CN111304106A - Bacillus clausii and method for producing tetrahydropyrimidine by using same - Google Patents
Bacillus clausii and method for producing tetrahydropyrimidine by using same Download PDFInfo
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- CN111304106A CN111304106A CN201811509078.0A CN201811509078A CN111304106A CN 111304106 A CN111304106 A CN 111304106A CN 201811509078 A CN201811509078 A CN 201811509078A CN 111304106 A CN111304106 A CN 111304106A
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- bacillus clausii
- tetrahydropyrimidine
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- fermentation
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- 241001328122 Bacillus clausii Species 0.000 title claims abstract description 75
- OTPDWCMLUKMQNO-UHFFFAOYSA-N 1,2,3,4-tetrahydropyrimidine Chemical compound C1NCC=CN1 OTPDWCMLUKMQNO-UHFFFAOYSA-N 0.000 title claims abstract description 67
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 22
- 150000003839 salts Chemical class 0.000 claims abstract description 19
- 238000004321 preservation Methods 0.000 claims abstract description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 62
- 235000002639 sodium chloride Nutrition 0.000 claims description 49
- 239000002609 medium Substances 0.000 claims description 36
- 239000011780 sodium chloride Substances 0.000 claims description 31
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 23
- 239000001963 growth medium Substances 0.000 claims description 17
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 14
- 238000010564 aerobic fermentation Methods 0.000 claims description 13
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 12
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 11
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- 230000008569 process Effects 0.000 claims description 11
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- 235000019270 ammonium chloride Nutrition 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
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- 235000013312 flour Nutrition 0.000 claims description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 6
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- 150000005326 tetrahydropyrimidines Chemical class 0.000 claims description 6
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
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- 235000019800 disodium phosphate Nutrition 0.000 claims description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 2
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 2
- 239000001488 sodium phosphate Substances 0.000 claims description 2
- 238000000855 fermentation Methods 0.000 abstract description 32
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- 238000009655 industrial fermentation Methods 0.000 abstract 1
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- 238000001514 detection method Methods 0.000 description 9
- VTGOHKSTWXHQJK-UHFFFAOYSA-N pyrimidin-2-ol Chemical compound OC1=NC=CC=N1 VTGOHKSTWXHQJK-UHFFFAOYSA-N 0.000 description 9
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- WQXNXVUDBPYKBA-YFKPBYRVSA-N ectoine Chemical compound CC1=[NH+][C@H](C([O-])=O)CCN1 WQXNXVUDBPYKBA-YFKPBYRVSA-N 0.000 description 3
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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
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Abstract
The invention relates to a bacillus clausii ET01 for producing tetrahydropyrimidine, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 16310. The invention also relates to a method for producing tetrahydropyrimidine by using the strain through fermentation. The bacillus clausii can produce tetrahydropyrimidine with high yield under the stimulation of low salt concentration, can be used for producing tetrahydropyrimidine by industrial fermentation, and has good application prospect.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to Bacillus clausii for producing tetrahydropyrimidine, and application and a method for producing tetrahydropyrimidine by using the Bacillus clausii.
Background
Tetrahydropyrimidine (Ectoine) is a compatible solute generated in cells by many salt-tolerant and halophilic microorganisms to maintain osmotic pressure balance, and can provide protection for cells, proteins, cell membranes, nucleic acids and the like under the stimulation of extreme conditions such as external high temperature, freezing, ray, drying and the like. In addition, the tetrahydropyrimidine has certain curative effect on neurological diseases such as Alzheimer's disease, Parkinson's disease and the like, and recent research finds that the tetrahydropyrimidine can improve the regeneration capability of skin and delay the aging of the skin. Therefore, the tetrahydropyrimidine has wide application prospect in the industries of fine chemical engineering, biological medicine and the like.
At present, the production method of tetrahydropyrimidine is mainly obtained by high-density fermentation of halophilic microorganisms (particularly, halomonas). Generally, the process is carried out by adopting a mode called 'bacteria milking/lactation', namely culturing bacteria under high osmotic pressure, then hypotonic impacting to release solute, then carrying out hypertonic culturing on the thalli again, hypotonic impacting to release solute, and circulating for 8-9 times in sequence to obtain the product. The method has higher requirements on the stability of reactor materials, the difficulty of a downstream purification process is increased due to the discontinuous production flow and the high-concentration salt, in addition, the high-concentration salt is easy to corrode equipment, the growth of thalli is influenced, the yield of the tetrahydropyrimidine is influenced, the production cost is increased, and the large-scale application of the tetrahydropyrimidine is influenced.
