WO2023000618A1 - Bacillus xiaoxiensis and application thereof - Google Patents

Bacillus xiaoxiensis and application thereof 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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French (fr)
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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Priority to JP2022538391A priority Critical patent/JP2023538160A/en
Publication of WO2023000618A1 publication Critical patent/WO2023000618A1/en

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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

Provided are a Bacillus xiaoxiensis STB08 for producing a β-cyclodextrin glucosyltransferase, and a method for preparing the β-cyclodextrin glucosyltransferase by using the Bacillus xiaoxiensis STB08. Also provided is the β-cyclodextrin glucosyltransferase derived from the Bacillus xiaoxiensis STB08, the β-cyclodextrin glucosyltransferase has an amino acid sequence as shown in SEQ ID NO.1, and has a nucleotide sequence of a coding gene thereof as shown in SEQ ID NO.2.

Description

一种小溪芽孢杆菌及其应用A kind of bacillus creek and application thereof 技术领域technical field
本发明涉及一种小溪芽孢杆菌及其应用，属于微生物技术领域。The invention relates to a bacillus creek and application thereof, belonging to the technical field of microbes.
背景技术Background technique
环糊精是由6个及6个以上葡萄糖单元经α-1,4-糖苷键连接而成的环状聚合物，略呈锥状的中空圆筒形。最常见的有α-、β-和γ-环糊精，分别由6、7和8个葡萄糖单元连接形成，也是工业上应用最多的，尤其是β-环糊精。环糊精因其葡萄糖羟基-OH围绕外圈、醚键C-O-C向内围绕排列，具有亲水的表面和疏水的空腔结构，常被用来包合疏水性客体分子进而改善它们的理化性质，如溶解度、挥发性和化学性能等，在食品、医药、化妆品等行业得到了广泛的应用。Cyclodextrin is a cyclic polymer composed of 6 or more glucose units connected by α-1,4-glucosidic bonds, which is slightly cone-shaped hollow cylinder. The most common ones are α-, β-, and γ-cyclodextrin, which are formed by linking 6, 7, and 8 glucose units respectively, and are the most widely used in industry, especially β-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.
目前，环糊精的生产主要是通过环糊精葡萄糖基转移酶(CGT酶)作用于淀粉或相关衍生物进行酶法合成，主要产物为α-、β-和γ-环糊精的混合物。根据反应初级阶段主产物的不同，分别命名为α-CGT酶、β-CGT酶和γ-CGT酶，其中，β-CGT酶是研究最多的。CGT酶通过野生菌或基因工程菌表达后，往往酶液中存在大量的杂酶，会造成以下问题：(1)杂酶多，需要对酶进行分离纯化，但工艺往往繁琐复杂，对其酶学性质和产物分析的研究带来了很大的困难。(2)环糊精生产过程中，副产物多，对于环糊精的生产效率以及产物的分离纯化均带来一定的影响。Rosso等人通过筛选分离，得到高产β-CGT酶的菌株Bacillus circulans DF9R，其发酵液中酶活为5.8U/mL，电泳图中存在多条杂条带，但均存在发酵周期长、特定发酵条件、胞外产酶少且纯度低、难于分离纯化、热稳定性差的缺陷，使其在工业应用中受到限制。因此筛选纯度高且稳定性强的高活力β-环糊精葡萄糖基转移酶具有广大的应用前景。At present, the production of 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 α-, β- and γ-cyclodextrin. According to the different main products in the primary stage of the reaction, they are named α-CGTase, β-CGTase and γ-CGTase, among which β-CGTase is the most studied. After CGTase is expressed by wild bacteria or genetically engineered bacteria, there are often a large number of miscellaneous enzymes in the enzyme solution, which will cause the following problems: (1) There are many miscellaneous enzymes, and the enzymes need to be separated and purified, but the process is often cumbersome and complicated. The study of chemical properties and product analysis has brought great difficulties. (2) During the production process of cyclodextrin, there are many by-products, which have a certain impact on the production efficiency of cyclodextrin and the separation and purification of products. Through screening and isolation, Rosso et al. obtained the strain Bacillus circulans DF9R with high production of β-CGTase. 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 β-cyclodextrin glucosyltransferase with high purity and strong stability has broad application prospects.
