A mature polypeptide of synthetic oligosaccharide

Abstract

The present invention relates to a mature polypeptide and / or a mature polypeptide mutant, whose amino acid sequence has at least 60% sequence identity with SEQ ID NO: 1 and / or SEQ ID NO: 2 and / or SEQ ID NO: 3 and / or SEQ ID NO: 4 and / or SEQ ID NO: 5 and / or SEQ ID NO: 6 and / or SEQ ID NO: 7 and / or SEQ ID NO: 8 and / or SEQ ID NO: 9 and / or SEQ ID NO: 10 and / or SEQ ID NO: 11 and / or SEQ ID NO: 12 and / or SEQ ID NO: 13 and / or SEQ ID NO: 14 and / or SEQ ID NO: 15 and / or SEQ ID NO: 16 and / or SEQ ID NO: 17 and / or SEQ ID NO: 18, and which has the ability to synthesize LNT and LNnT.

Description

Technical Field

[0001] The present invention relates to a mature polypeptide with oligosaccharide synthesis activity, and its expression in microorganisms and application in synthesizing human milk oligosaccharides, belonging to the technical field of enzyme engineering / genetic engineering. Background Art

[0002] Breast milk plays a vital role in the growth of infants and young children. Compared with breastfed infants, bottle-fed infants have a lower survival rate and a higher chance of infection, which has aroused scientists' interest in the study of breast milk components. Since the 21st century, researchers have found that the concentration of total HMOs (acidic and neutral oligosaccharides) in human colostrum is as high as 24g / L, but it decreases with the extension of lactation period; it is also found that HMOs are very important for the growth and development of infants and their health in the later period. It can promote the balance of infant intestinal microecology, promote the proliferation of beneficial bacteria in the intestine, inhibit the growth of harmful bacteria, resist pathogen infection, regulate the immune system and promote the cognitive development of infants. Many beneficial effects of breast milk are attributed to human milk oligosaccharides (HMOs).

[0003] With the continuous advancement of analytical tools and technologies for structural characterization, scientists are working to identify the components of breast milk, especially the rich and diverse HMOs, of which more than 200 have been identified. Among them, 2-fucosyllactose (2'-FL), lactose-N-triose (Lacto-N--triose II, LNTII), lactose-N-tetraose (Lacto-N-tetraose, LNT) and lactose-N-neotetraose (LNnT) were found to be the main components of HMOs, playing the role of bifidus factors. Studies have shown that adding HMOs to infant formula milk powder with cow's milk or goat's milk as the main raw material and feeding infants can approach the effect of breastfeeding in terms of nutrition and immunity of infants. Therefore, adding HMOs with physiological functions to infant formula, some artificial milk, dairy products, baby food, functional health products or special population foods will greatly improve the physical fitness of the corresponding population and improve their quality of life, which has obvious social and economic value.

[0004] Infant formula and dietary supplements added with HMOs are already sold in the markets of the United States, the European Union, Australia, New Zealand and other countries.

[0005] At present, both the EU EFSA and the US FDA have approved the marketing of lactose-N-tetraose (Lacto-N-tetraose, LNT) and lactose-N-neotetraose (LNnT). The approved lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT) are produced by microbial fermentation.

[0006] LNT and LNnT have similar structures, and their molecular weights are both 707.63. The chemical structure of LNT is as follows:

[0007]

[0008] Formula 1. Chemical formula of lactose-N-tetraose (LNT)

[0009] The chemical structure of LNnT is as follows:

[0010]

[0011] Formula 2. Chemical formula of lactose-N-neotetraose (LNnT)

[0012] 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. Although 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. At the same time, 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.

[0013] Chinese invention patent 201810206004.3 discloses a recombinant Bacillus subtilis and a method for preparing lacto-N-neotetraose. The lactose permease gene and the corresponding genes of β-1,3-N-glucosaminyltransferase and β-1,4-galactosyltransferase were recombinantly integrated into the genome of Bacillus subtilis. After 24 hours of fermentation, the obtained recombinant Bacillus subtilis contained 1071 mg / L lactose-N-neotetraose in the supernatant of the fermentation broth.

[0014] At the same time, enzymatic reactions are widely used to synthesize oligosaccharides due to their simple operation, product specificity, and environmental friendliness. Chinese patent 201180071512.1 reports a method for producing lactose-N-tetraose and lactose-N-neotetraose from lactose-N-triose using active cell extracts.

[0015] However, in order to achieve large-scale production of enzymatically synthesized LNT, and to promote the application of HMOs in various foods such as dairy products, baked goods, and beverages more quickly, although the existing technology provides various HMOs production methods, the efficiency of enzymatic reactions needs to be further improved in order to benefit the public and product iteration, and people are still studying HMOs production methods with more advantages. Summary of the invention

[0016] Purpose of the invention: In order to meet the demand for product iteration, the applicant has studied the catalytic synthesis process of HMOs and discovered a series of mature polypeptides that can simultaneously catalyze the synthesis of lactose-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), their preparation methods, and methods for using them to produce oligosaccharides that specifically exist in human breast milk. At present, no biotechnology that can simultaneously synthesize lactose-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) has been retrieved.

[0017] Technical solution:

[0018] The present invention describes a mature polypeptide and mutants thereof, which have the ability to synthesize lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT).

[0019] The mature polypeptide is β-galactosidase (EC3.2.1.23).

[0020] The β-galactosidase is an enzyme that hydrolyzes β-D-galactosidic bonds to free D-galactose, and is widely present in microorganisms, animals and plants. Its species source can be: Aspergillus oryzae, Kluyveromyces lactis, Kluyveromyces marxinus, Bacillus subtilis, Bacillus circulans, etc., and the further preferred species source is Bacillus circulans or Bacillus subtilis.

