CN110885809A - α -L-fucosidase and related biological material and application thereof - Google Patents

Abstract

The invention discloses α -L-fucosidase and related biological materials and application thereof, and discloses a protein, namely, A1) protein with an amino acid sequence of SEQ ID No.4, A2) protein with an amino acid sequence of SEQ ID No.3, A3) fusion protein obtained by connecting the N end or/and the C end of the protein of A1) or A2) with protein labels, and A4) protein obtained by substituting and/or deleting and/or adding one or more amino acid residues on the protein of SEQ ID No.3 or the protein of SEQ ID No.4, which has more than 90% of identity and the same function with the protein shown by A1) or A2).

Description

α -L-fucosidase and related biological material and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to α -L-fucosidase and related biological materials and application thereof.

Background

Fucosyllactose consists of one molecule of fucose and one molecule of lactose, the fucose residue can be linked to the galactose residue of lactose via α (1, 2) glycosidic bond, or glucose linked to the reducing end of lactose via α (1, 3) glycosidic bond Fucosyllactose is present in Human Milk in large amounts and has a variety of functional activities such as regulating intestinal flora, resisting adhesion of pathogenic bacteria, immunomodulation and promoting brain development (Yvan et al, Human Milk lipids: 2 ' -Fusosyllase (2 ' -FL) and Lact-N-Neotreose LNnT) in Infant formula, Nutrients, 2018, 10: 1161.) at present, 2 ' -glycosyllactose, either chemically or biosynthesized, has been approved by the European and US FDA for addition to Infant foods (Bych et al, products of HMOs-dietary-20156, biological Production, biological C.56. development of Fucosyllactose has therefore increased importance.

The enzymatic synthesis of oligosaccharides has the advantages of mild reaction conditions, good controllability and the like, and has been widely noticed in recent years (Bojarov & ltet al, Glycosidases in carbohydrate synthesis: while organic chemical sugars short. Chimia (Arau), 2011, 65, 65-70.) glycoside hydrolases and glycosyltransferases are two types of enzymes which are more widely used in oligosaccharide synthesis. α -L-fucosidase is an exoglycosidase which can specifically hydrolyze fucosyl oligosaccharides or fucose residues linked to fucosyl compounds. compared with fucosyltransferase, α -L-fucosidase has a wide source and high activity and can synthesize oligosaccharides using an economical raw material as a glycosyl donor (Lezyk, et al, Noconodal α -L-fucoidases Sofa for Production of oligosaccharides, and the yield of fucosyl oligosaccharides is limited by the main enzyme family of fucosidase, such as the polysaccharide family of polysaccharide oxidase, fucose-peroxidase, fucose-degrading, fucose-12, fucose-glucose-oxidase, fucose-glucose-12, fucose-glucose-oxidase, glucose-oxidase, glucose-oxidase, glucose-glucose.

3 '-fucosyllactose there is no report of natural occurrence of Fuc- α (1, 3) -Gal glycosidic bond at the end of a Human milk oligosaccharide nonasaccharide (Yamashita et al, Oligosaccharides of Human milk Mill.isolation and purification of Three New Disialylsystem Hexasaccharoides.Arch.Biochemi.Biophys, 1976, 174: 582-591.) there is a study of obtaining 3' -fucosyllactose by six steps of Chemical Modification of lactose, but using a large amount of toxic reagents, steps and low yield of products during the preparation process (Takamura.9, Chemical Modification of lactose. XV.Synthesis of O- α -andO- β -L-fucosyllactose- (1-3) -O-34-D-galactose-glycosyl-168- (fucose-3-galactose-168-galactose) and the results of the enzyme synthesis of fucosyllactose-3-fucose-3-lactose-3-galactose-34-galactose-3-carbohydrate-168-derived from fucosyllactose-fucose-2, and fucose-lactose-3-lactose-derived from Escherichia-lactose-2, No. 7, No.

To date, no study report on the probiotic activity of 3' -fucosyllactose exists.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a protein with transglycosidic activity participating in the synthesis of fucosyl compounds, so as to synthesize or prepare 3' -fucosyl lactose.

In order to solve the above problems, the present invention provides a protein, derived from geobacter (pedobacter sp.) and represented by any one of the following a1) -a 4):

A1) a protein having an amino acid sequence of SEQ ID No. 3;

A2) protein with an amino acid sequence of SEQ ID No. 4;

A3) connecting the protein of SEQ ID No.3 or the N end or/and the C end of the protein of SEQ ID No.4 with a protein tag to obtain a fusion protein;

A4) the protein with more than 90% of identity and function same as the protein shown in A1) or A2) is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in SEQ ID No.3 or the protein shown in SEQ ID No. 4.

Wherein, A1) is named as α -L-fucosidase (PbFuc), and SEQ ID No.3 consists of 422 amino acid residues.

A2) The protein shown is named as recombinant α -L-fucosidase (PbFuc-His), which is a fusion protein obtained by connecting MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEF at the N end of PbFuc of SEQ ID No.3, and SEQ ID No.4 consists of 458 amino acid residues.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

Among the above proteins, protein-tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate the expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

The invention further provides the application of the protein.

The application is as described in any one of the following:

D1) the application of the protein as α -L-fucosidase (PbFuc);

D2) the application of the protein in preparing or synthesizing fucosyl compounds;

D3) the use of the above protein in catalyzing the formation of 3' -fucosyllactose from a glycosyl donor and lactose.

Specifically, the fucosyl compound can be fucosyl-containing oligosaccharide, fucosyl-containing protein, fucosyl-containing lipid, or any other fucosyl-containing compound;

the glycosyl donor is any fucosyl-containing compound, such as any chain length saccharide, alcohol, lipid, amino acid or protein containing fucosyl, and specifically can be 2-chloro-4-Nitrophenyl- α -L-fucoside (4-Nitrophenyl- α -L-fucopyranoside, abbreviated as pNP-FUC).

The related biological materials of the above proteins are also within the scope of the present invention.

The related biological material of the protein provided by the invention is any one of the following C1) to C8):

C1) nucleic acid molecules encoding the above proteins;

C2) an expression cassette comprising the nucleic acid molecule of C1);

C3) a recombinant vector comprising the nucleic acid molecule of C1);

C4) a recombinant vector comprising the expression cassette of C2);

C5) a recombinant microorganism comprising the nucleic acid molecule of C1);

C6) a recombinant microorganism comprising the expression cassette of C2);

C7) a recombinant microorganism comprising the recombinant vector of C3);

C8) a recombinant microorganism comprising the recombinant vector of C4).

