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WO2023016387A1 - Bacillus amyloliquefaciens et son utilisation dans la préparation de 1-désoxynojirimycine - Google Patents

Bacillus amyloliquefaciens et son utilisation dans la préparation de 1-désoxynojirimycine Download PDF

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Publication number
WO2023016387A1
WO2023016387A1 PCT/CN2022/110772 CN2022110772W WO2023016387A1 WO 2023016387 A1 WO2023016387 A1 WO 2023016387A1 CN 2022110772 W CN2022110772 W CN 2022110772W WO 2023016387 A1 WO2023016387 A1 WO 2023016387A1
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bacillus amyloliquefaciens
fermentation
preparation
culture
medium
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PCT/CN2022/110772
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Chinese (zh)
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彭湘屏
郑玲辉
朱进伟
孙琼
高祥
石磊
张敏
陈世敏
汪超
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浙江珲达生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring

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  • the invention belongs to the technical field of industrial biology, and in particular relates to a strain of Bacillus amyloliquefaciens capable of producing 1-deoxynojirimycin, and a method and application for fermenting and producing 1-deoxynojirimycin using the strain.
  • 1-Deoxynojirimycin (1-Deoxynojirimycin, DNJ) is a piperidine alkaloid, the chemical name is 3,4,5-trihydroxy-2-hydroxymethyltetrahydropyridine, which is present in plants, microorganisms
  • the natural sugar analogues in silkworm body the highest content in mulberry tree in nature, is a powerful sugar metabolism enzyme inhibitor (such as ⁇ -glucosidase, hexokinase, glucuronidase and glycogen phosphatase, etc.), It can significantly delay the degradation process of polysaccharides, reduce the peak of postprandial blood sugar, and stabilize fasting blood sugar.
  • DNJ can also be used in the food field. Foods made from mulberry leaves, as functional hypoglycemic products, have been allowed to be sold in the market in Japan and other East Asian countries, showing the broad prospects of DNJ in the field of health food.
  • DNJ used in medicine is basically extracted from mulberry leaves.
  • the content of DNJ in natural products is relatively low; .
  • the synthesis of artificial DNJ is difficult and costly, and it is not suitable for large-scale production.
  • microorganisms that can produce DNJ have been reported at home and abroad as Streptomyces lavendulae, Monascus purpureus, Bacillus subtilis, Escherichia coli, and Bacillus amyloliquefaciens. .
  • Yohji Ezure et al. (1985) obtained the highest yield of Streptomyces lavender DNJ by mutation, which can reach 4-5g/L.
  • CN105296565A discloses that the purity of DNJ obtained by solid-state fermentation of Bacillus subtilis is relatively low. Gu Chengchen (2016) reported that the fermentation level of Monascus purple DNJ was 0.0279g/L. The fermentation level of Escherichia coli DNJ reported by KR1020190041680 was 0.264g/L. Kenji Yamagishi et al. (2016) and CN201810125101.X reported a strain of Bacillus amyloliquefaciens producing DNJ at a level of about 1.1 g/L.
  • the microorganisms that can produce 1-deoxynojirimycin reported in the prior art generally have the disadvantages of low yield of 1-deoxynojirimycin and long fermentation time. Therefore, it is of great significance to screen high-yield 1-deoxynojirimycin strains.
  • one of the objects of the present invention is to provide a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HDCC00252 with a high yield of 1-deoxynojirimycin, which is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee (CGMCC), the deposit number is CGMCC NO.22781, and the deposit date is June 25, 2021.
  • CGMCC General Microorganism Center of China Microbiological Culture Collection Management Committee
  • the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HDCC00252 of the present invention is obtained by screening the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HDCC00031 through NTG mutagenesis and ARTP-ultraviolet compound mutagenesis.