[0021] A mature polypeptide having the ability to synthesize HMOs, in particular the ability to synthesize LNT and LNnT, comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1.

[0022] The present invention also describes a series of mature polypeptide mutants, which are obtained by modifying one or more amino acids at positions 65, 150, 412, 414, 476, 535, and 566 of the mature polypeptide shown in SEQ ID NO:1.

[0023] The mature polypeptide mutant has a higher ability to catalyze the synthesis of LNT and LNnT.

[0024] The modification refers to any chemical modification of the polypeptide or its homologous sequence consisting of the mature polypeptide, as well as genetic manipulation of the DNA encoding the polypeptide. The modification can be the replacement, deletion and / or insertion of one or more (several) amino acids, and the replacement of one or more (several) amino acid side chains. The replacement is an amino acid replacement.

[0025] The deletion refers to the removal of an amino acid occupying one position, or the removal of multiple amino acids occupying multiple positions.

[0026] The mature polypeptide mutant is preferably a mature polypeptide shown in SEQ ID NO: 1, in which the amino acid residues at positions 65, 150, 412, 414, 476, 535 and 566 are replaced by phenylalanine residues, leucine residues or alanine residues.

[0027] The mature polypeptide mutant is preferably a mature polypeptide shown in SEQ ID NO: 1, in which the glutamic acid residue at position 65 is replaced by a phenylalanine residue, a leucine residue, or an alanine residue, and its amino acid sequence is shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

[0028] The mature polypeptide mutant is preferably a mature polypeptide shown in SEQ ID NO:1, in which the arginine residue at position 150 is replaced by a phenylalanine residue, and its amino acid sequence is shown in SEQ ID NO:5.

[0029] The mature polypeptide mutant is preferably a mature polypeptide shown in SEQ ID NO: 1, in which the tyrosine residue at position 414 is replaced by a phenylalanine residue, a leucine residue, or an alanine residue, and its amino acid sequence is shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

[0030] The mature polypeptide mutant is preferably a mature polypeptide shown in SEQ ID NO:1 in which the tyrosine residue at position 476 is replaced by a phenylalanine residue, and its amino acid sequence is shown in SEQ ID NO:9.

[0031] The mature polypeptide mutant is preferably a mature polypeptide shown in SEQ ID NO: 1, in which the tryptophan residue at position 535 is replaced by a phenylalanine residue, a leucine residue, or an alanine residue, and its amino acid sequence is shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

[0032] The mature polypeptide mutant is preferably a mature polypeptide shown in SEQ ID NO:1 in which the glutamic acid residue at position 566 is replaced by a leucine residue, and its amino acid sequence is shown in SEQ ID NO:13.

[0033] The mature polypeptide mutant is preferably a mature polypeptide shown in SEQ ID NO:1, in which the glutamic acid residue at position 65 is replaced by a phenylalanine residue, and the tyrosine residue at position 414 is replaced by a leucine residue, and its amino acid sequence is shown in SEQ ID NO:14.

[0034] The mature polypeptide mutant is preferably a mature polypeptide shown in SEQ ID NO:1, in which the glutamic acid residue at position 65 is replaced by a phenylalanine residue, and the tyrosine residue at position 414 is replaced by an alanine residue, and its amino acid sequence is shown in SEQ ID NO:15.

[0035] The mature polypeptide mutant is preferably a mature polypeptide shown in SEQ ID NO:1, in which the glutamic acid residue at position 65 is replaced by a phenylalanine residue, and the tryptophan residue at position 535 is replaced by an alanine residue, and its amino acid sequence is shown in SEQ ID NO:16.

[0036] The mature polypeptide mutant is preferably a mature polypeptide shown in SEQ ID NO:1, in which the glutamic acid residue at position 65 is replaced by a phenylalanine residue, the tyrosine residue at position 414 is replaced by an alanine residue, and the glutamic acid residue at position 566 is replaced by a leucine residue, and its amino acid sequence is shown in SEQ ID NO:17.

[0037] Preferably, the mature polypeptide and / or mature polypeptide mutant comprises a polypeptide having an amino acid sequence with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and / or SEQ ID NO: 2 and / or SEQ ID NO: 3 and / or SEQ ID NO: 4 and / or SEQ ID NO: 5 and / or SEQ ID NO: 6 and / or SEQ ID NO: 7 and / or SEQ ID NO: 8 and / or SEQ ID NO: 9 and / or SEQ ID NO: 10 and / or SEQ ID NO: 11 and / or SEQ ID NO: 12 and / or SEQ ID NO: 13 and / or SEQ ID NO: 14 and / or SEQ ID NO: 15 and / or SEQ ID NO: 16 and / or SEQ ID NO: 17, and has the ability to catalyze the synthesis of lacto-N-neotetraose (LNnT).

[0038] One embodiment of the present invention describes a composition comprising the mature polypeptide of the present invention for preparing LNT and LNnT.

[0039] Preferably, the composition contains one or more polypeptides having an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and / or SEQ ID NO: 2 and / or SEQ ID NO: 3 and / or SEQ ID NO: 4 and / or SEQ ID NO: 5 and / or SEQ ID NO: 6 and / or SEQ ID NO: 7 and / or SEQ ID NO: 8 and / or SEQ ID NO: 9 and / or SEQ ID NO: 10 and / or SEQ ID NO: 11 and / or SEQ ID NO: 12 and / or SEQ ID NO: 13 and / or SEQ ID NO: 14 and / or SEQ ID NO: 15 and / or SEQ ID NO: 16 and / or SEQ ID NO: 17.

