WO2021109022A1 - 岩藻糖苷酶及其相关生物材料与应用 - Google Patents

岩藻糖苷酶及其相关生物材料与应用 Download PDF

Info

Publication number
WO2021109022A1
WO2021109022A1 PCT/CN2019/122945 CN2019122945W WO2021109022A1 WO 2021109022 A1 WO2021109022 A1 WO 2021109022A1 CN 2019122945 W CN2019122945 W CN 2019122945W WO 2021109022 A1 WO2021109022 A1 WO 2021109022A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
seq
fucosyl
fucosyllactose
fucosidase
Prior art date
Application number
PCT/CN2019/122945
Other languages
English (en)
French (fr)
Inventor
江正强
史然
马俊文
刘军
闫巧娟
刘海杰
Original Assignee
中国农业大学
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 中国农业大学 filed Critical 中国农业大学
Priority to PCT/CN2019/122945 priority Critical patent/WO2021109022A1/zh
Publication of WO2021109022A1 publication Critical patent/WO2021109022A1/zh

Links

Images

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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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
    • C12P19/14—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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

Definitions

  • the invention belongs to the field of genetic engineering, and specifically relates to fucosidase and related biological materials and applications.
  • Fucosyllactose is composed of one molecule of fucose and one molecule of lactose. Fucose residues can be linked to the galactose residues of lactose through ⁇ (1,2) glycosidic bonds, or through ⁇ (1,3) The glycosidic bond is connected to the glucose at the reducing end of lactose.
  • Fucosyllactose is abundantly present in human milk, and has various functional activities such as regulating intestinal flora, resisting the adhesion of pathogenic bacteria, immune regulation and promoting brain development (Yvan et al., Human Milk Oligosaccharides: 2'-Fucosyllactose) (2'-FL) and Lacto-N-Neotetraose (LNnT) in Infant Formula. Nutrients, 2018, 10:1161.).
  • Yvan et al. Human Milk Oligosaccharides: 2'-Fucosyllactose) (2'-FL) and Lacto-N-Neotetraose (LNnT) in Infant Formula. Nutrients, 2018, 10:1161.
  • LNnT Lacto-N-Neotetraose
  • Enzymatic synthesis of oligosaccharides has the advantages of mild reaction conditions and good controllability, which has attracted wide attention in recent years (Bojarov et al., Glycosidases in carbohydrate synthesis: when organic chemistry falls short. Chimia (Aarau), 2011, 65, 65-70 .).
  • Glycoside hydrolases and glycosyltransferases are two types of enzymes that are widely used in the synthesis of oligosaccharides.
  • ⁇ -L-Fucosidase is a type of exoglycoside hydrolase, which can specifically hydrolyze fucose residues attached to fucosyl oligosaccharides or fucosyl compounds.
  • ⁇ -L-fucosidase Compared with fucosyltransferase, ⁇ -L-fucosidase has a wide range of sources, high activity and can use more economical raw materials as glycosyl donors for the synthesis of oligosaccharides (Lezyk, et al., Novel ⁇ -L -Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides.PLoS ONE, 2016:1-18.).
  • ⁇ -L-fucosidase is mainly divided into 4 families, namely glycoside hydrolase (GH) 29, 95, 141 and 151 families. According to the reaction mechanism, ⁇ -L-fucosidase can be divided into retention type and reverse type.
  • GH glycoside hydrolase
  • 3'-fucosyllactose can be used as an antibody probe to specifically bind to the corresponding antigens in adenocarcinoma and embryonic cancer cells (Miyauchi et al., A new fucosyl antigen expressed on colon adenocarcinoma and embryonal carcinoma cells. Nature) , 1982, 299: 168-169.).
  • the technical problem to be solved by the present invention is to provide a protein with transglycosidic activity involved in the synthesis of fucosyl compounds to synthesize or prepare 3'-fucosyllactose.
  • the present invention first provides a protein, derived from Pedobacter sp., as shown in any of the following A1)-A4):
  • A3 A fusion protein obtained by tagging the N-terminal or/and C-terminal protein of the protein of SEQ ID No. 3 or the protein of SEQ ID No. 4;
  • the protein of SEQ ID No. 3 or the protein of SEQ ID No. 4 is obtained by substitution and/or deletion and/or addition of one or several amino acid residues, and the protein shown in A1) or A2) has 90 Proteins with more than% identity and the same function.
  • the protein shown in A1) is named ⁇ -L-fucosidase (PbFuc), and SEQ ID No. 3 consists of 422 amino acid residues.
  • the protein shown in A2) is named recombinant ⁇ -L-fucosidase (PbFuc-His), which is the fusion protein obtained by connecting the N-terminal of PbFuc of SEQ ID No. 3 to MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEF, SEQ ID No. 4 is 458 Consists of three amino acid residues.
  • the above-mentioned protein can be synthesized artificially, or the coding gene can be synthesized first, and then obtained by biological expression.
  • a protein-tag refers to a polypeptide or protein expressed by fusion with the target protein by using DNA in vitro recombination technology to facilitate the expression, detection, tracing and/or purification of the target protein.
  • the protein tag can be Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag, etc.
  • identity refers to the identity of amino acid sequence.
  • the homology search site on the Internet can be used to determine the identity of the amino acid sequence, such as the BLAST page of the NCBI homepage.
  • the Expect value is set to 10
  • all Filters are set to OFF
  • BLOSUM62 is used as the Matrix
  • Gap existence cost Per resistance gap cost and Lambda ratio are respectively set to 11, 1 and 0.85 (default value) and search for the identity of a pair of amino acid sequences to calculate, and then the identity value (%) can be obtained.
  • the identity of more than 90% may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.
  • the present invention further provides the application of the above-mentioned protein.
  • the fucosyl compound may be a fucosyl-containing oligosaccharide, a fucosyl-containing protein, a fucosyl-containing lipid, or any other fucosyl-containing compound;
  • the glycosyl donor is any fucosyl-containing compound, such as sugars, alcohols, lipids, amino acids or proteins of any chain length containing fucosyl, specifically 2-chloro-4-nitro Nitrophenyl- ⁇ -L-fucopyranoside (4-Nitrophenyl- ⁇ -L-fucopyranoside, abbreviated as pNP-FUC).
  • the relevant biological material provided by the present invention for the above-mentioned protein is any one of the following C1) to C8):
  • C2 An expression cassette containing the nucleic acid molecule of C1);
  • nucleic acid molecules in C1) are as shown in B1) or B2) or B3) as follows:
  • the coding sequence is the DNA molecule shown in positions 10-1278 of SEQ ID No. 1;
  • the coding sequence is the DNA molecule shown in SEQ ID No. 2;
  • SEQ ID No. 1 consists of 1294 nucleotides, of which the 10-1278th position is named ⁇ -L-fucosidase gene (PbFuc gene), which encodes the ⁇ -L-rock of SEQ ID No. 3 Alcosidase (PbFuc).
  • SEQ ID No. 2 consists of 1377 nucleotides and is named the recombinant ⁇ -L-fucosidase gene (PbFuc-His gene), which encodes the recombinant ⁇ -L-fucosidase (PbFuc) of SEQ ID No. 4 -His).
  • the stringent conditions are hybridization in a solution of 2 ⁇ SSC, 0.1% SDS at 68°C and washing the membrane twice, 5 min each time, and hybridization in a solution of 0.5 ⁇ SSC, 0.1% SDS at 68°C And wash the membrane 2 times, 15min each time.
  • the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA, and the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.
  • the expression cassette (PbFuc gene expression cassette) containing the nucleic acid molecule encoding the above-mentioned protein described in C2) refers to the DNA capable of expressing PbFuc in the host cell, and the DNA may not only include the promoter that initiates the transcription of PbFuc The promoter may also include a terminator that terminates the transcription of PbFuc. Further, the expression cassette may also include an enhancer sequence.
  • the vector can be a plasmid, a cosmid, a phage or a virus vector.
  • the microorganisms may be yeast, bacteria, algae or fungi, such as Agrobacterium.
  • the present invention further provides applications of the above-mentioned related biological materials.
  • the fucosyl compound may be a fucosyl-containing oligosaccharide, a fucosyl-containing protein, a fucosyl-containing lipid, or any other fucosyl-containing compound;
  • the glycosyl donor is any fucosyl-containing compound, such as sugars, alcohols, lipids, amino acids or proteins of any chain length containing fucosyl, specifically 2-chloro-4-nitro Nitrophenyl- ⁇ -L-fucopyranoside (4-Nitrophenyl- ⁇ -L-fucopyranoside, abbreviated as pNP-FUC).
  • the present invention also provides a method for preparing the above-mentioned protein ( ⁇ -L-fucosidase).
  • the method for preparing the above-mentioned protein of the present invention includes introducing the gene of the above-mentioned protein (that is, the gene encoding the protein) into a recipient microorganism to obtain a recombinant microorganism expressing the above-mentioned protein, culturing the recombinant microorganism, and expressing the above-mentioned protein ( ⁇ -L-Fucosidase).
  • the recipient microorganism is a prokaryotic microorganism.
  • the prokaryotic microorganism is Escherichia coli. More specifically, the Escherichia coli is Escherichia coli BL21 (DE3).
  • the gene of the protein can be introduced into the E. coli expression strain BL21(DE3) through the recombinant plasmid pET-28a(+)-PbFuc to obtain the recombinant strain BL21(DE3)-pET-28a(+)-PbFuc;
  • the recombinant plasmid pET-28a(+)-PbFuc replaces the small DNA fragments between the NheI and XhoI restriction sites of the vector pET-28a(+) with the nucleotide sequence of SEQ ID No.