发明内容Contents of the invention
本发明的目的是为了获得纯度高的β-环糊精葡萄糖基转移酶，解决了研究和生产中的难题，省却了分离纯化步骤，有利于环糊精的制备。The purpose of the present invention is to obtain high-purity β-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.
本发明提供了一种小溪芽孢杆菌(Bacillus xiaoxiensis)，所述小溪芽孢杆菌的编号为STB08，保藏号为CGMCC NO.22625，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏日期为2021年5月28日。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.
本发明还提供了一种含有上述小溪芽孢杆菌的微生物制剂。The present invention also provides a microbial preparation containing the above-mentioned Bacillus creek.
本发明还提供了小溪芽孢杆菌来源的β-环糊精葡萄糖基转移酶，所述β-环糊精葡萄糖基转移酶具有如SEQ ID NO.1所示的氨基酸序列。The present invention also provides β-cyclodextrin glucosyltransferase derived from Bacillus creek, said β-cyclodextrin glucosyltransferase has the amino acid sequence shown in SEQ ID NO.1.
本发明还提供编码所述β-环糊精葡萄糖基转移酶的基因，所述基因具有SEQ ID NO.2所示的核苷酸序列。The present invention also provides the gene encoding the β-cyclodextrin glucosyltransferase, the gene has the nucleotide sequence shown in SEQ ID NO.2.
本发明还提供了一种上述小溪芽孢杆或上述的微生物制剂在生产β-环糊精葡萄糖基转移酶中的应用。The present invention also provides an application of the above-mentioned Bacillus xiaoxie or the above-mentioned microbial preparation in the production of β-cyclodextrin glucosyltransferase.
本发明还提供了一种制备β-环糊精葡萄糖基转移酶的方法，将上述的小溪芽孢杆菌在25-30℃发酵至少72h。The present invention also provides a method for preparing β-cyclodextrin glucosyltransferase, comprising fermenting the above-mentioned Bacillus creek at 25-30° C. for at least 72 hours.
在一种实施方式中，用于发酵的培养基以酵母粉为碳源。In one embodiment, the medium used for fermentation uses yeast powder as a carbon source.
在一种实施方式中，用于发酵的培养基以鱼蛋白胨为氮源。In one embodiment, the medium used for fermentation uses fish peptone as nitrogen source.
在一种实施方式中，用于发酵的发酵培养基含有玉米浆干粉、Na 2CO 3、MgSO 4·7H 2O、KH 2PO 4和木薯淀粉。 In one embodiment, 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.
本发明还提供了上述小溪芽孢杆菌在生产环糊精方面的应用。The present invention also provides the application of the above bacillus creek in the production of cyclodextrin.
本发明还提供了一种生产环糊精的方法，用上述的小溪芽孢杆菌生产β-环糊精葡萄糖基转移酶，再将所述β-环糊精葡萄糖基转移酶按2U/g (麦芽糊精干基)的量加入至含麦芽糊精的体系中，于45℃反应24h，煮沸灭酶10min，后加入2U/g (麦芽糊精干基)的糖化酶，在30℃下糖化1h。 The present invention also provides a method for producing cyclodextrin, using the above-mentioned bacillus creek to produce β-cyclodextrin glucosyltransferase, and then using the β-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.
本发明还提供了上述小溪芽孢杆菌在食品领域分解麦芽糊精中的应用。The present invention also provides the application of the above-mentioned Bacillus creek in decomposing maltodextrin in the food field.
本发明的有益效果：Beneficial effects of the present invention:
(1)本发明提供的小溪芽孢杆菌STB08可以分泌纯度较高的β-环糊精葡萄糖基转移酶，热稳定性较好，在50℃及以下保温2h，酶活保持在80％以上，60℃和65℃时的半衰期分别为42min和9min，具有潜在的工业应用价值，有利于环糊精的制备，分解麦芽糊精得到的β-环糊精达11.6g/L，占总环糊精的74.3％，具有较高的β-环糊精产物特异性。(1) Bacillus creek STB08 provided by the present invention can secrete β-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 β-cyclodextrin obtained by decomposing maltodextrin reaches 11.6g/L, accounting for 11.6g/L of the total cyclodextrin. 74.3% of β-cyclodextrin product specificity.