  • Another object of the present invention is to provide the application of the Bacillus amyloliquefaciens HDCC00252 or its fermentation liquid in the preparation of 1-deoxynojirimycin.
  • Another object of the present invention is to provide a method for preparing 1-deoxynojirimycin, which is prepared by fermentation with the Bacillus amyloliquefaciens described in claim 1.
  • the fermentation process includes performing aerobic fermentation in a fermentation medium containing assimilable carbon source and/or nitrogen source.
  • the carbon source is selected from glucose, glycerol, sucrose, fructose, lactose, maltose, dextrin, starch, mannitol, sorbitol; preferably glucose, sucrose, lactose or any combination thereof;
  • the nitrogen source is selected from corn steep liquor (powder), yeast extract, yeast extract powder, yeast peptone, soybean peptone, bovine bone peptone, meat peptone, fish powder peptone, nitrate, ammonium salt; preferably Yeast extract powder, nitrates, ammonium salts or any combination thereof.
  • the fermentation medium further includes inorganic salts, preferably sulfates, phosphates, ferrous salts, more preferably ammonium sulfate, dipotassium hydrogen phosphate, and ammonium ferrous sulfate.
  • inorganic salts preferably sulfates, phosphates, ferrous salts, more preferably ammonium sulfate, dipotassium hydrogen phosphate, and ammonium ferrous sulfate.
  • the fermentation medium contains 0.1-2% of glucose, 0.5-4% of sucrose, 0.5-10% of lactose, 2-5% of yeast extract powder, 0-4% of ammonium sulfate, and 0-4% of sodium nitrate. 3%, ferrous ammonium sulfate 0.1-0.8%, dipotassium hydrogen phosphate 0.2-0.6%.
  • the fermentation temperature is 28-40° C.
  • the pH of the culture medium is 5.0-9.0
  • the culture time is 12-100 hours.
  • the Bacillus amyloliquefaciens is fermented by inoculating seed liquid into the fermentation medium;
  • the seed liquid is obtained by culturing Bacillus amyloliquefaciens HDCC00252 in a seed medium.
  • the conditions of the seed cultivation are as follows: the temperature of the seed cultivation is 28-40° C., the pH of the medium is 5.0-9.0; the cultivation time is 4-24 hours;
  • the seed medium contains 0.2-4% of glucose, 0.2-1% of yeast extract powder, 0.2-2% of sodium chloride and 0.2-4% of peptone.
  • Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HDCC00252 of the present invention has high production capacity, and its ability to produce 1-deoxynojirimycin has been greatly improved compared with other strains in the prior art.
  • the titer can reach 5.31g/L. And the fermentation cycle is short, which is beneficial to realize industrial production.
  • Fig. 1 is the HPLC collection of illustrative plates of departure bacterial strain fermented liquid
  • Fig. 2 is the LCMS collection of illustrative plates of departure bacterial strain fermented liquid
  • Fig. 3 is the morphological figure of bacterial colonies starting from
  • Fig. 4 is the microscopic examination picture of the starting strain.
  • Embodiment 1 departure bacterial strain source
  • the original DNJ-producing strain was isolated from soil samples of mulberry roots in Xiniuqiao Village, Tongxiang City, Zhejiang province.
  • the composition of the liquid primary screening medium is: lactose 2.5%, ammonium sulfate 0.4%, the pH is adjusted to 7.5 before disinfection, and the disinfection conditions are 121-123°C for 30min.
  • Embodiment 2 DNJ original production strain (HDCC00031) morphological examination and physiological and biochemical tests
  • the colonies are irregular round or oval, white, with rough surface and bulges.
  • the colony morphology is shown in Figure 3, and the microscope photo is shown in Figure 4.
  • the measured 16S rDNA sequence (SEQ ID NO: 1) of the bacterial strain (HDCC00031) is compared with the sequences of related species and genus in the GenBank database by BLAST after proofreading, as shown in Table 5 (only the homologous It was found that the classification parameters of this strain were very close to those of Bacillus amyloliquefaciens. Therefore, the strain HDCC00031 was identified as a strain of Bacillus amyloliquefaciens.
