WO2018099366A1 - 一种枯草芽孢杆菌及其培养方法与应用 - Google Patents

一种枯草芽孢杆菌及其培养方法与应用 Download PDF

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WO2018099366A1
WO2018099366A1 PCT/CN2017/113344 CN2017113344W WO2018099366A1 WO 2018099366 A1 WO2018099366 A1 WO 2018099366A1 CN 2017113344 W CN2017113344 W CN 2017113344W WO 2018099366 A1 WO2018099366 A1 WO 2018099366A1
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culture
strain
medium
fructose
progeny
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PCT/CN2017/113344
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French (fr)
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窦光朋
邵先豹
杜倩
干昭波
李方华
张明站
杨腾腾
刘双双
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山东百龙创园生物科技股份有限公司
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Priority to US16/318,358 priority Critical patent/US10968424B2/en
Priority to CA3045528A priority patent/CA3045528C/en
Priority to EP17875315.8A priority patent/EP3550011A4/en
Publication of WO2018099366A1 publication Critical patent/WO2018099366A1/zh

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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
    • 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
    • C12N1/205—Bacterial isolates
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y501/00—Racemaces and epimerases (5.1)
    • C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)

Definitions

  • the invention relates to the field of microbial technology.
  • the present invention relates to a Bacillus subtilis strain or a progeny thereof, a culture method and application thereof.
  • the invention also relates to cultures and lysates of the B. subtilis strain or a progeny thereof.
  • the present invention also relates to a method for producing D-psicose epimerase using the Bacillus subtilis strain or a progeny thereof, and a method for producing D-psicose using the Bacillus subtilis strain or a progeny thereof.
  • Bacillus subtilis belonging to the genus Bacillus.
  • the bacterium is a Gram-positive bacterium, the single cell is 0.7-0.8 ⁇ 2 ⁇ 3 micron, and the spore is 0.6-0.9 ⁇ 1.0-1.5 micron. It is elliptical to columnar and is located in the center of the cell or slightly biased. After the spores are formed, the cells do not expand. The colony of Bacillus subtilis is rough and opaque, staining white to pale yellow.
  • the bacterium is an aerobic bacterium.
  • D-psicose is an important rare sugar found in nature in very small amounts in sugar cane molasses, dried fruit, sugar products, wheat and thorn plants. Its name originates from the antibiotic apyrone glucoside, and a small amount of D-psicose can also be isolated from the antibiotic psicose adenosine. D-psicose is absorbed into the blood circulation through the small intestine in the human body, is not metabolized into energy after absorption in the small intestine, and has low fermentation utilization for intestinal microorganisms. D-psicose has a variety of important physiological functions: neuroprotective, hypoglycemic, lipid-lowering, scavenging reactive oxygen species, anti-oxidation, inhibiting cancer cell proliferation, and as a low-calorie sweetener.
  • D-psicose is the C-3 epimer of D-fructose.
  • the early D-psicose synthesis method was to convert common monosaccharide or non-sugar precursors by multi-stage chemical, but multiple protection and deprotection operations resulted in low yield and complicated synthesis process. Big limitations.
  • D-psulose has been used as a raw material to prepare D-psicose by biotransformation, but the microorganisms which have been reported so far have poor conversion ability of D-psicose epimerase. The separation and purification process is difficult.
  • Chinese patent document CN105602879A (Application No. 201610051547.3) discloses a genetically engineered strain for efficiently secreting D-psicose 3-epimerase and a method for constructing the same.
  • the invention realizes RDPE in Bacillus subtilis by constructing a recombinant expression plasmid pMA5-RDPE by using D-psicose 3-epimerase gene rdpe derived from Ruminococcus sp. 5_1_39B_FAA, and then transforming Bacillus subtilis The constitutive secretory expression.
  • the optimal inducible promoter PxylA was obtained and the secretion level of RDPE was significantly increased.
  • Bacillus subtilis was blocked by knocking out the xylose utilization gene xylAB (xylA and xylB)
  • the xylose metabolic pathway further increased the secretion of RDPE and reduced the optimal induction concentration of the inducer xylose from 4.0% to 0.5%.
  • the engineered strain 1A751SD-XR was evaluated in a 7.5L fermentor by means of fed-batch, and the secretion level of RDPE was up to 95U/mL and 2.6g/L.
  • the enzyme activity of the enzyme is still low and cannot meet the needs of actual production.
  • the term "medium” is any known solid or liquid medium known in the art for supporting the growth of cells, particularly bacteria, such as Bacillus bacteria, such as Bacillus subtilis.
  • a medium eg, a first medium, a second medium, a solid medium, a seed medium, or a fermentation medium
  • the medium typically comprises a carbon source, a nitrogen source, an inorganic salt, and any other nutrients needed to maintain the bacteria in a viable state.
  • culture of a strain refers to a product obtained after culturing the strain in a medium.
  • lysate of a strain refers to a product obtained by disrupting the cell wall and/or cell membrane of a strain, which comprises an intracellular substance.
  • the lysate of the strain can be obtained by various techniques known in the art, such as sonication, homogenization, osmotic shock, freeze-thaw disruption, enzymatic dissolution, and the like.
  • the inventors of the present application have, after extensive experimentation and repeated exploration, unexpectedly found that the activity of a D-psicose epimerase produced by a specific mutagenized Bacillus subtilis strain is significantly higher than that of wild type hay Bacillus strain. Based on this finding, the inventors have developed a new Bacillus subtilis strain, a culture method thereof, and a preparation method of D-psicose based on the strain.
  • the invention provides a strain of Bacillus subtilis BLCY-005 or its progeny, the strain BLCY-005 was deposited on October 26, 2016 at the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the deposit number is CGMCC No. 13152.
  • the activity of the D-psicose epimerase produced by the strain or its progeny is compared to the D-psicose epimerase produced by the wild type strain , increased by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%.
  • the activity of the D-psicose epimerase produced by the strain or its progeny is compared to the D-psicose epimerase produced by the wild type strain , increased by at least 90%, or at least 100%.
  • the invention provides a culture comprising a Bacillus subtilis strain as described herein or a progeny thereof.
  • the culture comprises a medium, such as a solid medium or a liquid medium.
  • the activity of the D-psicose epimerase in the culture is increased compared to the D-psicose epimerase produced by the wild type strain. At least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. In certain preferred embodiments, the activity of the D-psicose epimerase in the culture is increased compared to the D-psicose epimerase produced by the wild type strain. At least 90%, or at least 100%.
  • the culture can be obtained by culturing the strain of the present invention or its progeny in a medium and under culture conditions, and those skilled in the art know how to select a suitable medium and culture conditions depending on the nature of the strain. .
  • the culture is obtained by culturing the strain of the invention or its progeny by the methods described above.
  • the invention provides a lysate of a strain, which is a Bacillus subtilis strain as described herein or a progeny thereof.
  • the lysate comprises a D-psicose epimerase.