(2)本发明通过平板和种子培养条件的优化，实现了小溪芽孢杆菌STB08的快速正常生长，有利于菌的保存及后续利用；(2) 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;
(3)本发明通过发酵培养条件的优化，实现了β-环糊精葡萄糖基转移酶的高分泌和高纯度表达。(3) The present invention realizes high secretion and high purity expression of β-cyclodextrin glucosyltransferase through optimization of fermentation and culture conditions.
生物材料保藏biological material deposit
小溪芽孢杆菌(Bacillus xiaoxiensis)STB08，分类命名为Bacillus xiaoxiensis，已于2021年5月28日保藏于中国微生物菌种保藏管理委员会，保藏号为CGMCC NO.22625，保藏地址为北京市朝阳区北辰西路1号院3号。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.
附图说明Description of drawings
图1为β-环糊精葡萄糖基转移酶的SDS-PAGE凝胶电泳图；Fig. 1 is the SDS-PAGE gel electrophoresis pattern of β-cyclodextrin glucosyltransferase;
图2为β-环糊精葡萄糖基转移酶的热稳定性分析图，其中，横坐标是时间，纵坐标是相对活力；Fig. 2 is the thermal stability analysis diagram of β-cyclodextrin glucosyltransferase, wherein, the abscissa is time, and the ordinate is relative activity;
图3为β-环糊精葡萄糖基转移酶作用于麦芽糊精(DE＝4)生产环糊精的过程图，其中，横坐标是时间，纵坐标是环糊精产量。Fig. 3 is a diagram showing the process of producing cyclodextrin by β-cyclodextrin glucosyltransferase acting on maltodextrin (DE=4), wherein the abscissa is time, and the ordinate is cyclodextrin yield.
具体实施方式detailed description
测定酶活的方法：取适当稀释的酶液0.1mL，加入装有0.9mL预先用10mM磷酸缓冲液(pH 6.5)配制的1％(w/v)麦芽糊精(DE＝4)溶液的试管中，在50℃下反应10min后，加入3.5mL 30mM NaOH使反应停止，再加入0.5mL由5mM Na 2CO 3溶液配制的0.02％(w/v)酚酞溶液，在室温下显色20min，在550nm下测定吸光度。以失活的酶作为空白。一个酶活单位定义为在上述条件下每分钟生成1μmolβ-环糊精所需的酶量。 Method for measuring enzyme activity: Take 0.1 mL of appropriately diluted enzyme solution and add to a test tube containing 0.9 mL of 1% (w/v) maltodextrin (DE=4) solution prepared in advance with 10 mM phosphate buffer (pH 6.5) After reacting at 50°C for 10 min, add 3.5 mL of 30 mM NaOH to stop the reaction, then add 0.5 mL of 0.02% (w/v) phenolphthalein solution prepared from 5 mM Na 2 CO 3 solution, develop color at room temperature for 20 min, and Absorbance was measured at 550 nm. The inactivated enzyme was used as a blank. One enzyme activity unit is defined as the amount of enzyme required to produce 1 μmol β-cyclodextrin per minute under the above conditions.
β-环糊精葡萄糖基转移酶纯度检测方法如下：The method for detecting the purity of β-cyclodextrin glucosyltransferase is as follows:
环糊精葡萄糖基转移酶纯度的检测采用SDS-PAGE凝胶电泳进行：参照碧云天的SDS-PAGE凝胶快速配制试剂盒说明书制备SDS-PAGE凝胶电泳，所用浓缩胶浓度为5％，分离胶浓度为10％，以0.125％考马斯亮蓝G-250进行染色。最终，采用凝胶成像分析仪进行电泳分析。The detection of the purity of cyclodextrin glucosyltransferase was carried out by SDS-PAGE gel electrophoresis: 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.
实施例1 小溪芽孢杆菌(Bacillus xiaoxiensis)分离和鉴定Example 1 Bacillus xiaoxiensis (Bacillus xiaoxiensis) isolation and identification
1.分离：采集淀粉厂附近的土壤，装入无菌瓶中，按常规法在平皿中分离出15株菌株。1. 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.