[0040] The embodiments of the present invention also describe a method for expressing the mature polypeptide and / or the mature polypeptide mutant, comprising the following steps:

[0041] (a) culturing a host cell under conditions suitable for expressing the mature polypeptide and / or mature polypeptide mutant;

[0042] Preferably, the method further comprises: (b) a step of recovering the mature polypeptide and / or mature polypeptide mutant.

[0043] In the step (a), preferably, a recombinant plasmid encoding the above-mentioned mature polypeptide and / or mature polypeptide mutant gene is first constructed and then introduced into a host cell;

[0044] The step (a) preferably further comprises the step of culturing the obtained recombinant engineered strain.

[0045] The step (b) preferably further comprises a step of separating and purifying the mature polypeptide and / or the mature polypeptide mutant.

[0046] Preferably, in the method for expressing the mature polypeptide and / or mature polypeptide mutant, the host cell is selected from yeast cells, bacterial cells or molds.

[0047] The bacterial cells preferably include: Bacillus and Escherichia; further preferably, include Escherichia coli, Escherichia hermanii, Escherichia fergusonii, Bacillus mycoides, Bacillus cereus, Bacillus polymyxa, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus brevis, Bacillus circulans or Bacillus subtilis; further preferably, the host cell is Bacillus circulans, Bacillus subtilis or Bacillus licheniformis.

[0048] The mold preferably includes: Aspergillus and Trichoderma; further preferably, Trichoderma viride, Trichoderma reesei, Trichoderma harzianum, Aspergillus oryzae, Aspergillus fumigatu, Aspergillus niger, or Aspergillus flavus cells; further preferably, the host cell is Aspergillus oryzae cells.

[0049] The yeast cells preferably include: cells of the genera Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia; further preferred are cells of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxinus, Yarrowia lipolytica, Rhodotorula graminis, Saccharomyces pastorianus, etc.; further preferred are cells of Saccharomyces cerevisiae, Kluyveromyces lactis, and Kluyveromyces marxinus.

[0050] In the method for expressing the mature polypeptide and / or the mature polypeptide mutant, the host cell includes a wild strain isolated from nature, a mutant strain after physical and chemical mutagenesis, or an engineered bacterium after genetic engineering.

[0051] Host cells are cultivated in a nutrient medium suitable for producing mature polypeptide and / or mature polypeptide mutants using methods known in the art. For example, it is possible to culture by shaking bottles, or in a suitable medium and under conditions allowing mature polypeptide and / or mature polypeptide mutants to express and / or separate, in a laboratory or industrial fermentor tank, carry out small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, batch-fed fermentation or solid-state fermentation) to cultivate cells. Cultivation is to use procedures known in the art, and occurs 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 composition.

[0052] The mature polypeptide and / or mature polypeptide mutant can be recovered using methods known in the art. For example, variants can be recovered from a nutrient medium by a variety of conventional procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation.

[0053] Mature polypeptides and / or mature polypeptide mutants can be purified by various procedures known in the art to obtain substantially pure mature polypeptides and / or mature polypeptide mutants, 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 for separation or purification. Further preferably, purification can also be performed by Ni column affinity chromatography.

[0054] In one embodiment, the use of mature polypeptides and / or mature polypeptide mutants, or combinations thereof, in the production of LNT and LNnT is described.

[0055] The application preferably comprises the steps of generating LNT and LNnT.

[0056] In the application, preferably, the reaction substrate is carbohydrates such as lactose and its derivatives.

[0057] In the application, preferably, the reaction substrates are lactose and lactose-N-triose (Lacto-N-triose, LNTII).

[0058] In the application, preferably, the reaction solvent is an aqueous solvent or a solution containing solids.

[0059] The application, preferably, can directly use the culture fluid containing the mature polypeptide and / or the mature polypeptide mutant;

[0060] Preferably, the mature polypeptide and / or mature polypeptide mutant is purified and used as a catalyst.

[0061] For the application, preferably, process parameters such as temperature, enzyme addition amount, reaction time, pH and the like can be studied or selected according to the method disclosed in the present invention.

[0062] definition:

[0063] The terms "mature polypeptide" and "mature polypeptide mutant" refer to any post-translational sequence or sequence modification, such as a known sequence, its N-terminal processing, truncation and / or C-terminal truncation. In one aspect, "mature polypeptide" and "mature polypeptide mutant" are both "mature polypeptides", and the mature polypeptides and / or mature polypeptide mutants of the present invention include proteins shown in SEQ ID NO: 1-17.

[0064] The term "sequence identity": The relatedness between two amino acid sequences is described by the parameter "sequence identity".

[0065] The term "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, such as, for example, measuring the concentration or activity of mRNA and / or translated polypeptide.

[0066] The term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, etc. with an expression vector. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to substitutions that occur during replication.

[0067] Beneficial Effects

[0068] The present invention provides a series of novel mature polypeptides and / or mature polypeptide mutants and expression methods. These mature polypeptides and / or mature polypeptide mutants can catalyze the synthesis of LNT and LNnT.

[0069] The present invention also provides a method for generating LNT and LNnT using lactose and lactose-N-triose (LNTII) as substrates, wherein the reaction is catalyzed by the novel mature polypeptide and / or the mature polypeptide mutant. The present invention provides for the first time a technical solution for generating LNT and LNnT by catalysis of the mature polypeptide and / or the mature polypeptide mutant, which has positive significance for the industrial production of human milk oligosaccharides.