In the biological material, the nucleic acid molecule C1) is represented by the following B1) or B2) or B3):

B1) the coding sequence is DNA molecule shown as 10 th-1278 th site of SEQ ID No. 1;

B2) the coding sequence is DNA molecule shown in SEQ ID No. 2;

B3) a DNA molecule which hybridizes with the DNA molecule defined by B1) or B2) under strict conditions and codes the protein.

Wherein, SEQ ID No.1 consists of 1294 nucleotides, wherein, the 10 th to 1278 th positions are named α -L-fucosidase gene (PbFuc gene), and α -L-fucosidase (PbFuc) of SEQ ID No.3 is coded.

SEQ ID No.2 consists of 1377 nucleotides and is designated recombinant α -L-fucosidase gene (PbFuc-His gene) encoding recombinant α -L-fucosidase (PbFuc-His) of SEQ ID No. 4.

The stringent conditions are hybridization and washing of the membrane 2 times 5min at 68 ℃ in a solution of 2 XSSC, 0.1% SDS and 2 times 15min at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS.

The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA, or the nucleic acid molecule may be RNA, such as mRNA or hnRNA.

In the above-mentioned related biological materials, the expression cassette containing a nucleic acid molecule encoding α -L-fucosidase (PbFuc gene expression cassette) described in C2) refers to a DNA capable of expressing PbFuc in a host cell, and the DNA may include not only a promoter that initiates transcription of PbFuc but also a terminator that terminates transcription of PbFuc.

In the above-mentioned related biological materials, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

In the above-mentioned related biological materials, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.

The invention further provides application of the related biological material.

The application is as described in any one of the following:

E1) the application of the related biological material in preparing or synthesizing α -L-fucosidase;

E2) the application of the related biological material in preparing or synthesizing fucosyl compound;

E3) the use of the above-mentioned related biomaterials in catalyzing the formation of 3' -fucosyllactose from a glycosyl donor and lactose.

Specifically, the fucosyl compound can be fucosyl-containing oligosaccharide, fucosyl-containing protein, fucosyl-containing lipid, or any other fucosyl-containing compound;

the glycosyl donor is any fucosyl-containing compound, such as any chain length saccharide, alcohol, lipid, amino acid or protein containing fucosyl, and specifically can be 2-chloro-4-Nitrophenyl- α -L-fucoside (4-Nitrophenyl- α -L-fucopyranoside, abbreviated as pNP-FUC).

The invention also provides a method for preparing the protein (α -L-fucosidase).

The method for producing the protein of the present invention comprises introducing a gene of the protein into a recipient microorganism to obtain a recombinant microorganism expressing the protein, and culturing the recombinant microorganism to express the protein (α -L-fucosidase).

In the above method, the recipient microorganism is a prokaryotic microorganism. Specifically, the prokaryotic microorganism is escherichia coli. More specifically, the Escherichia coli is Escherichia coli (Escherichia coli) BL21(DE 3).

In the method, the α -L-fucosidase gene can be introduced into an escherichia coli expression strain BL21(DE3) through a recombinant plasmid pET-28a (+) -PbFuc to obtain a recombinant strain BL21(DE3) -pET-28a (+) -PbFuc, wherein the recombinant plasmid pET-28a (+) -PbFuc is a recombinant expression vector obtained by replacing a DNA fragment between NheI and XhoI restriction sites of a vector pET-28a (+) by a DNA molecule with a nucleotide sequence shown in SEQ ID No.1, positions 10-1278, and keeping the other sequence of the pET-28a (+) vector unchanged, wherein the recombinant plasmid pET-28a (+) -PbFuc contains a PbFuc-His gene of SEQ ID No.2, encodes a fusion protein PbFuc-His of SEQ ID No.4, and the PbFuc-His is a fusion protein obtained by connecting MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEF with the N terminal of the PbFuc shown in SEQ ID No. 3.

In the method, the obtained α -L-fucosidase has the optimum pH of 5.0, is stable in the pH range of 4.0-11.0, has the residual enzyme activity of more than 80 percent, shows good pH stability, has the optimum temperature of 35 ℃, is relatively stable at the temperature of below 40 ℃, can keep the enzyme activity of more than 90 percent, rapidly decreases the enzyme activity after the temperature exceeds 45 ℃, and has half-lives of 555, 51 and 2.8min at the temperature of 35 ℃, 40 ℃ and 45 ℃ respectively.

The invention further provides a method for preparing 3' -fucosyllactose.

The method for preparing 3' -fucosyllactose according to the present invention comprises a step of catalyzing pNP-FUC and lactose with α -L-fucosidase.

In the above method, the pH value of the α -L-fucosidase catalyzed reaction may 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 a range between any two of the above values, such as 3.5-7.0, 6.5-9.5, or 7-9, or 8-10, or 8-8.5, or a pH value within 8.5-9;

in the method, the temperature of the α -L-fucosidase catalyzed reaction is 20-50 deg.C, specifically 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, or the range between any two of the above values is 20-35 deg.C, or 35-50 deg.C, or 25-50 deg.C, or 30-40 deg.C.

In the method, the time of the catalytic reaction of the α -L-fucosidase can be more than 0s, specifically 5, 10, 15, 30 and 45min, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24 and 48h, or the time between the two values is within the range of 5-60 min and 1-48 h.

In the method, the optimum temperature of the catalytic reaction of α -L-fucosidase is 35 ℃, the optimum pH is 8.5, the optimum reaction time is 3h, the optimum concentration of lactose is 700mM, the optimum enzyme addition amount is 0.5U/mL, the concentration of pNP-FUC is 10mM, 3' -fucosyllactose can be efficiently synthesized under the condition, and the conversion rate is 85%.

The invention further provides application of the 3' -fucosyllactose in promoting proliferation of probiotics.

In the application, the probiotics are lactobacillus delbrueckii, lactobacillus casei and bifidobacterium longum.

Specifically, the lactobacillus delbrueckii is lactobacillus delbrueckii NRRL B-548, the lactobacillus casei is lactobacillus casei AS.1.2435 and lactobacillus casei NRRL B-1922, and the bifidobacterium longum is bifidobacterium longum NRRL B-41409.