2.鉴定：对分离出的菌株进行菌株鉴定和理化性质的分析。2. Identification: carry out strain identification and analysis of physical and chemical properties on the isolated strains.
(1)理化性质分析(1) Analysis of physical and chemical properties
将平板上的菌株接种在种子培养基中活化，然后将活化液接入到发酵培养基中发酵，将发酵液离心收集并测定酶活，挑选出酶活最高的菌株进行菌株鉴定。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.
(2)菌株鉴定方法如下：(2) The strain identification method is as follows:
采用PCR扩增16S rRNA基因序列进行菌株鉴定。菌株16S rRNA PCR扩增引物序列为：27F(5’-AGAGTTTGATCCTGGCTCAG-3’，如SEQ ID NO:3所示)；1492R(5’-AAGTCGTAACAAGGTAACC-3’，如SEQ ID NO:4所示)。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前先将活化的菌液8000×g离心3min进行富集，倒掉上清液，加入蒸馏水1mL悬浮，再次离心富集，倒掉上清液，再加入500μL蒸馏水悬浮，待用。PCR反应体系(25μL)：2×Taq Plus MasterMix(Dye)12.5μL，ddH 2O 10.5μL，引物27F(100μM)0.5μL，引物1492R(100μM)0.5μL，模板(菌液)1μL。PCR反应参数如下：95℃预变性10min，95℃变性30s，55℃退火30s，72℃延伸30s，循环34次，72℃延伸10min。将PCR产物送样测序，PCR扩增片段约为1500bp，测序工作由华大基因完成。 Before PCR, centrifuge the activated bacterial liquid at 8000×g for 3 min to enrich, pour off the supernatant, add 1 mL of distilled water to suspend, centrifuge again for enrichment, pour off the supernatant, then add 500 μL of distilled water to suspend, and set aside. PCR reaction system (25 μL): 12.5 μL of 2×Taq Plus MasterMix (Dye), 10.5 μL of ddH 2 O, 0.5 μL of primer 27F (100 μM), 0.5 μL of primer 1492R (100 μM), 1 μL 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.
结果与分析：利用Blast(https://blast.ncbi.nlm.nih.gov/Blast.cgi)，将所测菌株的16S rRNA序列与数据库中已知菌株的16S rRNA比对进行分析。测序结果显示，该菌株的16S rRNA序列共1413bp。Bacillus xiaoxiensis STB08的16S rRNA序列如SEQ ID NO:5所示。使用Blast比较鉴定，同源性最高的5个菌种被列在表1，能够发现：它与小溪芽孢杆菌(Bacillus xiaoxiensis)的16S rRNA序列同源性最高，只有6个碱基的差别。同时，满足与某一种模式菌株序列或非模式菌株序列同源性≥99％且高于其他种0.8％，因此可鉴定推断出该菌株来源于小溪芽孢杆菌(Bacillus xiaoxiensis)，命名为Bacillus xiaoxiensis STB08。Results and analysis: Using Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi), the 16S rRNA sequences of the tested strains were compared with the 16S rRNA sequences of known strains in the database for analysis. The sequencing results showed that the 16S rRNA sequence of the strain was 1413bp in total. The 16S rRNA sequence of Bacillus xiaoxiensis STB08 is shown in SEQ ID NO:5. Using Blast comparative identification, the 5 strains with the highest homology are listed in Table 1, and it can be found that it has the highest homology with the 16S rRNA sequence of Bacillus xiaoxiensis, with only 6 base differences. At the same time, 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.
表1菌株的16S rRNA序列的同源性分析The homology analysis of the 16S rRNA sequence of bacterial strain of table 1
Figure PCTCN2021143099-appb-000001
Figure PCTCN2021143099-appb-000001
3.培养小溪芽孢杆菌STB08最适的pH3. Optimum pH for culturing Bacillus creek STB08
将小溪芽孢杆菌STB08按下述方式进行培养：Bacillus creek STB08 is cultivated in the following manner:
(1)平板划线：用接种环蘸取小溪芽孢杆菌STB08的菌液，在配制好的平板培养基(酵母粉6g/L，鱼蛋白胨6g/L，K 2HPO 4·3H 2O 1g/L，木薯淀粉12g/L，MgSO 4·7H 2O 0.2g/L，琼脂15g/L，用NaOH调节至pH 10.0)上划线，放入30℃培养箱培养12h。 (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.