[0070] The technical solution of the present invention 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 .The HPLC spectrum (210nm wavelength) of the reaction liquid after catalysis by M8 in Example 2 showed a peak time of 23.800min.

[0072] Figure 2 . Figure 1 LC-MS graph of the substance at rt=23.800min in the HPLC spectrum.

[0073] Figure 3 . Figure 1 Ion chromatogram of the substance at rt=23.800min in the HPLC spectrum. DETAILED DESCRIPTION

[0074] The following are examples of the preparation of the mature polypeptide and / or mature polypeptide mutant of the present invention, and their uses.

[0075] The experimental methods used in the following examples are conventional methods unless otherwise specified; all materials, reagents, etc., are commercially available unless otherwise specified.

[0076] The pET-32a plasmid was purchased from Sangon Biotechnology Co., Ltd.

[0077] The present invention is further described in detail below through examples. It should be understood that the specific embodiments described herein are

[0078] The embodiments are only used to explain the present invention and are not used to limit the present invention.

[0079] Modifications or substitutions to the details and forms of this technical solution all fall within the protection scope of the present invention.

[0080] Unless otherwise defined, all technical terms and scientific terms used in this specification have the same meanings as those commonly understood by those skilled in the art. In general, the nomenclature used in this specification and the experimental methods described below are well known and commonly used in the art.

[0081] In the following examples and test examples, the products obtained from the catalytic reaction of the mature polypeptide and / or the mature polypeptide mutant were analyzed and identified using HPLC analysis method and ion chromatography.

[0082] HPLC analysis conditions are as follows: using a 4.6×250 mm, 5 μm Asahipak NH2P-504E amino column, isocratic elution with 75% acetonitrile aqueous solution as the mobile phase; flow rate is 0.7 mL / min, column temperature is 30°C, injection volume is 10 μL, UV detector, detection wavelength is 210 nm.

[0083] The LC-MS analysis conditions are as follows, and the analysis results show:

[0084] Chromatographic column model: 4.6×250mm, 5μm 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 10μL, 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.

[0085] The ion chromatography analysis conditions were as follows: chromatographic column: MetroSep Carb2 (4.0 mm × 250 mm), eluent: 140 mM NaOH + 20 mM NaAc, isocratic elution, flow rate: 0.500 mL / min, amperometric detector, column temperature 40 °C, injection volume 20 μL, running time 50 min.

[0086] Lactose-N-triose (LNTⅡ), lactose-N-tetraose (LNT), and lactose-N-neotetraose (LNnT) standards were produced by ELICITYL, France.

[0087] It should be noted that the terms used here are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are to describe specific embodiments, rather than to limit the scope of protection of the present invention. If the experimental methods of specific conditions are not indicated in the following specific embodiments, the conventional methods and conditions of molecular biology within the art are generally followed, and such techniques and conditions are fully explained in the literature. See, for example, Sambrook et al., the techniques and conditions described in "Molecular Cloning: A Laboratory Manual", or according to the conditions recommended by the manufacturer.

[0088] Example 1. Expression of mature polypeptides and / or mature polypeptide mutants shown in SEQ ID NOs: 1-17 in Escherichia coli.

[0089] Through codon optimization and DNA synthesis, the genes corresponding to the mature polypeptide sequences and / or mature polypeptide mutants SEQ ID NO: 1-17 were obtained. According to the method of the Molecular Cloning Experiment Guide, the pET32a-BgaD-MX recombinant plasmid series (pET32a-BgaD-M1-17) were constructed, as shown in Table 1 (Table 1-1, Table 1-2). The above recombinant plasmid was transformed into Escherichia coli BL21 (DE3) according to the following steps: the prepared Escherichia coli BL21 (DE3) competent cells were taken and placed on ice for 30 minutes to melt, 100 μL of competent cells were mixed with 10 μL of pET32a-BgaD-MX recombinant plasmid (concentration 50 ng / μL), placed in a 42°C water bath for heat shock for 45 seconds, and then immediately cooled in an ice bath for 2 minutes, 1 mL of fresh LB medium (LB medium: peptone 1.0%, yeast extract 0.5%, NaCl 1.0%, and 1.5% agar powder on the plate) was added, and the culture was resuscitated at 37°C and 100 rpm for 1 hour, and then 100 μL of the bacterial solution was spread on an LB plate containing ampicillin (100 μg / mL), and after culturing in a 37°C constant temperature incubator for 12 hours, a single colony was picked for colony PCR [PCR amplification primers are shown in Table 1 (Table 1-1, Table 1-2)] to screen out positive transformants. Furthermore, the plasmid was extracted after culturing the correct transformant, and PCR amplification, double enzyme digestion and gene sequencing were used to verify whether the pET32a-BgaD-MX recombinant plasmid was successfully introduced into Escherichia coli.

[0090] Then, the correct transformant was inoculated into LB liquid medium, placed on a shaker at 37°C and 200rpm for 12h to obtain seed liquid; the seed liquid was then inoculated into fresh LB medium at a 1% (v / v) inoculation amount, shaken and cultured at 37°C until OD 600 was 0.8, and induced by isopropyl-β-D-thiogalactopyranoside (IPTG), with a final concentration of 0.1mmol / L of IPTG, induced at 16°C for 12h, and a speed of 200rpm. After induction of expression, the fermentation broth was centrifuged at 5000r / min for 30min at 4°C to collect the bacteria. After the bacteria were resuspended in 20mM pH7.4 PBS buffer, they were ultrasonically broken at a frequency of plus on 5s / off 5s for 30min to break the bacteria; the broken liquid was centrifuged at 13000×g and 4°C for 30min to remove cell debris and collect the supernatant.

