Bacillus subtilis and application thereof

Abstract

The invention discloses bacillus subtilis BETA-117, which can stably and efficiently utilize a renewable carbon source to ferment and produce enzyme, is used for catalyzing acrylic acid to produce beta-alanine under a high-temperature condition, reduces the energy consumption cost, enables the acrylic acid conversion rate to reach more than 98%, and shortens the production period, thereby reducing the production cost of beta-alanine and improving the yield of beta-alanine. And the production efficiency is improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a Bacillus subtilis strain and its applications. Background Technology

[0002] β-Alanine, also known as 3-aminopropionic acid, with the molecular formula C3H7NO2, is a non-natural amino acid. β-Alanine is a precursor to vitamin B5 and an important component of coenzyme A. Studies have shown that β-alanine can increase the concentration of carnosine in human muscles. Carnosine can scavenge reactive oxygen species and act as an effective buffer, preventing the formation of fatigue toxins in working muscles. Therefore, β-alanine has attracted much attention in food, health products, biomedicine, and animal feed, leading to a significant increase in market demand.

[0003] Biological methods for preparing β-alanine have become a development trend in recent years, mainly including fermentation and biocatalysis. Fermentation uses glucose as a starting material, which is inexpensive and readily available, and has always been a research hotspot. For example, patents CN112662609A and CN112625985A disclose the production of β-alanine by fermentation using engineered Escherichia coli. However, fermentation methods have problems such as more by-products, complex extraction processes, and high costs. There are two main biocatalytic methods: one uses L-aspartic acid as a substrate, with L-aspartic acid-α-decarboxylase (ADC) catalyzing decarboxylation to produce β-alanine. For example, patent CN108546697A uses this route. This method has high catalytic efficiency, mild conditions, and a simple and environmentally friendly extraction process, making it a long-standing research hotspot in the enzymatic synthesis of β-alanine. However, this method requires the relatively expensive substrate aspartic acid, resulting in high process costs and significantly limiting industrialization. The second method uses acrylic acid as a substrate, with aspartic acid lyase (also known as aspartate aminotransferase (AspB) or aspartate lyase) catalyzing ammonia addition for synthesis. For example, the literature (Vogel A, Schmiedel R, Hofmann U, et al. Converting Aspartase into aβ-Amino Acid Lyase by Cluster) shows this process. Screening[J].ChemCatChem,2014,6(4).DOI:10.1002 / cctc.201300986.), patents CN109385415A and CN110791493A all adopt this route. This method has been rapidly promoted to industrial development and application because the substrate is cheap and readily available, the enzyme catalysis reaction and extraction process are relatively simple, and the production cycle is short. However, in the bioenzymatic conversion method using acrylic acid as the substrate, an acid-base neutralization reaction occurs when the substrate acrylic acid and ammonia are mixed, releasing a large amount of heat. The system temperature can reach 65-75℃, while the applicable temperature of existing aspartic acid enzyme conversion is mostly 50℃. Therefore, in order to provide a suitable conversion temperature for aspartic acid enzyme to improve conversion efficiency, the process needs to introduce condensate water for temperature control, which will lead to an increase in energy consumption costs. In addition, at low enzyme conversion temperatures, the system is also easily contaminated by microorganisms in the environment, resulting in a high risk of microbial contamination. Bacillus subtilis exhibits strong adaptability to temperature and pH levels, enabling it to survive and grow under a wide range of environmental conditions. Therefore, developing a Bacillus subtilis strain capable of fermenting and producing an enzyme for catalyzing the ammonia-to-β-alanine synthesis from acrylic acid would have significant industrial value. Furthermore, the resulting enzyme would possess superior high-temperature tolerance, making it more suitable for industrial production and leading to greater economic benefits. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Bacillus subtilis BETA-117, which has high temperature tolerance and can reduce the risk of contamination during the fermentation and enzyme production process. At the same time, the whole cell or enzyme solution containing the enzyme can catalyze the production of β-alanine from acrylic acid under high temperature conditions, reducing energy consumption costs. The acrylic acid conversion rate can reach more than 98%, and the production cycle is shortened, thereby reducing the production cost of β-alanine and improving production efficiency.