(2)种子培养：从平板上挑取单菌落至种子培养基中，于30℃、200rpm下培养。(2) 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.
种子培养基配方如下：酵母粉6g/L，鱼蛋白胨6g/L，K 2HPO 4·3H 2O 1g/L，木薯淀粉12g/L，MgSO 4·7H 2O 0.2g/L，用NaOH分别调节至pH 7.0、8.0、9.0、10.0、11.0、12.0。 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.
在不同pH的种子培养基中进行种子培养，菌液生长情况见表2。能够发现，小溪芽孢杆菌STB08适合在pH 10.0～11.0条件下进行生长。在pH 10.0种子培养基中，菌液在12h已生长混浊，说明菌液的最适生长pH为10.0。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.
表2不同pH种子培养基对菌株生长情况的影响The influence of table 2 different pH seed culture media on bacterial strain growth
Figure PCTCN2021143099-appb-000002
Figure PCTCN2021143099-appb-000002
实施例2 小溪芽孢杆菌(Bacillus xiaoxiensis)分泌β-环糊精葡萄糖基转移酶Example 2 Bacillus xiaoxiensis secretes β-cyclodextrin glucosyltransferase
将小溪芽孢杆菌STB08按下述方式进行培养：Bacillus creek STB08 is cultivated in the following manner:
(1)平板划线：用接种环蘸取小溪芽孢杆菌STB08的菌液，在配制好的平板培养基(酵母粉6g/L，鱼蛋白胨6g/L，K 2HPO 4·3H 2O 1g/L，木薯淀粉12g/L，MgSO 4·7H 2O 0.2g/L，琼脂15g/L，用NaOH调节至pH 10.0)上划线，放入30℃培养箱培养12h。 (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.
(2)种子培养：从平板上挑取单菌落至种子培养基(酵母粉6g/L，鱼蛋白胨6g/L，K 2HPO 4·3H 2O 1g/L，木薯淀粉12g/L，MgSO 4·7H 2O 0.2g/L，用NaOH调节至pH 10.0)中，于30℃、200rpm下培养12h。 (2) 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.
(3)发酵培养：将种子培养液以4％(v/v)的接种量接入装有50mL发酵培养基的250mL三角瓶中，于25℃、200rpm下培养72h。(3) 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.
发酵培养基配方如下：玉米浆干粉26.6g/L，Na 2CO 3 4.14g/L，MgSO 4·7H 2O 0.21g/L，KH 2PO 4 1.63g/L，木薯淀粉12g/L，用NaOH分别调节至初始pH 7.0、8.0、9.0、10.0、11.0。 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.
在不同pH的发酵培养基中进行发酵产环糊精葡萄糖基转移酶，结果见表3。小溪芽孢杆菌STB08在25℃、pH 9.0条件下，酶活可达10.7U/mL。该分泌水平在野生菌生产环糊精葡萄糖基转移酶中处于较高水平。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.
表3 pH对环糊精葡萄糖基转移酶发酵的影响Table 3 The effect of pH on the fermentation of cyclodextrin glucosyltransferase
Figure PCTCN2021143099-appb-000003
Figure PCTCN2021143099-appb-000003
(4)酶液收集：将步骤(3)获得的培养后的菌液于4℃、10000×g离心15min，得到上清液即为β-环糊精葡萄糖基转移酶。所述发酵培养基配方如下：玉米浆干粉26.6g/L，Na 2CO 34.14g/L，MgSO 4·7H 2O 0.21g/L，KH 2PO 4 1.63g/L，木薯淀粉12g/L，pH＝9。 (4) Collection of enzyme solution: Centrifuge the cultured bacterial solution obtained in step (3) at 4° C. and 10,000×g for 15 minutes to obtain a supernatant that is β-cyclodextrin glucosyltransferase. The formula of the fermentation medium is as follows: dry corn steep liquor 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 , pH=9.
