WO2024103822A1 - Polypeptide mature pour la synthèse d'oligosaccharides - Google Patents

Polypeptide mature pour la synthèse d'oligosaccharides Download PDF

Info

Publication number
WO2024103822A1
WO2024103822A1 PCT/CN2023/108971 CN2023108971W WO2024103822A1 WO 2024103822 A1 WO2024103822 A1 WO 2024103822A1 CN 2023108971 W CN2023108971 W CN 2023108971W WO 2024103822 A1 WO2024103822 A1 WO 2024103822A1
Authority
WO
WIPO (PCT)
Prior art keywords
residue
seq
amino acid
acid sequence
sequence shown
Prior art date
Application number
PCT/CN2023/108971
Other languages
English (en)
Chinese (zh)
Inventor
方诩
牛康乐
唐琪
韩丽娟
Original Assignee
山东恒鲁生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 山东恒鲁生物科技有限公司 filed Critical 山东恒鲁生物科技有限公司
Publication of WO2024103822A1 publication Critical patent/WO2024103822A1/fr

Links

Classifications

    • 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
    • C12P19/26—Preparation of nitrogen-containing carbohydrates

Definitions

  • the present application belongs to the field of enzyme engineering/genetic engineering technology, and specifically relates to a polypeptide capable of synthesizing oligosaccharides, a preparation method thereof, and its application in synthesizing oligosaccharides (especially human milk oligosaccharides).
  • HMOs human milk oligosaccharides
  • infant intestinal microecology can promote the balance of infant intestinal microecology, including: promoting the proliferation of beneficial bacteria in the intestine, inhibiting the growth of harmful bacteria, resisting pathogen infection, regulating the immune system and promoting the cognitive development of infants.
  • beneficial effects of breast milk are attributed to HMOs.
  • HMOs 2-fucosyllactose
  • lactose-N-triose Lacto-N-triose II, LNTII
  • lactose-N-tetraose Lacto-N-tetraose
  • LNnT lactose-N-neotetraose
  • lactose-N-tetraose lactose-N-tetraose
  • lactose-N-neotetraose LNT
  • lactose-N-neotetraose LNnT
  • LNT lactose-N-tetraose
  • LNnT lactose-N-neotetraose
  • LNT and LNnT have similar structures, and their molecular weights are both 707.63.
  • the chemical structure of LNT is shown in the following formula 1:
  • HMOs are approved for use in a variety of foods, including dairy products, baked goods, and beverages.
  • the scope of application approved by the European Union has been expanded to dietary supplements, special medical formula foods, and milk-based beverages or similar products for infants only. Since the supply of HMOs that meet the requirements is limited, effective industrial production is very much needed. Direct extraction of HMOs from natural breast milk is certainly not an ideal approach.
  • researchers have developed some chemical methods and bioenzymatic methods for HMOs.
  • the preparation methods disclosed in existing literature have the problems of high production cost, low yield, and high price.
  • the chemical synthesis method can obtain oligosaccharides with defined structures through lengthy multiple protection-deprotection operations, it has the problems of cumbersome work, large workload, and low product yield.
  • the use of heavy metals increases food safety risks. Therefore, there is an urgent need for HMOs synthesis technology with stable process, green safety, low cost, high production efficiency, and large-scale industrial production to meet the public's demand for HMOs.
  • Chinese invention patent 201810206004.3 discloses a recombinant Bacillus subtilis and a method for preparing lacto-N-neotetraose using the same, which comprises: recombinantly integrating a lactose permease gene into the genome of Bacillus subtilis 168, and transforming a plasmid containing a ⁇ -1,3-N-glucosaminyltransferase gene and a ⁇ -1,4-galactosyltransferase gene into Bacillus subtilis 168. After 24 hours of fermentation, the obtained recombinant Bacillus subtilis has a fermentation broth supernatant containing 1071 mg/L lactose-N-neotetraose.
  • Chinese patent 201180071512.1 reports a method for producing lactose-N-tetraose and lactose-N-neotetraose from lactose-N-triose using active cell extracts.