The invention utilizes high-efficiency thermal asymmetric cross PCR (hiTAIL-PCR) to obtain a strain containing α -L-fucosidase gene (PbFuc) shown AS 10 th to 1278 th positions of SEQ ID No.1 by amplification from Geobacillus (Pedobacter sp.) and transfers the strain into E.coli BL21(DE3) to obtain a recombinant strain, and α -L-fucosidase (PbFuc) can be prepared by the recombinant strain, and further, the invention utilizes PbFuc to catalyze pNP-FUC and lactose to synthesize 3 '-fucosyllactose, the conversion rate of reaction 3h is 85%, and the strain has important significance for preparing 3' -fucosyllactose in large quantity, in addition, the invention utilizes the synthesized 3 '-fucosyllactose to culture four strains of probiotics in vitro (Lactobacillus delbrueckii NRRL B-548, Lactobacillus casei AS.1.2435, Lactobacillus casei NRRL-1922 and Bifidobacterium longum RL-41409) with the synthesized 3' -fucosyllactose AS a carbon source, the amounts of the four strains of probiotics (NRRL B-548, NRRL-41409, NO: 0.35, NO: 85, NO: 3650, NO: 1.2, NO: 3670: NO: 1: 11: NO: 1: NO: 1: NO: 1: 11: 1.

The α -L-fucosidase (PbFuc) provided by the invention can efficiently synthesize 3 '-fucosyllactose, has good application prospect in oligosaccharide synthesis, and the 3' -fucosyllactose has a better proliferation effect on four probiotics than fructo-oligosaccharide, and has a lower growth amount on four harmful bacteria than fructo-oligosaccharide, thereby showing excellent probiotic activity.

Drawings

FIG. 1 is a diagram showing the result of agarose gel electrophoresis of a conserved sequence obtained using genomic DNA of Geobacillus (Pedobacter sp.) as a template.

FIG. 2 shows the result of agarose gel electrophoresis of a DNA fragment obtained using the hitAIL-PCR fragment as a template.

FIG. 3 is a diagram showing multiple sequence alignments of PbFuc with amino acids of family 29 α -L-fucosidase.

FIG. 4 is a diagram showing the results of polyacrylamide gel electrophoresis (SDS-PAGE) of a crude enzyme solution of a culture solution of a recombinant bacterium; wherein, 1 is a control group crude enzyme solution without IPTG; 2 is crude enzyme solution of experimental group added with IPTG.

FIG. 5 shows SDS-PAGE patterns of crude PbFuc enzyme solution (1) and pure enzyme solution (2) before and after nickel column purification.

FIG. 6 shows the pH optimum (A) and pH stability (B) of PbFuc, wherein the pH of citric acid-trisodium citrate (■) is 3.0-6.0, the pH of PB buffer (□) is 6.0-8.0, the pH of Tris-HCl buffer (◆) is 6.0-9.0, the pH of CHES buffer (. DELTA.) is 8.0-10.0, the pH of CAPS buffer (. DELTA.) is 10.0-11.0, and Na₂HPO₄NaOH buffer (. cndot.) pH 11.0-12.0.

FIG. 7 shows the optimum temperature (A), temperature stability (B) and half-life (C) of PbFuc.

FIG. 8 is an HPLC chart of PbFuc catalyzed transglycosidation reaction.

FIG. 9 is a 3' -fucosyllactose primary mass spectrum.

FIG. 10 is 3' -fucosyllactose NMR Primary¹H spectrum (A) and¹³c spectrum (B) diagram.

FIG. 11 shows the optimum temperature (A), optimum pH (B), optimum enzyme amount (C), optimum reaction time (D) and optimum receptor concentration (E) for PbFuc transglycosidation activity.

FIG. 12 is a graph showing growth curves of Lactobacillus delbrueckii NRRL B-548(A), Lactobacillus casei NRRL B-1922(B), Lactobacillus casei AS.1.2435(C) and Bifidobacterium longum NRRL B-41409(D) cultured with 3' -fucosyllactose as a sole carbon source.

FIG. 13 is a growth curve of E.coli ATCC 11775(A), Salmonella typhimurium AS1.1552 (B), Listeria monocytogenes CICC 21635(C) and Staphylococcus aureus AS 1.1861(D) grown on 3' -fucosyllactose AS the sole carbon source.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1: construction of recombinant a-L-fucosidase encoding gene expression plasmid

1. Genomic DNA of geobacillus (Pedobacter sp.) was extracted and used as a template, and the DNA of the extracted geobacillus (geobacillus sp.) was purified using an artificially synthesized degenerate primer fuDP-F: 5 '-ACNACNAARCAYCAYGAYGGNTTY-3' and fuDP-R: 5 '-RTTNACNARCATRTTNCCNCC-3' as primer, and performing gradient PCR amplification (annealing temperature of 40-60 deg.C), detecting the amplification product by 1% agarose gel electrophoresis to obtain conserved sequence of about 700bp, the result is shown in FIG. 1.

The gradient PCR amplification system is as follows: 10 × LA buffer 5.0 μ l, dNTP mix (2.5mmol/1)4.0 μ l, fuDP-F/R (10pmol/μ l) each 8.0 μ l, genomic DNA 1.0 μ l, LA Taq (5.0U/pl)0.5 μ l, ddH₂O up to 50.0. mu.l; the gradient PCR amplification procedure was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 40-60 ℃ for 30s, extension at 72 ℃ for 40s, and 34 cycles; 5min at 72 ℃.

2. Connecting the conserved sequence obtained by amplification to a pMD19-T vector, and sequencing to obtain the nucleotide sequence of the conserved sequence.

3. According to the nucleotide sequence of the conserved sequence, amplifying the unknown sequence of the upstream 5 'end part and the unknown sequence of the downstream 3' end part of the gene by adopting a TAIL-PCR method, and respectively designing three specific primer pairs sp1 (comprising Fsp1 and Rsp1), sp2 (comprising Fsp2 and Rsp2) and sp3 (comprising Fsp3 and Rsp3) of the upstream and downstream of the gene. The primers are nested, the sp2 primer pair is positioned inside sp1, the sp3 primer pair is positioned inside sp2, the distance between the sp1 and the sp2 and the distance between the sp2 and the sp3 are about 200 bp. Meanwhile, according to the high-efficiency thermal asymmetric staggered PCR (hitAIL-PCR) method, four LAD primers (LAD1, LAD2, LAD3 and LAD4) and a nested specific primer AC were used to amplify flanking sequences of a known sequence fragment by three rounds of PCR. First round (TAIL I) PCR amplification was performed using Geobacillus (Pedobacter sp.) genomic DNA as a template. And properly diluting the PCR product of TAILI to be used as a template for the second round (TAIL II) of PCR amplification reaction, properly diluting the PCR product of TAIL II to be used as a template for the third round (TAIL III) of PCR amplification reaction, and finally obtaining the hitAIL-PCR fragment. TAIL II and TAIL III used the same PCR amplification parameters. The designed primer sequences are shown in Table 1, and the hitAIL-PCR reaction conditions are shown in Table 2.

