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WO2024021455A1 - GÈNE DE β-GALACTOSIDASE ET UTILISATION DE CE GÈNE DANS LE CODAGE D'UNE ENZYME - Google Patents

GÈNE DE β-GALACTOSIDASE ET UTILISATION DE CE GÈNE DANS LE CODAGE D'UNE ENZYME Download PDF

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WO2024021455A1
WO2024021455A1 PCT/CN2022/140522 CN2022140522W WO2024021455A1 WO 2024021455 A1 WO2024021455 A1 WO 2024021455A1 CN 2022140522 W CN2022140522 W CN 2022140522W WO 2024021455 A1 WO2024021455 A1 WO 2024021455A1
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galactosidase
seq
recombinant
lactose
enzyme
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Chinese (zh)
Inventor
李兆丰
张梓芊
李才明
顾正彪
班宵逢
程力
洪雁
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Jiangnan University
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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • 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
    • 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
    • C12P19/12—Disaccharides
    • 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
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • 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
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention relates to a ⁇ -galactosidase gene and the application of its encoding enzyme, specifically to a ⁇ -galactosidase gene that produces galacto-oligosaccharide and its application method, and belongs to the technical fields of genetic engineering and enzyme engineering.
  • ⁇ -galactooligosaccharides is a functional oligosaccharide with a degree of polymerization of 2 to 8, which uses galactose or glucose as the reducing end and is connected through ⁇ -glycosidic bonds 1 to 7
  • ⁇ -GOS has good taste, low sweetness, high solubility and strong moisturizing properties. It is an excellent food sweetener.
  • ⁇ -GOS has good anti-digestive properties and can resist degradation by digestive enzymes in the small intestine, maintaining a relatively intact structure and reaching the large intestine, thereby exerting many probiotic functions.
  • the specific performance is as follows: (1) It can selectively promote the proliferation of beneficial intestinal bacteria, especially Bifidobacterium and Lactobacillus, and at the same time inhibit the growth of putrefactive bacteria (such as some Clostridium). (2) Improve intestinal barrier function and relieve colitis.
  • Supplementing ⁇ -GOS in early life can help babies establish a healthy colon environment, increase the content of short-chain fatty acids (SCFAs) in the intestine, and reduce the risk of colitis; at the same time, dietary supplementation of ⁇ -GOS can also accelerate wound healing, which is beneficial to the treatment of colitis. Postoperative recovery. (3) Improve metabolism and delay aging. Synbiotics containing ⁇ -GOS can alleviate intestinal flora imbalance and significantly enhance the antioxidant capacity of the liver, exerting anti-aging effects through the liver-gut axis. (4) Improve diabetes symptoms. Due to its excellent antioxidant capacity and ability to balance intestinal flora, ⁇ -GOS has also been proven to reduce the levels of diabetes-related markers in the blood and delay the development of type II diabetes.
  • SCFAs short-chain fatty acids
  • ⁇ -GOS has no adverse effects on bodies of different ages or different organisms, and is safe and reliable. Therefore, ⁇ -GOS, as a prebiotic with rich nutritional value, can be used in infant food or dietary supplements for dietary treatment of special patients, and has broad application prospects in the fields of food, medicine and health. my country approved the use of ⁇ -GOS as a food additive in 2008. As its application market increases year by year, research on the functional development and production technology of ⁇ -GOS is of great significance.
  • ⁇ -GOS mainly relies on the enzymatic process, which uses ⁇ -galactosidase with transglycosidic activity to act on high-concentration lactose.
  • Japan prepared the first commercial product of ⁇ -GOS, and then its preparation process was introduced to Europe. The two monopolized the current production of high-purity ⁇ -GOS.
  • my country's ⁇ -GOS production started late and has not yet reached the industrial scale of 1,000 tons.
  • the main limiting factor is the lack of ⁇ -galactosidase with good properties.
  • ⁇ -galactosidase for commercial preparation of ⁇ -GOS: Aspergillus oryzae, Kluyveromyces lactis and Bacillus circulans.
  • the price of ⁇ -galactosidase derived from Aspergillus oryzae is relatively low, the conversion rate is about 30%, and the main product is galactooligosaccharide (about 18%); ⁇ -galactosidase derived from Kluyveromyces lactis
  • the highest conversion rate of glycosidase is also about 30%, and the disaccharide content in the product is the highest, but the probiotic activity of the disaccharide has not been confirmed; the conversion rate of ⁇ -galactosidase derived from Bacillus circulans is higher, up to 40% Around 26%, the product is mainly trisaccharide (about 26%).