[0005] To achieve the above objectives, in a first aspect, the present invention provides a Bacillus subtilis BETA-117, which has the accession number CCTCC NO: M20232501 at the China Center for Type Culture Collection.

[0006] To achieve the above objectives, a second aspect of the present invention provides a microbial agent, the active ingredient of which is the aforementioned Bacillus subtilis BETA-117.

[0007] To achieve the above objectives, a third aspect of the present invention provides the use of Bacillus subtilis BETA-117, or the inoculum, in any of the following A1)-A3):

[0008] A1) Production of β-alanine;

[0009] A2) Preparation for the production of β-alanine products;

[0010] A3) Produces aspartate aminotransferase.

[0011] Furthermore, the β-alanine is produced by catalyzing the addition of ammonia to acrylic acid using aspartic acid enzyme; the aspartic acid enzyme is produced by the above-mentioned Bacillus subtilis strain BETA-117 or by a fungal agent.

[0012] Furthermore, the aspartate enzyme catalyzes reactions in the form of enzyme solution or whole cells.

[0013] Furthermore, the whole cells can be prepared by centrifuging the fermentation broth of Bacillus subtilis BETA-117 and collecting the bacterial cells, which are the whole cells.

[0014] Furthermore, the enzyme solution can be a crude enzyme solution or a pure enzyme solution;

[0015] The crude enzyme solution can be prepared by the following method: centrifuge the fermentation broth of Bacillus subtilis BETA-117, collect the bacterial cells, then break the bacterial cells, centrifuge again, and collect the cell breakage supernatant as the crude enzyme solution.

[0016] Further, the crude enzyme solution is purified to obtain a pure enzyme solution;

[0017] The purification methods include, but are not limited to, various precipitation methods, various column chromatography methods (adsorption chromatography, ion exchange chromatography, gel filtration) and affinity chromatography (Ni column affinity chromatography), as long as they can achieve the purification purpose.

[0018] Furthermore, the catalytic reaction is carried out at 38-62°C;

[0019] Furthermore, the catalytic reaction is carried out at 40-62°C;

[0020] Furthermore, the catalytic reaction is carried out at 50-62°C;

[0021] Furthermore, the catalytic reaction is carried out at 60-62°C.

[0022] Furthermore, the product used to produce β-alanine contains aspartate enzyme, which is obtained by Bacillus subtilis BETA-117.

[0023] The phrase "contains aspartate enzyme" indicates that the aspartate enzyme contained in the product may be entirely produced by Bacillus subtilis BETA-117, or may be partially produced by Bacillus subtilis BETA-117.

[0024] Furthermore, the aspartate enzyme exists in the form of whole cells containing the enzyme, crude enzyme solution, or pure enzyme solution.

[0025] Furthermore, the aspartate enzyme is obtained by fermentation of Bacillus subtilis BETA-117 under shaking and aeration conditions.

[0026] Furthermore, the fermentation production method of the aspartic acid enzyme includes:

[0027] (1) Culture of strain: Bacillus subtilis BETA-117 was inoculated into seed culture medium and cultured for 12-16 hours at 35-50℃, aeration rate of 0.5-1 vvm and rotation speed of 200-300 rpm to obtain seed liquid;

[0028] (2) Fermentation enzyme production: Transfer the seed liquid to the fermentation medium at an inoculation rate of 10-20% (v / v) and culture at 35-50℃ with shaking and aeration for 24-48 hours to obtain the enzyme.

[0029] Among them, Bacillus subtilis BETA-117 utilizes a renewable carbon source to ferment and produce aspartic acid enzymes.

[0030] Furthermore, the renewable carbon source is any one or more of sugars, sugar alcohols, and low-carbon alcohols;

[0031] It can further be any one or more of monosaccharides, disaccharides, polysaccharides, sugar alcohols, and glycerol;

[0032] Furthermore, it can be any one or more of glucose, fructose, sucrose, maltose, xylose, and glycerol.

[0033] Furthermore, in step (1) of bacterial culture, the seed culture medium comprises the following components: glucose 10-30 g·L⁻¹ -1 5-10g / L of yeast powder -1 5-15 g / L of peptone -1 pH 6.0-7.0.