  • the purpose of the present application is to provide a series of polypeptides that can simultaneously catalyze the synthesis of lactose-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), methods for preparing the same, and their use in producing oligosaccharides specifically present in human breast milk (i.e., human milk oligosaccharides (HMOs)), as well as methods for producing HMOs based thereon.
  • LNT lactose-N-tetraose
  • LNnT lacto-N-neotetraose
  • HMOs human milk oligosaccharides
  • the present application also provides related nucleic acid products, enzyme products of the polypeptide, and preparation methods and applications thereof.
  • the present invention uses computer-aided design technology to screen out for the first time a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1, which is a derivative of ⁇ -galactosidase.
  • ⁇ -galactosidase is widely present in microorganisms, animals and plants, and its species source can be: Aspergillus oryzae, Kluyveromyces lactis, Kluyveromyces marxinus, Bacillus subtilis, Bacillus circulans, etc. More preferably, its species source is Bacillus circulans or Bacillus subtilis.
  • polypeptide shown in SEQ ID NO:1 has the ability to catalyze the synthesis of HMOs, in particular, it can simultaneously catalyze the synthesis of LNT and LNnT.
  • the inventors of the present application have carried out a series of modifications to the polypeptide shown in SEQ ID NO:1, and obtained a derivative polypeptide with greatly improved ability to catalyze the synthesis of LNT and LNnT, which has more excellent performance in the ability to simultaneously catalyze the synthesis of LNT and LNnT.
  • the present application provides a polypeptide capable of simultaneously catalyzing the synthesis of lactose-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), wherein the polypeptide has an amino acid sequence selected from the following:
  • amino acid sequence formed by replacing one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1, which also has the activity of catalyzing the synthesis of LNT and LNnT simultaneously;
  • the replacement of one or more amino acids is selected from:
  • the glutamic acid residue at position 100 is replaced by a phenylalanine residue
  • the tyrosine residue at position 449 is replaced by an alanine residue
  • the glutamic acid residue at position 601 is replaced by a leucine residue, thereby obtaining the amino acid sequence shown in SEQ ID NO:17.
  • the present application provides a polynucleotide encoding the polypeptide as described in the first aspect above.
  • the present application provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above, and one or more regulatory sequences operably linked thereto, wherein the regulatory sequences can guide the production of the polypeptide in a suitable expression host.
  • the present application provides an expression vector comprising the polynucleotide as described in the second aspect above, or comprising the nucleic acid construct as described in the third aspect above.
  • the present application provides a transformed host cell, into which the polynucleotide as described in the second aspect above, or the nucleic acid construct as described in the third aspect above, or the expression vector as described in the fourth aspect above is transformed.
  • the host cell is any one of bacteria, mold or yeast;
  • the bacteria are selected from: bacteria of the genera Bacillus and Escherichia;
  • Bacillus bacteria include Bacillus mycoides, Bacillus cereus, Bacillus polymyxa, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus brevis, Bacillus circulans and Bacillus subtilis;
  • Escherichia bacteria include Escherichia coli, Escherichia hermanii and Escherichia fergusonii;
  • the host cell is Bacillus circulans, Bacillus subtilis or Bacillus licheniformis.
  • the mold is selected from: Aspergillus and Trichoderma molds;
  • the mold is selected from: Trichoderma viride, Trichoderma reesei, Trichoderma harzianum, Aspergillus oryzae, Aspergillus fumigatu, Aspergillus niger and Aspergillus flavus;
  • the host cell is an Aspergillus oryzae cell.
  • the yeast is selected from the group consisting of Candida, Hansenula, Kluyveromyces, Pichia, yeasts of the genera Saccharomyces, Schizosaccharomyces, and Yarrowia;
  • the yeast is selected from: Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxinus, Yarrowia lipolytica, Rhodotorula graminis, Saccharomyces pastorianus and the like;