  • the activity of the D-psicose epimerase in the lysate is increased compared to the D-psicose epimerase produced by the wild type strain. At least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. In certain preferred embodiments, the activity of the D-psicose epimerase in the lysate is compared to that produced by a wild type strain D-psicose epimerase, increased by at least 90%, or at least 100%.
  • the invention provides a composition comprising fructose, and at least one component selected from the group consisting of: (1) a strain of the invention or a progeny thereof; (2) a culture of the invention; (3) The lysate of the present invention.
  • the fructose is obtained or provided in the form of a crystalline fructose aqueous solution, liquid fructose, fructose syrup (eg, high fructose corn syrup), dried fructose or fructose syrup.
  • the invention provides a method of culturing a Bacillus subtilis strain, or a progeny thereof, as described herein, comprising the step of seeding said strain or progeny thereof in a culture medium.
  • the medium is any known solid or liquid medium known in the art for supporting the growth of bacteria, particularly Bacillus bacteria, such as Bacillus subtilis.
  • the medium comprises a carbon source, a nitrogen source, an inorganic salt, and any other nutrients required to maintain the bacteria in a viable state.
  • the method comprises the steps of:
  • the strain in the first medium is inoculated into a second medium for culture.
  • step (1) at 25-40 ° C (eg, 25-38 ° C, 28-40 ° C, 28-38 ° C, 28-35 ° C, or 30-38 ° C; for example
  • the culture was carried out at a temperature of 35 ° C).
  • the cultivation is carried out at a temperature of 30-38 °C.
  • the cultivation is carried out at a temperature of 35 °C.
  • step (2) at 25-40 ° C (eg, 25-38 ° C, 28-40 ° C, 28-38 ° C, 28-35 ° C, or 30-38 ° C; for example
  • the culture was carried out at a temperature of 35 ° C).
  • the cultivation is carried out at a temperature of 30-38 °C.
  • the cultivation is carried out at a temperature of 35 °C.
  • step (3) at 25-40 ° C (eg, 25-38 ° C, 28-40 ° C, 28-38 ° C, 28-35 ° C, or 30-38 ° C; for example
  • the culture was carried out at a temperature of 35 ° C).
  • the cultivation is carried out at a temperature of 30-38 °C.
  • the cultivation is carried out at a temperature of 35 °C.
  • the first medium is a seed medium.
  • the second medium is a fermentation medium.
  • the method comprises the steps of:
  • the strain of the present invention or a progeny thereof is inoculated in a solid medium, and cultured at a temperature of 25 to 40 ° C for 6 to 12 hours to obtain an activated strain;
  • the seed liquid obtained in the step (2) is inoculated in a fermentation medium at a ratio of 1-10% by volume, at 25-40 ° C (for example, 25-38 ° C, 28-40 ° C, 28-38 ° C,
  • the fermentation broth is obtained by culturing at a temperature of 28-35 ° C, or 30-38 ° C; for example, 35 ° C, for 30-48 hours.
  • the seed medium comprises: 1% peptone, 0.5% yeast dipping powder, 1% sodium chloride, 0.01% anhydrous magnesium sulfate, 0.02% phosphoric acid Potassium hydrogen, and the balance of water (all percentages are by weight).
  • the pH of the seed medium is from 6.0 to 7.0 in step (2).
  • the fermentation medium comprises: 3% yeast extract, 2% corn flour, 1% glucose, 0.01% anhydrous magnesium sulfate, 0.02% hydrogen phosphate Diammonium, 0.02% ammonium sulfate, and the balance of water.
  • the pH of the fermentation medium is from 6.0 to 7.0 in step (3).
  • the solid medium is LB medium.
  • the invention provides a method of preparing a D-psicose epimerase comprising lysing a Bacillus subtilis strain of the invention or a progeny thereof and isolating D-psicose differentially
  • the method comprises the steps of: (1) cultivating the strain to obtain a fermentation broth of the strain under conditions that allow growth of the strain of the invention or a progeny thereof; and (2) The D-psicose epimerase is isolated from the fermentation broth obtained in the step (1).
  • step (1) the strain is cultured by a method as described herein.
  • step (2) the D-psicose epimerase is isolated from the fermentation broth by centrifugation, filtration, dialysis or chromatography. In certain exemplary embodiments, in step (2), the D-psicose epimerase is separated from the fermentation broth by centrifugation.
  • step (2) the D-psicose epimerase is isolated from the fermentation broth by the following steps:
  • step (2c) The product of the step (2b) is dried (for example, vacuum freeze-dried) to obtain a D-psicose epimerase.
  • step (2b) the precipitate obtained in step (2a) is thermally denatured at a temperature of 50-60 °C. In certain preferred embodiments, in step (2b), the precipitate obtained in step (2a) is thermally denatured at a temperature of 50-60 ° C for 40-60 min.
  • step (2b) is subjected to vacuum freeze drying in step (2c). In certain preferred embodiments, in step (2c), the product of step (2b) is subjected to vacuum freeze drying at -40 to -60 ° C, -20 to -60 kPa.
  • step (2a) the fermentation broth is centrifuged at 4 ° C, 10000 r/min for 10 min.
  • step (2) the D-psicose epimerase is separated from the fermentation broth by the following steps: (i) obtaining step (1) Centrifuging the fermentation broth, discarding the supernatant; (ii) washing the product of step (i); (iii) centrifuging the product of step (ii) to obtain a precipitate, which is a crude enzyme preparation; (vi) The crude enzyme preparation obtained in the step (iii) is placed in a water bath at 50-60 ° C for heat denaturation for 40-60 min; (v) the product of the step (vi) is at -40 to -60 ° C, -20 to -60 kPa. Vacuum freeze-drying was carried out under the conditions.
  • step (ii) the strain is washed with 50 mmol/L Tris-HCl buffer (pH 8.0).
  • the method comprises the step of isolating the D-psicose epimerase from the culture or lysate of the invention.
  • the D-psicose epimerase is isolated from the culture or lysate by centrifugation, filtration, dialysis or chromatography.
  • the D-psicose epimerase is isolated from the culture by centrifugation.
  • the invention provides a method of preparing D-psicose comprising any of the following steps:
  • the culture of the invention is contacted with fructose
  • the D-psicose epimerase is isolated from the strain of the present invention or its progeny, culture or lysate, and the D-psicose epimerase is contacted with fructose.
  • the fructose is obtained or provided in the form of a crystalline fructose aqueous solution, liquid fructose, fructose syrup (eg, high fructose corn syrup), dried fructose or fructose syrup.
  • the method comprises the steps of: (1) isolating a D-psicose epimerase from a strain of the invention or a progeny, culture or lysate thereof; (2) The D-psicose epimerase obtained in the step (1) is contacted with fructose; (3) D-psicose is optionally recovered from the product of the step (2).
  • step (2) the D-psicose epimerase is reacted under conditions suitable for D-psicose epimerase activity Fructose is in contact. In certain preferred embodiments, in step (2), the D-psicose epimerase is contacted with fructose at a pH of 5.5-6.5.