(5)酶活检测：将离心得到的环糊精葡萄糖基转移酶进行纯度检测和性质分析，结果见图1和2。图1中可以看出，小溪芽孢杆菌STB08制备的β-环糊精葡萄糖基转移酶经SDS-PAGE分析，呈单一条带，纯度较高。同时，该酶热稳定性较好，在50℃及以下保温2h，图2显示酶活保持在80％以上，60℃和65℃时的半衰期分别为42min和9min。本发明的环糊精葡萄糖基转移酶为一个条带对应一个酶蛋白，条带单一就说明就这一个酶，纯度就高。(5) Enzyme activity detection: The cyclodextrin glucosyltransferase obtained by centrifugation was subjected to purity detection and property analysis, and the results are shown in FIGS. 1 and 2 . It can be seen from Figure 1 that the β-cyclodextrin glucosyltransferase prepared by Bacillus creek STB08 was analyzed by SDS-PAGE and showed a single band with high purity. At the same time, the enzyme has good thermal stability. It is incubated at 50°C or below for 2 hours. Figure 2 shows that the enzyme activity remains above 80%, and the half-lives are 42min and 9min at 60°C and 65°C, respectively. In the cyclodextrin glucosyltransferase of the present invention, one band corresponds to one enzyme protein, and a single band means that only one enzyme has high purity.
实施例3 小溪芽孢杆菌来源的β-环糊精葡萄糖基转移酶鉴定Example 3 Identification of β-cyclodextrin glucosyltransferase derived from Bacillus creek
对实施例1筛选的小溪芽孢杆菌进行全基因组测序，分析获得如SEQ ID NO.2所示的编码β-环糊精葡萄糖基转移酶的基因序列，经分析，该基因转录翻译的β-环糊精葡萄糖基转移酶的氨基酸序列如SEQ ID NO.1所示。The Bacillus creek screened in Example 1 is carried out whole-genome sequencing, and the analysis obtains the gene sequence encoding β-cyclodextrin glucosyltransferase shown in SEQ ID NO.2. After analysis, the β-cyclodextrin glucosyltransferase of the gene transcription and translation The amino acid sequence of cyclodextrin glucosyltransferase is shown in SEQ ID NO.1.
实施例4 应用小溪芽孢杆菌分泌的β-环糊精葡萄糖基转移酶分解麦芽糊精Example 4 Application of β-cyclodextrin glucosyltransferase secreted by Bacillus creek to decompose maltodextrin
以5％(干基，w/v，5g/100mL)麦芽糊精(DE＝4)为底物，加入2U/g (麦芽糊精干基)的实施例2得到的β-环糊精葡萄糖基转移酶，置于45℃下反应，分别于，不同时间点(0、1、2、3、6、9、12、24h)取样，煮沸灭酶10min后加入2U/g (麦芽糊精干基)的糖化酶，在30℃下糖化1h，煮沸灭酶10min，10000r/min离心20min后，取上清液经0.45μm超滤膜过滤，用高效液相色谱法(HPLC)进行产物分析。 With 5% (dry basis, w/v, 5g/100mL) maltodextrin (DE=4) as the substrate, add 2 U/g (maltodextrin dry basis) of the β-cyclodextrin glucosyl obtained in Example 2 Transferase, reacted at 45°C, sampled at different time points (0, 1, 2, 3, 6, 9, 12, 24h), boiled to kill the enzyme for 10min, then added 2U/g (maltodextrin dry basis) 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 μm ultrafiltration membrane, and the product was analyzed by high performance liquid chromatography (HPLC).
HPLC测定条件：Waters 600高效液相色谱仪(配示差折光检测器)，色谱柱Lichrosorb NH 2(4.6mm×150mm)，流动相为乙腈-水(68％-32％)，柱温为30℃，流速为1mL/min。 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.
结果：实施例2得到的β-环糊精葡萄糖基转移酶作用于麦芽糊精(DE＝4)24h后，图3显示主产物β-环糊精达11.6g/L，占总环糊精的74.3％，具有较高的β-环糊精产物特异性。Result: After the β-cyclodextrin glucosyltransferase obtained in Example 2 acted on maltodextrin (DE=4) for 24 hours, Figure 3 shows that the main product β-cyclodextrin reached 11.6 g/L, accounting for the total cyclodextrin 74.3% of β-cyclodextrin product specificity.