[0091] The soluble mature polypeptide sequence was purified by nickel column affinity chromatography, and the process was as follows: deionized water was added to the nickel column to the top of the column, and after natural elution, it was eluted with 5 volumes of Binding buffer; the crude enzyme solution filtered by 0.45 μm filter membrane was then loaded onto the column, and the sample was fully combined with the nickel column at a flow rate of 1.5 mL / min. After the sample was dried, 5 columns of Washing buffer were used for continuous gradient elution to remove impurities, and finally the target protein was eluted with 5 times Elution buffer, and the eluate was collected. Then SDS-PAGE was used to analyze the expression of the target protein. After induction, the genetically engineered bacteria had obvious specific expression bands, and the molecular weight of the bands was basically consistent with the expected molecular weight of 110 kDa, and the mature polypeptides and / or mature polypeptide mutants shown in SEQ ID NO: 1-17 were obtained, as shown in Table 2.

[0092] Table 1-1. Primers used for recombinant plasmids

[0093]

[0094]

[0095] Table 1-2. Primers used for recombinant plasmids (continued from Table 1-1)

[0096]

[0097] Table 2. Correspondence between plasmid, mature polypeptide sequence and amino acid sequence number

[0098]

[0099]

[0100] Example 2. The mature polypeptides and / or mature polypeptide mutants (after purification) shown in SEQ ID NOs: 1-17 catalyze the synthesis of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT).

[0101] The purified mature polypeptide obtained in Example 1 was used for enzyme catalysis, specifically: 20 mM lactose, 20 mM lactose-N-trisaccharide (Lacto-N-trioseⅡ, LNTII), 0.13 mg / mL of mature polypeptide and / or mature polypeptide mutant, 10 mmol / L MgCl2, 100 mM phosphate buffer (pH 7) were respectively added to 1 mL, mixed, reacted at 45°C for 0.5 hour, the reaction was terminated, and purified by gel column method.

[0102] Identification of catalytic synthesis products:

[0103] High performance liquid chromatography (HPLC) and LC-MS analysis methods, and ion chromatography, were used to analyze the products of the catalytic reaction of lactose-N-tetraose (LNT), lactose-N-neotetraose (LNnT) standards and the mature polypeptides and / or mature polypeptide mutants shown in the above SEQ ID NOs: 1-17, respectively.

[0104] The HPLC analysis conditions were as shown above, and the analysis results showed:

[0105] (1) The peak time rt of LNT and LNnT standards is 23.800 min.

[0106] (2) In this example, the mature polypeptide and / or the mature polypeptide mutant were used to catalyze the reaction solution, and a strong absorption peak appeared near 23.800 min at a wavelength of 210 nm, which was consistent with the peak time of the LNT and LNnT standards, indicating that lactose-N-tetraose (LNT) and / or lactose-N-neotetraose LNnT may have been synthesized in the reaction solution.

[0107] LC-MS analysis

[0108] The product with rt=23.800 min in the HPLC spectrum was subjected to LC-MS analysis.

[0109] The mass spectra of the lactose-N-neotetraose (LNnT) standard are the same as those of the lactose-N-tetraose (LNT) standard.

[0110] As attached Figure 2 As shown, the molecular weight of 708.2567 should be LNnT / LNT+H; the molecular weight of 730.2393 should be LNnT / LNT+Na. The mass spectrometry results can be retrieved to obtain the following results: Compound CID:440993, Chemical Formula:C 26 H 45 NO 21 , Extract Mass:707.63.

[0111] In this example, the mass spectrometry results of the reaction solution of the mature polypeptide and / or the mature polypeptide mutant at rt = 23.800 min were consistent with those of the attached Figure 2 Therefore, it is speculated that the mature polypeptide and / or the mature polypeptide mutants (SEQ ID NOs: 1-17) all synthesized a substance with a molecular weight of about 707, which may be lactose-N-neotetraose (LNnT) and / or lactose-N-tetraose (LNT).

[0112] The results of ion chromatography analysis showed:

[0113] (1) The peak time of lactose-N-neotetraose (LNnT) standard is 26.99 min; the peak time of lactose-N-tetraose (LNT) standard is 30.92 min.

[0114] (2) In this example, the substance with rt=23.800 min in the reaction solution of the mature polypeptide and / or the mature polypeptide mutants M1-M17 (SEQ ID NO: 1-17) was respectively intercepted and analyzed by ion chromatography, and the following results were obtained:

[0115] (1) The reaction solutions using mature polypeptides and / or mature polypeptide mutants M1-M4, M6-M17 (SEQ ID NOs: 1-4, 6-17) all had strong absorption peaks at around 26.99 min and 30.92 min, which were consistent with the peak times of lactose-N-neotetraose (LNnT) standard and lactose-N-tetraose (LNT) standard, indicating that lactose-N-neotetraose (LNnT) and lactose-N-tetraose (LNT) were synthesized by the catalytic reaction.

[0116] (2) The reaction solution using the mature polypeptide mutant M5 (SEQ ID NO: 5) had a strong absorption peak only around 30.92 min, which was consistent with the peak time of the lactose-N-tetraose (LNT) standard, indicating that lactose-N-tetraose (LNT) was catalytically synthesized, but lactose-N-neotetraose (LNnT) was not synthesized.

[0117] in conclusion

[0118] As shown in the above HPLC, LC-MS and ion chromatography analysis results, the catalytic reactions catalyzed by mature polypeptides and / or mature polypeptide mutants M1 to M17 with lactose and lactose-N-triose (Lacto-N-trioseⅡ) as substrates, lactose-N-neotetraose (LNnT) and lactose-N-tetraose (LNT) were synthesized by the catalytic reactions of M1 to M4 and M6 to M17; lactose-N-tetraose (LNT) was synthesized by the catalytic reactions of M5, but lactose-N-neotetraose (LNT) was not synthesized.