  • the current enzymes used for ⁇ -GOS production have problems such as low conversion rate and low proportion of main products.
  • the composition of the transglycoside products of ⁇ -galactosidase is complex and contains different types of glycosidic bonds, which makes subsequent separation more difficult and is not conducive to functional research on ⁇ -GOS with different structures. Therefore, in order to reduce preparation and separation costs, it is an important research direction to find ⁇ -galactosidase with high transglycosylation efficiency and strong product specificity.
  • the purpose of the present invention is to make up for the shortcomings of the current enzymatic synthesis of ⁇ -GOS and provide a gene encoding ⁇ -galactosidase.
  • the gene is derived from Paenibacillus macquariensis, and the nucleotide sequence is as shown in SEQ ID NO.1 Show.
  • the present invention also provides a ⁇ -galactosidase encoded by the above nucleotide sequence, whose amino acid sequence is shown in SEQ ID NO. 2.
  • the present invention also provides a recombinant plasmid carrying the above-mentioned ⁇ -galactosidase gene.
  • the recombinant plasmid uses E. coli expression plasmid pET-20b(+) as a vector.
  • the present invention also provides a microbial cell carrying the above-mentioned ⁇ -galactosidase gene or the recombinant plasmid.
  • the microbial cell is recombinant E. coli.
  • the recombinant Escherichia coli uses Escherichia coli BL21 (DE3) as the expression host.
  • the construction method of the recombinant E. coli is: using a seamless cloning method, the ⁇ -galactosidase gene whose nucleotide sequence is shown in SEQ ID NO.1 is spliced into the expression vector pET -20b(+), construct the recombinant plasmid pmgal/pET-20b(+) and transform it into E.coli BL21(DE3).
  • the present invention also provides a method for producing ⁇ -galactosidase.
  • the method uses lactose as a substrate and utilizes the ⁇ -galactosidase with an amino acid sequence as shown in SEQ ID NO.2 to catalyze the substrate to generate oligomers.
  • Galactose is a method for producing ⁇ -galactosidase.
  • the ⁇ -galactosidase is added in an amount of no less than 500 U/g substrate.
  • the ⁇ -galactosidase is added in an amount of 500 to 1000 U/g substrate.
  • the ⁇ -galactosidase is added in an amount of 1000 U/g substrate.
  • the substrate is lactose, and the lactose concentration is 200-400g/L.
  • the lactose concentration is 400g/L.
  • the reaction is carried out at 45-55°C and pH 5.0-7.0 for 48-72 hours.
  • the reaction is carried out at 50°C.
  • reaction is carried out at pH 6.5 and 50°C for 60 hours.
  • the nucleotide shown in SEQ ID NO. 1 is connected to an expression vector and transferred into E. coli to obtain recombinant E. coli.
  • the fermentation involves inserting a certain amount of recombinant cells or recombinant E. coli into an LB medium containing ampicillin, culturing it at 37°C to the logarithmic growth phase, and preparing a seed liquid.
  • the seed liquid is used Proceed with fermentation.
  • the seed liquid is inoculated into TB medium containing ampicillin and 0-15% (w/v) lactose at an inoculation amount of 2% to 5% (v/v), and the seed liquid is inoculated at 25 to 37 Incubate in a shake flask at °C for 24 to 72 hours, and centrifuge to obtain the supernatant, which is the ⁇ -galactosidase crude enzyme solution.
  • the present invention also provides the ⁇ -galactosidase gene, the recombinant plasmid containing the ⁇ -galactosidase gene, and the E. coli expressing the ⁇ -galactosidase in the production of ⁇ -galactooligosaccharide.
  • the application uses ⁇ -galactosidase or an enzyme preparation containing the enzyme as a catalyst and a lactose solution as a substrate to convert lactose into functional galactooligosaccharides.
  • the enzyme preparation is the ⁇ -galactosidase crude enzyme solution or the pure enzyme obtained after separation and purification, and is added to the reaction system in the form of a solution or dry powder.
  • the concentration of the lactose solution is 200-400g/L.
  • the present invention screened out a ⁇ -galactosidase with a specific amino acid sequence, and successfully achieved heterologous expression in E. coli, which can be used in food and pharmaceutical production.
  • the expressed ⁇ -galactosidase The catalytic activity can reach 12378.6U/mg;
  • the ⁇ -galactosidase of the present invention has a specific enzyme activity higher than most of the currently reported similar enzymes, and can maintain a high level in a wide temperature range. Vitality, adaptable to different reaction temperature conditions;
  • the ⁇ -galactosidase of the present invention has obvious advantages in preparing galacto-oligosaccharides, such as higher substrate conversion rate, stronger product specificity, and can effectively improve The yield of galactooligosaccharide reduces its preparation difficulty and subsequent separation and purification costs.