  • Bacillus subtilis BLCY-005 was inoculated in a solid medium, and activated at 6 to 12 hours under conditions of 30 to 38 ° C to prepare an activated strain;
  • the seed liquid obtained in the step (2) is inoculated into the fermentation medium at a ratio of 1 to 10% by volume, and the culture medium is expanded at 30 to 38 ° C for 30 to 48 hours to obtain a bacterial fermentation broth.
  • Peptone 1% peptone 1%, yeast dipping powder 0.5%, sodium chloride 1%, anhydrous magnesium sulfate 0.01%, potassium dihydrogen phosphate 0.02%, balance water, pH 6.0-7.0;
  • Yeast extract powder 3% corn syrup powder 2%, glucose 1%, anhydrous magnesium sulfate 0.01%, diammonium phosphate 0.02%, ammonium sulfate 0.02%, balance water, pH 6.0-7.0.
  • step (b) The crude enzyme preparation prepared in the step (a) is subjected to heat denaturation in a water bath of 50 to 60 ° C for 40 to 60 minutes, and then used in a vacuum freeze dryer at a working pressure of -40 to -60 ° C, -20 to -60 kPa. Drying is to prepare a dry powder of D-psicose epimerase.
  • washing cell of the step (a) is a Tris-HCl buffer solution having a pH of 8.0 and a concentration of 50 mmol/L, and then separated by centrifugation to retain a precipitate to obtain a coarse phase. Enzyme.
  • the D-psicose epimerase produced by the Bacillus subtilis strain BLCY-005 of the present invention has remarkably improved enzymatic activity.
  • the enzyme activity of D-psicose epimerase in the culture or lysate of the strain of the present invention can reach 143 U/ml, which is more D-psicose epimerase than the wild type strain. , nearly doubled, which can significantly reduce production costs.
  • the optimum pH of the D-psicose epimerase produced by the Bacillus subtilis strain BLCY-005 of the present invention is 5.5 to 6.5, and the D-psicose produced by the wild type strain
  • the optimum pH neutrality of the epimerase is advantageous for the control of pollution in production.
  • the D-psicose epimerase produced by the Bacillus subtilis strain BLCY-005 of the present invention is an intracellular enzyme, and the separation process is simple, and can be obtained by simple centrifugation, washing, inactivation and drying. Dry powder enzyme preparation saves production cost and reduces power loss.
  • D-psicose can be prepared by directly contacting D-psicose epimerase produced by the strain of the present invention with D-fructose, and the conventional fermentation solution is passed. Compared with the method of preparing D-psicose by contacting D-fructose, the difficulty and cost of subsequent D-psicose purification are greatly reduced, and the quality of the D-psicose finished product can be remarkably improved.
  • the present invention relates to the following biological materials that have been deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee (No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing):
  • a scribing separation method Using a scribing separation method, a large test tube containing 5 ml of sterile water was taken, and 2 ml of the bacterial solution enriched in 1.1 was placed therein and diluted, and shaken well. Use the inoculating loop to pick up the diluent in a sterile operation. First, make a first parallel scribe line 3-4 on the side of the plate medium, and then rotate the culture dish at an angle of about 60 degrees to burn off the residue on the inoculation ring. After cooling, the same scribing method is used for the second scribing, and the third and fourth scribing are performed in the same manner.
  • the dish cover is covered, the culture dish is inverted, and cultured at 28-38 ° C for 24 h, a single colony is picked and inoculated on 10 slant medium, numbered 01 to 10, respectively.
  • 01 ⁇ 10 slant seeds were inoculated in shake flask medium and cultured at 28 ⁇ 38°C for 24h.
  • the enzyme activity of D-psicose against D-fructose was measured in 01 ⁇ 10 shake flask fermentation broth. No. 06 shake flask enzyme The highest activity, reaching 75U/ml.
  • the plate medium components are as follows, all in weight percentage:
  • the slant medium components are as follows, all in weight percentage:
  • the shake flask medium components are as follows, all in weight percentage:
  • Yeast extract powder 3% corn syrup powder 2%, glucose 1%, anhydrous magnesium sulfate 0.01%, diammonium hydrogen phosphate 0.02%, ammonium sulfate 0.02%, balance water, pH 6.0 ⁇ 7.0;
  • UV-induced mutagenesis of No. 06 strain UV-induced mutagenesis was carried out by using a 15W ultraviolet lamp at 20cm, and the irradiation time was 120s.
  • the obtained high-yield strain was subjected to nitrosoguanidine mutagenesis treatment to finally obtain high conversion D-alloxone.
  • the saccharide isomerase producing strain was named BLCY-005.
  • the strain was deposited on October 26, 2016 in the General Microbiology Center of the China Microbial Culture Collection Management Committee (Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing), with the accession number CGMCC No.13152. .
  • the strain produced D-psicose 3-epimerase under optimal conditions and the enzyme activity reached 143 U/ml.
  • Enzyme activity assay method In a 1 ml reaction system, 800 ⁇ l of a reaction substrate solution is added, and the reaction substrate solution is a solution obtained by dissolving D-fructose in 50 ml of phosphate buffer solution (pH 7.0) to obtain a D-fructose concentration of 100 g/L. 200 ⁇ l of the fermentation broth was incubated at 55 ° C for 10 min and then boiled for 10 min to terminate the enzyme reaction.
  • phosphate buffer solution pH 7.0
  • Enzyme unit (U) The amount of enzyme required to catalyze the production of 1 ⁇ mol D-psicose per minute.
  • Example 1 The method for culturing Bacillus subtilis BLCY-005 described in Example 1 is as follows:
  • Bacillus subtilis BLCY-005 was inoculated into LB medium, and activated at 35 ° C for 12 h to prepare an activated strain;
  • the seed medium components are as follows, all in weight percentage:
  • the fermentation medium components are as follows, all in weight percentage:
  • Yeast extract powder 3% corn syrup powder 2%, glucose 1%, anhydrous magnesium sulfate 0.01%, diammonium hydrogen phosphate 0.02%, ammonium sulfate 0.02%, balance water, pH 6.8.
  • the dry enzyme preparation is prepared.
  • the enzyme activity is 143 U/ml under the optimum pH value of 5.5-6.5, and the activity of the enzyme is much higher than the original strain of 75 U/ml. .
  • Example 1 The method for culturing Bacillus subtilis BLCY-005 described in Example 1 is as follows:
  • Bacillus subtilis BLCY-005 was inoculated into LB medium, and activated at 28 ° C for 12 h to prepare an activated strain;
  • the seed medium components are as follows, all in weight percentage:
  • Peptone 1% Peptone 1%, yeast dipping powder 0.5%, sodium chloride 1%, anhydrous magnesium sulfate 0.01%, potassium dihydrogen phosphate 0.02%, balance water, pH 5.0.
