WO2023000618A1 - Bacillus xiaoxiensis et son application - Google Patents

Bacillus xiaoxiensis et son application Download PDF

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WO2023000618A1
WO2023000618A1 PCT/CN2021/143099 CN2021143099W WO2023000618A1 WO 2023000618 A1 WO2023000618 A1 WO 2023000618A1 CN 2021143099 W CN2021143099 W CN 2021143099W WO 2023000618 A1 WO2023000618 A1 WO 2023000618A1
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bacillus
cyclodextrin
creek
cyclodextrin glucosyltransferase
glucosyltransferase
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PCT/CN2021/143099
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Chinese (zh)
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李才明
李兆丰
陈双娣
顾正彪
程力
洪雁
班宵逢
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江南大学
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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1074—Cyclomaltodextrin glucanotransferase (2.4.1.19)
    • 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
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • 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
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • 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
    • C12P19/20—Preparation of compounds containing saccharide radicals produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01019—Cyclomaltodextrin glucanotransferase (2.4.1.19)

Definitions

  • the invention relates to a bacillus creek and application thereof, belonging to the technical field of microbes.
  • Cyclodextrin is a cyclic polymer composed of 6 or more glucose units connected by ⁇ -1,4-glucosidic bonds, which is slightly cone-shaped hollow cylinder. The most common ones are ⁇ -, ⁇ -, and ⁇ -cyclodextrin, which are formed by linking 6, 7, and 8 glucose units respectively, and are the most widely used in industry, especially ⁇ -cyclodextrin. Cyclodextrin has a hydrophilic surface and a hydrophobic cavity structure because of its glucose hydroxyl-OH around the outer ring and ether bond C-O-C inward. It is often used to include hydrophobic guest molecules to improve their physical and chemical properties. Such as solubility, volatility and chemical properties, etc., have been widely used in food, medicine, cosmetics and other industries.
  • cyclodextrin is mainly enzymatically synthesized by the action of cyclodextrin glucosyltransferase (CGTase) on starch or related derivatives, and the main product is a mixture of ⁇ -, ⁇ - and ⁇ -cyclodextrin.
  • CCTase cyclodextrin glucosyltransferase
  • the main product is a mixture of ⁇ -, ⁇ - and ⁇ -cyclodextrin.
  • ⁇ -CGTase ⁇ -CGTase
  • ⁇ -CGTase ⁇ -CGTase
  • ⁇ -CGTase among which ⁇ -CGTase is the most studied.
  • the enzyme activity in the fermentation broth was 5.8 U/mL. Conditions, low extracellular enzyme production and low purity, difficulty in separation and purification, and poor thermal stability limit its industrial application. Therefore, screening high-activity ⁇ -cyclodextrin glucosyltransferase with high purity and strong stability has broad application prospects.
  • the purpose of the present invention is to obtain high-purity ⁇ -cyclodextrin glucosyltransferase, which solves the difficult problems in research and production, omits the separation and purification steps, and is beneficial to the preparation of cyclodextrin.
  • the invention provides a kind of Bacillus xiaoxiensis (Bacillus xiaoxiensis), the serial number of said Bacillus xiaoxiensis is STB08, and the preservation number is CGMCC NO.22625, which is preserved in the General Microorganism Center of China Microbial Strain Preservation Management Committee, and the preservation date is May 28, 2021.
  • the present invention also provides a microbial preparation containing the above-mentioned Bacillus creek.
  • the present invention also provides ⁇ -cyclodextrin glucosyltransferase derived from Bacillus creek, said ⁇ -cyclodextrin glucosyltransferase has the amino acid sequence shown in SEQ ID NO.1.
  • the present invention also provides the gene encoding the ⁇ -cyclodextrin glucosyltransferase, the gene has the nucleotide sequence shown in SEQ ID NO.2.
  • the present invention also provides an application of the above-mentioned Bacillus xiaoxie or the above-mentioned microbial preparation in the production of ⁇ -cyclodextrin glucosyltransferase.