The existing production strain and the method for producing tetrahydropyrimidine by using the same seriously restrict the industrial production and large-scale application of tetrahydropyrimidine, so that a novel tetrahydropyrimidine high-yield strain is developed to simplify the production process, improve the synthesis efficiency and reduce the production cost, and has important practical significance for the application of tetrahydropyrimidine.
The bacillus clausii is a probiotic strain, can meet the production with higher safety requirement, has mature fermentation process and simple fermentation culture requirement, and can meet the industrial application conditions of simple and convenient fermentation operation and low cost, but the existing bacillus clausii is mostly used for the industrial production of protease, and related reports of the bacillus clausii for the industrial production of tetrahydropyrimidine are not found.
Disclosure of Invention
The existing tetrahydropyrimidine production process is complex, low in synthesis efficiency and high in production cost, and the industrial production and large-scale application of tetrahydropyrimidine are severely restricted. Therefore, the invention aims to provide the strain for producing the tetrahydropyrimidine and the method for producing the tetrahydropyrimidine by fermenting the strain, which have the advantages of high biological safety, high yield of the tetrahydropyrimidine, simple and convenient fermentation operation and low culture cost, so as to overcome the defects of the prior art.
In a first aspect, the invention provides a strain of Bacillus clausii ET01(Bacillus clausii ET01) for producing tetrahydropyrimidine, with the preservation number of CGMCC NO. 16310. The strain of the invention can grow in a liquid culture medium with inorganic salt concentration of 0-100g/L and can produce tetrahydropyrimidine with high yield (up to 25g/L) under low salt concentration (less than 100g/L and more than 0g/L, such as 50 g/L).
In a second aspect, the present invention provides the use of bacillus clausii ET01 as described in the first aspect for the production of tetrahydropyrimidine.
In a third aspect, the present invention provides a process for the production of tetrahydropyrimidine, the process comprising:
aerobic fermentation of the first aspect of Bacillus clausii ET01 in a Bacillus clausii medium with an inorganic salt concentration of less than 100g/L to obtain tetrahydropyrimidine.
The invention has the following beneficial effects: compared with other strains (such as halopyrimidines in CN 103451137A) for producing tetrahydropyrimidine in the prior art, the Bacillus clausii ET01 has higher tetrahydropyrimidine synthesis capacity (25g/L), can be fermented and produced well under the culture condition of low salt concentration (less than 100g/L), and reduces the risks of fermentation equipment corrosion and environmental pollution caused by overhigh salinity of other halophilic strains in the fermentation and production; after aerobic fermentation is carried out by using the bacillus clausii ET01, the content of bacterial strain fermentation byproducts and other impurities is lower, no hydroxypyrimidine is generated, the purity of tetrahydropyrimidine obtained by simple separation and purification can reach more than 98%, the separation and purification cost is reduced, and the bacillus clausii ET01 has a good industrial application prospect.
Drawings
FIG. 1 shows Bacillus clausii ET01 observed under a 100-fold microscope.
Detailed Description
In the invention, the inventor adopts a gradient dilution method to separate and screen a strain of bacteria with high yield of tetrahydropyrimidine from salt field mud near the sea of the Weihai. Culturing the strain on LB solid culture medium (10g/L NaCl) at 38 deg.C for 24 hr to form milky colony with irregular edge and raised surface; the cells were observed under a microscope at 100 times, and were in the form of long rods (as shown in FIG. 1); gram-positive bacteria are positive by gram staining. This strain was identified and named Bacillus Claussonensis ET01(Bacillus clausii ET01) by reference to the standard described in Bergey's Manual of bacteria identification (SEQ ID NO:1) and 1984. The strain is preserved in China general microbiological culture Collection center (CGMCC) (China general microbiological culture Collection center) (Siro 1 institute of Chaoyang district, Beijing) with the preservation date of 2018, 8 months and 17 days, and the classified name of the strain is Bacillus clausii (Bacillus clausii), and the preservation number is CGMCC NO. 16310.