  • the substrate conversion rate can reach 70.9%.
  • the galactooligosaccharide content in the product accounts for approximately 63.1% of the total sugar.
  • the conversion rate and galactooligosaccharide content All have reached the highest level in the existing technology and have high industrial application value.
  • Figure 1 shows the SDS-PAGE analysis of purified recombinant ⁇ -galactosidase.
  • Figure 2 shows the thermal stability of recombinant ⁇ -galactosidase.
  • Figure 3 shows the effect of pH on the thermal stability of recombinant ⁇ -galactosidase.
  • Figure 4 shows HPAEC-PAD analysis when using recombinant ⁇ -galactosidase to prepare galactooligosaccharides.
  • the ⁇ -galactosidase enzyme activity assay method involved in the following examples is as follows:
  • Enzyme activity is defined as: under certain conditions, the amount of enzyme that hydrolyzes oNPG and releases 1 ⁇ mol oNP per minute by ⁇ -galactosidase is one unit of enzyme activity (U).
  • galacto-oligosaccharide content (%) 100% ⁇ mass of galacto-oligosaccharide in the product/mass of all sugars in the product.
  • the mass of galactooligosaccharide is the sum of the masses of transfer disaccharide to pentasaccharide.
  • the homologous recombination reaction system is: 1 ⁇ L of purified ⁇ -galactosidase fragment (50 ng/ ⁇ L), 1 ⁇ L of purified pET-20b(+) vector PCR fragment (50 ng/ ⁇ L), 4 ⁇ L of 5 ⁇ CEIIBuffer, 2 ⁇ L of ExnaseII, ddH 2 O 12 ⁇ L.
  • Example 2 Expression, isolation and purification of recombinant ⁇ -galactosidase
  • Example 1 Inoculate the genetically engineered bacteria preservation solution prepared in Example 1 into LB liquid culture medium containing 100 ⁇ g/mL ampicillin, and culture it at 37°C for 8 to 10 hours to prepare a seed solution;
  • the crude enzyme solution is purified by a nickel ion affinity chromatography column.
  • the buffers are A (10mmol/L Tris-HCl, 500mmol/L NaCl, pH 7.5) and B (10mmol/L). Tris-HCl, 500mmol/L NaCl, 500mmol/L imidazole, pH 7.5), the flow rate is 2mL/min. Equilibrate the nickel column with 25 to 30 mL of buffer A until it is stable, load the sample, and then use buffer A to elute the unbound protein in the purification column.
  • the thermal stability determination method of recombinant ⁇ -galactosidase pure enzyme is as follows: dilute the pure enzyme in 10mmol/L K 2 HPO 4 -KH 2 PO 4 buffer (pH 6.0), and incubate at 50°C and 55°C , incubate at 60°C for 60 minutes, take samples at different time points, and measure the enzyme activity. Taking the activity without incubation as 100%, calculate the relative residual enzyme activity at different times. The results are shown in Figure 2. The half-life of the enzyme at 50°C is approximately 50 minutes.
  • Recombinant ⁇ -galactosidase is used to prepare galactooligosaccharides.
  • the reaction process is as follows: prepare 200-400g/L lactose as substrate, adjust the pH to 5.0-7.0, add 500-1000U/g of pure enzyme as substrate, and 48 to 72 hours at 50°C (react until the residual lactose in the solution no longer degrades). Take part of the reaction solution and put it in a boiling water bath for 10 minutes to terminate the reaction, centrifuge at 8000r/min for 5 minutes, take the supernatant and dilute it to an appropriate multiple, and pass it through a 0.22 ⁇ m water filter membrane for testing.
  • HPAEC-PAD was used to determine the content of each component in the enzymatic hydrolyzate.
  • a ternary gradient elution program was adopted, eluent A was 0.25M sodium hydroxide, eluent B was 1.0M sodium acetate, and eluent C was ultrapure water.
  • the flow rate was 0.5 mL/min, the column temperature was 35°C, the injection volume was 10 ⁇ L, and the sugar four-potential waveform was used for detection.
  • the chromatographic analysis results are shown in Figure 4.
  • the substrate conversion rate is about 58% to 70%, and the galacto-oligosaccharide content is about 46% to 63% (% total sugar).
  • the substrate conversion rate is approximately 70.9%, and the galactooligosaccharide content in the product accounts for approximately 63.1% of total sugars.
  • the specific implementation is the same as in Examples 1 to 2 and 4. The difference is that the gene derived from Paenibacillus macquariensis is replaced with the ⁇ -galactosidase gene from other sources reported in the past. According to the steps in Examples 1 to 2 Methods Genetically engineered bacteria were constructed and cultured to prepare pure enzyme liquid. The product was analyzed according to the method in Example 4. The comparison results are shown in Table 1. It can be seen from the data in the table that the ⁇ -galactosidase encoded by the gene shown in SEQ ID NO.1 is superior to most ⁇ -galactosidase in existing reports in terms of substrate conversion rate and galactooligosaccharide yield. , has very broad application prospects.