  • the fermentation medium components are as follows, all in weight percentage:
  • Yeast extract powder 3% corn syrup powder 2%, glucose 1%, anhydrous magnesium sulfate 0.01%, diammonium hydrogen phosphate 0.02%, ammonium sulfate 0.02%, balance water, pH 5.0.
  • the dry enzyme preparation was prepared to have an enzyme activity of 107 U/ml under the optimum pH value of 5.5-6.5.

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Abstract

一种枯草芽孢杆菌菌株或其后代、其培养方法与应用。还涉及该枯草芽孢杆菌菌株或其后代的培养物及裂解物以及使用该枯草芽孢杆菌菌株或其后代制备D-阿洛酮糖差向异构酶和制备D-阿洛酮糖的方法。

Description

一种枯草芽孢杆菌及其培养方法与应用 技术领域
本发明涉及微生物技术领域。特别地，本发明涉及一种枯草芽孢杆菌菌株或其后代、其培养方法与应用。本发明还涉及该枯草芽孢杆菌菌株或其后代的培养物及裂解物。本发明还涉及使用该枯草芽孢杆菌菌株或其后代制备D-阿洛酮糖差向异构酶的方法，以及使用该枯草芽孢杆菌菌株或其后代制备D-阿洛酮糖的方法。
背景技术
枯草芽孢杆菌，属于芽孢杆菌属。该菌为革兰氏阳性菌，单个细胞为0.7～0.8×2～3微米，芽孢为0.6～0.9×1.0～1.5微米，呈椭圆到柱状，位于菌体中央或稍偏。芽孢形成后菌体不膨大。枯草芽孢杆菌的菌落表面粗糙不透明，污白色至淡黄色。该菌为需氧菌。
D-阿洛酮糖是一种重要的稀有糖，在自然界中极少量地存在于甘蔗糖蜜、水果干、糖制品、小麦和鼠刺属植物中。其名称起源于抗菌素阿洛酮糖腺苷，并且从抗菌素阿洛酮糖腺苷中也可以分离到少量的D-阿洛酮糖。D-阿洛酮糖在人体内是通过小肠吸收进入血液循环中，在小肠吸收后不会被代谢成能量，且对于肠道微生物具有较低的发酵利用度。D-阿洛酮糖具有多种重要的生理功能：神经保护作用，降血糖、减脂，清除活性氧簇，抗氧化，抑制癌细胞增殖，作为低卡路里甜味剂等。
D-阿洛酮糖是D-果糖的C-3差向异构体。早期的D-阿洛酮糖合成方法是通过多级化学转化普通单糖或非糖的前体物质，但多次的保护和脱保护操作会导致收率偏低，且合成过程繁杂，存在很大的局限性。目前，已有以D-果糖为原料，可通过生物转化法制备D-阿洛酮糖，但是目前已报道的微生物，产生的D-阿洛酮糖差向异构酶转化能力较差，且分离纯化过程较难。
中国专利文献CN105602879A(申请号201610051547.3)公开了一株高效分泌D-阿洛酮糖3-差向异构酶的基因工程菌株及其构建方法。该发明通过利用由瘤胃菌Ruminococcus sp.5_1_39B_FAA来源的D-阿洛酮糖3-差向异构酶基因rdpe构建重组表达质粒pMA5-RDPE，然后转化枯草芽孢杆菌，实现了RDPE在枯草芽孢杆菌中组成型分泌表达。通过比较三个糖诱导型启动子，得到最优诱导型启动子PxylA，并明显提高了RDPE的分泌水平。通过敲除木糖利用基因xylAB(xylA和xylB)，阻断了枯草芽孢杆菌 的木糖代谢途径，进一步提高了RDPE的分泌量，并使诱导剂木糖的最优诱导浓度由4.0％降低为0.5％。最终，采用分批补料方式，在7.5L发酵罐中对工程菌株1A751SD-XR进行评价，RDPE的分泌水平最高可以达到95U/mL和2.6g/L。但该酶的酶活仍然较低，无法满足实际生产的需要。
因此，寻找一种高转化率的D-阿洛酮糖差向异构酶生产菌株成为解决D-阿洛酮糖大规模生产应用的关键。
发明内容
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的微生物培养、生物化学、细胞生物学等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“培养基”是本领域已知用于支持细胞(特别是细菌，例如芽孢杆菌属细菌，例如枯草芽孢杆菌)生长的任何已知的固体或液体培养基。在某些优选的实施方案中，如本文所述的培养基(例如，第一培养基、第二培养基、固体培养基、种子培养基或发酵培养基)能够支持细菌(例如芽孢杆菌属细菌，例如枯草芽孢杆菌)的生长。在此类实施方案中，所述培养基通常包含碳源、氮源、无机盐以及维持该细菌处于存活状态所需的任何其他营养物质。
如本文中所使用的，术语菌株的“培养物”是指，将菌株在培养基中培养后获得的产物。
如本文中所使用的，术语菌株的“裂解物”是指，菌株的细胞壁和/或细胞膜经破碎后得到的产物，其包含胞内物质。菌株的裂解物可通过本领域已知的各种技术获得，例如超声破碎法、均质法、渗透压冲击法、冻融破碎法、酶溶法等。
本申请的发明人经过大量实验和反复摸索，出人意料地发现，一种经特定诱变后的枯草芽孢杆菌菌株所产生的D-阿洛酮糖差向异构酶的活性显著高于野生型枯草芽孢杆菌菌株。基于这一发现，本发明人开发了新的枯草芽孢杆菌菌株、其培养方法以及基于该菌株的D-阿洛酮糖制备方法。
因此，在一个方面，本发明提供了一种枯草芽孢杆菌(Bacillus subtilis)菌株 BLCY-005或其后代，所述菌株BLCY-005于2016年10月26日保藏于中国微生物菌种保藏管理委员会普通微生物中心、保藏号为CGMCC No.13152。
在某些优选的实施方案中，所述菌株或其后代产生的D-阿洛酮糖差向异构酶的活性，相比于野生型菌株产生的D-阿洛酮糖差向异构酶，提高了至少30％，至少35％，至少40％，至少45％，至少50％，至少60％，至少70％，至少80％，至少90％，或至少100％。在某些优选的实施方案中，所述菌株或其后代产生的D-阿洛酮糖差向异构酶的活性，相比于野生型菌株产生的D-阿洛酮糖差向异构酶，提高了至少90％，或至少100％。
在另一个方面，本发明提供了一种培养物，其包含如本文所述的枯草芽孢杆菌菌株或其后代。
在某些优选的实施方案中，所述培养物包含培养基，例如固体培养基或液体培养基。