[0034] Furthermore, the seed culture medium consists of: 20 g / L glucose. -1 5g / L of yeast powder -1 10g / L of peptone -1 pH 6.5.

[0035] Furthermore, during the enzyme production process in step (2), dissolved oxygen is controlled at 30-50%.

[0036] Furthermore, when the renewable carbon source in the fermentation medium is glucose, and the initial glucose in the fermentation medium is depleted, dissolved oxygen is replenished, and the replenished feed is glucose.

[0037] Furthermore, the fermentation medium comprises the following components: glucose 10-30 g·L⁻¹ -1 5-15g / L of yeast powder -1 , KH2PO4 4-7g·L -1 3-7 g / L of ammonium sulfate -1Vitamin B1 0.2-1g / L -1 FeSO4 0.5-1 mg / L -1 MgSO4 2-4 g / L -1 pH 6.0-7.5.

[0038] Furthermore, the fermentation medium is: glucose 30 g·L⁻¹ -1 5g / L of yeast powder -1 4 g·L KH2PO4 -1 4 g / L of ammonium sulfate -1 Vitamin B1 1g.L -1 FeSO4 0.5 mg / L -1 MgSO4 2g.L -1 pH 6.9.

[0039] Furthermore, when dissolved oxygen rapidly recovers, glucose is added in a flow, wherein the concentration of the added glucose is 50%-80%, preferably 60%.

[0040] Furthermore, the feed is supplied in a feed-feed manner at a rate of 8-12 g / L / h.

[0041] Furthermore, the shaking and aeration culture method is either shake flask culture or fermenter stirring and aeration culture.

[0042] Furthermore, the shaking speed of the shake flask culture is 200-250 rpm.

[0043] Furthermore, the ventilation volume of the fermenter for agitation and ventilation is 2-4 vvm, and the stirring speed is 500-1000 rpm.

[0044] Furthermore, dissolved oxygen can be controlled between 30-50% in the following ways: when dissolved oxygen is below 30%, control the feed flow rate to 8 g / L / h; when dissolved oxygen is above 50%, control the feed flow rate to 12 g / L / h; when 12 g / L / h still cannot effectively reduce dissolved oxygen, the rotation speed can be reduced within the range of 500-1000 prm.

[0045] Furthermore, the culture temperature is 38-50℃, and the culture time is 20-30h.

[0046] Furthermore, the culture temperature was 40℃ and the culture time was 24h.

[0047] Furthermore, the product used to produce β-alanine also contains acrylic acid and ammonia.

[0048] The beneficial effects of this invention are:

[0049] A strain of Bacillus subtilis BETA-117 was screened from nature. This strain can tolerate high temperatures of 38-50℃, reducing the risk of contamination during enzyme fermentation. Furthermore, this strain can directly ferment enzymes using renewable carbon sources such as glucose, making the raw materials inexpensive and readily available, aligning with green production principles. Simultaneously, the whole cell or enzyme solution containing the enzyme can catalyze the synthesis of β-alanine from acrylic acid with ammonia under high-temperature conditions (38-62℃, especially 50-62℃), reducing the requirements for cooling systems and the consumption of cooling water, thereby lowering production costs. The acrylic acid conversion rate can reach 98%, demonstrating high catalytic efficiency, which is beneficial for the industrial production of β-alanine.

[0050] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0051] Instructions for the Preservation of Biological Materials

[0052] Abbreviation of depositary institution: CCTCC

[0053] Name of depositary institution: China Center for Type Culture Collection (CCTCCC)

[0054] Address of the institution: Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China

[0055] Deposit date: December 8, 2023

[0056] Collection Center Registration Number: CCTCC NO: M 20232501

[0057] Classification and nomenclature: Bacillus subtilis

[0058] Strain number: BETA-117 Attached Figure Description

[0059] Figure 1 The image shows the HPLC detection results of the β-alanine standard in the examples.

[0060] Figure 2 The strain morphology of strain 12 is shown.