In the present invention, the term "Bacillus clausii medium" refers to a medium known to those skilled in the art to be useful for culturing Bacillus clausii, for example, a nutrient medium (e.g., LB medium or nutrient broth medium) containing a carbon source, a nitrogen source, and inorganic salts. The formulation of the medium may be a formulation known in the art, for example the formulation of LB medium commonly used is: 0.8-1 wt% of peptone, 0.5-0.8 wt% of yeast powder, 1-1.5 wt% of sodium chloride, and the pH value is 6.8-7.0; the nutrient broth culture medium has a formula of 0.8-1 wt% of peptone, 0.3-0.5 wt% of beef extract, 0.5-0.8 wt% of sodium chloride, and pH of 7.2-7.6. However, the skilled person can select and adjust the specific carbon source, nitrogen source and inorganic salts and their respective contents in the culture medium as required, especially the kind and concentration of inorganic salts, to be suitable for the cultivation and application of the Bacillus clausii of the present invention, for example, adjusting the sodium chloride concentration in LB medium to 5 wt% for producing tetrahydropyrimidine.
In the present invention, the term "carbon source" refers to a nutrient that provides carbon elements necessary for growth and reproduction of microorganisms. Examples of such carbon sources include, but are not limited to, glucose, dextrin, sucrose, monosodium glutamate, molasses, glycerol, lactic acid, maltodextrin, maltose. In some embodiments, the "carbon source" is preferably glucose.
In the present invention, the term "nitrogen source" refers to a nutrient that provides nitrogen elements necessary for growth and reproduction of microorganisms. Examples of the nitrogen source include, but are not limited to, yeast extract, tryptone, monosodium glutamate, ammonium chloride, ammonium sulfate, soybean peptone, soybean flour, corn flour, peanut cake, cottonseed meal. In some embodiments, the "nitrogen source" is preferably yeast extract, tryptone, ammonium chloride or ammonium sulfate, or any combination thereof.
In the present invention, the term "inorganic salt" refers to mineral salts of inorganic compounds, such as sodium salt, potassium salt, calcium salt, phosphate, sulfate, and the like. Examples of the inorganic salt include, but are not limited to: sodium chloride, dipotassium phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, ferric sulfate heptahydrate, calcium chloride dihydrate, potassium chloride, sodium sulfate and disodium phosphate.
In the present invention, the term "seed medium" refers to a medium used when a strain is activated, and the seed medium may be a general medium conventionally used in the art, such as an LB medium or a nutrient broth medium. The particular ingredients and the respective contents of the culture medium can be selected by the person skilled in the art as required to be suitable for the activation of the Bacillus clausii strain of the invention, in particular the type and concentration of the inorganic salts.
Therefore, the invention provides a strain of Bacillus clausii ET01(Bacillus clausii ET01) for producing tetrahydropyrimidine, and the preservation number is CGMCC NO. 16310. The strain grows in a liquid culture medium with the inorganic salt concentration of 0-100g/L, and the tetrahydropyrimidine is produced with high yield under the condition of low salt concentration (less than 100g/L and more than 0 g/L). In a preferred embodiment, the Bacillus clausii ET01 can be grown in a liquid medium with an inorganic salt concentration of 0 to 100g/L, preferably 30 to 70g/L, more preferably 50 g/L.
In one embodiment, the invention also provides the use of bacillus clausii ET01 of the invention for the production of tetrahydropyrimidine.
In one embodiment, the present invention provides a process for producing tetrahydropyrimidine, the process comprising: aerobic fermentation is carried out on the Bacillus clausii ET01 in a Bacillus clausii culture medium with inorganic salt concentration less than 100g/L, so as to obtain tetrahydropyrimidine.
In a preferred embodiment, the Bacillus clausii medium is a liquid medium used in the art for culturing Bacillus clausii, for example a nutrient medium containing carbon sources, nitrogen sources and inorganic salts, such as LB medium or nutrient broth medium. The skilled person will be able to select and adjust the specific components and amounts of the medium as required, and such selection and adjustment schemes are also within the scope of the invention.