TABLE 1 primer sequences

TABLE 2 HITAIL-PCR reaction conditions

Note: PCR product^aThe amplification products of the previous round are shown.

4. The hitAIL-PCR fragment is inquired about the open reading frame of the encoded protein (ATG start, TAA or TGA stop) through the ORF project of NCBI database, and a plurality of encoded protein sequences are subjected to sequence alignment analysis in the BLAST project, and one gene sequence which has higher homology with the reported α -L-fucosidase and better novelty is selected for cloning and expressing.

5. Designing a primer P1 according to the coding sequence of the amplified hitAIL-PCR fragment: 5' -CCGGAATTCCAGGATTACACACCTACAGCCGC-3' (restriction sites for the restriction enzyme EcoRI are underlined) and P2: 5' -ATAAGAATGC GGCCGCCTATCCAATCTCCAAAACAATCACCTG-3' (underlined is the restriction site of the restriction enzyme NotI), PCR amplification was performed, and 1% agarose gel electrophoresis was performed to detect the amplified product, resulting in FIG. 2, which gave a 1294bp DNA fragment as shown in SEQ ID No. 1.

The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 58 ℃ for 20s, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 10 min.

6. The target fragment was recovered by a gel recovery kit (AxyGen, America), and the DNA fragment was double-digested with restriction enzymes EcoRI and NotI, and the digested product was recovered by cleaning. Meanwhile, the vector pET-28a (+) (product of Novagen, catalog No. 69864-3CN) was double-digested with restriction enzymes EcoRI and NotI, and a vector fragment of about 5300bp was recovered.

7. The enzyme digestion product is connected with the carrier fragment, and escherichia coli DH5 α is transformed to obtain a recombinant plasmid pET-28a (+) -PbFuc.

According to the sequencing result, the structure of the recombinant plasmid pET-28a (+) -PbFuc is described as follows: the DNA fragment between the EcoRI and NotI restriction sites of the vector pET-28a (+) was replaced with a DNA molecule having the nucleotide sequence shown in positions 10-1278 of SEQ ID No.1, encoding the protein of SEQ ID No. 3. In the recombinant plasmid pET-28a (+) -PbFuc, DNA molecules shown in 10 th to 1278 th sites of SEQ ID No.1 are fused with DNA molecules containing a coding sequence (1 st to 108 th sites of SEQ ID No. 2) of a His-tag label (consisting of 6 histidine residues) on a vector to form DNA molecules of SEQ ID No.2, and finally expressed protein is fusion protein of SEQ ID No. 4.

Further alignment of the protein of SEQ ID No.3 with the known structural protein sequences of the PDB database gave a multiple sequence alignment of the protein of SEQ ID No.3 with α -L-fucosidase from the GH29 family, as a result of which the protein of SEQ ID No.3 was found to have the highest similarity (36.41%) to the sequence of the GH29 family α -L-fucosidase (PDB accession No.: 6GN6) derived from Paenibacillus thiolyticus, followed by Bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron) derived GH29 family α -L-fucosidase (BtFuc2970, PDB: 2WVT, 29.6%). furthermore, the protein of SEQ ID No.3 was also found to have a similarity to the protein of the ProtKB/oiss-Protissprot database (BtKB 2970, PDB: 8652 WVT, 29.6%) derived from human (Homopis found to have a homology: Swallowing the protein of SEQ ID No.3 with the protein of the human (Homopis) derived from Homopis initially assigned from Homopis found to have a 23, whereas the sequence of the fucosidase from the fucoidan 7-20, 9-H04031, 9-20-H9-20-fucosidase (Mac accession No. 7) derived from the fucoidan) derived from human fucoidan 9-31, 9-20) derived from the Homophilus family 4631, 9-20, 9-20-fucoidan.

The alignment analysis of the protein of SEQ ID No.3 by BLAST project shows that the protein has the highest homology with α -L-fucosidase of GH29 family, so it is assumed to be α -L-fucosidase of GH29 family primarily, namely, the protein of SEQ ID No.3 is named α -L-fucosidase (PbFuc), the coding gene of which is shown in 10 th-1278 th position of SEQ ID No.1 and is named α -L-fucosidase gene (PbFuc gene), and the N-terminal of PbFuc of SEQ ID No.3 is connected with MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEF to obtain the fusion protein of SEQ ID No.4 (named recombinant α -L-fucosidase, namely PbFuc-His), the coding gene of which is shown in SEQ ID No.2 and is named recombinant α -L-fucosidase gene, namely PbFuc-His gene.

Example 2: expression of recombinant a-L-fucosidase Gene

Construction of recombinant strain and expression of recombinant α -L-fucosidase

The recombinant plasmid pET-28a (+) -PbFuc is transformed into Escherichia coli BL21(DE3) to obtain a recombinant bacterium, and the recombinant bacterium is named as BL21(DE3) -pET-28a (+) -PbFuc. BL21(DE3) -pET-28a (+) -PbFuc was inoculated into LB liquid medium containing kanamycin (50. mu.g mL) for seed culture^-1) The inoculum size of the seed liquid was 1.5% (w/v), and the solid medium was an LB solid plate containing agar. Selecting positive transformant from solid culture medium plate, culturing at 37 deg.C for 12 hr, transferring to 200mL LB medium at 1%, culturing at 37 deg.C, and culturing in OD₆₀₀When reaching 0.6-0.8, IPTG (with IPTG added as experimental group and without IPTG added as control group) was added to a final concentration of 1mmol L^-1The cells were then harvested by centrifugation after 16h induction at 20 ℃. The cells were then resuspended in buffer (20mmol L)^-1pH8.0Tris-HCl buffer, 0.5mol L^-1NaCl，20mmol L^-1Imidazole), and performing ultrasonic wall breaking centrifugation to obtain supernatant, namely crude enzyme liquid.