[0061] Figure 3 The results of tank fermentation of Bacillus subtilis BETA-117 in Example 2 are shown. Detailed Implementation

[0062] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following embodiments are commercially available. All quantitative experiments in the following embodiments were performed in at least three replicates, and the results were averaged.

[0063] The detection methods involved in the following embodiments:

[0064] 1. Cell concentration: Take an appropriate amount of fermentation broth, dilute it, and measure the absorbance (OD) at 550 nm using a UV spectrophotometer. 550 .

[0065] 2. Glucose concentration: Dilute the fermentation broth appropriately and use the SBA-40E biosensor to measure the glucose concentration.

[0066] 3. β-Alanine content: The β-alanine content in the reaction product was determined by HPLC. The detection method was as follows:

[0067] The reaction product (conversion solution) was diluted 500 times with sterile water, filtered through a 0.22 μm filter membrane, and then injected into a high-performance liquid chromatograph.

[0068] Among them, the HPLC detection conditions are as follows:

[0069] Detection instrument: P-100 liquid chromatograph; chromatographic column: Primesp 100 4.6*250mm, 5μm;

[0070] Mobile phase: Acetonitrile: Ultrapure water: Trifluoroacetic acid (30:70:0.1, V / V / V);

[0071] Column temperature: 30℃;

[0072] Flow rate: 0.5 mL / min;

[0073] Ultraviolet detector, wavelength 210nm;

[0074] Injection volume: 20 μL.

[0075] When β-alanine standard (Yuanye Company, catalog number B21970) was used for injection and detection, the retention time of the characteristic peak of β-alanine was approximately 25.89 min (see...). Figure 1 ).

[0076] 4. Definition of terms:

[0077] Enzyme activity is defined as the production of 1 μmol of β-alanine within 1 minute through the catalytic reaction of acrylic acid with ammonia.

[0078] The definition of enzyme activity per unit cell: 1 U / OD is defined as the amount of enzyme that can catalyze the reaction of acrylic acid with ammonia to produce 1 μmol of β-alanine within 1 minute at a unit cell concentration.

[0079] Conversion rate = (moles of β-alanine produced / initial moles of acrylic acid) × 100%.

[0080] 5. Enzyme activity detection methods

[0081] Acrylic acid and 14.8% ammonia were mixed at a volume ratio of 1.7:5.3 to prepare the substrate. 25 mL of the substrate was taken, and enzyme-containing bacterial solution (OD = 120) was added. The concentration of bacteria in the transformation system was controlled at 10 OD. The transformation reaction was carried out at 50°C, and samples were taken for analysis.

[0082] Example 1: Screening and identification of thermostable Bacillus subtilis producing aspartate enzyme

[0083] The aspartate-producing Bacillus subtilis BETA-117 of this invention was obtained by sampling from the discharge point of acrylic acid ammonia conversion liquid in a β-alanine plant, and through enrichment, separation and purification, primary screening, and secondary screening, including the following steps:

[0084] 1. Screening of thermostable strains producing aspartic acid enzymes

[0085] S1. Sampling: Sampling was carried out at the discharge point of acrylic acid ammonia conversion liquid in Huaheng Biotechnology Plant, Shanhaiguan District, Qinhuangdao City, Hebei Province (the temperature of the waste liquid is 45-60℃);

[0086] S2. Initial screening of aspartic acid-producing strains includes the following steps:

[0087] ① Spread the sample taken in step S1 onto the enrichment medium and incubate at 38°C for 16 hours;

[0088] The enrichment culture medium was water as the solvent, and the solute and its concentration were: glucose 20 g·L⁻¹ -1 10g / L of yeast powder -1 15g / L of peptone -1 15 g·L sodium chloride -1 20g / L agar -1 pH 7.2.

[0089] ② The bacterial strains enriched on the slant were inoculated into seed culture medium in shake flasks and incubated at 200 rpm and 38°C for 16 h.

[0090] The seed culture medium in shake flasks is water as the solvent, and the solute and its concentration are: glucose 20 g·L⁻¹ -1 5g / L of yeast powder -1 10g / L of peptone -1pH 6.5.