  • Embodiment 4 DNJ high-yield strain (HDCC00252) mutagenesis screening
  • the original strain (HDCC00031) as the starting strain, collect its fresh bacterial lawn, wash it with sterile physiological saline, add glass beads to oscillate to disperse, and obtain a bacterial suspension.
  • the bacterial suspension was mixed with 500 ⁇ g/ml NTG mother solution 1:1 (v/v), placed on a shaker at 30°C for 30 minutes, centrifuged at 14,000 rpm, resuspended with normal saline, and washed repeatedly for 3 times before gradient dilution.
  • aqueous acetic acid solution 1.5mL 0.1% (V/V) at last, carry out liquid chromatography analysis after filtering through 0.45 ⁇ m microporous membrane, select titer to be higher than the bacterial strain more than 100% of contrast and carry out double screening, confirm that production capacity is stable and reliable Repeated strains were used as starting strains for further mutagenesis screening.
  • the composition of the liquid primary screening medium is: lactose 2.5%, ammonium sulfate 0.4%, the pH is adjusted to 7.5 before disinfection, and the disinfection conditions are 121-123°C for 30min.
  • the strain with the highest DNJ content was selected and preserved after three consecutive rounds of streaking, isolation and purification, and the preservation number was HDCC00252. (The strain was then deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on June 25, 2021, with a preservation number of CGMCC NO.22781).
  • Bacteria recovery and activation Take the original strain HDCC00031, thaw it at room temperature, inoculate 0.1ml of the bacterial suspension onto the LB solid plate, spread it evenly, and culture it in a 37°C incubator for 24 hours to obtain activated and recovered bacteria. moss.
  • the liquid seed culture medium consists of 1.5% glucose, 0.5% yeast extract powder, 1% peptone and 1% sodium chloride. Adjust the pH to 7.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
  • Fermentation culture take the liquid seeds qualified for cultivation, inoculate them into a 250ml Erlenmeyer flask with 30ml optimized liquid fermentation medium at a ratio of 4% (V/W), wrap them up and place them on a shaker at 37°C and 220rpm The shaking culture was completed after 4 days.
  • the formula of the liquid fermentation medium is as follows: 0.4% of glucose, 2% of sucrose, 2% of lactose, 3% of yeast extract powder, 0.1% of ammonium sulfate, 0.05% of sodium nitrate, 0.25% of ferrous ammonium sulfate, and 0.28% of dipotassium hydrogen phosphate. Adjust the pH to 8.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
  • Chromatographic column Kromasil C18 analytical column (4.6mm ⁇ 250mm, 5 ⁇ m)
  • Detection conditions fluorescence detector, excitation wavelength 254nm, emission wavelength 322nm
  • the sample derivatized in the same way as the known concentration DNJ reference substance as a standard calculate the sample concentration according to the standard concentration, peak area, and sample peak area, and the result confirms that the DNJ content in the fermented liquid obtained by this embodiment is 0.12g/L.
  • Bacterial recovery and activation take the bacterial strain HDCC00252, thaw it at room temperature, inoculate 0.1ml of the bacterial suspension onto the LB solid plate, spread it evenly, and culture it in a 37°C incubator for 24 hours to obtain an activated and recovered bacterial lawn .
  • the liquid seed culture medium consists of 1.5% glucose, 0.5% yeast extract powder, 1% peptone and 1% sodium chloride. Adjust the pH to 7.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
  • Fermentation culture take the liquid seeds qualified for cultivation, inoculate them into a 250ml Erlenmeyer flask with 30ml optimized liquid fermentation medium at a ratio of 4% (V/W), wrap them up and place them on a shaker at 37°C and 220rpm The shaking culture was completed after 4 days.
  • the formula of the liquid fermentation medium is as follows: 0.4% of glucose, 2% of sucrose, 2% of lactose, 3% of yeast extract powder, 0.1% of ammonium sulfate, 0.05% of sodium nitrate, 0.25% of ferrous ammonium sulfate, and 0.28% of dipotassium hydrogen phosphate. Adjust the pH to 8.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