In a preferred embodiment, the inorganic salt is selected from the group consisting of sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, iron sulfate heptahydrate, calcium chloride dihydrate, potassium chloride, sodium sulfate, disodium hydrogen phosphate, or any combination thereof. In a preferred embodiment, the inorganic salt is sodium chloride. In a preferred embodiment, the salt concentration of the inorganic salt is less than 100g/L and greater than 0g/L, preferably 30-70g/L, more preferably 50 g/L.
In a preferred embodiment, the carbon source is selected from glucose, dextrin, sucrose, monosodium glutamate, molasses, glycerol, lactic acid, maltodextrin, maltose, or any combination thereof.
In a preferred embodiment, the nitrogen source is selected from yeast extract, tryptone, monosodium glutamate, ammonium chloride, ammonium sulfate, soy peptone, soy flour, corn flour, peanut cake, cottonseed meal, or any combination thereof. In a preferred embodiment, the nitrogen source is selected from yeast extract, tryptone, ammonium chloride or ammonium sulfate, or any combination thereof.
In a preferred embodiment, the Bacillus clausii culture medium with an inorganic salt concentration of less than 100g/L is LB culture medium with a NaCl concentration of less than 100 g/L.
In some embodiments, aerobic fermentation may be performed using aerobic fermentation equipment or methods commonly used in the art. The skilled person can select suitable fermentation equipment and fermentation conditions according to the actual fermentation situation. For example, in the case of aerobic shake flask fermentation, the fermentation conditions are such that the culture is carried out at 28-38 ℃, preferably 37 ℃, 150-. It should be noted that any optimization and adjustment of aerobic fermentation conditions by a person skilled in the art on the basis of the present invention is also within the scope of the present invention.
In a preferred embodiment, the process for the production of tetrahydropyrimidines comprises culturing Bacillus clausii ET01 according to the invention in LB medium or nutrient broth with a sodium chloride concentration of less than 100g/L, preferably 30-70g/L, more preferably 50g/L, at 28-38 ℃, preferably 37 ℃, 150-220rpm, preferably 200rpm, for 12-24 h.
In a preferred embodiment, the tetrahydropyrimidines are obtained by isolation and/or purification of a fermentation broth of aerobic fermentation (also referred to as "aerobic fermentation broth"). The tetrahydropyrimidine in the fermentation broth may be isolated and/or purified using equipment or methods commonly used in the art for isolation and/or purification, such as centrifugation, filtration, extraction, and the like. In a preferred embodiment, the tetrahydropyrimidines are obtained by extraction, for example by extraction with 80% ethanol, of the aerobic fermentation broth.
In a further preferred embodiment, said extracting tetrahydropyrimidine in said aerobic fermentation broth with 80% ethanol comprises the steps of: and (3) standing the aerobic fermentation liquor for 12-24h by using an equal volume of 80% ethanol water solution, centrifuging, and taking the supernatant to obtain the tetrahydropyrimidine. Before adding 80% ethanol water solution, the fermentation liquid may be centrifuged to separate the thallus and/or bacterial liquid for extraction.
In a preferred embodiment, the method further comprises activating the bacillus clausii. In a preferred embodiment, the activating comprises: inoculating the Bacillus clausii ET01 into a seed culture medium for shake cultivation, and culturing at 28-38 deg.C and 150-220rpm for 12-24 h.
In a preferred embodiment, the seed medium is LB medium (0.8-1 wt% peptone, 0.5-0.8 wt% yeast powder, 1-1.5 wt% sodium chloride; pH 6.8-7.0) or nutrient broth medium (0.8-1 wt% peptone, 0.3-0.5 wt% beef extract, 0.5-0.8 wt% sodium chloride; pH 7.2-7.6).
In a preferred embodiment, the tetrahydropyrimidine prepared by the process of the invention has a purity of 98% or more.
Examples
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and the detailed description. It should be understood that the detailed description and specific examples, while indicating the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. The experimental methods and equipment described in the following examples are conventional methods and equipment unless otherwise specified. The experimental materials used in the following examples, all reagents were purchased from OXOID, Inc., unless otherwise specified.