  • the present invention also provides a method for preparing ⁇ -cyclodextrin glucosyltransferase, comprising fermenting the above-mentioned Bacillus creek at 25-30° C. for at least 72 hours.
  • the medium used for fermentation uses yeast powder as a carbon source.
  • the medium used for fermentation uses fish peptone as nitrogen source.
  • the fermentation medium used for fermentation contains corn steep steep powder, Na 2 CO 3 , MgSO 4 ⁇ 7H 2 O, KH 2 PO 4 and tapioca starch.
  • the present invention also provides the application of the above bacillus creek in the production of cyclodextrin.
  • the present invention also provides a method for producing cyclodextrin, using the above-mentioned bacillus creek to produce ⁇ -cyclodextrin glucosyltransferase, and then using the ⁇ -cyclodextrin glucosyltransferase at 2U/g ( maltodextrin dry basis) into the maltodextrin-containing system, react at 45°C for 24 hours, boil to inactivate the enzyme for 10 minutes, then add 2U/g (maltodextrin dry basis) glucoamylase, and saccharify at 30°C for 1 hour.
  • the present invention also provides the application of the above-mentioned Bacillus creek in decomposing maltodextrin in the food field.
  • Bacillus creek STB08 provided by the present invention can secrete ⁇ -cyclodextrin glucosyltransferase with higher purity and better thermal stability, and the enzyme activity is kept above 80% at 50°C or below for 2 hours,
  • the half-lives at 60°C and 65°C are 42min and 9min respectively, which has potential industrial application value and is beneficial to the preparation of cyclodextrin.
  • the ⁇ -cyclodextrin obtained by decomposing maltodextrin reaches 11.6g/L, accounting for 11.6g/L of the total cyclodextrin. 74.3% of ⁇ -cyclodextrin product specificity.
  • the present invention realizes the rapid and normal growth of Bacillus creek STB08 through the optimization of plate and seed culture conditions, which is beneficial to the preservation and subsequent utilization of bacteria;
  • the present invention realizes high secretion and high purity expression of ⁇ -cyclodextrin glucosyltransferase through optimization of fermentation and culture conditions.
  • Bacillus xiaoxiensis STB08 classified as Bacillus xiaoxiensis, was deposited in the China Committee for the Collection of Microorganisms on May 28, 2021.
  • the preservation number is CGMCC NO.22625, and the preservation address is Beichen, Chaoyang District, Beijing No. 3, Yard No. 1, West Road.
  • Fig. 1 is the SDS-PAGE gel electrophoresis pattern of ⁇ -cyclodextrin glucosyltransferase
  • Fig. 2 is the thermal stability analysis diagram of ⁇ -cyclodextrin glucosyltransferase, wherein, the abscissa is time, and the ordinate is relative activity;
  • One enzyme activity unit is defined as the amount of enzyme required to produce 1 ⁇ mol ⁇ -cyclodextrin per minute under the above conditions.
  • the method for detecting the purity of ⁇ -cyclodextrin glucosyltransferase is as follows:
  • SDS-PAGE gel electrophoresis was prepared according to the instructions of Beyond’s SDS-PAGE gel rapid preparation kit, the concentration of the stacking gel used was 5%, and the separation The gel concentration was 10%, and stained with 0.125% Coomassie Brilliant Blue G-250. Finally, electrophoresis analysis was performed using a gel imaging analyzer.
  • Example 1 Bacillus xiaoxiensis (Bacillus xiaoxiensis) isolation and identification
  • Isolation Collect the soil near the starch factory, put it into a sterile bottle, and isolate 15 strains in a plate according to the conventional method.
  • the strains on the plate were inoculated into the seed medium for activation, then the activation solution was added to the fermentation medium for fermentation, the fermentation broth was collected by centrifugation and the enzyme activity was determined, and the strain with the highest enzyme activity was selected for strain identification.
  • the 16S rRNA gene sequence was amplified by PCR for strain identification.
  • the primer sequence for 16S rRNA PCR amplification of the bacterial strain is: 27F (5'-AGAGTTTGATCCTGGCTCAG-3', as shown in SEQ ID NO:3); 1492R (5'-AAGTCGTAACAAGGTAACC-3', as shown in SEQ ID NO:4).