[0091] ③ Transfer the seed culture from the shake flask to the shake flask fermentation medium at an inoculation rate of 10% (v / v) and ferment at 38°C for 24 hours;

[0092] The shake-flask fermentation medium is water as the solvent, and the solute and its concentration are: glucose 30 g·L⁻¹ -1 5g / L of yeast powder -1 4 g·L KH2PO4 -1 4 g / L of ammonium sulfate -1 Vitamin B1 1g.L -1 FeSO4 0.5 mg / L -1 MgSO4 2g.L -1 pH 6.9.

[0093] ④ Centrifuge the above shake-flask fermentation culture medium, remove some of the supernatant, and then perform molecular identification, biomass and aspartate enzyme activity detection on the obtained enzyme-containing bacterial solution to select the strains with enzyme activity as aspartate enzyme-producing strains.

[0094] S3, High-Temperature Acclimation:

[0095] ① Take a loopful of the shake flask seed culture of the aspartate enzyme-producing strain, dilute it, spread it on the acclimatization medium, and incubate at 40℃ for 24h; select single colonies with good growth and excellent biological morphology, take a loopful and inoculate them into the shake flask seed culture medium, and incubate at 200rpm and 40℃.

[0096] ②After the bacteria from step ① have grown to the logarithmic phase, take a loopful, dilute it, and spread it on the acclimatization medium. Incubate at 45℃ for 24 hours. Select single colonies with good growth and excellent biological morphology, take a loopful, and inoculate them into the shake flask seed medium. Incubate at 45℃ for 200 rpm.

[0097] ③ After the bacteria from step ② have grown to the logarithmic phase, take one loopful, dilute it, and spread it on the acclimatization medium. Incubate at 50°C for 24 hours. Select single colonies with good growth and superior biological morphology, take one loopful, and inoculate them into the shake flask seed culture medium. Incubate at 200 rpm and 50°C for 24 hours. Select 20 strains with high bacterial concentration for secondary screening.

[0098] The solvent for the above acclimatization culture medium is: 20 g / L agar added to the shake flask seed culture medium. -1 .

[0099] S4. Screening for heat-resistant strains producing high levels of aspartate aminotransferase: Twenty strains of aspartate aminotransferase produced by the high-temperature acclimatization screening in step S3 were inoculated into shake-flask fermentation medium and cultured at 50°C for 24 hours. The fermentation broth was centrifuged, and the enzyme-containing bacterial solution was tested for biomass and aspartate aminotransferase activity as described in step S2 ④. It was found that strain 12 had the highest OD550 value of 20.61 (sample diluted 50 times) and an enzyme activity of 4.63 U / OD.

[0100] Therefore, strain 12 was identified as a heat-resistant strain that produces high levels of aspartic acid enzymes.

[0101] 2. Detection of the genetic stability of thermostable strain 12 producing aspartate aminotransferase

[0102] The selected strain 12 was passaged 10 times on plates, and each batch of strain was inoculated into shake flask fermentation medium. The fermentation broth was centrifuged, and the enzyme-containing bacterial solution was taken for biomass and aspartate enzyme activity detection. The results are shown in Table 1.

[0103] Table 1: Stability of strain 12

[0104]

[0105] As shown in Table 1, the aspartate enzyme activity of the selected strain 12 remained stable after 10 consecutive passages at the end of fermentation culture, indicating good genetic stability.

[0106] 3. Strain identification

[0107] The selected strain 12, exhibiting high enzyme activity, was inoculated onto LB liquid medium and cultured at 38°C for 24 hours. Gram staining revealed a blue-purple color; the cells were short, rod-shaped, slightly curved, with blunt ends, arranged singly, in pairs, or in a figure-eight pattern, with prominent spores visible in the center of the cells (see...). Figure 2 The morphological characteristics of this strain are consistent with Bacillus subtilis.

[0108] Based on the above colony morphology identification, strain 12 was identified as Bacillus subtilis and classified as Bacillus subtilis BETA-117. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on December 8, 2023, with accession number CCTCC NO: M 20232501. The deposit address is Wuhan University, Wuhan, China.