Reagents used in examples:
LB medium: tryptone 10 g/L; 5g/L of yeast extract powder; 0-100g/L of sodium chloride; sterilizing at 121 deg.C for 15min and pH 7.0.
LB solid medium: tryptone 10 g/L; 5g/L of yeast extract powder; 15g/L of agar; 20-100g/L of sodium chloride; sterilizing at 121 deg.C for 15min and pH 7.0.
Example 1 obtaining of Bacillus clausii ET01
Diluting 1g of a mud sample in a Weihai offshore salt farm to 100mL by using sterile water, coating 200 mu L of diluent on a LB solid culture medium of 20g/L, placing the mixture in a constant temperature incubator at 37 ℃ for culturing for 24h, selecting a single colony, performing multi-round streak culture, and finally separating to obtain a purified strain.
And carrying out bacterial morphology identification on the strain. Culturing the strain on an LB solid culture medium of 10g/L NaCl at 38 ℃ for 24 hours to form milky colony with irregular edge and a protrusion on the surface; when observed under a microscope at a magnification of 100 times, the cells were long-rod-shaped (as shown in FIG. 1), and gram-positive bacteria were obtained by gram-positive staining. According to the strain morphology and 16S rDNA sequencing comparison, the strain is identified and named as Bacillus clausii ET01(Bacillus clausii ET01), and the strain is preserved with the preservation number of CGMCC NO. 16310.
Example 2 culture conditions and product detection of Bacillus clausii
The deposited Bacillus clausii ET01 in glycerol tube was inoculated in an amount of 2 v/v% to 60mL of 10g/L NaCl LB medium and activated by overnight culture at 200rpm on a shaker at 37 ℃. The absorbance (OD) of the fermentation broth at a wavelength of 600nm was measured using a spectrophotometer600) Is 2.3.
The activated Bacillus clausii ET01 was inoculated in an amount of 1 v/v% to 60mL of LB medium containing 0g/L, 30g/L, 50g/L, 70g/L and 100g/L of NaCl, respectively, and cultured on a shaker at 37 ℃ and 200rpm for 24 hours.
In order to examine the growth of the Bacillus clausii ET01 of the present invention at various salt concentrations, the OD of the fermentation broth after 24h of cultivation was measured using a spectrophotometer600. Detecting and identifying fermentation products (tetrahydropyrimidine and hydroxypyrimidine) in fermentation liquor of bacillus clausii cultured for 24h by HPLC, and detecting by using an Agilent 1260 high-performance liquid phase detection system under the following detection conditions:
a) liquid chromatography column: zorbax SB C18 column (column length 150mm, column inner diameter 4.6 mm; packing particle size 5 μm);
b) mobile phase: 2% acetonitrile: 98% of water;
c) flow rate: 0.2 mL/min;
d) column temperature: 30 ℃;
e) sample introduction amount: 20 mu L of the solution;
f) detection wavelength: 210 nm.
g)0.1mg/mL of tetrahydropyrimidine standard aqueous solution and hydroxypyrimidine standard aqueous solution: both from Sigma-Aldrich, tetrahydropyrimidine, cat # E2271, hydroxypyrimidine, cat # 70709.
The detection is carried out on the standard products of the ectoine and the hydroxypyrimidine, the peak time of the ectoine is 7.8min, and the peak time of the hydroxypyrimidine is about 7.0min (HPLC peak figure is not shown).
The identification of tetrahydropyrimidine and hydroxypyrimidine in fermentation liquor cultured for 24h under different NaCl concentrations comprises the following steps: respectively taking 1mL of fermentation liquor, adding an equal volume of 80% ethanol solution for extraction, centrifuging at 8000rpm for 10min, and taking the supernatant for the HPLC detection. Results no significant hydroxypyrimidine product peak was detected at 7.0min for all samples (HPLC peak not shown), indicating that bacillus clausii of the present invention produces little hydroxypyrimidine; in addition, in all samples except the sample of 0g/L NaCl, a distinct tetrahydropyrimidine product peak was detected at 7.8min, and the results of HPLC peak patterns were numerically analyzed, and the data results are shown in Table 1.