  • the host cell is a Saccharomyces cerevisiae, Kluyveromyces lactis or Kluyveromyces marxinus cell.
  • the host cell may be a wild strain isolated from nature, a mutant strain induced by physical or chemical mutation, or an engineered bacterium transformed by genetic engineering, etc.
  • the transformed host cell is a genetically engineered bacterium into which the polynucleotide as described in the second aspect, or the nucleic acid construct as described in the third aspect, or the expression vector as described in the fourth aspect (thereby expressing the polypeptide as described in the first aspect) is transformed, preferably Kluyveromyces lactis, Bacillus subtilis or Aspergillus oryzae containing a gene encoding the polypeptide as described in the first aspect.
  • the present application provides an enzyme agent or enzyme composition, which comprises the polypeptide as described in the first aspect above.
  • the present application provides a method for producing the polypeptide as described in the first aspect above, comprising:
  • the step (1) comprises: firstly introducing a nucleic acid construct or a recombinant expression vector encoding the polypeptide as described in the first aspect above into a host cell to construct an engineered host cell expressing the polypeptide; then, culturing the engineered host cell and inducing it to express the polypeptide.
  • the step (2) includes the steps of isolating and purifying the polypeptide from the culture.
  • Host cells can be cultivated in a nutrient medium suitable for producing polypeptides using methods known in the art.
  • cells can be cultured by shaking bottles, or in a suitable medium and under conditions that allow polypeptide expression and/or separation, small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, batch-fed fermentation or solid-state fermentation) can be carried out in a laboratory or industrial fermentor tank to cultivate cells.
  • Cultivation is to use procedures known in the art, which occur in a suitable nutrient medium, and the medium comprises carbon and nitrogen sources and inorganic salts.
  • Suitable medium can be purchased through commercial channels, or prepared according to disclosed compositions.
  • the polypeptide may be recovered from the culture using methods known in the art.
  • the variant may be recovered from the nutrient medium by a variety of conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation.
  • polypeptide can be purified by various procedures known in the art to obtain substantially pure polypeptides, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, gel filtration chromatography), electrophoresis procedures (e.g., preparative isoelectric focusing), differential solubility methods (e.g., ammonium sulfate precipitation), SDS-PAGE, salting out, etc. or a combination thereof; further preferably, purification can be performed by Ni column affinity chromatography.
  • chromatography e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, gel filtration chromatography
  • electrophoresis procedures e.g., preparative isoelectric focusing
  • differential solubility methods e.g., ammonium sulfate precipitation
  • SDS-PAGE salting out, etc. or a combination thereof
  • purification can be performed by Ni column affinity
  • the present application provides a method for simultaneously producing lactose-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), comprising: using the polypeptide described in the first aspect above or the transformed host cell (especially the transformed engineered bacteria) described in the fifth aspect above, or the enzyme or enzyme composition described in the sixth aspect above as a catalyst, and lactose and its derivatives as reaction substrates, to generate LNT and LNnT through catalytic or fermentation reactions.
  • LNT lactose-N-tetraose
  • LNnT lacto-N-neotetraose
  • the transformed host cell is Kluyveromyces lactis, Bacillus subtilis or Aspergillus oryzae into which the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect or the expression vector as described in the fourth aspect is transformed;
  • the reaction substrate is a mixture of lactose and lactose-N-triose (Lacto-N-triose, LNTII);
