CN110885809B

CN110885809B - alpha-L-fucosidase and related biological material and application thereof

Info

Publication number: CN110885809B
Application number: CN201911231875.1A
Authority: CN
Inventors: 江正强; 史然; 马俊文; 刘军; 闫巧娟; 刘海杰
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-12-04
Filing date: 2019-12-04
Publication date: 2021-08-17
Anticipated expiration: 2039-12-04
Also published as: CN110885809A

Abstract

The invention discloses an alpha-L-fucosidase and a related biological material and application thereof. The invention discloses a protein: 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) a fusion protein obtained by connecting protein tags to the N end or/and the C end of the protein of A1) or A2); 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). The invention further discloses a related biological material of the protein and application thereof. The protein provided by the invention can efficiently synthesize 3' -fucosyllactose, and has good application prospect in oligosaccharide synthesis.

Description

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

Technical Field

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

Background

Fucosyllactose is composed of one molecule of fucose and one molecule of lactose, and the fucose residue can be linked to the galactose residue of lactose through an alpha (1, 2) glycosidic bond, or to the glucose at the reducing end of lactose through an alpha (1, 3) glycosidic bond. Fucosyllactose is abundantly present in Human Milk and has a variety of functional activities such as regulation of intestinal flora, resistance to adhesion of pathogenic bacteria, immunomodulation and promotion of brain development (Yvan et al, Human Milk Oligosaccharides: 2 '-Fucosyllactone (2' -FL) and Lacto-N-Neotetraose (LNnT) in animal formulae Nutrients, 2018, 10: 1161). Currently, chemically synthesized or biosynthesized 2' -fucosyllactose has been approved by the FDA of the European Union and the United states for addition to infant food (Bych et al, Production of HMOs using microbial hosts-from cell engineering to large scale Production. Current Opinion in Biotechnology, 2019, 56C: 130-. Therefore, the development of a method for synthesizing fucosyllactose is of great significance.

The enzymatic synthesis of oligosaccharides has the advantages of mild reaction conditions, good controllability and the like, and has attracted much attention in recent years (Bojarov et al, Glycosidases in carbohydrate synthesis: hen organic chemistry wells short. Chimia (Aarau), 2011, 65, 65-70.). Glycoside hydrolases and glycosyltransferases are two classes of enzymes that are more commonly used in oligosaccharide synthesis. alpha-L-fucosidase is one kind of exo-glycoside hydrolase and can hydrolyze fucose residue connected to fucosyl oligosaccharide or fucosyl compound specifically. Compared with fucosyltransferase, alpha-L-fucosidase has a wide source, high activity and can be used as an economical raw material for oligosaccharide synthesis as a glycosyl donor (Lezyk, et al., Novel alpha-L-fucoidases from a Soil reagent for Production of functionalized Human Milk oligosaccharides. PLoS ONE, 2016: 1-18.). Currently, α -L-fucosidases are mainly classified into 4 families, which are Glycoside Hydrolases (GH)29, 95, 141 and 151 families, respectively. Depending on the reaction mechanism, α -L-fucosidases can be divided into retention and reversal types, with only the fucosidases of the GH29 and GH151 families having transglycosidic activity (birgite et al: substrate specificity and transduction activity of GH29 α -L-fucosidases for enzymatic products of human milk oligosaccharides, New Biotechnology, 2018, 41: 34-45; Elise et al, SYNTHESIS OF NEW FUCOSE-CONTA. International patent No. US20140228554A 1). To date, the main limiting factor in the synthesis of fucosyllactose using the transglycosidic activity of alpha-L-fucosidase is the low product yield. Therefore, it is of great significance to discover α -L-fucosidase having excellent properties and high transglycosidic activity.

3' -fucosyllactose there is no naturally occurring report that Fuc-alpha (1, 3) -Gal glycosidic linkages are present at the end of a Human milk oligosaccharide nonaose (Yamashita et al, Oligosaccharides of Human milk. isolation and catalysis of New Disialylsusystem Hexasaccharoids. Arch. biochem. Biophys., 1976, 174: 582-591). There are studies on obtaining 3 ' -fucosyllactose via six steps of reaction by Chemical Modification of lactose, but the preparation process uses a large amount of toxic reagents, the steps are complicated, and the product yield is low (Takamura.et al, Chemical Modification of lactose. XV. Synthesis of O- α -and O- β -L-glucopyranosyl- (1 → 3) -O- β -D-galactopyranosyl- (1 → 4) -D-glucopyranoses (3 ' -O-a-and3 ' -O- β -L-glucopyranosyls). CarbohydratedResearch, 1980: 84: 53-60). There are few reports on the Enzymatic synthesis of 3 ' -fucosyllactose, and only Alcaligenes sp. derived α -L-fucosidase has been used to synthesize 3 ' -fucosyllactose by its transglycosidic activity, with a yield of 34% (Murata et al,. enzymic synthesis of alpha-L-fucosyl-N-acetyl-lac-tosamides and3 ' -O-alpha-L-fucosylation utilization alpha-L-fucosidases. carbohydrate research.1999, 320: 192-. There are also few reports of functional activity of 3' -fucosyllactose. 3' -fucosyllactose was found to bind specifically as an antibody probe to the corresponding antigen in adenocarcinoma and embryonic carcinoma cells (Miyauchi et al, Anew fucosyl amplified on colon adenocarinoma and embryonic carcinoma cells Nature, 1982, 299: 168-169.).