[0109] Example 2: Fermentation of aspartic acid enzyme by Bacillus subtilis BETA-117

[0110] Aspartate enzyme was produced by fermentation using Bacillus subtilis BETA-117 screened in Example 1, with the following steps:

[0111] S1. Inoculate one loop of Bacillus subtilis BETA-117 cultured on the slant culture medium into the seed culture medium, and culture for 16 h at 38℃, aeration rate of 0.5 vvm and stirring speed of 200 rpm to obtain the seed liquid.

[0112] The seed culture medium uses water as the solvent, and the solute and its concentration are: glucose 20 g·L⁻¹ -1 5g / L of yeast powder -1 10g / L of peptone -1 pH 6.5.

[0113] S2. Transfer the seed culture at a volume ratio of 10% to a 5L fermenter containing 3L of fermentation medium. Control the dissolved oxygen at 30-50%, fermentation temperature at 38℃, ventilation rate at 2vvm, and stirring speed at 500-1000rpm for 24 hours. During the fermentation process, adjust the pH to 6.9 with ammonia.

[0114] The fermentation medium solvent is water, and the solute and its concentration are: glucose 30 g·L⁻¹ -1 5g / L of yeast powder -1 , KH2PO4 4g·L -1 4 g / L of ammonium sulfate -1 Vitamin B1 1g.L -1 FeSO4 0.5 mg / L -1 MgSO4 2g.L -1 pH 6.9.

[0115] During fermentation, dissolved oxygen is controlled as follows:

[0116] When the initial sugar (glucose) in the fermentation medium is depleted, dissolved oxygen is controlled by feeding. Glucose (60% concentration) is added at a flow rate of 8-12 g / L / h. If the initial dissolved oxygen is 100%, when the dissolved oxygen is below 30%, the feeding flow rate is 8 g / L / h. When the dissolved oxygen is above 50%, the feeding flow rate is controlled at 12 g / L / h. If 12 g / L / h still cannot effectively reduce the dissolved oxygen, the rotation speed is reduced to control oxygen in the range of 500-1000 prm.

[0117] Detection of unit enzyme activity and OD of aspartate in fermentation broth 550 Values, results are shown below Figure 3 It is evident that Bacillus subtilis BETA-117 can directly ferment aspartate enzyme using glucose as the sole carbon source, and the unit enzyme activity of aspartate enzyme can reach 8 U / OD.

[0118] The enzyme activity detection method is as follows: centrifuge the fermentation broth, discard part of the supernatant, and collect the cells to obtain the enzyme-containing bacterial solution or whole cells; mix acrylic acid and 14.8% ammonia water at a volume ratio of 1.7:5.3 to prepare the substrate; take 25 mL of substrate, add the enzyme-containing bacterial solution (OD=120) to it, control the concentration of the bacteria in the transformation system to 10OD, and carry out the transformation reaction at 50℃, and take samples for detection.

[0119] Example 3: Enzyme-containing whole-cell catalysis of ammonia addition to acrylic acid to synthesize β-alanine

[0120] Cells from the fermentation broth in Example 2 were collected by centrifugation. Specifically, the cell culture medium was centrifuged at 4000 rpm for 10 minutes, a portion of the supernatant was discarded, and the cells were collected to obtain an enzyme-containing bacterial suspension. The OD of the bacterial suspension was measured using a spectrophotometer. 550 Value, bacterial culture OD 550 =120 (adjusted by adding water).

[0121] The substrate was prepared by mixing the enzyme-containing bacterial solution, acrylic acid, and 14.8% ammonia in a volume ratio of 1:4.2:6. The concentration of acrylic acid in the substrate was 4.6 mol / L. Then, the enzyme-containing bacterial solution was added, and the concentration of the bacterial solution in the transformation system was controlled at 10 OD. The transformation system was then controlled at 50-62℃ for 3 h, and samples were taken for testing. The results are shown in Table 2.

[0122] Table 2: Conversion results of aspartic acidase-containing bacterial cultures at different temperatures

[0123] Conversion temperature ℃ Reaction time / h Acrylic acid conversion rate % 50 3.7 96.53 55 3.5 97.16 60 3.1 98.12 61 3 98.15 62 2.8 98.34 63 3.6 96.87 64 4.1 95.34 65 4.2 95.20

[0124] Acrylic acid conversion rate = (number of moles of β-alanine generated / initial number of moles of acrylic acid) × 100%.