Table 1: OD of fermentation liquor of bacillus clausii ET01 under different NaCl concentration conditions600And tetrahydropyrimidine assay results
| NaCl(g/L) | OD600 | Tetrahydropyrimidines (g/L) |
| 0 | 2.3 | 0 |
| 30 | 5.7 | 13.2 |
| 50 | 6.1 | 25.0 |
| 70 | 2.8 | 14.8 |
| 100 | 0.6 | 4.9 |
From the above results, it can be seen that: under the condition that no NaCl is added (0g/L NaCl), the strain of the Bacillus clausii grows normally, but no tetrahydropyrimidine is generated; in the case of too high a NaCl addition (100g/L NaCl), the OD of the strain600The yield is low, which indicates that the strain grows poorly, only weak tetrahydropyrimidine is accumulated, and the yield is low as a whole; in the case of 30-70g/L, the strain not only grows normally, but also has higher tetrahydropyrimidine constant, especially in the case of 50g/L NaClOD600The highest value indicates that the strain grows well, and the yield of the tetrahydropyrimidine is the highest when the strain is cultured in comparison with other salt concentrations and reaches 25 g/L.
Example 3 cultivation and product detection of Bacillus clausii TDB1-4
The fermentation culture was carried out using Bacillus clausii TDB1-4(CCTCC NO: M2015430) and Bacillus clausii ET01 of the present invention in LB medium with NaCl concentration of 0g/L and 50g/L, respectively. The strains were cultured according to the method of example 2 and OD 24 hours after fermentation culture was measured600And tetrahydropyrimidine, the results are shown in Table 2.
TABLE 2 OD of B.clausii ET01 and TDB1-4600And tetrahydropyrimidine detection results
As can be seen from Table 2, the growth of Bacillus clausii ET01 of the present invention is similar to that of Bacillus clausii TDB1-4 without NaCl addition; however, in LB medium with 50g/L NaCl, the Bacillus clausii ET01 of the present invention grows well and produces tetrahydropyrimidine; while Bacillus clausii TDB1-4 grows poorly and does not produce tetrahydropyrimidine. This demonstrates that the Bacillus clausii ET01 of the present invention has some salt tolerance and is capable of producing tetrahydropyrimidine. The ability of synthesizing tetrahydropyrimidine of the bacillus clausii ET01 of the invention can reach 25.0g/L, compared with other tetrahydropyrimidine producing strains (such as halomonas in CN103451137A, 18g/L) reported in the prior art, the invention has obvious improvement and has a prospect for industrial production of tetrahydropyrimidine.
Example 4 detection of other metabolites in B.clausii ET01 fermentation broth
According to GB/T18246-2000 and GB5009.157-2016, amino acids, organic acids and other products in the Bacillus clausii fermentation liquor cultured for 24h in LB culture medium of 50g/L NaCl are detected, and the results are shown in Table 3.
TABLE 3 amino acid and organic acid contents in the culture media before and after fermentation
As can be seen from Table 3, the fermentation of Bacillus clausii ET01 according to the invention does not result in the accumulation of a large number of other metabolites, which facilitates the subsequent isolation and purification of tetrahydropyrimidines.
The bacillus clausii ET01 is aerobically fermented and cultured by selecting LB culture medium of 50g/L NaCl to obtain fermentation liquor, then the fermentation liquor is extracted by 80% ethanol, and the tetrahydropyrimidine with the purity of more than 98% can be obtained after drying and crystallization, without high separation and purification processes with high cost, such as ion exchange chromatography, molecular sieve and the like, the cost can be saved, the operation is simple, and the method has good industrial application prospect.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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gaaggcgact ctctggtctg taactgacgc tgaggcgcga aagcgtgggg agcaaacagg 660
attagatacc ctggtagtcc acgccgtaaa cgatgagtgc taggtgttag gggtttcgat 720
gcccgtagtg ccgaagttaa cacattaagc actccgcctg gggagtacgg ccgcaaggct 780
gaaactcaaa ggaattgacg gggacccgca caagcagtgg agcatgtggt ttaattcgaa 840
gcaacgcgaa gaaccttacc aggtcttgac atcctttgac cacccaagag attgggcttc 900
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Claims (10)
1. A strain of Bacillus clausii ET01(Bacillus clausii ET01) for producing tetrahydropyrimidine has a preservation number of CGMCC NO. 16310.