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Abstract

La présente invention concerne le domaine technique du génie génétique et du génie enzymatique, et présente une β-galactosidase, un gène la codant, et un procédé de préparation de galactooligosaccharide. La séquence d'acides aminés de la β-galactosidase est représentée dans SEQ ID NO : 2, et la séquence nucléotidique du gène codant pour la β-galactosidase est telle que représentée dans SEQ ID NO : 1. L'expression hétérologue est réalisée sur la β-galactosidase en utilisant Escherichia coli, et l'activité catalytique d'une solution enzymatique brute de β-galactosidase exprimée peut atteindre 12 378,6 U/mg. La β-galactosidase peut catalyser le lactose pour générer du galactooligosaccharide.
PCT/CN2022/140522 2022-07-26 2022-12-21 GÈNE DE β-GALACTOSIDASE ET UTILISATION DE CE GÈNE DANS LE CODAGE D'UNE ENZYME Ceased WO2024021455A1 (fr)

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CN120574912A (zh) * 2025-08-06 2025-09-02 中国农业科学院北京畜牧兽医研究所 乳糖酶dt1降解乳糖的应用

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CN102154239A (zh) * 2011-01-06 2011-08-17 中山大学 一种具有高效转糖基β-半乳糖苷酶的新基因及应用
CN106957832A (zh) * 2016-01-08 2017-07-18 中国农业大学 一种细菌β-半乳糖苷酶及其编码基因与应用
CN111235132A (zh) * 2019-12-23 2020-06-05 浙江工业大学 一种β-半乳糖苷酶、基因、工程菌及其应用
CN113774073A (zh) * 2021-10-25 2021-12-10 中国水产科学研究院黄海水产研究所 一种深海宏基因组来源β-半乳糖苷酶、编码基因和应用

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CN106957832A (zh) * 2016-01-08 2017-07-18 中国农业大学 一种细菌β-半乳糖苷酶及其编码基因与应用
CN111235132A (zh) * 2019-12-23 2020-06-05 浙江工业大学 一种β-半乳糖苷酶、基因、工程菌及其应用
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120574912A (zh) * 2025-08-06 2025-09-02 中国农业科学院北京畜牧兽医研究所 乳糖酶dt1降解乳糖的应用

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