Polyacrylamide gel electrophoresis (SDS-PAGE) showed that the crude enzyme solution obtained from the control group without IPTG had no expression of the target protein, while the crude enzyme solution obtained from the experimental group with IPTG had a higher expression of the target protein (FIG. 4). further, the crude enzyme solution from the experimental group was experimentally determined to have the ability to hydrolyze pNP-FUC, indicating that the experimental group had a higher activity of α -L-fucosidase (the crude enzyme solution shown in Table 3 had the activity of α -L-fucosidase), but the control group had no activity at all.

Purification of recombinant α -L-fucosidase

Based on the sequence encoding the His-Tag protein contained in the pET-28a (+) plasmid, the recombinant protein was selected for purification using a Ni-IDA affinity column. First, 1.0mL min of equilibration buffer was used^-1Flow rate (20mmol L)^-1Tris-HCl buffer pH8.0，0.5mol L^-1NaCl，20mmol L^-1Imidazole) elute 10 column volumes (all 5-10 column volumes); the crude enzyme solution obtained in the first step is added for 0.5mL min^-1Sampling at a flow rate; then equilibration buffer for 1.0mL min^-1Eluting at flow rate to OD₂₈₀Less than 0.05, washing off foreign protein; finally, eluent B (20mmol L)^-1pH8.0Tris-HCl buffer, 0.5mol L^-1NaCl，100mmolL^-1Imidazole) and collecting the solution of the eluent B passing through the column, namely the purified recombinant protein solution is the pure enzyme solution.

The protein purity of the purified enzyme solution was examined by SDS-PAGE (Laemmli UK.1970. clean of structural protein analysis of the assembly of the head of bacterial protein T4.Nature 227: 680-685), and the results are shown in FIG. 5, wherein M is a low molecular weight standard protein, 1 is a crude enzyme solution, and 2 is a purified enzyme solution, the results show that the purified enzyme solution gave a distinct single band with a molecular weight of 50kDa and a specific enzyme activity of 26.3U/mL, recombinant α -L-fucosidase purification results are shown in Table 3, wherein the protein content was determined by the method of Lowry et AL (Lowry OH, Rosebrough NJ, Farr AL, Randall RJ.1951.protein assay with the protein enzyme in the assembly of the reagent. J Biochem 193: the method of determining the protein content by bovine serum 275, and the method of the albumin assay was used as a standard protein assay, and the following step 3.

TABLE 3 purification Table of Deuterorhizobium α -L-fucosidase (PbFuc)

a represents enzyme activity determination conditions: 5mM pNP-FUC was used as a substrate, and the reaction was carried out in 50mM citrate buffer pH 5.0 at 35 ℃ for 20 min.

b represents protein concentration determined using the Lowry method.

Example 3: enzymatic Properties of recombinant a-L-fucosidase (PbFuc)

Method for defining and measuring activity of PbFuc enzyme

α -L-fucosidase Activity measurement reference Janet et al (Janet et al, α -Fucosidaseswitch differential requirements experiments f)rom two species of fusarium. appl microbiol Biotechnol, 2013, 97: 5371-5380.). The reaction system was added with 100. mu.L of 10mM pNP-FUC, 100. mu. L0.05M pH 5.0 citric acid-trisodium citrate buffer, 10. mu.L of an appropriately diluted enzyme solution, reacted at 35 ℃ for 20min, and finally 200. mu.L of Na was added₂CO₃(1M) the reaction was stopped and shaken to homogeneity. mu.L of the suspension was added to a 96-well plate, and absorbance was measured at 405 nm. pNP standards were plotted as standard curves. Definition of enzyme activity: the amount of enzyme required to catalyze the production of pNP-FUC to 1. mu. mol pNP per minute was one unit of enzyme activity (U).

Enzymatic properties of PbFuc

Optimum reaction pH and pH stability of (I) PbFuc

Respectively diluting PbFuc with buffer systems with different pH values (citrate, 3.0-6.0; PB, 6.0-8.0; Tris-HCl, 6.0-9.0; CHES, 8.0-10.0), measuring the enzyme activity of α -L-fucosidase at 35 deg.C, and plotting with the highest point of enzyme activity as 100%.

PbFuc pH stability assay: the above buffer solution with different pH values and other two buffer solutions (CAPS, 10.0-11.0; Na)₂HPO₄NaOH, 11.0-12.0), treating the diluted enzyme solution in a water bath kettle at 25 ℃ for 30min respectively, and then rapidly placing the sample in an ice water bath for cooling for 30 min. After the treatment is finished, measuring the residual enzyme activity at the optimum temperature, taking untreated enzyme liquid as a reference, and finally calculating the percentage of the residual enzyme activity in the untreated reference enzyme activity.

The results showed that PbFuc had an optimum pH of 5.0 (see A in FIG. 6), remained stable in the pH range of 4.0 to 11.0, had a residual enzyme activity of more than 80%, and exhibited good pH stability (see B in FIG. 6).

(II) optimum reaction temperature, temperature stability and half-life

PbFuc was diluted by an appropriate factor to50 mmol L^-1The enzyme activity of the recombinant fucosidase was measured in a citrate buffer at pH 5.0, at different temperatures of 20-50 ℃ according to standard methods. The highest point of enzyme activity was plotted as 100%.

PbFuc temperature stability assay: with 50mmol L^-1Citric acid-citric acid III of pH 5.0Sodium is used for diluting pure enzyme solution by proper times, treating the pure enzyme solution for 30min at different temperatures respectively, then placing the pure enzyme solution in ice water bath for cooling for 30min, and finally determining the residual enzyme activity according to a standard method, wherein the enzyme activity of untreated enzyme is used as a reference.

Half-life measurement: the enzyme solution was diluted with 50mmol L^-1After appropriate dilution with citrate buffer at pH 5.0, the solutions were treated at 35, 40, 45 ℃ for 0-4h, respectively, and sampled at different time intervals. And (3) determining the residual enzyme activity by taking untreated enzyme liquid as a control, finally calculating the percentage of the residual enzyme activity in the control enzyme activity, and calculating the time for the enzyme to decay to 50% at different temperatures.

The result shows that the optimum temperature of PbFuc is 35 ℃ (shown as A in figure 7), the PbFuc is relatively stable at the temperature below 40 ℃, the enzyme activity can be kept more than 90%, and the enzyme activity is rapidly reduced after the temperature exceeds 45 ℃ (shown as B in figure 7). The half-lives of the enzyme at 35 deg.C, 40 deg.C and 45 deg.C were 555, 51 and 2.8min, respectively (see C in FIG. 7).