  • the recombinant plasmid pET-28a(+)-PbFuc contains the PbFuc-His gene of SEQ ID No. 2, encoding the fusion protein PbFuc-His of SEQ ID No. 4, and PbFuc-His is shown in SEQ ID No. 3.
  • the N-terminal of PbFuc is connected to the fusion protein obtained by MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEF.
  • the optimal pH of the obtained ⁇ -L-fucosidase is 5.0, which is stable in the pH range of 4.0-11.0, and the residual enzyme activity is greater than 80%, showing good pH stability; the optimal temperature is 35°C, it remains relatively stable below 40°C, and the enzyme activity can maintain more than 90%. After 45°C, the enzyme activity declines rapidly, and the half-life at 35°C, 40°C and 45°C are 555, 51 and 2.8 respectively. min.
  • the present invention further provides a method for preparing 3'-fucosyllactose.
  • the method for preparing 3'-fucosyllactose of the present invention includes the step of using ⁇ -L-fucosidase to catalyze pNP-FUC and lactose.
  • the pH value of the reaction catalyzed by the ⁇ -L-fucosidase can be 3.5-11.0, specifically 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11.0; or the range value between any two of the above-mentioned points, such as 3.5-7.0; 6.5-9.5; or 7-9; or 8-10; or 8-8.5; or 8.5- PH within 9;
  • the temperature of the ⁇ -L-fucosidase catalyzed reaction is 20-50°C; specifically 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C; or the above
  • the range value between any two said point values is for example 20-35°C; or 35-50°C; or 25-50°C; or temperature within 30-40°C.
  • the time for the ⁇ -L-fucosidase to catalyze the reaction may be greater than 0 s, and specifically may be 5, 10, 15, 30, 45 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24, 48h; or the range between the above two points, such as the time within 5-60min, 1 ⁇ 48h.
  • the optimal temperature for the reaction catalyzed by the ⁇ -L-fucosidase is 35°C
  • the optimal pH is 8.5
  • the optimal reaction time is 3h
  • the optimal concentration of lactose is 700mM
  • the optimal amount of enzyme added is
  • the concentration of pNP-FUC is 0.5U/mL and the concentration of pNP-FUC is 10mM. Under these conditions, 3'-fucosyllactose can be synthesized efficiently, and the conversion rate is 85%.
  • the present invention further provides the application of 3'-fucosyllactose in promoting the proliferation of probiotics.
  • the probiotics are Lactobacillus delbrueckii, Lactobacillus casei and Bifidobacterium longum.
  • the Lactobacillus delbrueckii is Lactobacillus delbrueckii NRRL B-548
  • the Lactobacillus casei is Lactobacillus casei AS.1.2435 and Lactobacillus casei NRRL B-1922
  • the Bifidobacterium longum is Bifidobacterium longum.
  • the present invention uses high-efficiency thermal asymmetry staggered PCR (hiTAIL-PCR) to amplify from Pedobacter sp. to obtain the ⁇ -L-fucosidase shown in SEQ ID No. 1 from 10-1278.
  • Gene PbFuc
  • E.coli BL21(DE3) E.coli BL21(DE3)
  • the recombinant bacteria can be used to prepare ⁇ -L-fucosidase (PbFuc).
  • the present invention utilizes PbFuc to catalyze pNP-FUC and lactose to synthesize 3'-fucosyllactose, with a conversion rate of 85% for 3 hours, which is of great significance for the large-scale preparation of 3'-fucosyllactose.
  • the present invention uses synthetic 3'-fucosyllactose as a carbon source to culture four probiotics in vitro (Lactobacillus delbrueckii NRRL B-548, Lactobacillus casei AS.1.2435, Lactobacillus casei NRRL B-1922 and Long Bifidobacterium NRRL (B-41409), the growth (OD 595) after 48 hours was 0.467, 0.362, 0.375, and 0.403, respectively. Under the same conditions, the OD 595 of these four probiotics were 0.182, 0.320, 0.290, and 0.333, respectively, indicating that 3'-fucosyllactose has a better proliferation effect on the above four probiotics.
  • Fructooligosaccharides in addition, four harmful bacteria (including Escherichia coli ATCC 11775, Salmonella typhimurium AS1.1552, Listeria monocytogenes CICC 21635 and Staphylococcus aureus AS 1.1861) fermentation
  • the growth (OD 595) after 24h was 0.091, 0.074, 0.086, and 0.095, which were close to the sugar-free group.
  • the growth (OD 595) of the four harmful bacteria after 24h fermentation was 0.179, respectively. 0.186, 0.199 and 0.109 indicate that the proliferation effect of 3'-fucosyllactose on the above four harmful bacteria is lower than that of oligofructose, showing excellent beneficial life.
  • Figure 1 is a graph of agarose gel electrophoresis results of conserved sequences obtained using Pedobacter sp. genomic DNA as a template.
  • Figure 2 is a graph of agarose gel electrophoresis results of DNA fragments obtained using hiTAIL-PCR fragments as templates.
  • Figure 3 is a multiple sequence alignment diagram of PbFuc and 29 family ⁇ -L-fucosidase amino acids.
  • Figure 4 shows the results of polyacrylamide gel electrophoresis (SDS-PAGE) of the crude enzyme solution of the recombinant bacterial culture solution; among them, 1 is the crude enzyme solution of the control group without IPTG; 2 is the crude enzyme solution of the experimental group with IPTG.
  • SDS-PAGE polyacrylamide gel electrophoresis
  • Fig. 5 is the SDS-PAGE chart of PbFuc crude enzyme solution (1) and pure enzyme solution (2) before and after purification by nickel column.
  • Figure 6 shows the optimum pH (A) and pH stability (B) of PbFuc; among them, citrate-trisodium citrate ( ⁇ ) pH 3.0-6.0; PB buffer ( ⁇ ) pH 6.0-8.0; Tris- HCl buffer ( ⁇ ) pH 6.0-9.0; CHES buffer pH ( ⁇ ) 8.0-10.0, CAPS buffer (*) pH 10.0-11.0, Na 2 HPO 4 -NaOH buffer ( ⁇ ) pH 11.0-12.0.
  • Figure 7 shows the optimum temperature (A), temperature stability (B) and half-life (C) of PbFuc.
  • Figure 8 is an HPLC chart of PbFuc-catalyzed transglycosidation reaction.
  • Figure 9 is the first-order mass spectrum of 3'-fucosyllactose.
  • Figure 10 is a 3'-fucosyllactose NMR primary 1 H spectrum (A) and 13 C spectrum (B) diagram.
  • Figure 11 shows the optimal temperature (A), optimal pH (B), optimal enzyme amount (C), optimal reaction time (D), and optimal receptor concentration (E) of PbFuc transglycoside activity.
  • Figure 12 shows the cultivation of Lactobacillus delbrueckii NRRL B-548 (A), Lactobacillus casei NRRL B-1922 (B), Lactobacillus casei AS.1.2435 (C) and long The growth curve of Bifidobacterium NRRL B-41409(D).
  • Figure 13 shows the cultivation of Escherichia coli ATCC 11775 (A), Salmonella typhimurium AS 1.1552 (B), Listeria monocytogenes CICC 21635 (C) and Staphylococcus aureus AS 1.1861 with the sole carbon source of 3'-fucosyllactose (D) Growth curve.
  • Example 1 Construction of recombinant ⁇ -L-fucosidase encoding gene expression plasmid
  • the gradient PCR amplification system is: 10 ⁇ LA buffer 5.0 ⁇ l, dNTP mix (2.5mmol/l) 4.0 ⁇ l, fuDP-F/R (10pmol/ ⁇ l) each 8.0 ⁇ l, genomic DNA 1.0 ⁇ l, LA Taq (5.0U/ ⁇ l) 0.5 ⁇ l, ddH 2 O up to 50.0 ⁇ l;
  • the gradient PCR amplification program is: 95°C pre-denaturation 3min; 95°C denaturation 30s, 40-60°C annealing 30s, 72°C extension 40s, 34 cycles; 72°C 5min .
  • hiTAIL-PCR high-efficiency thermal asymmetry staggered PCR
  • four LAD primers LAD1, LAD2, LAD3 and LAD4
  • a nested specific primer AC are used to amplify known sequence fragments through three rounds of PCR. Flanking sequence.
  • TAIL I Pedobacter sp. genomic DNA
  • TAIL II the first round PCR amplification was performed.
  • the PCR product of TAIL I is appropriately diluted and used as the template for the second round (TAIL II) PCR amplification reaction
  • the PCR product of TAIL II is appropriately diluted and used as the template for the third round (TAIL III) PCR amplification reaction
  • hiTAIL- PCR fragment TAIL II and TAIL III use the same PCR amplification parameters.
  • the designed primer sequences are shown in Table 1, and the reaction conditions of hiTAIL-PCR are shown in Table 2.
  • PCR product a represents the amplification product of the previous round.
  • Design primer P1 according to the coding sequence of the amplified hiTAIL-PCR fragment: 5′-CCG GAATTC CAGGATTACACACACCTACAGCCGC-3′ (underlined is the restriction enzyme EcoRI restriction site) and P2: 5′-ATAAGAAT GCGGCCGC CTATCCAATCTCCAAAACAATCACCTG-3' (underlined is the restriction enzyme NotI site), and PCR amplification was performed, and the amplified product was detected by 1% agarose gel electrophoresis. The result is shown in Figure 2, and the result is as SEQ ID No. 1 DNA fragment of 1294bp.
  • the reaction procedure is: 95°C pre-denaturation for 5min; 95°C denaturation for 20s, 58°C annealing for 20s, 72°C extension for 30s, 35 cycles; 72°C extension for 10min.
  • the structure of the recombinant plasmid pET-28a(+)-PbFuc is described as follows: replace the small DNA fragments between the EcoRI and NotI restriction sites of the vector pET-28a(+) with the nucleotide sequence It is the DNA molecule shown at positions 10-1278 of SEQ ID No. 1, and encodes the protein of SEQ ID No. 3.
  • the DNA molecule shown at positions 10-1278 of SEQ ID No. 1 and the coding sequence containing the His-tag tag (consisting of 6 histidine residues) on the vector (SEQ ID No. 2 No. 1-108) are fused to form the DNA molecule of SEQ ID No. 2, and the final expressed protein is the fusion protein of SEQ ID No. 4.
  • the protein of SEQ ID No. 3 is further compared with the known structural protein sequence in the PDB database to obtain a multiple sequence alignment diagram of the protein of SEQ ID No. 3 and the ⁇ -L-fucosidase of the GH29 family.
  • the results showed that the protein of SEQ ID No. 3 has the highest sequence similarity (36.41%) with the GH29 family ⁇ -L-fucosidase derived from Paenibacillus thiaminolyticus (PDB accession number: 6GN6), followed by Bacteroides thetaiotaomicron.
  • Source GH29 family ⁇ -L-fucosidase (BtFuc2970, PDB: 2WVT, 29.6%).
  • ⁇ -L-Fucosidase that is, the protein of SEQ ID No. 3 is named ⁇ -L-Fucosidase (PbFuc), and its coding gene is shown in SEQ ID No. 1, No. 10-1278, named as ⁇ -L-fucosidase gene (PbFuc gene); the fusion protein of SEQ ID No.