在某些优选的实施方案中，所述培养物中的D-阿洛酮糖差向异构酶的活性，相比于野生型菌株产生的D-阿洛酮糖差向异构酶，提高了至少30％，至少35％，至少40％，至少45％，至少50％，至少60％，至少70％，至少80％，至少90％，或至少100％。在某些优选的实施方案中，所述培养物中的D-阿洛酮糖差向异构酶的活性，相比于野生型菌株产生的D-阿洛酮糖差向异构酶，提高了至少90％，或至少100％。
在本发明中，所述培养物可以通过在培养基中且在培养条件下培养本发明的菌株或其后代来获得，并且本领域技术人员已知如何根据菌株性质选择合适的培养基及培养条件。
在某些优选的实施方案中，所述培养物通过如上所述的方法培养本发明的菌株或其后代来获得。
在另一个方面，本发明提供了一种菌株的裂解物，所述菌株为如本文所述的枯草芽孢杆菌菌株或其后代。
在某些优选的实施方案中，所述裂解物包含D-阿洛酮糖差向异构酶。
在某些优选的实施方案中，所述裂解物中的D-阿洛酮糖差向异构酶的活性，相比于野生型菌株产生的D-阿洛酮糖差向异构酶，提高了至少30％，至少35％，至少40％，至少45％，至少50％，至少60％，至少70％，至少80％，至少90％，或至少100％。在某些优选的实施方案中，所述裂解物中的D-阿洛酮糖差向异构酶的活性，相比于野生型菌株产生的 D-阿洛酮糖差向异构酶，提高了至少90％，或至少100％。
在另一个方面，本发明提供了一种组合物，其包含果糖，以及至少一种选自下列的组分：(1)本发明的菌株或其后代；(2)本发明的培养物；和(3)本发明的裂解物。
在某些优选的实施方案中，所述果糖是以结晶果糖水溶液、液体果糖、果葡糖浆(例如，高果糖玉米糖浆)、干果糖或果糖糖浆的形式得到或提供。
在另一个方面，本发明提供了一种培养如本文所述的枯草芽孢杆菌菌株或其后代的方法，其包括将所述菌株或其后代接种于培养基中的步骤。
在某些优选的实施方案中，所述培养基是本领域已知用于支持细菌(特别是芽孢杆菌属细菌，例如枯草芽孢杆菌)生长的任何已知的固体或液体培养基。一般地，所述培养基包含碳源、氮源、无机盐以及维持该细菌处于存活状态所需的任何其他营养物质。
在某些优选的实施方案中，所述方法包括以下步骤：
(1)将本发明的菌株或其后代接种于固体培养基中，进行培养；
(2)将所述固体培养基中的菌株接种于第一培养基中，进行培养；
(3)任选地将所述第一培养基中的菌株接种于第二培养基中，进行培养。
在某些优选的实施方案中，在步骤(1)中，在25-40℃(例如25-38℃，28-40℃，28-38℃，28-35℃，或30-38℃；例如，35℃)的温度下进行培养。在某些优选的实施方案中，在步骤(1)中，在30-38℃的温度下进行培养。在某些优选的实施方案中，在步骤(1)中，在35℃的温度下进行培养。
在某些优选的实施方案中，在步骤(2)中，在25-40℃(例如25-38℃，28-40℃，28-38℃，28-35℃，或30-38℃；例如，35℃)的温度下进行培养。在某些优选的实施方案中，在步骤(2)中，在30-38℃的温度下进行培养。在某些优选的实施方案中，在步骤(2)中，在35℃的温度下进行培养。
在某些优选的实施方案中，在步骤(3)中，在25-40℃(例如25-38℃，28-40℃，28-38℃，28-35℃，或30-38℃；例如，35℃)的温度下进行培养。在某些优选的实施方案中，在步骤(3)中，在30-38℃的温度下进行培养。在某些优选的实施方案中，在步骤(3)中，在35℃的温度下进行培养。
在某些优选的实施方案中，所述第一培养基是种子培养基。在某些优选的实施方案中，所述第二培养基是发酵培养基。
在某些优选的实施方案中，所述方法包括以下步骤：
(1)将本发明的菌株或其后代接种于固体培养基中，在25-40℃的温度下，培养6-12h，以获得活化菌株；
(2)将步骤(1)获得的活化菌株接种于种子培养基中，在25-40℃的温度下，培养6-12h，以获得种子液；
(3)将步骤(2)获得的种子液按体积比1-10％的比例接种于发酵培养基中，在25-40℃(例如25-38℃，28-40℃，28-38℃，28-35℃，或30-38℃；例如，35℃)的温度下，培养30-48h，从而获得菌体发酵液。
在某些优选的实施方案中，在步骤(2)中，所述种子培养基包含：1％蛋白胨，0.5％酵母浸粉，1％氯化钠，0.01％无水硫酸镁，0.02％磷酸二氢钾，以及余量的水(所有百分比均为重量百分比)。在某些优选的实施方案中，在步骤(2)中，所述种子培养基的pH为6.0-7.0。
在某些优选的实施方案中，在步骤(3)中，所述发酵培养基包含：3％酵母浸粉，2％玉米浆粉，1％葡萄糖，0.01％无水硫酸镁，0.02％磷酸氢二铵，0.02％硫酸铵，以及余量的水。在某些优选的实施方案中，在步骤(3)中，所述发酵培养基的pH为6.0-7.0。
在某些优选的实施方案中，在步骤(1)中，所述固体培养基是LB培养基。
在另一个方面，本发明提供了一种制备D-阿洛酮糖差向异构酶的方法，其包括裂解本发明的枯草芽孢杆菌菌株或其后代并分离D-阿洛酮糖差向异构酶的步骤，或者从本发明的培养物或裂解物中分离D-阿洛酮糖差向异构酶的步骤。
在某些优选的实施方案中，所述方法包括以下步骤：(1)在允许本发明的菌株或其后代生长的条件下，培养所述菌株，以获得所述菌株的发酵液；和(2)从步骤(1)获得的发酵液中分离所述D-阿洛酮糖差向异构酶。
在某些优选的实施方案中，在步骤(1)中，通过如本文所述的方法培养所述菌株。
在某些优选的实施方案中，在步骤(2)中，通过离心、过滤、透析或层析从所述发酵液中分离所述D-阿洛酮糖差向异构酶。在某些示例性实施方案中，在步骤(2)中，通过离心从所述发酵液中分离所述D-阿洛酮糖差向异构酶。
在某些优选的实施方案中，在步骤(2)中，通过下述步骤从所述发酵液中分离所述D-阿洛酮糖差向异构酶：
(2a)将步骤(1)获得的发酵液离心，以获得沉淀物；
(2b)将步骤(2a)获得的沉淀物进行热变性；
(2c)将步骤(2b)的产物进行干燥(例如，真空冷冻干燥)，从而获得D-阿洛酮糖差向异构酶。
在某些优选的实施方案中，在步骤(2b)中，将步骤(2a)获得的沉淀物在50-60℃的温度下进行热变性。在某些优选的实施方案中，在步骤(2b)中，将步骤(2a)获得的沉淀物在50-60℃的温度下进行热变性，持续40-60min。