实施例5 应用小溪芽孢杆菌发酵生产β-环糊精葡萄糖基转移酶Example 5 Production of β-cyclodextrin glucosyltransferase by fermentation of Bacillus creek
将小溪芽孢杆菌STB08按下述方式进行培养：Bacillus creek STB08 is cultivated in the following manner:
(1)平板划线：用接种环蘸取小溪芽孢杆菌STB08的菌液，在配制好的平板培养基(酵母粉6g/L，鱼蛋白胨6g/L，K 2HPO 4·3H 2O 1g/L，木薯淀粉12g/L，MgSO 4·7H 2O 0.2g/L，琼脂15g/L，用NaOH调节至pH 10.0)上划线，放入30℃培养箱培养12h。 (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.
(2)种子培养：从平板上挑取单菌落至种子培养基中，于30℃、200rpm下培养。(2) 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.
种子培养基配方如下：The seed medium formula is as follows:
种子培养基A：酵母粉6g/L，鱼蛋白胨6g/L，K 2HPO 4·3H 2O 1g/L，木薯淀粉12g/L，MgSO 4·7H 2O 0.2g/L，用NaOH调节至pH 10.0。 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.
种子培养基B：酵母粉6g/L，大豆蛋白胨6g/L，K 2HPO 4·3H 2O 1g/L，木薯淀粉12g/L，MgSO 4·7H 2O 0.2g/L，用NaOH调节至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.
种子培养基C：酵母粉6g/L，胰蛋白胨6g/L，K 2HPO 4·3H 2O 1g/L，木薯淀粉12g/L，MgSO 4·7H 2O 0.2g/L，用NaOH调节至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.
种子培养基D：酵母粉6g/L，酪蛋白胨6g/L，K 2HPO 4·3H 2O 1g/L，木薯淀粉12g/L，MgSO 4·7H 2O 0.2g/L，用NaOH调节至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.
种子培养基D：酵母粉6g/L，牛骨蛋白胨6g/L，K 2HPO 4·3H 2O 1g/L，木薯淀粉12g/L，MgSO 4·7H 2O 0.2g/L，用NaOH调节至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.
在不同种子培养基中进行种子培养，菌液生长情况见表4。能够发现，鱼蛋白胨对于小溪芽孢杆菌STB08的正常生长至关重要。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.
表4不同种类蛋白胨对菌株生长情况的影响Table 4 Effects of different kinds of peptones on the growth of bacterial strains
Figure PCTCN2021143099-appb-000004
Figure PCTCN2021143099-appb-000004
Figure PCTCN2021143099-appb-000005
Figure PCTCN2021143099-appb-000005
实施例6 应用小溪芽孢杆菌发酵生产β-环糊精葡萄糖基转移酶Example 6 Production of β-cyclodextrin glucosyltransferase by fermentation of Bacillus creek
将小溪芽孢杆菌STB08按下述方式进行培养：Bacillus creek STB08 is cultivated in the following manner:
(1)平板划线：用接种环蘸取小溪芽孢杆菌STB08的菌液，在配制好的平板培养基(酵母粉6g/L，鱼蛋白胨6g/L，K 2HPO 4·3H 2O 1g/L，木薯淀粉12g/L，MgSO 4·7H 2O 0.2g/L，琼脂15g/L，用NaOH调节至pH 10.0)上划线，放入30℃培养箱培养12h。 (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.
(2)种子培养：从平板上挑取单菌落至种子培养基中，于30℃、200rpm下培养。(2) 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.
种子培养基配方如下：The seed medium formula is as follows:
A：酵母粉6g/L，鱼蛋白胨6g/L，K 2HPO 4·3H 2O 1g/L，木薯淀粉12g/L，MgSO 4·7H 2O0.2g/L，用NaOH调节至pH 10.0。 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 with NaOH.
B：酵母浸膏6g/L，鱼蛋白胨6g/L，K 2HPO 4·3H 2O 1g/L，木薯淀粉12g/L，MgSO 4·7H 2O0.2g/L，用NaOH调节至pH 10.0。 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 .
在这2种种子培养基中进行种子培养，结果发现含有酵母粉的种子培养基在培养12h后已混浊，而含有酵母浸膏的种子培养基需培养24h后才混浊。因此，酵母粉对于菌株的快速生长起着重要的作用，有利于工业上环糊精葡萄糖基转移酶的高效生产。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.