  • Example 2 Expression of recombinant ⁇ -L-fucosidase gene
  • the recombinant plasmid pET-28a(+)-PbFuc was transformed into E. coli BL21(DE3) to obtain a recombinant bacteria, which was named BL21(DE3)-pET-28a(+)-PbFuc. Insert BL21(DE3)-pET-28a(+)-PbFuc into LB liquid medium for seed liquid culture.
  • the medium contains kanamycin (50 ⁇ g mL -1 ), and the seed liquid inoculum is 1.5% (w/ v), the solid medium is an LB solid plate containing agar.
  • the cells were resuspended in buffer (20mmol L -1 pH 8.0 Tris-HCl buffer, 0.5 mol L -1 NaCl, 20 mmol L -1 imidazole), and the supernatant was obtained by ultrasonic breaking and centrifugation to obtain the crude enzyme solution.
  • Ni-IDA affinity column was selected to purify the recombinant protein.
  • equilibration buffer to elute 10 column volumes (5-10 columns) at a flow rate of 1.0 mL min -1 (20 mmol L -1 pH 8.0 Tris-HCl buffer, 0.5 mol L -1 NaCl, 20 mmol L -1 imidazole) The volume can be used); load the crude enzyme solution in the above step 1 at a flow rate of 0.5 mL min -1; then use the equilibration buffer to elute at a flow rate of 1.0 mL min -1 until the OD 280 is less than 0.05 to wash away the impurities; Eluent B (20mmol L -1 pH 8.0 Tris-HCl buffer, 0.5mol L -1 NaCl, 100mmol L -1 imidazole) elute, collect the solution after the eluate B passes through
  • the pure enzyme solution was tested for protein purity by SDS-PAGE (Laemmli UK.1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685), and the results are shown in Figure 5, where M is Low molecular weight standard protein; 1 is crude enzyme solution; 2 is pure enzyme solution. The results showed that the pure enzyme solution obtained an obvious single band with a molecular weight of 50kDa and a specific enzyme activity of 26.3U/mL. See Table 3 for the purification results of recombinant ⁇ -L-fucosidase.
  • the protein content determination refers to the method of Lowry et al.
  • a represents the conditions of enzyme activity determination: 5mM pNP-FUC as the substrate, reacted for 20min under the conditions of 50mM citrate buffer pH 5.0 and 35°C.
  • b represents the protein concentration measured by Lowry method.
  • ⁇ -L-fucosidase activity refers to the method of Janet et al. (Janet et al., ⁇ -Fucosidases with different substrate specificities from two species of Fusarium. Appl Microbiol Biotechnol, 2013, 97:5371-5380.).
  • Add 100 ⁇ L 10mM pNP-FUC, 100 ⁇ L 0.05M pH 5.0 citrate-trisodium citrate buffer to the reaction system, 10 ⁇ L appropriately diluted enzyme solution, react at 35°C for 20min, and finally add 200 ⁇ L Na 2 CO 3 (1M) to stop the reaction. Shake evenly. Take 200 ⁇ L into a 96-well plate, and measure the absorbance at 405nm. The pNP standard was used as a standard curve.
  • Enzyme activity definition The amount of enzyme required to catalyze pNP-FUC to produce 1 ⁇ mol pNP per minute is an enzyme activity unit (U).
  • PbFuc pH stability determination Use the above buffers with different pH values and two other buffers (CAPS, 10.0-11.0; Na 2 HPO 4 -NaOH, 11.0-12.0) to dilute the enzyme solution, and put the diluted enzyme solution at 25 Treat them in a water bath at °C for 30 minutes, and then quickly place the sample in an ice water bath to cool for 30 minutes. After the treatment, the residual enzyme activity was measured at the optimum temperature, and the untreated enzyme solution was used as a control. Finally, the percentage of the residual enzyme activity in the untreated control enzyme activity was calculated.
  • CAPS 10.0-11.0
  • Na 2 HPO 4 -NaOH 11.0-12.0
  • PbFuc temperature stability determination Use 50mmol L -1 pH 5.0 citric acid-trisodium citrate to dilute the pure enzyme solution to an appropriate multiple and treat it at different temperatures for 30 minutes, then cool it in an ice-water bath for 30 minutes, and finally follow the standard method The residual enzyme activity was determined, and the enzyme activity of the untreated enzyme was used as a control.
  • Half-life determination The enzyme solution was appropriately diluted with 50mmol L -1 pH 5.0 citrate buffer, and then placed at 35, 40, and 45°C for 0-4h, and samples were taken at different time intervals.
  • the untreated enzyme solution was used as a control to determine the residual enzyme activity, and finally the percentage of the residual enzyme activity to the control enzyme activity was calculated, and the time for the enzyme activity to decay to 50% at different temperatures was calculated.
  • the synthesis conditions are as follows: pNP-FUC is used as a fucosyl donor, lactose is used as an acceptor, an appropriate amount of recombinant ⁇ -L-fucosidase is added, and the reaction is carried out in a buffer of appropriate pH for an appropriate time.
  • the reaction solution was inactivated by boiling for 10 min, filtered through a 0.22 ⁇ m microporous membrane, and analyzed by HPLC.
  • the quantitative method refers to the method of Birgitte et al.
  • the molar conversion rate of the product is calculated based on the initial concentration of pNP-FUC as 100%, and the formula is as follows:
  • HPLC quantitative analysis conditions are as follows: Agilent 1260 high-phase liquid chromatograph with RID detector. Chromatographic conditions: Shodex-KS-802 gel chromatography column, mobile phase is ultrapure water, flow rate is 0.8mL/min, column temperature is 65°C, RID detector temperature is 35°C.
  • the product after the reaction was concentrated under vacuum and decompression, passed through anion and cation resin to remove salt and pNP, and then passed through a Bio-gel P2 column (1.2cm*110cm, ultrapure water, flow rate 0.3mL/min), and the product was 1mL/tube collection.
  • the sample was dissolved in pure water, and a Thermo Scientific TM Q Exactive TM mass spectrometer was used in the positive-ion mode of the ESI ion source to collect a high-resolution primary mass spectrum of the sample to determine the molecular weight of the transglycoside product.
  • the reaction conditions including temperature, pH, amount of enzyme, reaction time and lactose concentration are optimized.
  • the optimal preparation conditions for 3'-fucosyllactose were determined: the optimal temperature was 35°C (A in Figure 11), the optimal pH was 8.5 (B in Figure 11), and the optimal amount of enzyme added was 0.5 U/ mL (C in Figure 11), the optimal reaction time is 3h (D in Figure 11), and the optimal concentration of receptor is 700 mM (E in Figure 11).
  • the above reaction product was concentrated under vacuum and reduced pressure, passed through anion and cation resin to remove salt and pNP, and then passed through Bio-gel P2 column. The collected product was tested for purity by thin layer chromatography (TLC), and after freeze-drying, the final sample was white powder.
  • MRS medium supplemented with 1% 3'-fucosyllactose (0.5g/L L-cysteine salt should be added when cultivating bifidobacteria) to cultivate fifteen strains of probiotics (Lactobacillus rhamnosus).
  • a commercial prebiotic, fructooligosaccharide (FOS) was used as a positive control.
  • Four harmful bacteria E. coli ATCC 11775, Listeria monocytogenes CICC 21635, Salmonella typhimurium AS 1.1552 and gold
  • M 9 medium supplemented with 1% 3'-fucosyllactose (3'-FL).
  • Staphylococcus aureus AS 1.1861 was cultured on M 9 medium supplemented with 1% 3'-fucosyllactose ('-FL).
  • Staphylococcus aureus AS 1.1861 commercial prebiotic oligosaccharide (FOS) as a positive control.
  • ⁇ OD 595 Use a microplate reader to detect the growth (OD 595nm) after 12, 24, 48 and 72 hours of culture, and calculate the difference between the growth of the bacteria after supplementing 3'-fucosyllactose and the OD 595 of the sugar-free group ( ⁇ OD 595 ).
  • ⁇ OD 595 > 0.1 3'-fucosyllactose is considered to have a proliferation effect on the bacteria, and when ⁇ OD 595 ⁇ 0.1, it is considered that 3'-fucosyl lactose has no significant effect on the proliferation of the bacteria.
  • Lactobacillus casei AS 1.2435 (L.casei subsp.casei AS 1.2435, Zhang et al., D -Tagatose production by Lactococcus lactis NZ9000 Cells Harboring Lactobacillus plantarum L-arabinose Isomerase.Indian Journal of Pharmaceutical Education and Research.2017,51(2):288-294.
  • Bifidobacterium longum NRRL B-41409 Bifidobacterium longum NRRL B-41409
  • Bialonska et al. The Effect of Pomegranate (Punica granatum L.) Byproducts and Ellagitannins on the Growth of Human Gut Bacteria.J.Agric.Food Chem.2009,57,8344-8349) Proliferation ( ⁇ OD 595 > 0.1, see Figure 12).
  • the growth (OD 595) of the above four probiotics after 48 hours of fermentation with 3'-fucosyllactose as the sole carbon source were 0.467, 0.362, 0.375, and 0.403, respectively.
  • the growth (OD 595) of the four probiotic strains after 48 hours of proliferation by oligofructose was 0.182, 0.320, 0.290, and 0.333, respectively (see Table 4).
  • four harmful bacteria including E.
  • PbFuc can efficiently synthesize 3'-fucosyllactose and has a good application prospect in the synthesis of oligosaccharides.
  • the proliferation effect of 3'fucosyllactose on the four probiotic bacteria is better than that of fructooligosaccharides, and the proliferation effect on the four harmful bacteria is lower than that of fructooligosaccharides, showing excellent beneficial life.
  • the ⁇ -L-fucosidase (PbFuc) provided by the present invention can efficiently synthesize 3'-fucosyllactose, and has a good application prospect in the synthesis of oligosaccharides.
  • 3'-fucosyllactose has better proliferation effects on the four probiotic bacteria than fructooligosaccharides, while the growth of the four harmful bacteria is lower than that on fructooligosaccharides, showing excellent beneficial life properties.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

