGTTACCTTGTTACGACTT-3'SEQ ID NO.3) for amplifying prokaryotic microorganism 16S rDNA sequence for PCR reaction. The PCR reaction solution (25 μL) was composed of: 2×EasyTaq PCR SuperMix (-dye): 1 μL; Primer 1 (27F): 1 μL; Primer 2: (1492R): 1 μL; Sample DNA: 1 μL; ddH 2 O: 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 Bio-Tech for sequencing. The complete 16S rDNA sequence is shown in SEQ ID N.1. According to the similarity comparison of its 16S rDNA sequence, see Figure 6 The 16SrDNA of R19 bacteria has a high homology with other strains of the genus Brevibacterium. It was identified as a bacterium of the genus Brevibacterium and a new strain of Brevibacterium. The strain is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO:M20242906. The deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China.
[0029] The strain has the following specific characteristics: the strain is a Gram-positive bacillus, the colony is round, light yellow to white, the surface is smooth and moist, the edges are neat, and the bacteria are short rod-shaped; the strain is catalase and alkaline phosphatase positive; it can hydrolyze gelatin and casein, but not urea; it can utilize cellobiose, fructose, xylan, glucose, glycerol, and mannose; it does not utilize arabinose, mannitol, fucose, and lactose. The growth temperature range of the strain is 10-45°C, and the optimal growth temperature is 30°C; the pH growth range is 6-11, and the optimal growth pH is 7; the NaCl concentration range is 0-15%, and the optimal growth NaCl concentration is 3%.
[0030] Example 2, a method for producing D-pantoic acid lactone hydrolase by Brevibacterium sp. R19 from the deep sea as described in Example 1: inoculating Brevibacterium sp. R19 into a liquid seed culture medium with a liquid volume of 20%, culturing at 30°C and 200 r / min for 24 hours to obtain a seed solution; inoculating the seed solution into a liquid fermentation medium with an inoculation amount of 1% and a liquid volume of 40%, culturing at 30°C and 200 r / min for 48 hours, and centrifuging to obtain a supernatant, which is a crude D-pantoic acid lactone hydrolase enzyme solution.
[0031] Example 3, Optimization of the optimal fermentation medium for Brevibacterium marinum R19
[0032] Fermentation was carried out under the same conditions using a liquid fermentation medium containing different carbon sources, nitrogen sources, and inorganic salts. The crude enzyme solution was used to detect the esterase activity using the pNPP method. The detection method is as follows: 100 μl of the crude enzyme solution was added to 2.8 ml of Tris-HCl (pH = 7.0, containing 100 μl of 10 mM p-nitrophenyl acetate solution) buffer, the reaction was carried out in a water bath at 40°C for 15 min, and the absorbance was detected at 215 nm.
[0033] The results showed that the best carbon source, nitrogen source and inorganic salt were soluble starch, tryptone and potassium chloride, respectively. After the optimization, the relative enzyme activity reached 181.78% of the initial enzyme activity. Figure 1-3 .
[0034] Example 4, a method for preparing D-pantoic acid by transformation using crude enzyme solution of Brevibacterium R19, the steps are as follows: prepare 1.6 ml of 20 mM D-pantoic acid lactone substrate solution, add 0.4 ml of crude D-pantoic acid lactone hydrolase enzyme solution, place the reaction system at 40°C for sufficient reaction for 6 hours, filter with a 0.22 μm filter to obtain D-pantoic acid reaction solution, and detect it by high performance liquid chromatography. The peak time is consistent with the peak time of the standard, both of which are 4.48.
[0035] Liquid chromatography detection conditions: The ultraviolet detector equipped with Shimadzu high performance liquid chromatograph was set to a wavelength of 215 nm and Hypersil GOLD TM Chromatographic column (4.6×250mm). The mobile phase is a mixed solution of acetonitrile and potassium dihydrogen phosphate (pH=3.0), where the volume ratio of acetonitrile to potassium dihydrogen phosphate is 1:9, and the flow rate is 1.0mL / min. The column temperature is controlled at 25℃, and the injection volume is 20μL.
[0036] The HPLC spectrum of substrate D-pantolactone is as follows Figure 4 As shown in Figure 2, the peak times are 6.67 min. The HPLC spectrum of the product D-pantoic acid is shown in Figure 2. Figure 5As shown in the figure, the elution time is 4.48min. It can be seen from the chromatogram that D-pantoic acid lactone is hydrolyzed to produce D-pantoic acid under the action of D-pantoic acid lactone hydrolase.
[0037] 16S rDNA of Brevibacterium abyssinum R19D (SEQ ID NO.1):
[0038]
Claims
1. A strain of Brevibacterium brevis R19 producing D-pantolactone hydrolase, characterized in that: The deposit number of the deep-sea brevibacterium R19 is CCTCC M 20242906.
2. A method for preparing D-pantolactone hydrolase using the deep sea Brevibacterium R19 according to claim 1, characterized in that: The method comprises the steps of inoculating the deep-sea brevibacterium R19 described in claim 1 into a liquid seed culture medium, culturing at 30-37° C. and a shaking constant temperature for 18-24 hours, taking the culture fluid and inoculating it into a liquid fermentation culture medium at a volume ratio of 5-10%, culturing at a shaking constant temperature of 25-30° C. for 40-50 hours, and centrifuging to obtain a supernatant, which is a crude enzyme solution of D-pantolactone hydrolase.
3. The method according to claim 2, characterized in that The liquid seed culture medium comprises: 1% tryptone, 0.5% yeast extract, 1% sodium chloride, the above percentages are all mass percentages, and the remaining component is water.
4. The method according to claim 2, characterized in that: The liquid fermentation medium comprises: 0.5% soluble starch, 1.0% tryptone, and 1.0% potassium chloride, the above percentages are all mass percentages, and the remaining component is water.
5. The use of Brevibacterium abyssinum R19 according to claim 1, characterized in that: The application method is to use D-pantoic acid lactone as a substrate, add the fermentation liquid of deep-sea brevibacterium R19, and place the reaction system at 35-42° C. for full reaction for 5h-7h to obtain an enzymatic hydrolyzate containing D-pantoic acid.