Claims (12)

  1. 一种小溪芽孢杆菌(Bacillus xiaoxiensis)，于2021年5月28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.22625。A species of Bacillus xiaoxiensis was deposited in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on May 28, 2021, with the preservation number CGMCC NO.22625.
  2. 含有权利要求1所述小溪芽孢杆菌的微生物制剂。Containing the microorganism preparation of bacillus creek described in claim 1.
  3. 权利要求1所述的小溪芽孢杆或权利要求2所述的微生物制剂在生产β-环糊精葡萄糖基转移酶中的应用。The application of the Bacillus brook described in claim 1 or the microbial preparation described in claim 2 in the production of β-cyclodextrin glucosyltransferase.
  4. 一种制备β-环糊精葡萄糖基转移酶的方法，其特征在于，将权利要求1所述的小溪芽孢杆菌在25-30℃发酵至少72h。A method for preparing β-cyclodextrin glucosyltransferase, characterized in that the Bacillus creek described in claim 1 is fermented at 25-30° C. for at least 72 hours.
  5. 根据权利要求4所述的方法，其特征在于，用于发酵的培养基以酵母粉为碳源。The method according to claim 4, characterized in that, the medium used for fermentation is carbon source with yeast powder.
  6. 根据权利要求4所述的方法，其特征在于，用于发酵的培养基以鱼蛋白胨为氮源。The method according to claim 4, characterized in that, the medium used for fermentation uses fish peptone as nitrogen source.
  7. 根据权利要求4所述的方法，其特征在于，用于发酵的发酵培养基含有玉米浆干粉，Na 2CO 3、MgSO 4·7H 2O、KH 2PO 4和木薯淀粉。 The method according to claim 4, characterized in that the fermentation medium used for fermentation contains corn steep liquor dry powder, Na 2 CO 3 , MgSO 4 ·7H 2 O, KH 2 PO 4 and tapioca starch.
  8. 小溪芽孢杆菌来源的β-环糊精葡萄糖基转移酶，其特征在于，具有如SEQ ID NO.1所示的氨基酸序列。The β-cyclodextrin glucosyltransferase derived from Bacillus creek is characterized in that it has the amino acid sequence shown in SEQ ID NO.1.
  9. 编码权利要求8所述β-环糊精葡萄糖基转移酶的基因，其特征在于，具有SEQ ID NO.2所示的核苷酸序列。The gene encoding claim 8 β-cyclodextrin glucosyltransferase is characterized in that it has the nucleotide sequence shown in SEQ ID NO.2.
  10. 权利要求1所述的小溪芽孢杆菌或权利要求8所述的β-环糊精葡萄糖基转移酶在生产环糊精方面的应用。The application of the bacillus creek described in claim 1 or the β-cyclodextrin glucosyltransferase described in claim 8 in the production of cyclodextrin.
  11. 一种生产环糊精的方法，其特征在于，用权利要求1所述的小溪芽孢杆菌生产β-环糊精葡萄糖基转移酶，再将所述β-环糊精葡萄糖基转移酶按2U/g (麦芽糊精干基)的量加入至含麦芽糊精的体系中，于45℃反应24h，煮沸灭酶10min，后加入2U/g (麦芽糊精干基)的糖化酶，在30℃下糖化1h。 A kind of method for producing cyclodextrin, it is characterized in that, produce β-cyclodextrin glucosyltransferase with Bacillus creek described in claim 1, then described β-cyclodextrin glucosyltransferase is pressed 2U Add the amount of /g (maltodextrin dry basis) to the system containing maltodextrin, react at 45°C for 24h, boil to inactivate the enzyme for 10min, then add 2U/g (maltodextrin dry basis) of glucoamylase, at 30°C Saccharification for 1h.
  12. 权利要求1所述的小溪芽孢杆菌或权利要求8所述的β-环糊精葡萄糖基转移酶在食品领域分解麦芽糊精中的应用。Application of the bacillus creek described in claim 1 or the β-cyclodextrin glucosyltransferase described in claim 8 in decomposing maltodextrin in the food field.
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