Example 4: application of PbFuc in preparation of 3' -fucosyllactose

Synthesis and quantification of mono, 3' -fucosyllactose

The synthesis conditions were as follows, pNP-FUC as a fucosyl donor, lactose as an acceptor, an appropriate amount of recombinant α -L-fucosidase was added, the reaction was carried out for an appropriate time in a buffer solution of an appropriate pH, the reaction solution was boiled for 10min for inactivation, filtered through a 0.22 μm microporous membrane, and analyzed by HPLC, the quantitative method was referred to the method of Birgite et al (Birgite et al, Substrate specificity and transfection efficiency of GH29 a-L-fucoidases for enzyme production of human milk oligosaccharides [ J ] New Biotechnology, 2018, 41: 34-45.), the quantification of the product was carried out using purified 3 '-fucosyllactose as a standard, and a curve was established for the relationship between the 3' -fucosyllactose concentration and the corresponding HPLC signal peak area in the product.

The molar conversion of the product was calculated as pNP-FUC initial concentration of 100%, and the formula is as follows:

molar conversion (%). the amount of product material/amount of glycosyl donor material x 100% ═ fucosyllactose (mM)/pNP-fuc (mM) × 100%

The HPLC quantitative analysis conditions were as follows: an Agilent 1260 high-phase liquid chromatograph matched with an RID detector. Chromatographic conditions are as follows: Shodex-KS-802 gel chromatographic column, the mobile phase is ultrapure water, the flow rate is 0.8mL/min, the column temperature is 65 ℃, and the RID detector temperature is 35 ℃.

The results show that after recombinant α -L-fucosidase (PbFuc) is added into the reaction system for 3h, HPLC analysis shows that sugar with higher polymerization degree than that of fucose (monosaccharide) and lactose (disaccharide) is generated in the product (FIG. 8), which shows that PbFuc can synthesize a transglycosylation product with higher polymerization degree by using pNP-FUC and lactose as substrates, and the enzyme is proved to have transglycosylation activity, and further the transglycosylation product is subjected to separation and purification, molecular weight determination and structure identification.

Purification and structural identification of di, 3' -fucosyllactose

The reaction product was concentrated under reduced pressure in vacuo, desalted and pNP removed by anion and cation resin, and then subjected to Bio-gel column Bio-gel P2 (1.2 cm. multidot.110 cm, ultrapure water, flow rate 0.3mL/min) to collect the product at 1 mL/tube. The purity of the collected product was verified by TLC analysis (the spreading agent was n-butanol: ethanol: water 2: 1, the developing agent was methanol: sulfuric acid 95: 5) and the freeze-dried sample was a white powder.

The samples were dissolved in purified water using Thermo Scientific^TMQ Exactive^TMAnd (3) a mass spectrometer collects a high-resolution primary mass spectrogram of the sample in an ESI ion source positive-ion mode, and determines the molecular weight of the transglycosidation product.

Dissolving the sample in D₂O and transferred to a special nuclear magnetic test tube, DSS (3- (trimethylsilyl) -1-propanesulfonic acid sodium) is added as an internal standard, and 500MHz Varian VNMR SYSTEM is used^TMThe device collects signals under the condition of 298K temperature. One-dimensional acquisition of a sample at a resonance frequency of 499.9MHz¹H spectrum, one-dimensional of sample is obtained at resonance frequency of 125.7MHz¹³C spectrum, finally obtaining the chemical shift taking the signal of the DSS as the reference.

The results show that the purified product has a mass-to-charge ratio (M/z) [ M + Na ] in the first-order high-resolution mass spectrogram]⁺Is presented at 511.1611Single ion peak (figure 9) indicating molecular weight of the product is 488.1611, which is consistent with molecular weight of fucosyllactose (488.44), indicating that the oligosaccharide is fucosyllactose.

The structure of the fucosyllactose was further determined by NMR, from one dimension¹H and¹³the compound can be obtained by C pattern¹Chemical shifts of H protons (see A in FIG. 10) and¹³chemical shift of C (see B in FIG. 10). The compound was consistent with the Albaligenes sp. derived α -L-fucosidase transglycosidation product (Murata et al.,. enzymationcsynthesis of alpha-L-fucosylation-N-acetyl-desamines and 3' -O-alpha-L-fucosylation. carbohydrate research.1999, 320: 192-.

Preparation of tri, 3' -fucosyllactose

The preparation of 3' -fucosyllactose by PbFuc firstly optimizes the reaction conditions including temperature, pH, enzyme adding amount, reaction time and lactose concentration. The optimal preparation conditions of the 3' -fucosyllactose are determined: the optimum temperature was 35 ℃ (A in FIG. 11), the optimum pH was 8.5 (B in FIG. 11), the optimum enzyme addition amount was 0.5U/mL (C in FIG. 11), the optimum reaction time was 3h (D in FIG. 11), and the optimum acceptor concentration was 700mM (E in FIG. 11).

3' -fucosyllactose was prepared under the above conditions, and the molar conversion of the product (molar conversion (%) -amount of product substance/amount of glycosyl donor substance × 100% ═ fucosyllactose (mM)/pNP-fuc (mM) × 100%) was 85% as determined by HPLC (fig. 11, E). Vacuum concentrating the above reaction product, desalting with anion and cation resin and pNP, and purifying with biological gel column Bio-gel P2. The collected product was checked for purity by Thin Layer Chromatography (TLC), and after lyophilization, the sample was finally obtained as a white powder.

Example 5: probiotic activity of 3' -fucosyllactose

Fifteen probiotics (Lactobacillus rhamnosus AS 1.2466, Lactobacillus delbrueckii NRRL B-548, Lactobacillus casei NRRL B-1922, Lactobacillus acidophilus NRRL B-4495, Lactobacillus brevis NRRL B-4527, Lactobacillus coryniformis NRRL B-4391, Lactobacillus reuteri CICC6132, Lactobacillus casei AS.1.2435, Lactobacillus delbrueckii AS.1.2132, Bifidobacterium bifidum NRRL B-41410, Bifidobacterium breve NRRL B-41408, Bifidobacterium infantis NRRL B-41661, Bifidobacterium longum NRRLB-41409 and Bifidobacterium adolescentis ATCC15703) are respectively cultured by using MRS culture medium added with 1% 3' -fucosyllactose (0.5 g/L of L-cysteine is required when Bifidobacterium is cultured). Commercial prebiotic Fructooligosaccharides (FOS) was used as a positive control. Four harmful bacteria (escherichia coli ATCC 11775, listeria monocytogenes cic 21635, salmonella typhimurium AS1.1552, and staphylococcus aureus AS 1.1861) were cultured with M9 medium supplemented with 1% 3 '-fucosyllactose (3' -FL), respectively, with commercial prebiotic Fructooligosaccharide (FOS) AS a positive control.