[0125] As can be seen from the table above, the aspartic acid enzyme produced by Bacillus subtilis BETA-117 of this invention exhibits good catalytic activity for the ammonia addition of acrylic acid, with a gradually increasing activity across the temperature range of 50-62℃ followed by a decreasing activity, demonstrating high-temperature tolerance within this range. The enzyme exhibits the best catalytic activity for the ammonia addition of acrylic acid at 62℃, achieving a conversion rate of up to 98.34%, while also shortening the reaction time. This makes it more suitable for industrial production and possesses significant application value and industrial prospects.

[0126] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Bacillus subtilis BETA-117, whose accession number at the China Center for Type Culture Collection is CCTCC NO: M 20232501.

2. A microbial agent, the active ingredient of which is Bacillus subtilis BETA-117 as described in claim 1.

3. The use of Bacillus subtilis BETA-117 as described in claim 1, or the inoculum as described in claim 2, in any one of A1)-A3) below: A1) Production of β-alanine; A2) Preparation for the production of β-alanine products; A3) Produces aspartate aminotransferase.

4. The application according to claim 3, characterized in that: The β-alanine is produced by catalyzing the addition of ammonia to acrylic acid using aspartic acid enzyme; the aspartic acid enzyme is produced by the Bacillus subtilis strain BETA-117 as described in claim 1 or the inoculum as described in claim 2.

5. The application according to claim 4, characterized in that: The aspartate enzyme catalyzes the action in the form of enzyme solution or whole cell; Furthermore, the enzyme solution is a crude enzyme solution or a pure enzyme solution.

6. The application according to claim 4, characterized in that: The catalytic reaction was carried out at 38-62°C; Furthermore, the conversion reaction is carried out at 40-62°C; Furthermore, the conversion reaction is carried out at 50-62°C; Furthermore, the conversion reaction is carried out at 60-62°C.

7. The application according to claim 3, characterized in that: The product used to produce β-alanine contains aspartate enzyme, which is obtained by Bacillus subtilis BETA-117. Furthermore, the aspartate enzyme exists in the form of whole cells containing the enzyme, crude enzyme solution, or pure enzyme solution; Furthermore, the product used to produce β-alanine also contains acrylic acid and ammonia.

8. The application according to claim 3 or 4, characterized in that: The aspartate enzyme was obtained by fermentation of Bacillus subtilis BETA-117 under shaking and aeration conditions.

9. The application according to claim 8, characterized in that: The fermentation production method of the aspartic acid enzyme includes: (1) Culture of strain: Bacillus subtilis BETA-117 was inoculated into seed culture medium and cultured for 12-16 hours at 35-50℃, aeration rate of 0.5-1 vvm and rotation speed of 200-300 rpm to obtain seed liquid; (2) Fermentation enzyme production: Transfer the seed liquid to the fermentation medium at an inoculation rate of 10-20% (v / v) and culture at 35-50℃ with shaking and aeration for 24-48 hours to obtain the enzyme.

10. The application according to any one of claims 7-9, characterized in that: The Bacillus subtilis BETA-117 is used to produce aspartic acid enzymes through fermentation using a renewable carbon source. Furthermore, the renewable carbon source is any one or more of sugars, sugar alcohols, and low-carbon alcohols; Furthermore, it can be any one or more of monosaccharides, disaccharides, polysaccharides, sugar alcohols, and glycerol; It is further composed of one or more of glucose, fructose, sucrose, maltose, xylose, and glycerol; Furthermore, the fermentation medium comprises the following components: glucose 10-30 g·L⁻¹ -1 5-15g / L of yeast powder -1 , KH2PO4 4-7g·L -1 3-7 g / L of ammonium sulfate -1 Vitamin B1 0.2-1g / L -1 FeSO4 0.5-1 mg / L -1 MgSO4 2-4 g / L -1 pH 6.0-7.5; Furthermore, during the fermentation and enzyme production process, dissolved oxygen is controlled at 30-50%.

Images