本发明公开了一种岩藻糖苷酶及其相关生物材料与应用。本发明公开了一种蛋白质：A1)氨基酸序列为SEQ ID No.4的蛋白质；A2)氨基酸序列为SEQ ID No.3的蛋白质；A3)将A1)或A2)的蛋白质的N端或/和C端连接蛋白标签得到的融合蛋白；A4)将SEQ ID No.3的蛋白质或SEQ ID No.4的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与A1)或A2)所示的蛋白质具有90％以上的同一性且功能相同的蛋白质。本发明进一步公开了上述蛋白质的相关生物材料及其应用。本发明提供的蛋白质可高效合成3'-岩藻糖基乳糖，在寡糖合成中具有良好的应用前景。

Description

岩藻糖苷酶及其相关生物材料与应用 技术领域
本发明属于基因工程领域，具体涉及岩藻糖苷酶及其相关生物材料与应用。
背景技术
岩藻糖基乳糖由一分子岩藻糖和一分子乳糖构成，岩藻糖残基可通过α(1,2)糖苷键连接到乳糖的半乳糖残基上，或通过α(1,3)糖苷键连接到乳糖还原端的葡萄糖。岩藻糖基乳糖大量存在于人乳中，具有调节肠道菌群、抵抗病原菌的粘附、免疫调节及促进大脑发育等多种功能活性(Yvan et al.,Human Milk Oligosaccharides:2’-Fucosyllactose(2’-FL)and Lacto-N-Neotetraose(LNnT)in Infant Formula.Nutrients,2018,10:1161.)。目前，化学合成或生物合成的2’-岩藻糖基乳糖已被欧盟和美国FDA批准添加到婴幼儿食品中(Bych et al.,Production of HMOs using microbial hosts—from cell engineering to large scale production.Current Opinion in Biotechnology,2019,56C:130-137.)，市场对于岩藻糖基乳糖需求日益增加。因此，开发合成岩藻糖基乳糖的方法具有重要意义。
酶法合成寡糖具有反应条件温和、可控性良好等优势近年来受到广泛关注(Bojarová et al.,Glycosidases in carbohydrate synthesis:when organic chemistry falls short.Chimia(Aarau),2011,65,65-70.)。糖苷水解酶和糖基转移酶是在寡糖合成中应用较多的两类酶。α-L-岩藻糖苷酶是一类外切糖苷水解酶，能特异性水解岩藻糖基寡糖或岩藻糖基化合物上连接的岩藻糖残基。与岩藻糖基转移酶相比，α-L-岩藻糖苷酶来源广泛、活性高且能使用较为经济的原料作为糖基供体进行寡糖的合成(Lezyk,et al.,Novelα-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides.PLoS ONE,2016:1-18.)。目前，α-L-岩藻糖苷酶主要分为4个家族，分别是糖苷水解酶(GH)29、95、141和151家族。根据反应机制，α-L-岩藻糖苷酶可分为保留型和反转型，仅保留型GH29和GH151家族的岩藻糖苷酶具有转糖苷活性(Birgitte et al.Substrate specificity and transfucosylation activity of GH29 α-L-fucosidases for enzymatic production of human milk oligosaccharides,New Biotechnology,2018,41:34-45；Elise et al.,SYNTHESIS OF NEW FUCOSE-CONTA.国际专利:US20140228554A1)。迄今，利用α-L-岩藻糖苷酶的转糖苷活性合成岩藻糖基乳糖的主要限制因素是产物得率低。因此，发掘特性优良、高转糖苷活性的α-L-岩藻糖苷酶具有重要意义。
3’-岩藻糖基乳糖尚无天然存在报道，Fuc-α(1,3)-Gal糖苷键存在于一种人乳低聚九糖的末端(Yamashita et al.,Oligosaccharides of Human Milk.Isolation and Characterization of Three New Disialylfucosyl Hexasaccharides.Arch.Biochem.Biophys.,1976,174:582-591)。有研究通过对乳糖的化学修饰经六步反应获得3’-岩藻糖基乳糖，但制备过程中使用大量有毒试剂、步骤繁芜且产物得率低(Takamura.et al.,Chemical  Modification of Lactose.XV.Syntheses of O-α-and O-β-L-Fucopyranosyl-(1→3)-O-β-D-galactopyranosyl-(1→4)-D-glucopyranoses(3′-O-α-and3′-O-β-L-Fucopyranosyllactoses).Carbohydrate Research,1980:84:53-60)。酶法合成3’-岩藻糖基乳糖的报道很少，仅有Alcaligenes sp.来源的α-L-岩藻糖苷酶通过其转糖苷活性合成3’-岩藻糖基乳糖，得率为34％(Murata et al,.Enzymatic synthesis of alpha-L-fucosyl-N-acetyllac-tosamines and3′-O-alpha-L-fucosyllactose utilizing alpha-L-fucosidases.Carbohydrate Research.1999,320:192-199.)。3’-岩藻糖基乳糖功能活性的报道也很少。研究发现3’-岩藻糖基乳糖可作为抗体探针与腺癌和胚胎癌细胞中相应的抗原发生特异性结合(Miyauchi et al.,A new fucosyl antigen expressed on colon adenocarcinoma and embryonal carcinoma cells.Nature,1982,299:168-169.)。
迄今，尚无3’-岩藻糖基乳糖益生活性的研究报道。
发明公开
本发明所要解决的技术问题为提供一种参与岩藻糖基化合物合成的具有转糖苷活性的蛋白质，以合成或制备3’-岩藻糖基乳糖。
为解决上述问题，本发明首先提供了一种蛋白质，来源于地杆菌(Pedobacter sp.)，为如下A1)-A4)任一所示：
A1)氨基酸序列为SEQ ID No.3的蛋白质；
A2)氨基酸序列为SEQ ID No.4的蛋白质；
A3)将SEQ ID No.3的蛋白质或SEQ ID No.4的蛋白质的N端或/和C端连接蛋白标签得到的融合蛋白；
A4)将SEQ ID No.3的蛋白质或SEQ ID No.4的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与A1)或A2)所示的蛋白质具有90％以上的同一性且功能相同的蛋白质。
其中，A1)所示的蛋白质的命名为α-L-岩藻糖苷酶(PbFuc)，SEQ ID No.3由422个氨基酸残基组成。
A2)所示的蛋白质的命名为重组α-L-岩藻糖苷酶(PbFuc-His)，其为SEQ ID No.3的PbFuc的N端连接MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEF得到的融合蛋白，SEQ ID No.4由458个氨基酸残基组成。
上述蛋白质可人工合成，也可先合成其编码基因，再进行生物表达得到。
上述蛋白质中，蛋白标签(protein-tag)是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽或者蛋白，以便于目的蛋白的表达、检测、示踪和/或纯化。所述蛋白标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
上述蛋白质中，同一性是指氨基酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性，如NCBI主页网站的BLAST网页。例如，可在高级BLAST2.1中，通过使用blastp作为程序，将Expect值设置为10，将所有Filter设置为OFF，使用BLOSUM62作为Matrix，将Gap existence cost，Per residue gap cost 和Lambda ratio分别设置为11，1和0.85(缺省值)并进行检索一对氨基酸序列的同一性进行计算，然后即可获得同一性的值(％)。
上述蛋白质中，所述90％以上的同一性可为至少91％、92％、95％、96％、98％、99％或100％的同一性。
本发明进一步提供了上述蛋白质的应用。
所述应用如下述任一所述：
D1)上述蛋白质作为α-L-岩藻糖苷酶(PbFuc)的应用；
D2)上述蛋白质在制备或合成岩藻糖基化合物的应用；
D3)上述蛋白质在催化糖基供体和乳糖形成3’-岩藻糖基乳糖的应用。
具体的，所述岩藻糖基化合物可以为含岩藻糖基的寡糖、含岩藻糖基的蛋白质、含岩藻糖基的脂类、其他任意含岩藻糖基的化合物；
所述糖基供体为任何含岩藻糖基的化合物，如含岩藻糖基的任意链长的糖类、醇类、脂类、氨基酸或蛋白，具体可为2-氯-4-硝基苯基-α-L-岩藻糖苷(4-Nitrophenyl-α-L-fucopyranoside，简写为pNP-FUC)。
上述蛋白质的相关生物材料也在本发明的保护范围之内。
本发明提供上述蛋白质的相关生物材料为下述C1)至C8)中的任一种：
C1)编码上述蛋白质的核酸分子；
C2)含有C1)所述核酸分子的表达盒；
C3)含有C1)所述核酸分子的重组载体；
C4)含有C2)所述表达盒的重组载体；
C5)含有C1)所述核酸分子的重组微生物；
C6)含有C2)所述表达盒的重组微生物；
C7)含有C3)所述重组载体的重组微生物；
C8)含有C4)所述重组载体的重组微生物。
上述生物材料中，C1)所述核酸分子为如下B1)或B2)或B3)所示：
B1)编码序列为SEQ ID No.1第10-1278位所示的DNA分子；
B2)编码序列为SEQ ID No.2所示的DNA分子；
B3)在严格条件下与B1)或B2)限定的DNA分子杂交，且编码上述蛋白质的DNA分子。
其中，SEQ ID No.1由1294个核苷酸组成，其中，第10-1278位命名为α-L-岩藻糖苷酶基因(PbFuc基因)，编码SEQ ID No.3的α-L-岩藻糖苷酶(PbFuc)。
SEQ ID No.2由1377个核苷酸组成，命名为重组α-L-岩藻糖苷酶基因(PbFuc-His基因)，编码SEQ ID No.4的重组α-L-岩藻糖苷酶(PbFuc-His)。
所述严格条件是在2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次5min，又于0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次15min。