  • the reaction solvent is an aqueous solvent or a solution containing a solid.
  • a purified form of the polypeptide as described in the first aspect above can be used, or a culture containing it (i.e., a crude enzyme solution) can be used; in addition, the polypeptide can be immobilized and used, or mixed with other materials that can maintain or enhance its activity and used as an enzyme preparation.
  • process parameters such as the reaction temperature, the amount of catalyst added, the reaction time, and the pH value can be optimized or selected according to the method disclosed in the examples of the present application.
  • the reaction is carried out at 30-50°C, and more preferably, the reaction temperature is 45°C;
  • the reaction is carried out in a buffer solution at pH 4-11, preferably pH 6-8, more preferably pH 7;
  • the buffer is a phosphate buffer.
  • the present application provides the use of the polypeptide as described in the first aspect above, or the transformed host cell (especially the transformed engineered bacteria) as described in the fifth aspect above, or the enzyme or enzyme composition as described in the sixth aspect above as a catalyst in the production of lactose-N-tetraose (LNT) and lacto-N-neotetraose (LNnT);
  • the transformed host cell is Kluyveromyces lactis, Bacillus subtilis or Aspergillus oryzae into which the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, or the expression vector as described in the fourth aspect is transformed;
  • the method for producing lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) is as described in the eighth aspect above.
  • expression includes any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, etc.; expression can be measured by techniques known in the art, for example, measuring the concentration or activity of mRNA and/or translated polypeptide.
  • host cell refers to any cell type that is susceptible to transformation, transfection, transduction, etc. with an expression vector.
  • host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to substitutions that occur during replication.
  • the polypeptide provided in the present application can catalyze the synthesis of LNT and LNnT with extremely high efficiency.
  • the present application also provides polynucleotides encoding the polypeptide and related nucleic acid products, transformed host cells, enzymes containing the polypeptide, and methods for catalytically synthesizing LNT and LNnT using the polypeptide as a catalyst.
  • the technical solution of the present application has positive significance for the industrial production of human milk oligosaccharides.
  • the method is green, efficient, and sustainable, is conducive to the application of industrial large-scale production, and has important practical value.
  • FIG. 1 is a HPLC spectrum (wavelength 210 nm) of the reaction solution after the catalytic reaction using polypeptide M8 as described in Example 2.
  • the pET-32a plasmid was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.
  • HPLC analysis conditions were as follows: using a 4.6 ⁇ 250 mm, 5 ⁇ m Asahipak NH2P-50 4E amino column, isocratic elution with 75% acetonitrile aqueous solution as the mobile phase; flow rate was 0.7 mL/min, column temperature was 30°C, injection volume was 10 ⁇ L, UV detector, detection wavelength was 210 nm.
  • Chromatographic column model 4.6 ⁇ 250mm, 5 ⁇ m AsahipakNH2P-504E amino chromatographic column was used, and 75% acetonitrile aqueous solution was used as the mobile phase for isocratic elution; the column temperature was 30°C, the injection volume was 10 ⁇ L, the UV detector, the detection wavelength was 210nm, the flow rate was 1.0mL/min; H-ESI mode, the molecular weight scanning range was 400-900.
  • the ion chromatography analysis conditions are as follows:
  • Chromatographic column MetroSep Carb2 (4.0mm ⁇ 250mm), eluent: 140mM NaOH+20mM NaAc, isocratic elution, flow rate: 0.500mL/min, amperometric detector, column temperature 40°C, injection volume 20 ⁇ L, running time 50min.
  • Lactose-N-triose (LNTII), lactose-N-tetraose (LNT), and lactose-N-neotetraose (LNnT) standards were produced by ELICITYL, France.
  • polypeptides shown in SEQ ID NO: 2-17 involved in the following examples are a series of derivative polypeptides obtained by replacing the following amino acids with the polypeptide shown in SEQ ID NO: 1:
  • the glutamic acid residue at position 100 is replaced by a phenylalanine residue, a leucine residue, or an alanine residue to obtain polypeptides having amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively;
  • the tyrosine residue at position 449 is replaced by a phenylalanine residue, a leucine residue, or an alanine residue to obtain polypeptides with amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively;
  • the tryptophan residue at position 570 is replaced by a phenylalanine residue, a leucine residue, or an alanine residue to obtain polypeptides with amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively;
  • the glutamic acid residue at position 100 is replaced by a phenylalanine residue
  • the tyrosine residue at position 449 is replaced by an alanine residue
  • the glutamic acid residue at position 601 is replaced by a leucine residue to obtain a polypeptide with an amino acid sequence as shown in SEQ ID NO:17.
  • Example 1 Expression of polypeptides shown in SEQ ID NO: 1-17 in Escherichia coli
  • the pET32a-BgaD-MX recombinant plasmid series (pET32a-BgaD-M1-17) was constructed using the Fast Mutagenesis Kit; among them, the empty plasmid pET32a was purchased from Shanghai Biotechnology.
  • Example 2 Determination of the ability of the polypeptides shown in SEQ ID NO: 1-17 to catalyze the synthesis of lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT)
  • Example 1 The purified polypeptides obtained in Example 1 were used as catalysts, and lactose and lactose-N-triose were used as reaction substrates to catalyze the synthesis of LNT and LNnT.
  • the specific procedure was as follows:
  • lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) standards and the catalytic reaction products obtained by the above procedures were analyzed by high performance liquid chromatography (HPLC), LC-MS analytical method and ion chromatography, respectively.
  • LNnT lactose-N-neotetraose
  • the molecular weight of 708.2548 should be LNnT/LNT+H; the molecular weight of 730.2367 should be LNnT/LNT+Na.
  • lactose-N-tetraose (LNT) and/or lactose-N-neotetraose (LNnT) in the catalytic reaction solution of each polypeptide was determined by ion chromatography, and the results are recorded in Table 3.
  • Example 3 Expression of polypeptides M6 and M17 in Kluyveromyces lactis (CICC1773) and determination of their ability to catalyze the synthesis of lactose-N-neotetraose (LNnT) and lactose-N-tetraose (LNT)
  • the coding genes of the polypeptides M6 and M17 obtained in Example 1 were cloned into the pKLAC1 plasmid by restriction endonucleases and T4 ligase, respectively, to construct recombinant expression plasmids pKLAC1-M6 and pKLAC1-M17; specifically, the gene fragment encoding the polypeptide and the pKLAC1 plasmid were double-digested with restriction endonucleases EcoRI and NotI, respectively, and then ligated with T4 ligase, and the ligated plasmids were transformed into Escherichia coli DH5 ⁇ , and after screening and identification, the recombinant expression plasmids pKLAC1-M6 and pKLAC1-M17 were obtained.
  • KLAC1-M6 or KLAC1-M17 expression plasmid After linearization of KLAC1-M6 or KLAC1-M17 expression plasmid, transform it into Kluyveromyces lactis.
  • the specific operation is as follows: pick freshly cultured Kluyveromyces lactis colonies into 1 mL YPD medium, place it in a constant temperature oscillator, and culture it at 30°C and 250 rpm for 12-14 h; transfer it to 50 mL fresh YPD medium with an initial inoculation OD of 0.2, and culture it at 30°C and 200 rpm for 3-4 h until the OD is 0.8-1; place the bacterial culture on ice for 5-10 min, and at the same time, treat 25 ⁇ L salmon sperm DNA (2 mg/mL) at 95°C for 10 min, then quickly cool it in an ice water bath and place it on ice for use; prepare the expression plasmid to be transformed, add a total of 5-10 ⁇ g