在某些优选的实施方案中，在步骤(2c)中，将步骤(2b)的产物进行真空冷冻干燥。在某些优选的实施方案中，在步骤(2c)中，将步骤(2b)的产物在-40～-60℃，-20～-60kpa的条件下进行真空冷冻干燥。
在某些示例性实施方案中，在步骤(2a)中，将所述发酵液在4℃、10000r/min条件下离心10min。
在某些示例性实施方案中，在步骤(2)中，通过下述步骤从所述发酵液中分离所述D-阿洛酮糖差向异构酶：(i)将步骤(1)获得的发酵液离心，弃去上清液；(ii)洗涤步骤(i)的产物；(iii)将步骤(ii)的产物离心，以获得沉淀物，其即为粗酶制剂；(vi)将步骤(iii)获得的粗酶制剂放置于50-60℃水浴中进行热变性，持续40-60min；(v)将步骤(vi)的产物在-40～-60℃，-20～-60kpa的条件下进行真空冷冻干燥。
在某些示例性实施方案中，在步骤(ii)中，使用50mmol/L的Tris-HCl缓冲液(pH8.0)洗涤所述菌株。
在某些优选的实施方案中，所述方法包括以下步骤：从本发明的培养物或裂解物中分离D-阿洛酮糖差向异构酶。在某些优选的实施方案中，通过离心、过滤、透析或层析从所述培养物或裂解物中分离所述D-阿洛酮糖差向异构酶。在某些示例性实施方案中，通过离心从所述培养物中分离所述D-阿洛酮糖差向异构酶。
在另一个方面，本发明提供了一种制备D-阿洛酮糖的方法，其包括以下步骤中的任一项：
将本发明的菌株或其后代与果糖接触；
将本发明的培养物与果糖接触；
将本发明的裂解物与果糖接触；以及
从本发明的菌株或其后代、培养物或裂解物中分离D-阿洛酮糖差向异构酶，并将所述D-阿洛酮糖差向异构酶与果糖接触。
在某些优选的实施方案中，所述果糖是以结晶果糖水溶液、液体果糖、果葡糖浆(例如，高果糖玉米糖浆)、干果糖或果糖糖浆的形式得到或提供。
在某些优选的实施方案中，所述方法包括以下步骤：(1)从本发明的菌株或其后代、培养物或裂解物中分离D-阿洛酮糖差向异构酶；(2)将步骤(1)获得的D-阿洛酮糖差向异构酶与果糖接触；(3)任选地从步骤(2)的产物中回收D-阿洛酮糖。
在某些优选的实施方案中，在步骤(2)中，在适合于D-阿洛酮糖差向异构酶活性的条件下，将所述D-阿洛酮糖差向异构酶与果糖接触。在某些优选的实施方案中，在步骤(2)中，在pH 5.5-6.5的条件下，将所述D-阿洛酮糖差向异构酶与果糖接触。
在另一个方面，提供了本发明的菌株或其后代，本发明的培养物，或本发明的裂解物，在制备D-阿洛酮糖差向异构酶中的用途。
在另一个方面，提供了本发明的菌株或其后代，本发明的培养物，本发明的裂解物，或本发明的组合物，在制备D-阿洛酮糖中的用途。
本发明还包含以下示例性实施方案：
1、一株枯草芽孢杆菌(Bacillus subtilis)BLCY-005，2016年10月26日保存于中国微生物菌种保藏管理委员会普通微生物中心，保藏号CGMCC No.13152，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所。
2、如项目1所述的枯草芽孢杆菌(Bacillus subtilis)BLCY-005的培养方法，其特征在于，步骤如下：
(1)取枯草芽孢杆菌(Bacillus subtilis)BLCY-005接种于固体培养基中，在30～38℃的条件下，活化培养6～12h，制得活化菌株；
(2)取步骤(1)制得的活化菌株，接种于种子培养基中，在30～38℃的条件下，增殖培养6～12h，制得种子液；
(3)取步骤(2)制得的种子液，按体积比1～10％的比例接种于发酵培养基中，在30～38℃，扩大培养30～48h，即得菌体发酵液。
3、如项目2所述的培养方法，其特征在于，所述步骤(2)中的种子培养基组分如下，均为重量百分比：
蛋白胨1％，酵母浸粉0.5％，氯化钠1％，无水硫酸镁0.01％，磷酸二氢钾0.02％，余量水，pH6.0～7.0；
4、如项目2所述的培养方法，其特征在于，所述步骤(3)中的发酵培养基组分如下，均为重量百分比：
酵母浸粉3％，玉米浆粉2％，葡萄糖1％，无水硫酸镁0.01％，磷酸氢二铵0.02％，硫酸铵0.02％，余量水，pH6.0～7.0。
5、项目1所述枯草芽孢杆菌(Bacillus subtilis)BLCY-005在制备D-阿洛酮糖差向异构酶中的应用。
6、如项目5所述的应用，其特征在于，步骤如下：
(a)取上述制备的菌体发酵液经离心分离，洗涤菌体，二次离心，保留沉淀物，即为粗酶制剂；
(b)将步骤(a)制得的粗酶制剂置于50～60℃水浴中热变性40～60min，再用真空冷冻干燥机，于-40～-60℃，-20～-60kpa工作压力下干燥，即制得D-阿洛酮糖差向异构酶干粉。
7、如项目5所述的应用，其特征在于，所述步骤(a)的洗涤菌体采用pH 8.0、浓度50mmol/L的Tris-HCl缓冲液，然后经离心分离，保留沉淀，制得粗酶制剂。
8、如项目5所述的应用，其特征在于，所述步骤(a)、(b)中的离心分离均为在4℃、10000r/min条件下，离心10min。
9、如项目5所述制备的D-阿洛酮糖转移酶在制备D-阿洛酮糖中的应用。
发明的有益效果
与现有技术相比，本发明的技术方案至少具有以下有益效果：
(1)本发明的枯草芽孢杆菌菌株BLCY-005所产生的D-阿洛酮糖差向异构酶具有显著提高的酶活性。本发明的菌株的培养物或裂解物中的D-阿洛酮糖差向异构酶的酶活性可达到143U/ml，较野生型菌株所产生的D-阿洛酮糖差向异构酶，提高近一倍，从而能够显著降低生产成本。
(2)本发明的枯草芽孢杆菌菌株BLCY-005所产生的D-阿洛酮糖差向异构酶的最适pH值为5.5～6.5，与野生型菌株所产生的D-阿洛酮糖差向异构酶的最适pH偏中性相比，有利于生产中对污染的控制。
(3)本发明的枯草芽孢杆菌菌株BLCY-005所产生的D-阿洛酮糖差向异构酶为胞内酶，分离工艺简单，经简单的离心、洗涤、灭活、干燥即可得到干粉酶制剂，节约了生产成本，降低了动力损耗。
(4)在本发明中可以通过将本发明的菌株所产生的D-阿洛酮糖差向异构酶直接与D-果糖接触从而制备D-阿洛酮糖，与传统的通过将发酵液与D-果糖接触从而制备D-阿洛酮糖的方法相比，极大地降低后续D-阿洛酮糖提纯的难度和成本，并可以显著提高D-阿洛酮糖成品的质量。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