  • PCR reaction system 25 ⁇ L: 12.5 ⁇ L of 2 ⁇ Taq Plus MasterMix (Dye), 10.5 ⁇ L of ddH 2 O, 0.5 ⁇ L of primer 27F (100 ⁇ M), 0.5 ⁇ L of primer 1492R (100 ⁇ M), 1 ⁇ L of template (bacteria solution).
  • the PCR reaction parameters were as follows: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 34 cycles, and extension at 72°C for 10 min.
  • the PCR product was sent for sequencing.
  • the PCR amplified fragment was about 1500bp, and the sequencing work was completed by BGI.
  • the homology with a certain type strain or non-type strain sequence is ⁇ 99% and higher than that of other species by 0.8%, so it can be identified and inferred that the strain is derived from Bacillus xiaoxiensis and named Bacillus xiaoxiensis STB08.
  • Bacillus creek STB08 is cultivated in the following manner:
  • (1) Streaking on the plate Dip the bacterial solution of Bacillus creek STB08 with an inoculation loop, and put it on the prepared plate medium (yeast powder 6g/L, fish peptone 6g/L, K 2 HPO 4 3H 2 O 1g /L, tapioca starch 12g/L, MgSO 4 ⁇ 7H 2 O 0.2g/L, agar 15g/L, adjusted to pH 10.0 with NaOH) and placed in a 30°C incubator for 12h.
  • the prepared plate medium yeast powder 6g/L, fish peptone 6g/L, K 2 HPO 4 3H 2 O 1g /L, tapioca starch 12g/L, MgSO 4 ⁇ 7H 2 O 0.2g/L, agar 15g/L, adjusted to pH 10.0 with NaOH
  • Seed culture Pick a single colony from the plate and put it into the seed medium, and culture it at 30° C. and 200 rpm.
  • the formula of the seed medium is as follows: yeast powder 6g/L, fish peptone 6g/L, K 2 HPO 4 3H 2 O 1g/L, tapioca starch 12g/L, MgSO 4 7H 2 O 0.2g/L, and NaOH respectively Adjust to pH 7.0, 8.0, 9.0, 10.0, 11.0, 12.0.
  • Seed culture was carried out in seed media with different pH, and the growth of the bacterial solution is shown in Table 2. It can be found that Bacillus creek STB08 is suitable for growing under the condition of pH 10.0 ⁇ 11.0. In the seed medium with pH 10.0, the bacterial liquid had grown turbid within 12 hours, indicating that the optimum growth pH of the bacterial liquid was 10.0.
  • Bacillus creek STB08 is cultivated in the following manner:
  • (1) Streaking on the plate Dip the bacterial solution of Bacillus creek STB08 with an inoculation loop, and put it on the prepared plate medium (yeast powder 6g/L, fish peptone 6g/L, K 2 HPO 4 3H 2 O 1g /L, tapioca starch 12g/L, MgSO 4 ⁇ 7H 2 O 0.2g/L, agar 15g/L, adjusted to pH 10.0 with NaOH) and placed in a 30°C incubator for 12h.
  • the prepared plate medium yeast powder 6g/L, fish peptone 6g/L, K 2 HPO 4 3H 2 O 1g /L, tapioca starch 12g/L, MgSO 4 ⁇ 7H 2 O 0.2g/L, agar 15g/L, adjusted to pH 10.0 with NaOH
  • Seed culture Pick a single colony from the plate to the seed medium (yeast powder 6g/L, fish peptone 6g/L, K 2 HPO 4 3H 2 O 1g/L, tapioca starch 12g/L, MgSO 4 ⁇ 7H 2 O 0.2g/L, adjusted to pH 10.0 with NaOH), cultured at 30°C and 200rpm for 12h.