  • the shake flask fermentation was performed as follows: 1) Preparation of primary species: a single colony of the above-mentioned genetically engineered yeast strain was cultured overnight in 10 mL YGD liquid culture medium at 30°C and 200 rpm. The resulting strain was the primary species. 2) Preparation of secondary species: the primary species was inoculated in 1 L YGD liquid culture medium and cultured at 30°C and 200 rpm until OD600 reached about 7. 3) The YGD culture medium was centrifuged to obtain the crude enzyme solution of the target protein, and the protein was purified according to the method in Example 2. The resulting proteins were polypeptides M6 and M17, respectively, and their amino acid sequences were shown in SEQ ID NO: 6 and SEQ ID NO: 17, respectively.
  • polypeptides M6 and M17 obtained in this example were taken respectively, and the reaction system was placed in a water bath at 45° C. for 0.5 hour according to the catalytic synthesis method described in Example 2. The reaction was terminated and purified by gel column method. The yields of lactose-N-neotetraose (LNnT) and lactose-N-tetraose (LNT) in the reaction solution were measured by ion chromatography. The results are recorded in Table 4.
  • Table 4 shows that the polypeptides M6 and M17 expressed by Kluyveromyces lactis also have excellent ability to catalyze the synthesis of LNT and LNnT.
  • Example 4 Expression of polypeptides M6 and M17 in Bacillus subtilis 168 and determination of their ability to catalyze the synthesis of lactose-N-neotetraose (LNnT) and lactose-N-tetraose (LNT)
  • the coding genes of the polypeptides M6 and M17 obtained in Example 1 were cloned into the pEB03 plasmid by restriction endonucleases and T4 ligase, respectively, to construct recombinant expression plasmids pEB03-M6 and pEB03-M17; specifically, the gene fragment encoding the polypeptide and the plasmid pEB03 were double-digested with restriction endonucleases EcoR I and NotI, respectively, and then the two were connected with T4 ligase, and the connected plasmids were transformed into Escherichia coli DH5 ⁇ . After screening and identification, recombinant expression plasmids pEB03-M6 and pEB03-M17 were obtained.
  • the recombinant plasmid was transformed in Bacillus subtilis.
  • the specific transformation method was as follows: a full loop of Bacillus subtilis glycerol bacteria was streaked onto an LB plate (LB medium: 1.0% peptone, 0.5% yeast extract, 1.0% NaCl, and 1.5% agar powder on the plate), and cultured overnight at 37°C in an incubator; a single colony was picked and transferred to 1 mL of LB medium, and cultured overnight at 37°C, 200 rpm, and 200 ⁇ L of the culture solution was transferred to 200 mL of growth medium (growth medium: LB medium plus Add 0.5 M sorbitol), culture at 37°C, 200 rpm until OD600 reaches 0.85-0.95, ice bath for 10 min, centrifuge at 4°C, 5000 rpm for 5 min, then wash 4 times with pre-cooled electroporation medium (electroporation medium: LB medium with 0.5 M sorbitol and 10% glycerol
  • the treated bacterial solution was divided into 80 ⁇ L per tube, 1 ⁇ L (about 50 ng) of plasmid was added, and the solution was transferred to a pre-cooled electroporation cup and placed on ice for 1-1.5 min. Then, the electroporation instrument was used for electroporation.
  • the electroporation parameters were set as follows: voltage 2000 V, resistance 200 ⁇ , followed by an ice bath for 2 min, and then 1 mL of recovery medium (recovery medium: LB medium with 0.5 M sorbitol and 0.38 M mannitol) was added and cultured at 37 ° C and 200 rpm for 90 min.
  • recovery medium recovery medium: LB medium with 0.5 M sorbitol and 0.38 M mannitol
  • a positive transformation single colony was picked from an LB plate containing 5 ⁇ g/mL chloramphenicol and inoculated into 20 mL seed culture medium (containing: 0.5% yeast extract, 0.5% tryptone, 1% glucose, 1.8% K 2 HPO 4 , 5 ⁇ g/mL chloramphenicol), and cultured at 37°C and 220 rpm for 8 hours; 2.5 mL was inoculated into 50 mL fermentation culture medium (containing: 1.0% tryptone, 0.5% yeast powder, 1.0% NaCl), and cultured at 34°C and 250 rpm for 60 hours, and the supernatant was taken for SDS-PAGE electrophoresis detection.
  • the expression of the target protein was analyzed by SDS-PAGE, and polypeptides M6 and M17 were obtained, respectively, and their amino acid sequences were shown in SEQ ID NO:6 and SEQ ID NO:17, respectively.
  • polypeptides M6 and M17 obtained in this example were taken respectively, and the reaction system was placed in a water bath according to the catalytic synthesis method described in Example 2, and reacted at 45°C for 0.5 hour, the reaction was terminated, and the products were purified by gel column method.