关于生物材料保藏的说明
本发明涉及下列已在中国微生物菌种保藏管理委员会普通微生物中心(北京市朝阳区北辰西路1号院3号)进行保藏的生物材料：
枯草芽孢杆菌(Bacillus subtilis)菌株BLCY-005，其具有保藏号CGMCC No.13152，且保藏时间为2016年10月26日。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1.枯草芽孢杆菌的诱变及筛选
1.1富集培养
选取山东德州百龙创园研发中试车间附近的土壤，用小铲子除去表土，取离地面5～15cm处的土壤约10g，用无菌水稀释10倍，加入LB培养基进行富集培养，30-38℃培养24h。
1.2纯种分离
采用划线分离法，取一支盛有5ml无菌水的大试管，取1.1中富集培养后的菌液2ml放入其中稀释，充分振荡分散。用接种环以无菌操作挑取稀释液一环先在平板培养基一边做第一次平行划线3-4条，再转动培养皿约60度角，将接种环上剩余物烧掉，待冷却后同一次划线方法做第二次划线，同法依次做第三次和第四次划线。划线完毕，盖上皿盖，将培养皿倒置，28-38℃培养24h后，挑取单个菌落接种于10个斜面培养基上，分别编号01～10。
将01～10斜面种子接种于摇瓶培养基中培养28～38℃培养24h，对01～10摇瓶发酵液进行D-阿洛酮糖对D-果糖的酶活测定，06号摇瓶酶活最高，达到75U/ml。
所述平板培养基组分如下，均为重量百分比：
蛋白胨1％，酵母浸粉0.5％，氯化钠1％，余量水，pH自然；
所述斜面培养基组分如下，均为重量百分比：
蛋白胨1％，酵母浸粉0.5％，氯化钠1％，琼脂粉1％，余量水，pH自然；
所述摇瓶培养基组分如下，均为重量百分比：
酵母浸粉3％，玉米浆粉2％，葡萄糖1％，无水硫酸镁0.01％，磷酸氢二铵0.02％，硫酸铵0.02％，余量水，pH 6.0～7.0；
1.3诱变筛选
对06号菌种进行紫外线诱变，紫外线诱变采用15W紫外线灯20cm照射，照射时间为120s，得到的高产菌种再进行亚硝基胍诱变处理，最终得到高转化率D-阿洛酮糖差向异构酶的产生菌株命名为BLCY-005。该菌株于2016年10月26日保存于中国微生物菌种保藏管理委员会普通微生物中心(地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所)，保藏号为CGMCC No.13152。该菌株产D-阿洛酮糖3-差向异构酶在最适条件下，酶活达到143U/ml。
酶活测定方法：1ml的反应体系中，加入800μl反应底物溶液，反应底物溶液为由50ml磷酸盐缓冲液(pH7.0)溶解D-果糖制得D-果糖浓度为100g/L的溶液，200μl的发酵液，55℃保温10min，然后煮沸10min，终止酶反应。
用HPLC检测D-阿洛酮糖的生产量，计算酶活。酶活单位(U)：每分钟催化产生1μmol D-psicose所需要的酶的量。
实施例2.
实施例1所述的枯草芽孢杆菌(Bacillus subtilis)BLCY-005的培养方法，步骤如下：
(1)取枯草芽孢杆菌(Bacillus subtilis)BLCY-005接种于LB培养基中，在35℃的条件下，活化培养12h，制得活化菌株；
(2)取步骤(1)制得的活化菌株，接种于种子培养基中，在35℃的条件下，增殖培养12h，制得种子液；
所述种子培养基组分如下，均为重量百分比：
蛋白胨1％，酵母浸粉0.5％，氯化钠1％，无水硫酸镁0.01％，磷酸二氢钾0.02％，余量水，pH6.8；
(3)取步骤(2)制得的种子液，按体积比5％的比例接种于发酵培养基中，在35℃，扩大培养48h，即得菌体发酵液；
所述发酵培养基组分如下，均为重量百分比：
酵母浸粉3％，玉米浆粉2％，葡萄糖1％，无水硫酸镁0.01％，磷酸氢二铵0.02％，硫酸铵0.02％，余量水，pH6.8。
经离心、洗涤、灭菌、干燥后制得干酶制剂，在最适pH值为5.5～6.5的条件下，测定其酶活为143U/ml，该酶活远高于原始菌株的75U/ml。
对比例1.
实施例1所述的枯草芽孢杆菌(Bacillus subtilis)BLCY-005的培养方法，步骤如下：
(1)取枯草芽孢杆菌(Bacillus subtilis)BLCY-005接种于LB培养基中，在28℃的条件下，活化培养12h，制得活化菌株；
(2)取步骤(1)制得的活化菌株，接种于种子培养基中，在28℃的条件下，增殖培养12h，制得种子液；
所述种子培养基组分如下，均为重量百分比：
蛋白胨1％，酵母浸粉0.5％，氯化钠1％，无水硫酸镁0.01％，磷酸二氢钾0.02％，余量水，pH5.0。
(3)取步骤(2)制得的种子液，按体积比5％的比例接种于发酵培养基中，在28℃，扩大培养48h，即得菌体发酵液；
所述发酵培养基组分如下，均为重量百分比：
酵母浸粉3％，玉米浆粉2％，葡萄糖1％，无水硫酸镁0.01％，磷酸氢二铵0.02％，硫酸铵0.02％，余量水，pH5.0。
经离心、洗涤、灭活、干燥后制得干酶制剂在最适pH值为5.5～6.5的条件下，测定其酶活为107U/ml。
由上述结果可以看出，培养条件不在本发明所述权利要求范围内时，D-阿洛酮糖的的转化率大幅降低。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (10)

  1. 一种枯草芽孢杆菌(Bacillus subtilis)菌株BLCY-005或其后代，所述菌株BLCY-005于2016年10月26日保藏于中国微生物菌种保藏管理委员会普通微生物中心、保藏号为CGMCC No.13152；
    优选地，所述菌株或其后代产生的D-阿洛酮糖差向异构酶的活性，相比于野生型菌株产生的D-阿洛酮糖差向异构酶，提高了至少30％，至少35％，至少40％，至少45％，至少50％，至少60％，至少70％，至少80％，至少90％，或至少100％。
  2. 一种培养权利要求1的枯草芽孢杆菌菌株的方法，其包括将所述菌株或其后代接种于培养基中的步骤；
    优选地，所述方法包括以下步骤：
    (1)将所述菌株或其后代接种于固体培养基中，进行培养；
    (2)将所述固体培养基中的菌株接种于第一培养基中，进行培养；