[0119] The yield of lactose-N-tetraose (LNT) and / or lactose-N-neotetraose (LNnT) in the reaction solution was determined by ion chromatography, and the results are recorded in Table 3.

[0120] Table 3 Study on the catalytic synthesis of LNT and LNnT by mature polypeptide and / or mature polypeptide mutant

[0121]

[0122]

[0123] Table 3 Data Description:

[0124] (1) The mature polypeptide and / or the mature polypeptide mutants M1-M4, M6-M17 can catalyze LNTII and lactose to synthesize lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT).

[0125] (2) The mature polypeptide mutants M3-M6, M8, M11, M14, and M15-17 have a stronger ability to catalyze the synthesis of lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT).

[0126] Example 3. Expression of mature polypeptide mutants M6 and M17 (amino acid sequences are shown in SEQ ID NO: 6 and SEQ ID NO: 17, respectively) in Kluyveromyces lactis

[0127] The construction method of recombinant plasmids pKLAC1-M6 and pKLAC1-M17 is as follows:

[0128] According to the gene sequence of the mature polypeptide mutant M6 or M17, primers were designed for PCR amplification. The PCR reaction system was 50 μL: 1 μL DNA template, 25 μL 2×Phanta Max Buffer, 1 μL 10 pmol / μL dNTP Mix, 2 μL 10 pmol / μL forward and reverse primers, 1 μL 1U / μL Phanta Max Super-Fidelity DNA Polymerase, and ddH2O was added to 50 μL. PCR reaction program: 95℃3min; 95℃15s, 60℃15s, 72℃1.5min, 33 cycles; 72℃5min; 4℃ storage. The PCR fragment and the plasmid fragment were double-digested with restriction endonucleases XmajI and NotI, respectively, and then ligated with ligation high ligase, transformed into Escherichia coli DH5ɑ, and the recombinant plasmid pKLAC1-M6 or pKLAC1-M17 was obtained after screening and identification.

[0129] Then, the KLAC1-M6 or KLAC1-M17 expression cassette was amplified. According to the sequence of the pklac1 plasmid, primers were designed for PCR amplification. PCR reaction system 50 μL: DNA template (pklac1-BgaD-M6 or pklac1-BgaD-M17) 1 μL (about 50 ng), 2×Phanta Max Buffer 25 μL, 10 pmol / μL dNTP Mix 1 μL, 10 pmol / μL forward and reverse primers 2 μL each, 1U / μL Phanta Max Super-Fidelity DNA Polymerase DNA polymerase 1 μL, add ddH2O to 50 μL. PCR reaction program: 95℃, 3min; 95℃, 15s, 60℃, 15s, 72℃, 5min, 33 cycles; 72℃, 5min; 4℃ storage. After agarose gel electrophoresis, the gel was cut and recovered to obtain the KLAC1-M6 or KLAC1-M17 expression cassette.

[0130] Afterwards, the KLAC1-M6 or KLAC1-M17 expression cassette was transformed into Kluyveromyces lactis. The specific operation was as follows: freshly cultured Kluyveromyces lactis colonies were picked into 1 mL YPD medium, placed in a constant temperature oscillator, and cultured at 30°C and 250 rpm for 12-14 h; the initial inoculation OD was transferred to 50 mL fresh YPD medium at 0.2, and cultured at 30°C and 200 rpm for 3-4 h until the OD was 0.8-1; the bacterial culture was placed on ice for 5-10 min, and at the same time, 25 μL salmon sperm DNA (2 mg / mL) was placed in a PCR instrument at 95°C for 10 min and quickly cooled in an ice water bath, and placed on ice for use; the expression cassette solution to be transformed was prepared, a total amount of 5-10 μg was added to each tube, and the solution was filled to 50 μL with sterilized ddH2O. After standing on ice, centrifuge the culture solution at 4°C, 5000×g for 5 min, discard the supernatant, and collect the bacteria; resuspend the bacteria with sterilized ddH2O, centrifuge at 4°C, 5000×g for 5 min, discard the supernatant, collect the bacteria, and repeat once; add 1mL 0.1M LiCl solution to resuspend the bacteria, transfer to a new 1.5mL centrifuge tube, centrifuge at 4°C, 13000×g for 15s, and discard the supernatant; add 400μL of 0.1M LiCl solution to resuspend the bacteria, divide into 50μL per tube, centrifuge at 4°C, 13000×g for 15s, discard the supernatant, and place on ice for use; add 240μL of 50% PEG3350 solution and 1M 36μL of LiCl solution, 25μL of pre-treated salmon sperm DNA solution, 50μL of expression cassette solution to be transformed, vortex vigorously to mix until the bacteria are completely distributed (about 5min); the fully mixed system is placed in a 30℃ water bath for 30min; placed in a 42℃ constant temperature water bath, heat shock for 25min; at room temperature, centrifuge at 8000×g for 2min to collect the bacteria, resuspend the bacteria with 1mL YPD, and culture on a 30℃ shaker for 1~4h. Take 25~135μL of bacterial solution and apply it to the culture plate containing G418 resistance; place the plate in a 30℃ constant temperature incubator, invert and culture for 2-3 days, and pick the transformant to verify whether the expression cassette has been successfully transferred.