  • the yields of lactose-N-neotetraose (LNnT) and lactose-N-tetraose (LNT) in the reaction solution were measured by ion chromatography, and the results are recorded in Table 5.
  • Table 5 shows that the polypeptides M6 and M17 expressed by Bacillus subtilis also have excellent ability to catalyze the synthesis of LNT and LNnT.
  • Example 5 Expression of polypeptides M6 and M17 in Aspergillus oryzae (CCTCC AF 93036) and determination of their ability to catalyze the synthesis of lactose-N-neotetraose (LNnT) and lactose-N-tetraose (LNT)
  • genomic DNA was extracted from Aspergillus oryzae, and the Aspergillus oryzae genomic DNA was used as a template to amplify the Aspergillus oryzae amylase promoter and glycosidase terminator.
  • the promoter, terminator and expression vector backbone pCAMBIA1304 were respectively digested and ligated to connect the promoter PamyB and terminator TagdA to the corresponding digestion positions, and the Aspergillus oryzae reading frame was constructed to obtain the plasmid pCAMBIA001.
  • the coding genes of the polypeptides M6 and M17 obtained in Example 1 were cloned into the plasmid pCAMBIA001 by restriction endonucleases and T4 ligase.
  • the polypeptide coding gene fragment and the plasmid pCAMBIA001 were double-digested with restriction endonucleases EcoRI and NotI, respectively, and then the two were connected with T4 ligase, and the connected plasmids were transformed into Escherichia coli DH5 ⁇ , and positive clones were screened and identified and expanded, and plasmids were extracted and sequenced to obtain recombinant expression plasmids pCAMBIA002 and pCAMBIA003 (which encode polypeptides M6 and M17, respectively).
  • Aspergillus oryzae engineered bacteria were activated, they were cultured at a constant temperature of 30°C for 3 days, and then the spores were washed with sterile water to make a bacterial suspension for inoculation; different strains were inoculated into solid fermentation medium at 2%, and further, by using induced expression medium, they were cultured at 30°C and 200 rpm for 6 days, centrifuged at 12000 rpm, and the supernatant was collected and concentrated using a 10 kDa ultrafiltration membrane to obtain polypeptides M6 and M17, respectively, whose amino acid sequences are shown in SEQ ID NO:6 and SEQ ID NO:17, respectively.
  • polypeptides M6 and M17 obtained in this example were taken respectively, and the reaction system was placed in a water bath according to the catalytic synthesis method described in Example 2, and reacted at 45°C for 0.5 hour, the reaction was terminated, and the products were purified by gel column method.
  • the yields of lactose-N-neotetraose (LNnT) and lactose-N-tetraose (LNT) in the reaction solution were measured by ion chromatography, and the results are recorded in Table 6.
  • Table 5 shows that the polypeptides M6 and M17 expressed by Aspergillus oryzae also have excellent ability to catalyze the synthesis of LNT and LNnT.
  • the polypeptide with ⁇ -galactosidase activity provided in the present application can catalyze the synthesis of LNT and LNnT at the same time, and its efficiency in catalyzing the synthesis of LNT and LNnT is much higher than that of wild-type ⁇ -galactosidase (EC3.2.1.23); the method of catalyzing the synthesis of LNT and LNnT using the polypeptide as a catalyst is green, efficient, and sustainable, is conducive to the application of industrial large-scale production, and has important practical value.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne un polypeptide et un produit d'acide nucléique associé et un produit enzymatique correspondant, son procédé de préparation, et son application dans la production de lacto-N-tétraose (LNT) et de lacto-N-néotétraose (LNnT). Le polypeptide peut catalyser et générer efficacement LNT et LNnT, et l'efficacité du polypeptide est supérieure à celle de la β-galactosidase de type sauvage.
PCT/CN2023/108971 2022-11-20 2023-07-24 Polypeptide mature pour la synthèse d'oligosaccharides WO2024103822A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211451076.7 2022-11-20
CN202211451076.7A CN118056903A (zh) 2022-11-20 2022-11-20 一种合成寡糖的成熟多肽