其中，所述核酸分子可以为DNA，如cDNA、基因组DNA或重组DNA，所述核酸分子也可以为RNA，如mRNA或hnRNA。
上述相关生物材料中，C2)所述的含有编码上述蛋白质的核酸分子的表达盒(PbFuc基因表达盒)，是指能够在宿主细胞中表达PbFuc的DNA，该DNA不但可包括启动PbFuc转录的启动子，还可包括终止PbFuc转录的终止子。进一步，所述表达盒还可包括增强子序列。
上述相关生物材料中，所述载体可为质粒、黏粒、噬菌体或病毒载体。
上述相关生物材料中，所述微生物可为酵母、细菌、藻或真菌，如农杆菌。
本发明进一步还提供了上述相关生物材料的应用。
所述应用如下述任一所述：
E1)上述相关生物材料在制备或合成α-L-岩藻糖苷酶中的应用；
E2)上述相关生物材料在制备或合成岩藻糖基化合物的应用；
E3)上述相关生物材料在催化糖基供体和乳糖形成3’-岩藻糖基乳糖的应用。
具体的，所述岩藻糖基化合物可以为含岩藻糖基的寡糖、含岩藻糖基的蛋白质、含岩藻糖基的脂类、其他任意含岩藻糖基的化合物；
所述糖基供体为任何含岩藻糖基的化合物，如含岩藻糖基的任意链长的糖类、醇类、脂类、氨基酸或蛋白，具体可为2-氯-4-硝基苯基-α-L-岩藻糖苷(4-Nitrophenyl-α-L-fucopyranoside，简写为pNP-FUC)。
本发明还提供了制备上述蛋白质(α-L-岩藻糖苷酶)的方法。
本发明制备上述蛋白质的方法，包括将上述蛋白质的基因(即所述蛋白质的编码基因)导入到受体微生物中，得到表达上述蛋白质的重组微生物，培养所述重组微生物，表达得到上述蛋白质(α-L-岩藻糖苷酶)。
上述方法中，所述受体微生物为原核微生物。具体的，所述原核微生物为大肠杆菌。更具体的，所述大肠杆菌为大肠杆菌(Escherichia coli)BL21(DE3)。
上述方法中，所述蛋白质的基因可通过重组质粒pET-28a(+)-PbFuc导入到大肠杆菌表达菌株BL21(DE3)，得到重组菌BL21(DE3)-pET-28a(+)-PbFuc中；所述重组质粒pET-28a(+)-PbFuc是将载体pET-28a(+)的NheI和XhoI限制性酶切位点之间的DNA小片段替换为核苷酸序列是SEQ ID No.1第10-1278位所示的DNA分子，且保持pET-28a(+)载体其他序列不变得到的重组表达载体。其中，重组质粒pET-28a(+)-PbFuc中含有SEQ ID No.2的PbFuc-His基因，编码SEQ ID No.4的融合蛋白PbFuc-His，PbFuc-His是SEQ ID No.3所示的PbFuc的N端连接MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEF得到的融合蛋白。
上述方法中，得到的α-L-岩藻糖苷酶的最适pH为5.0，在pH 4.0-11.0范围内保持稳定，残余酶活大于80％，表现出良好的pH稳定性；最适温度为35℃，在40℃以下保持相对稳定，酶活能保持90％以上，超过45℃后酶活力迅速下降，且在35℃、40℃和45℃时的半寿期分别是555、51和2.8min。
本发明进一步提供了一种制备3’-岩藻糖基乳糖的方法。
本发明制备3’-岩藻糖基乳糖的方法，包括利用α-L-岩藻糖苷酶催化pNP-FUC和乳糖的步骤。
上述方法中，所述α-L-岩藻糖苷酶催化反应的pH值可为3.5-11.0，具体可为3.5、4、4.5、5、5.5、6、6.5、7、7.5、8、8.5、9、9.5、10、10.5、11.0；或上述任两个所述点值间的范围值如3.5-7.0；6.5-9.5；或7-9；或8-10；或8-8.5；或8.5-9内的pH值；
上述方法中，所述α-L-岩藻糖苷酶催化反应的温度为20-50℃；具体可为20℃、25℃、30℃、35℃、40℃、45℃、50℃；或上述任两个所述点值间的范围值如20-35℃；或35-50℃；或25-50℃；或30-40℃内的温度。
上述方法中，所述α-L-岩藻糖苷酶催化反应的时间，可为大于0s，具体可为5、10、15、30、45min；或1、2、3、4、5、6、7、8、9、10、24、48h；或上述两个所述点值间的范围如5～60min，1～48h内的时间。
上述方法中，所述α-L-岩藻糖苷酶催化反应的最适温度为35℃，最适pH为8.5，最适反应时间为3h，乳糖最适浓度为700mM，最适加酶量为0.5U/mL，pNP-FUC浓度为10mM，该条件下可高效合成3’-岩藻糖基乳糖，转化率为85％。
本发明进一步还提供了3’-岩藻糖基乳糖在促进益生菌增殖上的应用。
上述应用中，所述益生菌为德氏乳杆菌、干酪乳杆菌和长双歧杆菌。
具体的，所述德氏乳杆菌为德氏乳杆菌NRRL B-548，所述干酪乳杆菌为干酪乳杆菌AS.1.2435和干酪乳杆菌NRRL B-1922，所述长双歧杆菌为长双歧杆菌NRRL B-41409。
本发明利用高效热不对称性交错PCR(hiTAIL-PCR)从地杆菌属(Pedobacter sp.)扩增得到包含如SEQ ID No.1第10-1278位所示的α-L-岩藻糖苷酶基因(PbFuc)，并将其转入至E.coli BL21(DE3)得到一种重组菌，利用重组菌可制备得到α-L-岩藻糖苷酶(PbFuc)。进一步，本发明利用PbFuc催化pNP-FUC和乳糖合成3’-岩藻糖基乳糖，反应3h转化率为85％，对于大量制备3’-岩藻糖基乳糖具有重要的意义。另外，本发明以合成的3’-岩藻糖基乳糖为碳源体外培养四株益生菌(德氏乳杆菌NRRL B-548、干酪乳杆菌AS.1.2435、干酪乳杆菌NRRL B-1922和长双歧杆菌NRRL B-41409)，在48h后的生长量(OD 595)分别为0.467，0.362，0.375，和0.403。相同条件下低聚果糖对这四种益生菌的生长量(OD 595)分别为0.182，0.320，0.290，和0.333，表明3’-岩藻糖基乳糖对以上四株益生菌的增殖效果优于低聚果糖；另外，3’-岩藻糖基乳糖发酵四株有害菌(包括大肠杆菌ATCC 11775、鼠伤寒沙门氏菌AS1.1552、单增李斯特菌CICC 21635和金黄色葡萄球菌AS 1.1861)，发酵24h后的生长量(OD 595)分别为0.091，0.074，0.086和0.095，接近无糖组；而相同条件下低聚果糖发酵这四株有害菌24h后的生长量(OD 595)分别为0.179，0.186，0.199和0.109，表明3’-岩藻糖基乳糖对以上四株有害菌的增殖作用低于低聚果糖，表现出优良的益生活性。
附图说明
图1为以地杆菌(Pedobacter sp.)的基因组DNA为模板得到的保守序列的琼脂糖凝胶电泳结果图。
图2为以hiTAIL-PCR片段为模板得到的DNA片段的琼脂糖凝胶电泳结果图。
图3为PbFuc与29家族α-L-岩藻糖苷酶氨基酸的多重序列比对图。
图4为重组菌培养液的粗酶液的聚丙烯酰胺凝胶电泳(SDS-PAGE)结果图；其中，1为不加IPTG的对照组粗酶液；2为加IPTG实验组粗酶液。
图5为经镍柱纯化前后PbFuc粗酶液(1)和纯酶液(2)的SDS-PAGE图谱。
图6为PbFuc的最适pH(A)和pH稳定性(B)；其中，柠檬酸-柠檬酸三钠citrate(■)pH 3.0-6.0；PB缓冲液(□)pH 6.0-8.0；Tris-HCl缓冲液(◆)pH 6.0-9.0；CHES缓冲液pH(△)8.0-10.0,CAPS缓冲液(*)pH 10.0-11.0,Na 2HPO 4-NaOH缓冲液(●)pH 11.0-12.0。
图7为PbFuc的最适温度(A)、温度稳定性(B)和半寿期(C)。
图8为PbFuc催化转糖苷反应HPLC图。
图9为3’-岩藻糖基乳糖一级质谱图。
图10为3’-岩藻糖基乳糖NMR一级 1H谱(A)和 13C谱(B)图。
图11为PbFuc转糖苷活性的最适温度(A)、最适pH(B)、最适加酶量(C)、最适反应时间(D)和最适受体浓度(E)。
图12为以3’-岩藻糖基乳糖唯一碳源培养德氏乳杆菌NRRL B-548(A)、干酪乳杆菌NRRL B-1922(B)、干酪乳杆菌AS.1.2435(C)和长双歧杆菌NRRL B-41409(D)的生长曲线。
图13为以3’-岩藻糖基乳糖唯一碳源培养大肠杆菌ATCC 11775(A)、鼠伤寒沙门氏菌AS 1.1552(B)、单增李斯特菌CICC 21635(C)和金黄色葡萄球菌AS 1.1861(D)的生长曲线。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：重组α-L-岩藻糖苷酶编码基因表达质粒的构建
1、提取地杆菌(Pedobacter sp.)的基因组DNA并以其作为模板，以人工合成的简并引物fuDP-F：5′-ACNACNAARCAYCAYGAYGGNTTY-3′和fuDP-R：5′-RTTNACNARCATRTTNCCNCC-3′为引物，进行梯度PCR扩增(退火温度为40℃～60℃)，以1％的琼脂糖凝胶电泳对扩增产物进行检测，得到约700bp的保守序列，结果如图1。
梯度PCR扩增体系为：10×LA buffer 5.0μl，dNTP mix(2.5mmol/l)4.0μl，fuDP-F/R(10pmol/μl)各8.0μl，基因组DNA 1.0μl，LA Taq(5.0U/μl)0.5μl，ddH 2O up to 50.0μl；梯度PCR扩增程序为：95℃预变性3min；95℃变性30s，40-60℃退火30s，72℃延伸40s，34个循环；72℃ 5min。
2、将扩增得到的保守序列连接pMD19-T载体上，经测序得到保守序列的核苷酸序列。
3、根据得到保守序列的核苷酸序列，采用TAIL-PCR方法扩增基因上游5′端部分未知序列及下游3′端部分未知序列，分别设计基因上游与下游三条特异性引物对sp1(包 括Fsp1和Rsp1)，sp2(包括Fsp2和Rsp2)，sp3(包括Fsp3和Rsp3)。引物为嵌套式，sp2引物对位于sp1的内侧，而sp3引物对位于sp2的内侧，引物对sp1与sp2之间，sp2与sp3之间的距离为200bp左右。同时，根据高效热不对称性交错PCR(hiTAIL-PCR)方法，应用四条LAD引物(LAD1、LAD2、LAD3和LAD4)及一条嵌套的特异性引物AC通过三轮PCR扩增已知序列片段的侧翼序列。以地杆菌(Pedobacter sp.)基因组DNA为模板，进行第一轮(TAIL I)PCR扩增。TAIL I的PCR产物适当稀释后作为第二轮(TAIL II)PCR扩增反应的模板，TAIL II的PCR产物适当稀释后作为第三轮(TAIL III)PCR扩增反应的模板，最终得到hiTAIL-PCR片段。TAIL II与TAIL III使用相同的PCR扩增参数。设计的引物序列如表1所示，hiTAIL-PCR反应条件如表2所示。
表1 引物序列
Figure PCTCN2019122945-appb-000001
表2 hi TAIL-PCR反应条件
Figure PCTCN2019122945-appb-000002
Figure PCTCN2019122945-appb-000003
注：PCR product a表示上一轮的扩增产物。