  • the seed medium yeast powder 6g/L, fish peptone 6g/L, K 2 HPO 4 3H 2 O 1g/L, tapioca starch 12g/L, MgSO 4 ⁇ 7H 2 O 0.2g/L, adjusted to pH 10.0 with NaOH
  • Fermentation culture the seed culture liquid was transferred into a 250 mL Erlenmeyer flask containing 50 mL of fermentation medium at an inoculum size of 4% (v/v), and cultured at 25° C. and 200 rpm for 72 hours.
  • the formulation of the fermentation medium is as follows: corn steep liquor dry powder 26.6g/L, Na 2 CO 3 4.14g/L, MgSO 4 7H 2 O 0.21g/L, KH 2 PO 4 1.63g/L, tapioca starch 12g/L, with NaOH was adjusted to initial pH 7.0, 8.0, 9.0, 10.0, 11.0, respectively.
  • Cyclodextrin glucosyltransferase was produced by fermentation in fermentation medium with different pH, and the results are shown in Table 3.
  • the enzyme activity of Bacillus creek STB08 can reach 10.7U/mL at 25°C and pH 9.0.
  • the secretion level is at a higher level in wild fungus producing cyclodextrin glucosyltransferase.
  • Example 1 The Bacillus creek screened in Example 1 is carried out whole-genome sequencing, and the analysis obtains the gene sequence encoding ⁇ -cyclodextrin glucosyltransferase shown in SEQ ID NO.2. After analysis, the ⁇ -cyclodextrin glucosyltransferase of the gene transcription and translation The amino acid sequence of cyclodextrin glucosyltransferase is shown in SEQ ID NO.1.
  • the glucoamylase was saccharified at 30°C for 1 hour, boiled to inactivate the enzyme for 10 minutes, centrifuged at 10,000 r/min for 20 minutes, the supernatant was filtered through a 0.45 ⁇ m ultrafiltration membrane, and the product was analyzed by high performance liquid chromatography (HPLC).
  • HPLC measurement conditions Waters 600 high-performance liquid chromatography (equipped with differential refractive index detector), chromatographic column Lichrosorb NH 2 (4.6mm ⁇ 150mm), mobile phase is acetonitrile-water (68%-32%), column temperature is 30°C , the flow rate is 1 mL/min.
  • Bacillus creek STB08 is cultivated in the following manner:
  • (1) Streaking on the plate Dip the bacterial solution of Bacillus creek STB08 with an inoculation loop, and put it on the prepared plate medium (yeast powder 6g/L, fish peptone 6g/L, K 2 HPO 4 3H 2 O 1g /L, tapioca starch 12g/L, MgSO 4 ⁇ 7H 2 O 0.2g/L, agar 15g/L, adjusted to pH 10.0 with NaOH) and placed in a 30°C incubator for 12h.
  • the prepared plate medium yeast powder 6g/L, fish peptone 6g/L, K 2 HPO 4 3H 2 O 1g /L, tapioca starch 12g/L, MgSO 4 ⁇ 7H 2 O 0.2g/L, agar 15g/L, adjusted to pH 10.0 with NaOH
  • Seed culture Pick a single colony from the plate and put it into the seed medium, and culture it at 30° C. and 200 rpm.
  • the seed medium formula is as follows:
  • Seed medium A yeast powder 6g/L, fish peptone 6g/L, K 2 HPO 4 3H 2 O 1g/L, tapioca starch 12g/L, MgSO 4 7H 2 O 0.2g/L, adjusted to pH 10.0.
  • Seed medium B yeast powder 6g/L, soybean peptone 6g/L, K 2 HPO 4 3H 2 O 1g/L, tapioca starch 12g/L, MgSO 4 7H 2 O 0.2g/L, adjusted to pH 10.0.
  • Seed medium C yeast powder 6g/L, tryptone 6g/L, K 2 HPO 4 3H 2 O 1g/L, tapioca starch 12g/L, MgSO 4 7H 2 O 0.2g/L, adjusted to pH 10.0.
  • Seed medium D yeast powder 6g/L, casein peptone 6g/L, K 2 HPO 4 3H 2 O 1g/L, tapioca starch 12g/L, MgSO 4 7H 2 O 0.2g/L, adjusted to pH 10.0.