Respectively detecting the growth amounts (OD595 nm) after culturing for 12, 24, 48 and 72h by using an enzyme-labeling instrument, and calculating the growth amount of thallus and sugar-free group OD after supplementing 3' -fucosyllactose₅₉₅Difference (δ OD)₅₉₅). When delta OD₅₉₅If the ratio is more than 0.1, the 3' -fucosyllactose is considered to have a proliferation effect on the bacterium, and if the ratio is delta OD₅₉₅If the ratio is less than 0.1, the effect of 3' -fucosyllactose on the growth of the bacterium is not significant.

The results show that the synthesized 3' -fucosyllactose significantly promotes Lactobacillus delbrueckii NRRL B-548(L.delbrueckii subsp. Bulgaricus NRRL B-548, Burgos-Rubio et al, Kinetic Study of the Conversion of Difference Sustrates to Lactic Acid Using Lactobacillus Bulgaricus Biotechnol. prog.2000, 16, 305. 314.), Lactobacillus casei AS1.2435(L.casei subsp. casei AS1.2435, Zhang et al, D-Tagatose promoter byLactobacillus lactis NZ9000 Cells Harboring Lactobacillus casei strain L-Lactobacillus strain L-arabinosomerase L-1927, Maryland Biotech. Streptococcus strain NRRL 1089, Maryland et al, Maryland Biotech. Lactobacillus casei J.S.J.P.J.P.J.P.J.P.P.S.J.P.S.J.P.No. Ser. No. 7, Maryland No.2, Maryland S.S.J.P.P.S.J.J.S.J.S.J.S.No. 7, Maryland No. 7, Marylactinic Acid, Maryland No. 7, Maryland No. Mitsuba-Mitsuba et al, (L.S. Mitsuba et al, mani-

Biological availability and storage stability of yogurts and transformed mills prepared with a partial mixture of lactic acid bacteria.J. Dairy Sci.97: 2578-₅₉₅> 0.1, see fig. 12). The growth amounts (OD595) of the above four probiotics after fermentation for 48h with 3' -fucosyllactose as the sole carbon source were 0.467, 0.362, 0.375, and 0.403, respectively. Under the same conditions, the growth amounts (OD595) of the four probiotics after proliferation for 48h by fructo-oligosaccharide are respectively 0.182, 0.320, 0.290 and 0.333 (see Table 4). Meanwhile, four harmful bacteria (including escherichia coli ATCC 11775, salmonella typhimurium AS1.1552, listeria monocytogenes cic 21635, and staphylococcus aureus AS 1.1861, see fig. 13) were fermented with 3' -fucosyllactose, and the growth amounts (OD595) (see table 5) after 24 hours of fermentation were 0.091, 0.074, 0.086, and 0.095, respectively, which were close to the sugar-free group; under the same conditions, the growth amounts (OD595) of the four harmful bacteria after 24 hours of fructo-oligosaccharide fermentation are respectively 0.179, 0.186, 0.199 and 0.109 (see Table 5), which are all higher than that of 3' -fucosyllactose. The results show that the proliferation effect of the 3 '-fucosyllactose on four probiotics is superior to that of fructo-oligosaccharide, and the proliferation effect on four harmful bacteria is lower than that of fructo-oligosaccharide, so that the 3' -fucosyllactose shows excellent probiotic activity.

While 3' -fucosyllactose has no obvious proliferation promoting effect (delta OD) on eleven other probiotics₅₉₅< 0.1), including Lactobacillus rhamnosus AS 1.2466, Lactobacillus acidophilus NRRL B-4495, Lactobacillus delbrueckii AS.1.2132, Lactobacillus brevis NRRL B-4527, Lactobacillus reuteri CICC6132, Bifidobacterium infantis NRRL B-41661, Lactobacillus coryniformis NRRL B-4391, Bifidobacterium breve NRRL B-41408, Bifidobacterium lactis Bb-12, Bifidobacterium bifidum NRRL B-41410 and Bifidobacterium adolescentis ATCC15703 (see Table 5).

In conclusion, PbFuc can efficiently synthesize 3' -fucosyllactose, and has good application prospect in oligosaccharide synthesis. The 3' fucosyllactose has a better proliferation effect on four probiotics than fructo-oligosaccharide, and has a lower proliferation effect on four harmful bacteria than fructo-oligosaccharide, thereby showing excellent probiotic activity.

TABLE 4 growth amounts of four probiotic bacteria fermented at different times (OD595) with 3 '-fucosyllactose (3' FL) as sole carbon source

TABLE 5 growth amounts of four harmful bacteria (OD595) cultured with 3 '-fucosyllactose (3' FL) as a sole carbon source at different times

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

SEQUENCE LISTING

<110> university of agriculture in China

<120> α -L-fucosidase and related biological material and application thereof

<130>GNCFY191297

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<170>PatentIn version 3.5

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<213> Geobacillus (Pedobacter sp.)

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Pro Gly Ser Gly Glu Trp Val Met Asn Asp Arg Lys Leu Thr Val His