4、将hiTAIL-PCR片段通过NCBI数据库ORF项目查询编码蛋白的开放阅读框(ATG启始，TAA或TGA终止)，并将多个编码蛋白序列在BLAST项目进行序列比对分析，从中选择一个与已报道α-L-岩藻糖苷酶同源性较高，同时新颖性较好的基因序列进行克隆表达。
5、根据扩增得到的hiTAIL-PCR片段的编码序列设计引物P1：5′-CCG GAATTCCAGGATTACACACCTACAGCCGC-3′(下划线为限制性内切酶EcoRI的酶切位点)和P2：5′-ATAAGAAT GCGGCCGCCTATCCAATCTCCAAAACAATCACCTG-3′(下划线为限制性内切酶NotI的酶切位点)，并进行PCR扩增，1％的琼脂糖凝胶电泳对扩增产物进行检测，结果如图2，得到如SEQ ID No.1的1294bp的DNA片段。
反应程序为：95℃预变性5min；95℃变性20s，58℃退火20s，72℃延伸30s，35个循环；72℃延伸10min。
6、通过胶回收试剂盒(AxyGen,America)回收目的片段，并使用限制性内切酶EcoRI和NotI双酶切DNA片段，并进行清洁回收酶切产物。同时，用限制性内切酶EcoRI和NotI双酶切载体pET-28a(+)(Novagen公司产品，产品目录号为69864-3CN)，回收约5300bp的载体片段。
7、将酶切产物与载体片段连接，转化大肠杆菌DH5α得到重组质粒pET-28a(+)-PbFuc。
根据测序结果，对重组质粒pET-28a(+)-PbFuc进行结构描述如下：将载体pET-28a(+)的EcoRI和NotI限制性酶切位点之间的DNA小片段替换为核苷酸序列是SEQ ID No.1第10-1278位所示的DNA分子，编码SEQ ID No.3的蛋白质。重组质粒pET-28a(+)-PbFuc中，SEQ ID No.1第10-1278位所示的DNA分子与载体上的包含His-tag标签(由6个组氨酸残基组成)的编码序列(SEQ ID No.2第1-108)在内的DNA分子融合形成SEQ ID No.2的DNA分子，最终表达的蛋白为SEQ ID No.4的融合蛋白。
进一步将SEQ ID No.3的蛋白质与PDB数据库中已知结构蛋白序列进行比对，得到SEQ ID No.3的蛋白质与GH29家族的α-L-岩藻糖苷酶的多重序列比对图。结果发现SEQ ID No.3的蛋白质与Paenibacillus thiaminolyticus来源的GH29家族α-L-岩藻糖苷酶(PDB登录号：6GN6)序列相似度最高(36.41％)，其次为多形拟杆菌(Bacteroides  thetaiotaomicron)来源GH29家族α-L-岩藻糖苷酶(BtFuc2970，PDB：2WVT，29.6％)。另外，又将SEQ ID No.3的蛋白质与UniProtKB/Swiss-Prot(swissprot)数据库蛋白序列进行比对，发现SEQ ID No.3的蛋白质与来源于人(Homo sapiens)的GH29家族α-L-岩藻糖苷酶(登录号：Q9BTY2)相似性为30.54％。其次为另一个来源于人(Homo sapiens)的GH29家族α-L-岩藻糖苷酶(31.28％，登录号：P04066)和一个来源于食蟹猕猴(Macaca fascicularis)的α-L-岩藻糖苷酶(31.08％，登录号：Q60HF8)。而与其他蛋白相似度较低(见图3)。
通过BLAST项目对SEQ ID No.3的蛋白质进行比对分析，结果表明该蛋白与已报道的GH29家族的α-L-岩藻糖苷酶同源性最高，因此，初步将其假定为GH29家族的α-L-岩藻糖苷酶，即将SEQ ID No.3的蛋白质命名为α-L-岩藻糖苷酶(PbFuc)，其编码基因为SEQ ID No.1第10-1278位所示，命名为α-L-岩藻糖苷酶基因(PbFuc基因)；将SEQ ID No.3的PbFuc的N端连接MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEF得到的SEQ ID No.4的融合蛋白(命名为重组α-L-岩藻糖苷酶，即PbFuc-His)，其编码基因为SEQ ID No.2所示，命名为重组α-L-岩藻糖苷酶基因，即PbFuc-His基因。
实施例2：重组α-L-岩藻糖苷酶基因的表达
一、重组菌株的构建及重组α-L-岩藻糖苷酶的表达
将重组质粒pET-28a(+)-PbFuc转化至大肠杆菌BL21(DE3)得到重组菌，将该重组菌命名为BL21(DE3)-pET-28a(+)-PbFuc。将BL21(DE3)-pET-28a(+)-PbFuc接入LB液体培养基中进行种子液培养，培养基含卡那霉素(50μg mL -1)，种子液接种量为1.5％(w/v)，固态培养基为含琼脂的LB固体平板。从固态培养基平板上挑取阳性转化子到液体培养基中，37℃培养12h，以1％的接种量转接到200mL LB培养基中，37℃培养，当培养液OD 600达到0.6-0.8时，加入IPTG(以加IPTG作为实验组，以不加IPTG为对照组)至终浓度1mmol L -1，于20℃诱导16h，离心收集细胞。然后将细胞重悬于缓冲液(20mmol L -1pH 8.0 Tris-HCl缓冲液，0.5mol L -1NaCl，20mmol L -1咪唑)中，超声破壁离心取上清液即为粗酶液。
聚丙烯酰胺凝胶电泳(SDS-PAGE)显示不加IPTG的对照组得到的粗酶液无目的蛋白表达，而加IPTG的实验组得到的粗酶液中有较高的目的蛋白表达(图4)。进一步通过实验测定实验组粗酶液具有水解pNP-FUC的能力，表明实验组具有较高的α-L-岩藻糖苷酶活性(表3中所示粗酶液有α-L-岩藻糖苷酶活性)，但对照组完全没有活性。
二、重组α-L-岩藻糖苷酶的纯化
基于pET-28a(+)质粒中含有编码His-Tag标签蛋白的序列，选择使用Ni-IDA亲和柱纯化重组蛋白。先用平衡缓冲液以1.0mL min -1流速(20mmol L -1pH 8.0 Tris-HCl缓冲液，0.5mol L -1NaCl，20mmol L -1咪唑)洗脱10个柱体积(5-10个柱体积均可)；将上述步骤一中粗酶液以0.5mL min -1流速上样；再用平衡缓冲液以1.0mL min -1流速洗脱至OD 280小于0.05，洗去杂蛋白；最后用洗脱液乙(20mmol L -1pH 8.0 Tris-HCl缓冲液，0.5mol L -1NaCl，100mmol L -1咪唑)洗脱，收集洗脱液乙过柱后的溶液，即为纯化的 重组蛋白液即为纯酶液。
将纯酶液用SDS-PAGE法(Laemmli UK.1970.Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227:680-685)检测蛋白纯度，结果如图5所示，其中M为低分子量标准蛋白质；1为粗酶液；2为纯酶液。结果表明，纯酶液得到明显的单一条带，分子量大小为50kDa，比酶活为26.3U/mL。重组α-L-岩藻糖苷酶纯化结果见表3。其中蛋白含量测定参照Lowry等(Lowry OH,Rosebrough NJ,Farr AL,Randall RJ.1951.Protein measurement with the folin phenol reagent.J Biol Chem 193:265-275)的方法，以牛血清白蛋白作为标准蛋白，酶活力测定方法同下述实施例3中步骤一。
表3 地杆菌α-L-岩藻糖苷酶(PbFuc)的纯化表
Figure PCTCN2019122945-appb-000004
a代表酶活测定条件：5mM pNP-FUC为底物，在50mM citrate缓冲液pH 5.0、35℃条件下反应20min。
b代表蛋白浓度使用Lowry法测定。
实施例3：重组α-L-岩藻糖苷酶(PbFuc)的酶学性质
一、PbFuc酶活力的定义及测定方法
α-L-岩藻糖苷酶活力测定参考Janet等的方法(Janet et al.,α-Fucosidases with different substrate specificities from two species of Fusarium.Appl Microbiol Biotechnol,2013,97:5371-5380.)。反应体系加入100μL 10mM pNP-FUC，100μL 0.05M pH 5.0柠檬酸-柠檬酸三钠缓冲液，10μL适当稀释的酶液，在35℃反应20min，最后加入200μL Na 2CO 3(1M)终止反应并震荡均匀。取200μL加入96孔板中，在405nm测吸光值。pNP标准品作标准曲线。酶活力定义：每分钟催化pNP-FUC生成1μmol pNP所需要的酶量为一个酶活力单位(U)。
二、PbFuc的酶学性质
(一)PbFuc的最适反应pH及pH稳定性
将PbFuc分别用不同pH值的缓冲体系稀释(citrate，3.0-6.0；PB，6.0-8.0；Tris-HCl，6.0-9.0；CHES，8.0-10.0)，然后在35℃条件下测定α-L-岩藻糖苷酶的酶活力，以酶活力最高点作为100％作图。
PbFuc pH稳定性测定：使用以上不同pH值的缓冲液和其他两种缓冲液(CAPS，10.0-11.0；Na 2HPO 4-NaOH，11.0-12.0)稀释酶液，将稀释好的酶液在25℃水浴锅中分别处理30min，然后迅速将样品置于冰水浴中冷却30min。处理完毕后，在最适温度下测定残余酶活力，以未经处理的酶液作为对照，最后计算残余酶活力占未处理对照酶活力的百分比。
结果表明，PbFuc的最适pH为5.0(见图6中A)，在pH 4.0-11.0范围内保持稳 定，残余酶活大于80％，表现出良好的pH稳定性(见图6中B)。
(二)最适反应温度、温度稳定性和半寿期
将PbFuc稀释适当倍数于50mmol L -1pH 5.0的citrate缓冲液中，然后分别在20-50℃不同温度下按照标准方法测定重组岩藻糖苷酶的酶活力。以酶活力最高点作为100％作图。
PbFuc温度稳定性测定：用50mmol L -1pH 5.0的柠檬酸-柠檬酸三钠将纯酶液稀释适当倍数分别在不同的温度下处理30min，然后置于冰水浴中冷却30min，最后按照标准方法测定残余酶活力，以未经处理酶的酶活力作为对照。
半寿期测定：将酶液用50mmol L -1pH 5.0的citrate缓冲液适当稀释后，分别置于35,40,45℃下处理0-4h，不同时间间隔取样。以未经处理的酶液作为对照，测定残余酶活力，最后计算残余酶活力占对照酶活力的百分比，计算酶在不同温度下酶活衰变至50％的时间。
结果表明，PbFuc最适温度为35℃(见图7中A)，在40℃以下保持相对稳定，酶活能保持90％以上，超过45℃后酶活力迅速下降(见图7中B)。该酶在35℃、40℃和45℃时的半寿期分别是555、51和2.8min(见图7中C)。
实施例4：PbFuc在制备3’-岩藻糖基乳糖中的应用
一、3’-岩藻糖基乳糖的合成及定量