Publications (1)

Publication Number Publication Date
WO2024103822A1 true WO2024103822A1 (fr) 2024-05-23

Family

ID=91068951

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/108971 WO2024103822A1 (fr) 2022-11-20 2023-07-24 Polypeptide mature pour la synthèse d'oligosaccharides

Country Status (2)

Country Link
CN (1) CN118056903A (fr)
WO (1) WO2024103822A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120135468A1 (en) * 2009-06-05 2012-05-31 Amano Enzyme Inc. Beta-galactosidase derived from bacillus circulans
CN112351990A (zh) * 2018-04-05 2021-02-09 合同酒精株式会社 来自饲料类芽孢杆菌(Paenibacillus pabuli)的能够制造低聚半乳糖的酶及制造低聚半乳糖的方法
CN112574977A (zh) * 2020-09-29 2021-03-30 天津科技大学 一种低聚半乳糖生产专用酶及其制备与应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120135468A1 (en) * 2009-06-05 2012-05-31 Amano Enzyme Inc. Beta-galactosidase derived from bacillus circulans
CN112351990A (zh) * 2018-04-05 2021-02-09 合同酒精株式会社 来自饲料类芽孢杆菌(Paenibacillus pabuli)的能够制造低聚半乳糖的酶及制造低聚半乳糖的方法
CN112574977A (zh) * 2020-09-29 2021-03-30 天津科技大学 一种低聚半乳糖生产专用酶及其制备与应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI CHENXIA; XIANG ZHIXUAN; LIJING; JIANG ZHENGQIANG; YAN QIAOJUAN; MA JUNWEN: "Directed Evolution, High-Level Expression and Application of the β-Galactosidase from Aspergillus oryzae", SHIPIN YU SHENGWU JISHU XUEBAO - JOURNAL OF WUXI UNIVERSITY OF LIGHT INDUSTRY, JIANGNAN DAXUE ZAZHISHE, CN, vol. 41, no. 10, 15 October 2022 (2022-10-15), CN , pages 49 - 57, XP009554598, ISSN: 1673-1689 *
ZEUNER BIRGITTE; NYFFENEGGER CHRISTIAN; MIKKELSEN JøRN DALGAARD; MEYER ANNE S.: "Thermostable β-galactosidases for the synthesis of human milk oligosaccharides", NEW BIOTECHNOLOGY, ELSEVIER BV, NL, vol. 33, no. 3, 1 January 1900 (1900-01-01), NL , pages 355 - 360, XP029448274, ISSN: 1871-6784, DOI: 10.1016/j.nbt.2016.01.003 *

Also Published As

Publication number Publication date
CN118056903A (zh) 2024-05-21

Similar Documents

Publication Publication Date Title
CN110157654B (zh) 一种纳豆芽孢杆菌重组菌及其构建方法与应用
US11447760B2 (en) Special enzyme for galactooligosaccharide production as well as preparation and application thereof
CN115725530B (zh) 一种糖苷水解酶及制备方法和应用
CN111662831A (zh) 黑曲霉Rha-N1及其应用
CN113234699A (zh) α-1,2-岩藻糖基转移酶及其应用
CN113846024A (zh) 一种降低l-苹果酸发酵过程中副产物富马酸的方法、菌种及应用
WO2024103822A1 (fr) Polypeptide mature pour la synthèse d'oligosaccharides
CN114703117B (zh) 一种重组枯草芽孢杆菌、其构建方法及一种重组胶原酶
CN111518851B (zh) 一种固定化酶连续制备[14/15n]-l-瓜氨酸的方法
WO2024109169A1 (fr) Séquences polypeptidiques matures pour la synthèse de hmos et leur utilisation
CN115786319A (zh) 一种热稳定性提高的d-阿洛酮糖3-差向异构酶及突变体
CN107201375B (zh) 生产(r,r)-2,3-丁二醇基因工程菌株的构建方法及其应用
WO2024103825A1 (fr) Séquence polypeptidique mature pour synthétiser un oligosaccharide et utilisation
CN107083375B (zh) 一种中温α-淀粉酶及其基因和应用
CN111394295B (zh) 一种制备固定化精氨酸脱亚胺酶及生产[14/15n]-l-瓜氨酸的方法
WO2023151476A1 (fr) Glycoside hydrolase, son procédé de préparation et son application
CN116286575B (zh) 一种利用枯草芽孢杆菌高效表达生淀粉α-淀粉酶的方法
CN108342400A (zh) 重组表达草酸氧化酶的基因工程菌及其构建方法和应用
CN114107270B (zh) 一种L-天冬氨酸β-脱羧酶突变体
CN116875522B (zh) 含有醇脱氢酶突变体基因的工程菌及其应用
CN114806899B (zh) 一种生产l-苹果酸的里氏木霉工程菌及其应用
WO2023102816A1 (fr) Bactérie génétiquement modifiée et procédé de préparation de l-ornithine à partir d'une bactérie génétiquement modifiée
CN117965491A (zh) 一种烟酰胺核糖激酶突变体、重组基因工程菌及其菌剂以及由其制得的桑叶康普茶
CN116814576A (zh) 糖苷水解酶及其制备方法和应用
WO2021128432A1 (fr) Isomère de l-arabinose isomérase et son application

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23890263

Country of ref document: EP

Kind code of ref document: A1