35 40 45

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50 55 60

Glu Gln Trp Val Thr Met Ala Lys Asn Ala Gly Met Lys Tyr Ile Thr

65 70 75 80

Leu Ile Thr Arg His His Asp Gly Phe Ser Met Trp Asp Thr Lys Tyr

85 90 95

Ser Asp Phe Asn Ile Met Asn Thr Pro Tyr Lys Lys Asp Ile Val Lys

100 105 110

Met Met Ala Glu Glu Cys His Lys Gln Gly Ile Pro Leu Tyr Leu Tyr

115 120 125

Tyr Ser Leu Leu Asp Trp Arg Arg Glu Asp Tyr Pro His Glu Thr Gly

130 135 140

Arg Thr Gly Gln His Ser Gly Arg Thr Gly Lys Gly Asp Tyr Ala Ser

145 150 155 160

Tyr Leu Gln Phe Met Lys Asn Gln Leu Thr Glu Leu Leu Thr Asn Tyr

165 170 175

Gly Glu Ile Gly Gly Ile Trp Phe Asp Gly His Trp Asp Gln Thr Glu

180 185 190

Pro Glu Gly Ser Lys Asp Arg Thr Ser Arg Ile Asp Trp Lys Tyr Asn

195 200 205

Glu Ile Tyr Gly Leu Ile His Lys Leu Gln Pro Gln Cys Met Ile Gly

210 215 220

Asn Asn His His Leu Thr Pro Phe Ala Gly Glu Asp Phe Gln Met Phe

225 230 235 240

Glu Arg Asp Leu Pro Gly Glu Asn Lys Ser Gly Leu Ser Phe Gln Lys

245 250 255

Ala Ser Asp Lys Leu Pro Leu Glu Thr Cys Glu Thr Ile Ser Asn Ser

260 265 270

Trp Gly Tyr Asn Leu Ser Asp Thr Tyr Tyr Lys Ser Asn Lys Glu Leu

275 280 285

Val His Met Leu Val Lys Ala Ala Ser Leu Gly Ser Asn Leu Leu Leu

290 295 300

Asn Ile Gly Pro Met Pro Ser Gly Lys Ile Gln Pro Glu Phe Gln Asp

305 310 315 320

Arg Leu Ala Gly Leu Gly Asn Trp Leu Lys Ile Tyr Gly Glu Ser Ile

325 330 335

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340 345 350

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355 360 365

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370 375 380

Tyr Leu Leu Lys Asp Lys Ser Gln Val Asn Phe Thr Phe Lys Lys Ala

385 390 395 400

Lys Leu Ser Ile Thr Ser Ala Ala Ser Ala Ser Glu Pro Asp Gln Val

405 410 415

Ile Val Leu Glu Ile Gly

420

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50 55 60

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65 70 75 80

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85 90 95

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100 105 110

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115 120 125

Asp Thr Lys Tyr Ser Asp Phe Asn Ile Met Asn Thr Pro Tyr Lys Lys

130 135 140

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145 150 155 160

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165 170 175

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180 185 190

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195 200 205

Leu Thr Asn Tyr Gly Glu Ile Gly Gly Ile Trp Phe Asp Gly His Trp

210 215 220

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225 230 235 240

Trp Lys Tyr Asn Glu Ile Tyr Gly Leu Ile His Lys Leu Gln Pro Gln

245 250 255

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260 265 270

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275 280 285

Ser Phe Gln Lys Ala Ser Asp Lys Leu Pro Leu Glu Thr Cys Glu Thr

290 295 300

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305 310 315 320

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325 330 335

Asn Leu Leu Leu Asn Ile Gly Pro Met Pro Ser Gly Lys Ile Gln Pro

340 345 350

Glu Phe Gln Asp Arg Leu Ala Gly Leu Gly Asn Trp Leu Lys Ile Tyr

355 360 365

Gly Glu Ser Ile Tyr Gly Thr Lys Ala Gly Phe Ile Lys Pro Gln Ala

370 375 380

Trp Gly Ser Ile Thr Gln Lys Asp Asn Arg Ile Tyr Ile His Ile Val

385 390 395 400

Asp Gly Lys Thr Thr Ser Leu Glu Leu Glu Asn Val Pro Val Lys Lys

405 410 415

Ile Lys Lys Ala Tyr Leu Leu Lys Asp Lys Ser Gln Val Asn Phe Thr

420 425 430

Phe Lys Lys Ala Lys Leu Ser Ile Thr Ser Ala Ala Ser Ala Ser Glu

435 440 445

Pro Asp Gln Val Ile Val Leu Glu Ile Gly

450 455

Claims (10)

1. A protein, which is characterized in that the protein is any one of the following proteins:

A1) protein with an amino acid sequence of SEQ ID No. 4;

A2) a protein having an amino acid sequence of SEQ ID No. 3;

A3) connecting the protein of SEQ ID No.3 or the N end or/and the C end of the protein of SEQ ID No.4 with a protein tag to obtain a fusion protein;

A4) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues on the protein of SEQ ID No.3 or the protein of SEQ ID No.4, has more than 90 percent of identity and function with the protein shown by A1) or A2).

2. The protein-related biomaterial of claim 1, wherein the related biomaterial is any one of:

C1) a nucleic acid molecule encoding the protein of claim 1;

C2) an expression cassette comprising the nucleic acid molecule of C1);

C3) a recombinant vector comprising the nucleic acid molecule of C1);

C4) a recombinant vector comprising the expression cassette of C2);

C5) a recombinant microorganism comprising the nucleic acid molecule of C1);

C6) a recombinant microorganism comprising the expression cassette of C2);

C7) a recombinant microorganism comprising the recombinant vector of C3);

C8) a recombinant microorganism comprising the recombinant vector of C4).

3. The related biological material according to claim 2, wherein C1) the nucleic acid molecule is any one of:

B1) the coding sequence is DNA molecule shown as 10 th-1278 th site of SEQ ID No. 1;

B2) the coding sequence is DNA molecule shown in SEQ ID No. 2;

B3) a DNA molecule which hybridizes under stringent conditions with a DNA molecule defined in B1) or B2) and which encodes a protein according to claim 1.

4. Use of the protein of claim 1 in any one of the following:

D1) use of the protein of claim 1 as α -L-fucosidase;

D2) use of the protein of claim 1 for the preparation or synthesis of fucosyl compounds;

D3) use of a protein according to claim 1 for catalyzing the formation of 3' -fucosyllactose from a glycosyl donor and lactose.

5. Use of the relevant biomaterial of claim 2 in any of the following applications:

E1) use of the related biological material of claim 2 in the preparation or synthesis of α -L-fucosidase;

E2) use of the related biological material of claim 2 for the preparation or synthesis of fucosyl compounds;

E3) use of the related biomaterial of claim 2 to catalyze the formation of 3' -fucosyllactose from a glycosyl donor and lactose.

6. Use according to claim 4 or 5, characterized in that: the fucosyl compound is fucosyl-containing oligosaccharide, fucosyl-containing protein or fucosyl-containing lipid.

7. A method of producing the protein of claim 1, comprising: a recombinant microorganism expressing the protein of claim 1 obtained by introducing the gene of the protein of claim 1 into a recipient microorganism, and the protein of claim 1 is expressed by culturing the recombinant microorganism.

8. A method of preparing 3' -fucosyllactose, comprising: comprising the step of catalyzing pNP-FUC and lactose with the protein of claim 1.

Use of 3' -fucosyllactose for promoting proliferation of probiotic bacteria or for preparing product for promoting proliferation of probiotic bacteria.

10. Use according to claim 9, characterized in that: the probiotics are lactobacillus delbrueckii, lactobacillus casei and bifidobacterium longum.

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