合成条件如下：以pNP-FUC作为岩藻糖基供体，以乳糖为受体，加入适量重组α-L-岩藻糖苷酶，在适当pH的缓冲液中反应适当时间。反应液煮沸10min灭活，经0.22μm微孔滤膜过滤，利用HPLC进行分析。定量方法参考Birgitte等的方法(Birgitte et al.,Substrate specificity and transfucosylation activity of GH29 α-L-fucosidases for enzymatic production of human milk oligosaccharides[J].New Biotechnology,2018,41:34-45.)。产物的定量以纯化后的3’-岩藻糖基乳糖为标准品，建立3’-岩藻糖基乳糖浓度与相应HPLC信号峰面积之间的关系曲线。根据标准曲线计算产物中3’-岩藻糖基乳糖的浓度。
产物的摩尔转化率以pNP-FUC初始浓度为100％计算，公式如下：
摩尔转化率(％)＝产物物质的量/糖基供体物质的量×100％＝岩藻糖基乳糖(mM)/pNP-FUC(mM)×100％
HPLC定量分析条件如下：安捷伦1260高相液相色谱仪，配套RID检测器。色谱条件：Shodex-KS-802凝胶色谱柱，流动相为超纯水，流速为0.8mL/min，柱温65℃，RID检测器温度35℃。
结果表明，反应体系中加入重组α-L-岩藻糖苷酶(PbFuc)3h后，经HPLC分析发现产物中生成了比岩藻糖(单糖)和乳糖(二糖)聚合度更高的糖(图8)，表明PbFuc能够以pNP-FUC和乳糖为底物合成聚合度更高的转糖苷产物，证明该酶具有转糖苷活性，进一步对该转糖苷产物进行分离纯化、分子量测定和结构鉴定。
二、3’-岩藻糖基乳糖的纯化和结构鉴定
反应后的产物经真空减压浓缩，过阴阳离子树脂除盐和pNP，再经生物凝胶柱Bio-gel P2柱(1.2cm*110cm，超纯水，流速为0.3mL/min)，产物以1mL/管收集。 收集的产物经TLC分析验证纯度(展层剂为正丁醇：乙醇：水＝2:1:1，显色剂为甲醇：硫酸＝95:5)，冷冻干燥后的样品为白色粉末。
将样品溶解于纯水中，使用Thermo Scientific TMQ Exactive TM质谱仪，在ESI离子源positive-ion模式下，采集样品的高分辨率一级质谱图，测定该转糖苷产物的分子量。
将样品溶解于D 2O中并转移到核磁专用试管中，加入DSS(3-(三甲基硅基)-1-丙磺酸钠)作为内标，使用500MHz Varian VNMR SYSTEM TM设备，在298K温度条件下采集信号。在499.9MHz共振频率下获得样品的一维 1H谱，在125.7MHz共振频率获得样品的一维 13C谱，最终获得以DSS的信号为参考的化学位移。
结果表明，纯化后的产物在一级高分辨率质谱图中在质荷比(m/z)[M+Na] +为511.1611处呈现单一离子峰(图9)，表明该产物的分子量为488.1611，这与岩藻糖基乳糖的分子量(488.44)一致，表明该寡糖为岩藻糖基乳糖。
进一步通过NMR测定该岩藻糖基乳糖的结构，由一维 1H及 13C图谱可获得该化合物 1H质子的化学位移(见图10中A)和 13C的化学位移(见图10中B)。经数据比对，该化合物与Alcaligenes sp.来源的α-L-岩藻糖苷酶转糖苷产物一致(Murata et al.,.Enzymatic synthesis of alpha-L-fucosyl-N-acetyllac-tosamines and 3′-O-alpha-L-fucosyllactose utilizing alpha-L-fucosidases.Carbohydrate Research.1999,320:192-199.)，表明该化合物为3’-岩藻糖基乳糖(Fucα1,3-Galβ1,4-Glu)。
三、3’-岩藻糖基乳糖的制备
PbFuc制备3’-岩藻糖基乳糖，首先对反应条件包括温度、pH、加酶量、反应时间及乳糖浓度进行优化。确定了3’-岩藻糖基乳糖的最适制备条件：最适温度为35℃(图11中A)，最适pH为8.5(图11中B)，最适加酶量为0.5U/mL(图11中C)，最适反应时间为3h(图11中D)，受体最适浓度为700mM(图11中E)。
在以上条件下制备3’-岩藻糖基乳糖，经HPLC定量，产物的摩尔转化率(摩尔转化率(％)＝产物物质的量/糖基供体物质的量×100％＝岩藻糖基乳糖(mM)/pNP-FUC(mM)×100％)为85％(图11中E)。将以上反应产物经真空减压浓缩，过阴阳离子树脂除盐和pNP，再经生物凝胶柱Bio-gel P2柱。收集的产物经薄层层析法(TLC)检验纯度，经冷冻干燥后，最终获得样品为白色粉末。
实施例5：3’-岩藻糖基乳糖的益生活性
分别用添加1％3’-岩藻糖基乳糖的MRS培养基(培养双歧杆菌时需添加0.5g/L的L-半胱氨酸盐)培养十五株益生菌(鼠李糖乳杆菌AS 1.2466，德氏乳杆菌NRRL B-548，干酪乳杆菌NRRL B-1922，嗜酸乳杆菌NRRL B-4495，短乳杆菌NRRL B-4527，棒状乳杆菌NRRL B-4391，罗伊氏乳杆菌CICC6132，干酪乳杆菌AS.1.2435，德氏乳杆菌AS.1.2132，两歧双歧杆菌NRRL B-41410，短双歧杆菌NRRL B-41408，婴儿双歧杆菌NRRL B-41661，长双歧杆菌NRRL B-41409，青春双歧杆菌ATCC 15703)。以商业益生元低聚果糖(FOS)为阳性对照。分别用添加1％3’-岩藻糖基乳糖(3’-FL)的M 9培养基培养四株有害菌(大肠杆菌ATCC 11775、单增李斯特菌CICC 21635、鼠伤寒沙门氏菌AS 1.1552和金黄色葡萄球菌AS 1.1861)，以商业益生元低聚果糖(FOS)为 阳性对照。
使用酶标仪分别检测培养12，24，48和72h后的生长量(OD 595nm)，计算补充3’-岩藻糖基乳糖后菌体的增长量与无糖组OD 595的差值(δOD 595)。当δOD 595＞0.1，则认为3’-岩藻糖基乳糖对该菌有增殖作用，当δOD 595＜0.1时，则认为3’-岩藻糖基乳糖对该菌增殖效果不明显。
结果表明，与无糖组相比，合成的3’-岩藻糖基乳糖在体外可显著促进德氏乳杆菌NRRL B-548(L.delbrueckii subsp.bulgaricus NRRL B-548，Burgos-Rubio et al.,Kinetic Study of the Conversion of Different Substrates to Lactic Acid Using Lactobacillus bulgaricus.Biotechnol.Prog.2000,16,305-314.)、干酪乳杆菌AS 1.2435(L.casei subsp.casei AS 1.2435,Zhang et al.,D-Tagatose production by Lactococcus lactis NZ9000 Cells Harboring Lactobacillus plantarum L-arabinose Isomerase.Indian Journal of Pharmaceutical Education and Research.2017,51(2):288-294.董银苹,等.,乳酸杆菌及嗜热链球菌脉冲场凝胶电泳分子分型方法建立及应用.中华预防医学杂志,2011,45(12):1086-1089.)、干酪乳杆菌NRRL B-1922(L.casei subsp.casei NRRL B-1922,Mani-López et al.,Probiotic viability and storage stability of yogurts and fermented milks prepared with several mixtures of lactic acid bacteria.J.Dairy Sci.97:2578-2590)和长双歧杆菌NRRL B-41409(Bifidobacterium longum NRRL B-41409,Bialonska et al.,The Effect of Pomegranate(Punica granatum L.)Byproducts and Ellagitannins on the Growth of Human Gut Bacteria.J.Agric.Food Chem.2009,57,8344–8349)的增殖(δOD 595＞0.1，见图12)。以上四种益生菌在添加3’-岩藻糖基乳糖为唯一碳源发酵48h后的生长量(OD 595)分别为0.467，0.362，0.375，和0.403。相同条件下低聚果糖对这四株益生菌增殖48h后菌体的生长量(OD 595)分别为0.182，0.320，0.290，和0.333(见表4)。同时，利用3’-岩藻糖基乳糖发酵四株有害菌(包括大肠杆菌ATCC 11775、鼠伤寒沙门氏菌AS1.1552、单增李斯特菌CICC 21635和金黄色葡萄球菌AS 1.1861，见图13)，发酵24h后的生长量(OD 595)(见表5)分别为0.091，0.074，0.086和0.095，接近无糖组；而相同条件下低聚果糖发酵这四株有害菌24h后的生长量(OD 595)分别为0.179，0.186，0.199和0.109(见表5)，均高于3’-岩藻糖基乳糖。以上结果表明3’-岩藻糖基乳糖对四株益生菌的增殖效果优于低聚果糖，而对四株有害菌的增殖作用低于低聚果糖，表现出优良的益生活性。
而3’-岩藻糖基乳糖对另外十一株益生菌无明显促进增殖的作用(δOD 595＜0.1)，包括鼠李糖乳杆菌AS 1.2466、嗜酸乳杆菌NRRL B-4495、德氏乳杆菌AS.1.2132、短乳杆菌NRRL B-4527、罗伊氏乳杆菌CICC 6132、婴儿双歧杆菌NRRL B-41661、棒状乳杆菌NRRL B-4391、短双歧杆菌NRRL B-41408、乳双歧杆菌Bb-12，两歧双歧杆菌NRRL B-41410和青春双歧杆菌ATCC 15703(见表5)。
综上所述，PbFuc可高效合成3’-岩藻糖基乳糖，在寡糖合成中具有良好的应用前景。3’岩藻糖基乳糖对四株益生菌的增殖效果优于低聚果糖，而对四株有害菌的增殖作用低于低聚果糖，表现出优良的益生活性。
表4 以3’-岩藻糖基乳糖(3’FL)为唯一碳源发酵四株益生菌在不同时间的生长量(OD595)
Figure PCTCN2019122945-appb-000005
表5 以3’-岩藻糖基乳糖(3’FL)为唯一碳源发酵四株有害菌在不同时间的生长量(OD595)
Figure PCTCN2019122945-appb-000006
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范 围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
工业应用
本发明提供的α-L-岩藻糖苷酶(PbFuc)可高效合成3’-岩藻糖基乳糖，在寡糖合成中具有良好的应用前景。3’-岩藻糖基乳糖对四株益生菌的增殖作用优于低聚果糖，而对四株有害菌的增长量低于低聚果糖，表现出优良的益生活性。