2. Use of bacillus clausii ET01 according to claim 1 for the production of tetrahydropyrimidines.
3. A process for producing tetrahydropyrimidine, the process comprising: aerobic fermentation of Bacillus clausii ET01 according to claim 1 in a Bacillus clausii medium with a concentration of inorganic salts of less than 100g/L to obtain tetrahydropyrimidine.
4. The method according to claim 3, wherein the Bacillus clausii culture medium is a medium containing a carbon source, a nitrogen source, and inorganic salts for culturing Bacillus clausii; wherein,
the carbon source is preferably selected from glucose, dextrin, sucrose, monosodium glutamate, molasses, glycerol, lactic acid, maltodextrin, maltose, or any combination thereof;
the nitrogen source is preferably selected from yeast extract, tryptone, monosodium glutamate, ammonium chloride, ammonium sulfate, soybean peptone, soybean flour, corn flour, peanut cake, cottonseed meal, or any combination thereof;
the inorganic salt is preferably selected from sodium chloride, dipotassium phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, iron sulfate heptahydrate, calcium chloride dihydrate, potassium chloride, sodium sulfate, disodium phosphate, or any combination thereof.
5. The method of claim 4, wherein the carbon source is glucose; the nitrogen source is selected from yeast extract, tryptone, ammonium chloride or ammonium sulfate, or any combination thereof; the inorganic salt is sodium chloride.
6. The method according to claim 3 or 4, wherein the Bacillus clausii culture medium is LB medium or nutrient broth medium.
7. A process according to any one of claims 3 to 6, wherein the inorganic salt concentration is less than 100g/L, preferably 30 to 70g/L, more preferably 50 g/L.
8. The method as claimed in any one of claims 3 to 7, wherein the aerobic fermentation is carried out at a temperature of 28-38 ℃ and a speed of 150-220rpm for a period of 12-24 h.
9. The process according to claim 3, wherein the Bacillus clausii ET01 is cultivated for 12 to 24h at 28 to 38 ℃, preferably 37 ℃, 150-.
10. The method of any one of claims 3-9, wherein the method further comprises activating the bacillus clausii ET 01.
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| CN113308482B (en) * | 2021-05-28 | 2022-12-27 | 深圳中科欣扬生物科技有限公司 | Tetrahydropyrimidine synthetic gene cluster from Yunnan tengcong and application thereof |
| CN113621540A (en) * | 2021-08-11 | 2021-11-09 | 沈阳农业大学 | Bacillus clausii strain and screening method and application thereof |
| CN113621540B (en) * | 2021-08-11 | 2023-01-06 | 沈阳农业大学 | Bacillus clausii strain and screening method and application thereof |
| CN116333906A (en) * | 2022-07-13 | 2023-06-27 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Bacillus and method for preparing ectoin by using same |
| CN116333906B (en) * | 2022-07-13 | 2023-10-17 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Bacillus and method for preparing ectoin by using same |
| CN115261284A (en) * | 2022-08-30 | 2022-11-01 | 河北农业大学 | Halobacterium clausii PA21 microbial inoculum and preparation method thereof |
| CN115404184A (en) * | 2022-08-30 | 2022-11-29 | 河北农业大学 | Halobacterium clausii PA21 and application thereof in degradation of potato solanine |
| CN115261284B (en) * | 2022-08-30 | 2023-05-26 | 河北农业大学 | Alkaline halophiles clausii PA21 microbial inoculum and preparation method thereof |
| CN115404184B (en) * | 2022-08-30 | 2023-07-18 | 河北农业大学 | Alkaline halophiles clausii PA21 and application thereof in degradation of potato solanine |
| CN116200297A (en) * | 2022-12-27 | 2023-06-02 | 山东丰金美业科技有限公司 | High-yield tetrahydropyrimidine salt-addicted single island bacillus cereus and culture method and application thereof |
| CN116200297B (en) * | 2022-12-27 | 2023-10-03 | 山东丰金美业科技有限公司 | High-yield tetrahydropyrimidine salt-addicted single island bacillus cereus and culture method and application thereof |
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| Publication number | Publication date |
|---|---|
| CN111304106B (en) | 2021-07-30 |
| WO2020119828A1 (en) | 2020-06-18 |
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