  • Seed medium D yeast powder 6g/L, bovine bone peptone 6g/L, K 2 HPO 4 3H 2 O 1g/L, tapioca starch 12g/L, MgSO 4 7H 2 O 0.2g/L, adjusted with NaOH to pH 10.0.
  • Seed culture was carried out in different seed media, and the growth conditions of the bacterial solution are shown in Table 4. It could be found that fish peptone is essential for the normal growth of Bacillus creek STB08.
  • Bacillus creek STB08 is cultivated in the following manner:
  • (1) Streaking on the plate Dip the bacterial solution of Bacillus creek STB08 with an inoculation loop, and put it on the prepared plate medium (yeast powder 6g/L, fish peptone 6g/L, K 2 HPO 4 3H 2 O 1g /L, tapioca starch 12g/L, MgSO 4 ⁇ 7H 2 O 0.2g/L, agar 15g/L, adjusted to pH 10.0 with NaOH) and placed in a 30°C incubator for 12h.
  • the prepared plate medium yeast powder 6g/L, fish peptone 6g/L, K 2 HPO 4 3H 2 O 1g /L, tapioca starch 12g/L, MgSO 4 ⁇ 7H 2 O 0.2g/L, agar 15g/L, adjusted to pH 10.0 with NaOH
  • Seed culture Pick a single colony from the plate and put it into the seed medium, and culture it at 30° C. and 200 rpm.
  • the seed medium formula is as follows:
  • B Yeast extract 6g/L, fish peptone 6g/L, K 2 HPO 4 3H 2 O 1g/L, tapioca starch 12g/L, MgSO 4 7H 2 O 0.2g/L, adjusted to pH 10.0 with NaOH .
  • Seed culture was carried out in these two kinds of seed medium, and it was found that the seed medium containing yeast powder became turbid after 12 hours of cultivation, while the seed medium containing yeast extract was only cloudy after 24 hours of cultivation. Therefore, yeast powder plays an important role in the rapid growth of strains, which is conducive to the efficient production of cyclodextrin glucosyltransferase in industry.

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Abstract

La présente invention concerne un Bacillus xiaoxiensis STB08 pour produire une β-cyclodextrine glucosyltransférase, et un procédé pour préparer la β-cyclodextrine glucosyltransférase en utilisant le Bacillus xiaoxiensis STB08. La présente invention concerne également la β-cyclodextrine glucosyltransférase dérivée du Bacillus xiaoxiensis STB08, la β-cyclodextrine glucosyltransférase possédant une séquence d'acides aminés telle que représentée dans SEQ ID NO.1, et possédant une séquence nucléotidique d'un gène codant de celle-ci telle que représentée dans SEQ ID NO. 2.
PCT/CN2021/143099 2021-07-22 2021-12-30 Bacillus xiaoxiensis et son application WO2023000618A1 (fr)

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CHEN YI-GUANG, ZHANG YU-QIN, CHEN QI-HUI, KLENK HANS-PETER, HE JIAN-WU, TANG SHU-KUN, CUI XIAO-LONG, LI WEN-JUN: "Bacillus xiaoxiensis sp. nov., a slightly halophilic bacterium isolated from non-saline forest soil", INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, SOCIETY FOR GENERAL MICROBIOLOGY, GB, vol. 61, no. 9, 1 September 2011 (2011-09-01), GB , pages 2095 - 2100, XP093027573, ISSN: 1466-5026, DOI: 10.1099/ijs.0.026286-0 *
DATABASE NUCLEOTIDE ANONYMOUS : "Bacillus cereus strain LGQ01 CGTase (cgt) gene, complete cds", XP093027571, retrieved from NCBI *
ZHANG XINPING, ZHANG PEI; LI CUILING: "Properties of Cyclomaltodetrin Glucanotransferase and Production of Cyclodextrin", FIRST ACTA SCIENTIARUM NATURALIUM UNIVERSITATIS NANKAIENSIS, no. 4, 31 December 1994 (1994-12-31), XP093027575, ISSN: 0465-7942 *

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