    (3)任选地将所述第一培养基中的菌株接种于第二培养基中，进行培养；
    优选地，在步骤(1)中，在25-40℃(例如25-38℃，28-40℃，28-38℃，28-35℃，或30-38℃；例如，35℃)的温度下进行培养；优选地，在步骤(1)中，在30-38℃的温度下进行培养；优选地，在步骤(1)中，在35℃的温度下进行培养；
    优选地，在步骤(2)中，在25-40℃(例如25-38℃，28-40℃，28-38℃，28-35℃，或30-38℃；例如，35℃)的温度下进行培养；优选地，在步骤(2)中，在30-38℃的温度下进行培养；优选地，在步骤(2)中，在35℃的温度下进行培养；
    优选地，在步骤(3)中，在25-40℃(例如25-38℃，28-40℃，28-38℃，28-35℃，或30-38℃；例如，35℃)的温度下进行培养；优选地，在步骤(3)中，在30-38℃的温度下进行培养；优选地，在步骤(3)中，在35℃的温度下进行培养；
    优选地，所述第一培养基是种子培养基；
    优选地，所述第二培养基是发酵培养基。
  3. 权利要求2的方法，其中，所述方法包括以下步骤：
    (1)将权利要求1的枯草芽孢杆菌菌株或其后代接种于固体培养基中，在25-40℃的温度下，培养6-12h，以获得活化菌株；
    (2)将步骤(1)获得的活化菌株接种于种子培养基中，在25-40℃的温度下，培养6-12h，以获得种子液；
    (3)将步骤(2)获得的种子液按体积比1-10％的比例接种于发酵培养基中，在25-40℃(例如25-38℃，28-40℃，28-38℃，28-35℃，或30-38℃；例如，35℃)的温度下，培养30-48h，从而获得菌体发酵液；
    优选地，在步骤(2)中，所述种子培养基包含：1％蛋白胨，0.5％酵母浸粉，1％氯化钠，0.01％无水硫酸镁，0.02％磷酸二氢钾，以及余量的水(所有百分比均为重量百分比)；优选地，在步骤(2)中，所述种子培养基的pH为6.0-7.0；
    优选地，在步骤(3)中，所述发酵培养基包含：3％酵母浸粉，2％玉米浆粉，1％葡萄糖，0.01％无水硫酸镁，0.02％磷酸氢二铵，0.02％硫酸铵，以及余量的水；优选地，在步骤(3)中，所述发酵培养基的pH为6.0-7.0；
    优选地，在步骤(1)中，所述固体培养基是LB培养基。
  4. 一种培养物，其包含权利要求1的枯草芽孢杆菌菌株或其后代；
    优选地，所述培养物包含培养基，例如固体培养基或液体培养基；
    优选地，所述培养物通过使用权利要求2或3的方法培养权利要求1的枯草芽孢杆菌菌株或其后代来获得。
  5. 一种菌株的裂解物，所述菌株为权利要求1的枯草芽孢杆菌菌株或其后代。
  6. 一种组合物，其包含果糖，以及至少一种选自下列的组分：(1)权利要求1的菌株或其后代；(2)权利要求4的培养物；和(3)权利要求5的裂解物；
    优选地，所述果糖是以结晶果糖水溶液、液体果糖、果葡糖浆(例如，高果糖玉米糖浆)、干果糖或果糖糖浆的形式得到或提供。
  7. 一种制备D-阿洛酮糖差向异构酶的方法，其包括裂解权利要求1的枯草芽孢杆菌菌株或其后代并分离D-阿洛酮糖差向异构酶的步骤，或者从权利要求4的培养物或权利要求5的裂解物中分离D-阿洛酮糖差向异构酶的步骤；
    优选地，所述方法包括以下步骤：(1)在允许所述菌株或其后代生长的条件下，培养所述菌株，以获得所述菌株的发酵液；和(2)从步骤(1)获得的发酵液中分离所述D-阿 洛酮糖差向异构酶；
    优选地，在步骤(1)中，通过权利要求2或3的方法培养所述菌株；
    优选地，在步骤(2)中，通过离心、过滤、透析或层析从所述发酵液中分离所述D-阿洛酮糖差向异构酶；优选地，在步骤(2)中，通过离心从所述发酵液中分离所述D-阿洛酮糖差向异构酶；
    优选地，在步骤(2)中，通过下述步骤从所述发酵液中分离所述D-阿洛酮糖差向异构酶：
    (2a)将步骤(1)获得的发酵液离心，以获得沉淀物；
    (2b)将步骤(2a)获得的沉淀物进行热变性；
    (2c)将步骤(2b)的产物进行干燥(例如，真空冷冻干燥)，从而获得D-阿洛酮糖差向异构酶；
    优选地，在步骤(2b)中，将步骤(2a)获得的沉淀物在50-60℃的温度下进行热变性；优选地，在步骤(2b)中，将步骤(2a)获得的沉淀物在50-60℃的温度下进行热变性，持续40-60min；
    优选地，在步骤(2c)中，将步骤(2b)的产物进行真空冷冻干燥；优选地，在步骤(2c)中，将步骤(2b)的产物在-40～-60℃，-20～-60kpa的条件下进行真空冷冻干燥；
    优选地，在步骤(2a)中，将所述发酵液在4℃、10000r/min条件下离心10min；
  8. 一种制备D-阿洛酮糖的方法，其包括以下步骤中的任一项：
    将权利要求1的菌株或其后代与果糖接触；
    将权利要求4的培养物与果糖接触；
    将权利要求5的裂解物与果糖接触；以及
    从权利要求1的菌株或其后代、权利要求4的培养物或权利要求5的裂解物中分离D-阿洛酮糖差向异构酶，并将所述D-阿洛酮糖差向异构酶与果糖接触；
    优选地，所述果糖是以结晶果糖水溶液、液体果糖、果葡糖浆(例如，高果糖玉米糖浆)、干果糖或果糖糖浆的形式得到或提供；
    优选地，所述方法包括以下步骤：(1)从权利要求1的菌株或其后代、权利要求4的培养物或权利要求5的裂解物中分离D-阿洛酮糖差向异构酶；(2)将步骤(1)获得的D-阿洛酮糖差向异构酶与果糖接触；(3)任选地从步骤(2)的产物中回收D-阿洛酮糖；
    优选地，在步骤(2)中，在适合于D-阿洛酮糖差向异构酶活性的条件下，将所述D- 阿洛酮糖差向异构酶与果糖接触；
    优选地，在步骤(2)中，在pH 5.5-6.5的条件下，将所述D-阿洛酮糖差向异构酶与果糖接触。
  9. 权利要求1的枯草芽孢杆菌菌株或其后代，权利要求4的培养物，或权利要求5的裂解物，在制备D-阿洛酮糖差向异构酶中的用途。
  10. 权利要求1的枯草芽孢杆菌菌株或其后代，权利要求4的培养物，或权利要求5的裂解物，或权利要求6的组合物，在制备D-阿洛酮糖中的用途。
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