[0131] The shake flask fermentation was performed as follows: 1) Preparation of primary strain: a single colony of the above yeast genetically engineered strain was cultured overnight in 10 mL YGD liquid medium at 30°C and 200 rpm. The resulting strain was the primary strain. 2) Preparation of secondary strain: the primary strain was inoculated into 1 L YGD liquid medium and cultured at 30°C and 200 rpm until OD 600 3) The YGD 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 obtained proteins were mature polypeptide mutants M6 and M17, and their amino acid sequences were shown in SEQ ID NO: 6 and SEQ ID NO: 17, respectively.

[0132] Take M6 and M17 obtained in this example respectively, and according to the catalytic synthesis method described in Example 2, place the reaction system in a water bath at 45°C for reaction for 0.5 hour, terminate the reaction, and purify it by gel column method. The yield of lactose-N-neotetraose (LNnT) and lactose-N-tetraose (LNT) in the reaction solution is measured by ion chromatography. The results are recorded in Table 4.

[0133] Table 4

[0134]

[0135] Example 4. Expression of mature polypeptide mutants M6 and M17 (amino acid sequences are shown in SEQ ID NO: 6 and SEQ ID NO: 17, respectively) in Bacillus subtilis

[0136] According to the gene sequence of the mature polypeptide mutant M6 or M17, primers were designed for PCR amplification. The PCR reaction system was 50 μL: 1 μL DNA template (about 50 ng), 2×Phanta Max Buffer 25 μL, 10 pmol / μL dNTP Mix 1 μL, 10 pmol / μL forward and reverse primers 2 μL each, 1U / μLPhanta Max Super-Fidelity DNA Polymerase DNA polymerase 1 μL, and ultrapure water was added to 50 μL. PCR reaction program: 95℃, 3min; 95℃, 15s, 58℃, 15s, 72℃, 2.5min, 30 cycles; 72℃, 5min; 4℃ storage. After double digestion of the PCR fragment and plasmid pEB03 with restriction endonucleases EcoR I and Hind III, they were connected with T4 ligase and transformed into Escherichia coli DH5α. The recombinant plasmid was obtained after screening and identification.

[0137] The transformation method of the recombinant plasmid in Bacillus subtilis is as follows: take a full loop of Bacillus subtilis glycerol bacteria and streak it on LB plate (LB medium: peptone 1.0%, yeast extract 0.5%, NaCl 1.0%, plate plus 1.5% agar powder), and culture it in a 37°C incubator overnight. Pick a single colony to 1mL LB medium, culture it at 37°C, 200rpm overnight, take 200μL culture solution and transfer it to 200mL growth medium (growth medium: LB medium with 0.5M sorbitol), culture it at 37°C, 200rpm until OD600 reaches 0.85-0.95, ice bath for 10min, centrifuge at 4°C, 5000rpm for 5min, then wash it 4 times with pre-cooled electrotransfer medium (electrotransfer medium: LB medium with 0.5M sorbitol, 10% glycerol), and finally resuspend it with 2mL pre-cooled electrotransfer medium. The treated bacterial solution was divided into 80 μL per tube, 1 μL (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 electrotransformation. The electroporation parameters were set as follows: voltage 2000 V, resistance 200 Ω, 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. The bacterial solution was coated on a chloramphenicol (5 μg / mL) LB plate, and the transformant was picked to obtain the target Bacillus subtilis genetically engineered strain.

[0138] Pick a positive transformation single colony from the LB plate containing 5μg / mL chloramphenicol and inoculate it into 20mL seed culture medium (0.5% yeast extract, 0.5% tryptone, 1% glucose, 1.8% K2HPO4, 5μg / mL chloramphenicol), and culture it at 37°C and 220rpm for 8h. Take 2.5mL and inoculate it into 50mL fermentation medium (1.0% tryptone, 0.5% yeast powder, 1.0% NaCl), and culture it at 34°C and 250rpm for 60h. Take the supernatant for SDS-PAGE electrophoresis detection. The expression of the target protein was analyzed by SDS-PAGE, and the mature polypeptide mutants M6 and M17 were obtained, and their amino acid sequences are shown in SEQ ID NO:6 and SEQ ID NO:17, respectively.

[0139] Take M6 and M17 obtained in this example respectively, and according to the catalytic synthesis method described in Example 2, place the reaction system in a water bath, react at 45°C for 0.5 hour, terminate the reaction, purify it by gel column method, and measure the yield of lactose-N-neotetraose (LNnT) and lactose-N-tetraose (LNT) in the reaction solution by ion chromatography. The results are recorded in Table 5.

[0140] Table 5

[0141]

[0142] Example 5. Expression of mature polypeptide mutants M6 and M17 (amino acid sequences are shown in SEQ ID NO: 6 and SEQ ID NO: 17, respectively) in Aspergillus oryzae

[0143] In this experiment, 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 digested and connected respectively, and the promoter PamyB and terminator TagdA were connected to the corresponding restriction sites to construct the Aspergillus oryzae reading frame to obtain the plasmid pCAMBIA001. The genes of the mature polypeptide mutants M6 and M17 and the plasmid pCAMBIA001 were double-digested with SalI and SpeI, respectively, and the two sequences digested with the same restriction endonuclease were connected. The connected products were transformed into Escherichia coli competent cells DH5α, and the positive clones were screened and expanded for culture. The plasmids pCAMBIA002 and pCAMBIA003 were extracted, and the recombinant plasmids were sequenced for verification. The sequencing results showed that the target gene was correct.