  • Chromatographic column Kromasil C18 analytical column (4.6mm ⁇ 250mm, 5 ⁇ m)
  • Detection conditions fluorescence detector, excitation wavelength 254nm, emission wavelength 322nm
  • Bacterial recovery and activation take the bacterial strain HDCC00252, thaw it at room temperature, inoculate 0.1ml of the bacterial suspension onto the LB solid plate, spread it evenly, and culture it in a 37°C incubator for 24 hours to obtain an activated and recovered bacterial lawn .
  • the liquid seed medium consists of 4% glucose, 1% yeast extract powder, 4% peptone and 2% sodium chloride. Adjust the pH to 5.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
  • Fermentation culture take the liquid seeds qualified for cultivation, inoculate them into a 250ml Erlenmeyer flask with 30ml optimized liquid fermentation medium at a ratio of 4% (V/W), wrap them up and place them on a shaker at 28°C and 220rpm The shaking culture was completed after 4 days.
  • the liquid fermentation medium formula consists of: 0.1% of glucose, 0.5% of sucrose, 10% of lactose, 5% of yeast extract powder, 4% of ammonium sulfate, 0.1% of ferrous ammonium sulfate, and 0.6% of dipotassium hydrogen phosphate. Adjust the pH to 9.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
  • Chromatographic column Kromasil C18 analytical column (4.6mm ⁇ 250mm, 5 ⁇ m)
  • Detection conditions fluorescence detector, excitation wavelength 254nm, emission wavelength 322nm
  • the sample derivatized by the same method as the known concentration DNJ reference substance as a standard calculate the sample concentration according to the standard concentration, peak area, and sample peak area, and the result confirms that the DNJ content in the fermented liquid obtained by this embodiment is 5.01g/L.
  • Bacterial recovery and activation take the bacterial strain HDCC00252, thaw it at room temperature, inoculate 0.1ml of the bacterial suspension onto the LB solid plate, spread it evenly, and culture it in a 37°C incubator for 24 hours to obtain an activated and recovered bacterial lawn .
  • the liquid seed medium consists of 0.2% glucose, 0.2% yeast extract powder, 0.2% peptone and 0.2% sodium chloride. Adjust the pH to 9.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
  • Fermentation culture take the liquid seeds qualified for cultivation, inoculate them into a 250ml Erlenmeyer flask with 30ml optimized liquid fermentation medium at a ratio of 4% (V/W), wrap them up and place them on a shaker at 37°C and 220rpm The shaking culture was completed after 4 days.
  • the formula of the liquid fermentation medium is as follows: 2% of glucose, 4% of sucrose, 0.5% of lactose, 2% of yeast extract powder, 3% of sodium nitrate, 0.8% of ferrous ammonium sulfate, and 0.2% of dipotassium hydrogen phosphate. Adjust the pH to 5.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
  • Chromatographic column Kromasil C18 analytical column (4.6mm ⁇ 250mm, 5 ⁇ m)
  • Detection conditions fluorescence detector, excitation wavelength 254nm, emission wavelength 322nm
  • the sample derivatized by the same method as the known concentration DNJ reference substance as a standard calculate the sample concentration according to the standard concentration, peak area, and sample peak area, and the result confirms that the DNJ content in the fermented liquid obtained by this embodiment is 4.98g/L.

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Abstract

L'invention concerne un Bacillus amyloliquefaciens HDCC00252 dont le numéro de dépôt est CGMCC No 22781. L'invention concerne également un procédé de production de 1-désoxynojirimycine au moyen d'une culture par fermentation de Bacillus amyloliquefaciens HDCC00252.
PCT/CN2022/110772 2021-08-09 2022-08-08 Bacillus amyloliquefaciens et son utilisation dans la préparation de 1-désoxynojirimycine WO2023016387A1 (fr)

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CN113637605B (zh) * 2021-08-09 2023-05-05 浙江珲达生物科技有限公司 一种解淀粉芽孢杆菌及其在制备1-脱氧野尻霉素中的应用

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