Claims (11)

  1. 一种蛋白质，其特征在于，所述蛋白质为如下任一所示：
    A1)氨基酸序列为SEQ ID No.4的蛋白质；
    A2)氨基酸序列为SEQ ID No.3的蛋白质；
    A3)将SEQ ID No.3的蛋白质或SEQ ID No.4的蛋白质的N端或/和C端连接蛋白标签得到的融合蛋白；
    A4)将SEQ ID No.3的蛋白质或SEQ ID No.4的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与A1)或A2)所示的蛋白质具有90％以上的同一性且功能相同的蛋白质。
  2. 权利要求1所述的蛋白质的相关生物材料，其特征在于，所述相关生物材料为如下任一所示：
    C1)编码权利要求1所述的蛋白质的核酸分子；
    C2)含有C1)所述核酸分子的表达盒；
    C3)含有C1)所述核酸分子的重组载体；
    C4)含有C2)所述表达盒的重组载体；
    C5)含有C1)所述核酸分子的重组微生物；
    C6)含有C2)所述表达盒的重组微生物；
    C7)含有C3)所述重组载体的重组微生物；
    C8)含有C4)所述重组载体的重组微生物。
  3. 根据权利要求2所述的相关生物材料，其特征在于，C1)所述核酸分子为如下任一所示：
    B1)编码序列为SEQ ID No.1第10-1278位所示的DNA分子；
    B2)编码序列为SEQ ID No.2所示的DNA分子；
    B3)在严格条件下与B1)或B2)限定的DNA分子杂交，且编码权利要求1所述的蛋白质的DNA分子。
  4. 权利要求1所述的蛋白质在如下任一所示的应用：
    D1)权利要求1所述的蛋白质作为α-L-岩藻糖苷酶的应用；
    D2)权利要求1所述的蛋白质在制备或合成岩藻糖基化合物的应用；
    D3)权利要求1所述的蛋白质在催化糖基供体和乳糖形成3’-岩藻糖基乳糖的应用。
  5. 根据权利要求4所述的应用，其特征在于：所述岩藻糖基化合物为含岩藻糖基的寡糖、含岩藻糖基的蛋白质或含岩藻糖基的脂类。
  6. 权利要求2所述的相关生物材料在如下任一所示的应用：
    E1)权利要求2所述的相关生物材料在制备或合成α-L-岩藻糖苷酶中的应用；
    E2)权利要求2所述的相关生物材料在制备或合成岩藻糖基化合物的应用；
    E3)权利要求2所述的相关生物材料在催化糖基供体和乳糖形成3’-岩藻糖基乳糖的应用。
  7. 根据权利要求6所述的应用，其特征在于：所述岩藻糖基化合物为含岩藻糖基的寡糖、含岩藻糖基的蛋白质或含岩藻糖基的脂类。
  8. 制备权利要求1所述蛋白质的方法，其特征在于，所述方法包括：将权利要求1所述的蛋白质的基因导入到受体微生物中，得到表达权利要求1所述蛋白质的重组微生物，培养所述重组微生物，表达得到权利要求1所述的蛋白质。
  9. 一种制备3’-岩藻糖基乳糖的方法，其特征在于：包括利用权利要求1所述的蛋白质催化pNP-FUC和乳糖的步骤。
  10. 3’-岩藻糖基乳糖在促进益生菌增殖中或制备促进益生菌增殖产品中的应用。
  11. 根据权利要求10所述的应用，其特征在于：所述益生菌为德氏乳杆菌、干酪乳杆菌和长双歧杆菌。
PCT/CN2019/122945 2019-12-04 2019-12-04 岩藻糖苷酶及其相关生物材料与应用 WO2021109022A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2019/122945 WO2021109022A1 (zh) 2019-12-04 2019-12-04 岩藻糖苷酶及其相关生物材料与应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2019/122945 WO2021109022A1 (zh) 2019-12-04 2019-12-04 岩藻糖苷酶及其相关生物材料与应用

Publications (1)

Publication Number Publication Date
WO2021109022A1 true WO2021109022A1 (zh) 2021-06-10

Family

ID=76220932

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/122945 WO2021109022A1 (zh) 2019-12-04 2019-12-04 岩藻糖苷酶及其相关生物材料与应用

Country Status (1)

Country Link
WO (1) WO2021109022A1 (zh)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109640702A (zh) * 2016-09-07 2019-04-16 雀巢产品技术援助有限公司 用于婴儿和/或幼儿的包含低聚糖的营养组合物

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109640702A (zh) * 2016-09-07 2019-04-16 雀巢产品技术援助有限公司 用于婴儿和/或幼儿的包含低聚糖的营养组合物

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN 15 July 2019 (2019-07-15), ANONYMOUS: "alpha-L-fucosidase [Pedobacter sp. OV280]", XP055817857, retrieved from NCBI Database accession no. WP_116250522 *
MURATA, T. MORIMOTO, S. ZENG, X. WATANABE, S. USUI, T.: "Enzymatic synthesis of @a-l-fucosyl-N-acetyllactosamines and 3'-O-@a-l-fucosyllactose utilizing @a-l-fucosidases", CARBOHYDRATE RESEARCH, PERGAMON, GB, vol. 320, no. 3-4, 15 August 1999 (1999-08-15), GB, pages 192 - 199, XP004362845, ISSN: 0008-6215, DOI: 10.1016/S0008-6215(99)00156-1 *

Similar Documents

Publication Publication Date Title
JP5042431B2 (ja) Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍから単離された新たな酵素
JP6641179B2 (ja) トランスガラクトシル化活性を有するポリペプチド
Yin et al. Engineering of the Bacillus circulans β-galactosidase product specificity
Hung et al. Molecular and biochemical analysis of two β-galactosidases from Bifidobacterium infantis HL96
CN111133112A (zh) 唾液酸转移酶及其在生产唾液酸化低聚糖中的用途
Rhimi et al. Production of D-tagatose, a low caloric sweetener during milk fermentation using L-arabinose isomerase
CN110885809B (zh) α-L-岩藻糖苷酶及其相关生物材料与应用
CN109880813B (zh) 一种具有低聚半乳糖合成能力的β-葡萄糖苷酶及其表达菌株和应用
Park et al. Microbial production of palatinose through extracellular expression of a sucrose isomerase from Enterobacter sp. FMB-1 in Lactococcus lactis MG1363
CN104328098B (zh) 一种β-葡萄糖苷酶及其制备方法与应用
Huang et al. A novel β-galactosidase from Klebsiella oxytoca ZJUH1705 for efficient production of galacto-oligosaccharides from lactose
Neifar et al. A novel thermostable and efficient class II glucose isomerase from the thermophilic Caldicoprobacter algeriensis: biochemical characterization, molecular investigation, and application in high fructose syrup production
Delgado et al. Genetic and biochemical characterization of an oligo-α-1, 6-glucosidase from Lactobacillus plantarum
KR101091138B1 (ko) 류코노스톡 락티스로부터 유래된 글루칸수크라제 및 그 제조방법
Böhmer et al. Recombinant production of hyperthermostable CelB from Pyrococcus furiosus in Lactobacillus sp.
Zhu et al. Expression, characterization and structural profile of a heterodimeric β-galactosidase from the novel strain Lactobacillus curieae M2011381
Nishimoto et al. Identification of the putative proton donor residue of lacto-N-biose phosphorylase (EC 2.4. 1.211)
Zhang et al. A novel Lactococcus lactis l-arabinose isomerase for d-tagatose production from lactose
Zhang et al. Biotechnological production of D-tagatose from lactose using metabolically engineering Lactiplantibacillus plantarum
WO2021109022A1 (zh) 岩藻糖苷酶及其相关生物材料与应用
EP2211643B1 (en) L-arabinose isomerase for converting d-galactose into d-tagatose in a dairy product which contains d-galactose
CN113699087B (zh) 一种转化乳糖生成乳果糖的植物乳杆菌工程菌株及其构建方法与应用
Lu et al. Recent Progress on galactooligosaccharides synthesis by microbial β-galactosidase
CN112501049B (zh) 产转糖基活性β-半乳糖苷酶的开菲尔乳杆菌及制备的β-半乳糖苷酶生产低聚半乳糖的方法
Bıyıklı et al. Extracellular recombinant production of 4, 6 and 4, 3 α-glucanotransferases in Lactococcus lactis

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: 19954900

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19954900

Country of ref document: EP

Kind code of ref document: A1