[0144] Preparation of competent Agrobacterium tumefaciens EHA105: 1) Take Agrobacterium tumefaciens EHA105 activated on solid YEB plate and inoculate it into 20mL liquid YEB medium, to which rifampicin antibiotics have been added. Shake and culture at 180rpm for 24h at 28℃. 2) Inoculate Agrobacterium tumefaciens into 50mL liquid YEB medium with rifampicin added at 1% inoculum, shake and culture at 180rpm at 28℃ until OD600 is 0.8, then it can be used. 3) Take 50mL bacterial solution and place it on ice for 30min, centrifuge it at 5000rpm for 10min at 4℃, and discard the supernatant. 4) Add 10mL pre-cooled 0.1M CaCl2 solution, resuspend the bacteria, place it on ice for 30min; centrifuge it at 5000rpm for 10min at 4℃, and discard the supernatant. 5) Add 2 mL of pre-cooled 0.1 M CaCl2 and 2 mL of glycerol, resuspend the EHA105 cells, and dispense into 135 μL portions for use. Store in an ultra-low temperature refrigerator at -70°C until use.

[0145] The pCAMBIA002 and pCAMBIA003 plasmids were transformed into competent EHA105 by freeze-thaw method: 1) Thaw the competent EHA105 of Agrobacterium tumefaciens on ice, add 10 μL of recombinant plasmids pCAMBIA002 and pCAMBIA003, mix well, and place on ice for 30 minutes. 2) Quickly place in liquid nitrogen for cold stimulation for 5 minutes, and immediately place in a 28°C constant temperature water bath and incubate for 5 minutes. 3) Place on ice for 5 minutes. 4) Add 400 μL of YEB liquid culture medium without rifampicin to the mixture, and culture at 28°C with shaking at 180 rpm for 2 hours. 5) Take 200 mL of bacterial solution and spread on solid YEB plates containing rifampicin and kanamycin, and culture at 28°C upside down for 48 hours to observe the formation of colonies. 6) Pick a single colony and place it in 20 mL of YEB liquid medium containing rifampicin and kanamycin, and shake and incubate at 28°C and 180 rpm for 48 hours. Identify and screen positive clones using colony PCR. Extract plasmid DNA from positive clones and perform plasmid PCR detection.

[0146] Transformation of Aspergillus oryzae with expression vector mediated by Agrobacterium tumefaciens: 1) Inoculate the EHA105 bacterial solution containing different expression vectors into liquid LB medium supplemented with rifampicin and kanamycin, and culture at 28°C and 180rpm for 24h. 2) Take 2mL of bacterial solution and add it to a mixed medium consisting of 10mL of liquid LB medium and 10mL of basal medium, and culture at 28°C and 180rpm for 48h. 3) Take 2mL of bacterial solution and add it to 20mL of induction medium, and culture at 28°C and 180rpm until OD600 is 0.8, and then co-culture with Aspergillus oryzae. 4) Prepare conidia suspension of Aspergillus oryzae. 5) Take 135μL of spore suspension and 135μL of EHA105 bacterial solution, mix them evenly, and spread them on a solid induction medium covered with a microporous filter membrane, to which acetosyringone has been added, and co-culture at 28°C for 3 days. 6) Remove the microporous filter membrane and invert it onto Cha's medium containing hygromycin B and cephalosporin for antibiotic screening culture at 30°C for 3 days. 7) Remove the microporous filter membrane and continue culturing until resistant colonies grow. Transfer the selected resistant colonies to another selection medium and continue screening culture. Perform genomic PCR verification on the selected resistant transformants.

[0147] Expression of mature polypeptide mutants M6 and M17 (amino acid sequences are shown in SEQ ID NO:6 and SEQ ID NO:17, respectively): The constructed Aspergillus oryzae engineering bacteria were activated and cultured at 30°C for 3 days, the spores were washed with sterile water, and a bacterial suspension was prepared for inoculation. Different strains were inoculated into solid fermentation medium at 2%, and further, the induction expression medium was used to culture for 6 days at 30°C and 200rpm, and the supernatant was collected and concentrated using a 10kDa ultrafiltration membrane to obtain mature polypeptide mutants M6 and M17, whose amino acid sequences are shown in SEQ ID NO:6 and SEQ ID NO:17, respectively.

[0148] Take M6 and M17 obtained in this example respectively, and according to the catalytic synthesis method described in Example 2, place the reaction system in a water bath, react at 45°C for 0.5 hour, terminate the reaction, purify it by gel column method, and measure the yield of lactose-N-neotetraose (LNnT) and lactose-N-tetraose (LNT) in the reaction solution by ion chromatography. The results are recorded in Table 6.

[0149] Table 6

[0150]

[0151] The above description is only an embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention, as well as the research and selection of process parameters such as the temperature of the catalytic reaction, the amount of enzyme added, the reaction time, and the pH value, shall fall within the scope of the present invention.

[0152] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A polypeptide whose amino acid sequence is shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:

17.

2. A composition, characterized in that Contains one or more of the polypeptides described in claim 1.

3. The method for expressing the polypeptide according to claim 1, characterized in that: This includes culturing the host cell under conditions suitable for expression of the polypeptide.

4. The expression method according to claim 3, characterized in that: The host cell is selected from Aspergillus oryzae, Kluyveromyces lactis, Escherichia coli or Bacillus subtilis.

5. Use of the polypeptide according to claim 1 or the composition according to claim 2 in the production of lactose-N-neotetraose (LNnT) and lactose-N-tetraose (LNT).

6. A method for preparing lactose-N-tetraose and lactose-N-neotetraose, characterized in that: Lactose-N-tetraose and lactose-N-neotetraose are synthesized by using one of the polypeptides described in claim 1 or the composition described in claim 2 as a catalyst and lactose and lactose-N-triose as substrates.

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