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) -A4):

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) 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).

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

A2) The protein shown is named as recombinant alpha-L-fucosidase (PbFuc-His), which is a fusion protein obtained by connecting MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEF to the N terminal 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 alpha-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 compound containing fucosyl, such as saccharide, alcohol, lipid, amino acid or protein with any chain length containing fucosyl, and specifically can be 2-chloro-4-Nitrophenyl-alpha-L-fucoside (4-Nitrophenyl-alpha-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-1278 th position is named as alpha-L-fucosidase gene (PbFuc gene), and the alpha-L-fucosidase (PbFuc) of SEQ ID No.3 is coded.

SEQ ID No.2 consists of 1377 nucleotides and is designated as recombinant α -L-fucosidase gene (PbFuc-His gene) encoding the 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. Further, the expression cassette may also include an enhancer sequence.

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 alpha-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.

The invention also provides a method for preparing the protein (alpha-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, culturing the recombinant microorganism, and expressing the recombinant microorganism to obtain 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 gene of the alpha-L-fucosidase 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; the recombinant plasmid pET-28a (+) -PbFuc is a recombinant expression vector obtained by replacing a small DNA fragment between the NheI restriction sites and the XhoI restriction sites of a vector pET-28a (+) by a DNA molecule with the nucleotide sequence shown in the 10 th to 1278 th sites of SEQ ID No.1 and keeping other sequences 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 end of PbFuc shown in SEQ ID No. 3.

In the method, the optimal pH of the obtained alpha-L-fucosidase is 5.0, the alpha-L-fucosidase is stable within the pH range of 4.0-11.0, the residual enzyme activity is more than 80%, and good pH stability is shown; the optimum temperature is 35 ℃, the enzyme activity is kept relatively stable below 40 ℃, the enzyme activity can be kept above 90%, the enzyme activity is rapidly reduced after the temperature exceeds 45 ℃, and the half-lives at 35 ℃, 40 ℃ and 45 ℃ are 555, 51 and 2.8min 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 alpha-L-fucosidase.

In the above method, the pH of the reaction catalyzed by the α -L-fucosidase may be 3.5 to 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 foregoing values, e.g., 3.5-7.0; 6.5-9.5; or 7 to 9; or 8 to 10; or 8-8.5; or a pH within 8.5-9;

in the method, the temperature of the catalytic reaction of the alpha-L-fucosidase is 20-50 ℃; specifically, the temperature can be 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C; or a range between any two of the foregoing values, e.g., 20-35 ℃; or 35 to50 ℃; or 25 to50 ℃; or a temperature within 30-40 ℃.

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

In the method, the optimal temperature of the alpha-L-fucosidase catalyzed reaction is 35 ℃, the optimal pH is 8.5, the optimal reaction time is 3h, the optimal concentration of lactose is 700mM, the optimal enzyme addition amount is 0.5U/mL, and the concentration of pNP-FUC is 10mM, so that the 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 amplify from geobacillus (Pedobacter sp.) to obtain the gene (PbFuc) containing the alpha-L-fucosidase shown as the 10 th-1278 th site of SEQ ID No.1, and transfers the gene (PbFuc) into E.coli BL21(DE3) to obtain a recombinant bacterium, and the recombinant bacterium can be utilized to prepare the alpha-L-fucosidase (PbFuc). Further, the invention utilizes PbFuc to catalyze pNP-FUC and lactose to synthesize 3 '-fucosyllactose, the conversion rate of reaction for 3h is 85%, and the invention has important significance for preparing 3' -fucosyllactose in large scale. In addition, the invention uses the synthesized 3' -fucosyllactose as a carbon source to culture four probiotics (Lactobacillus delbrueckii NRRL B-548, Lactobacillus casei AS.1.2435, Lactobacillus casei NRRL B-1922 and Bifidobacterium longum NRRL B-41409) in vitro, and the growth amounts (OD595) after 48h are respectively 0.467, 0.362, 0.375 and 0.403. Under the same conditions, the growth amounts (OD595) of the fructo-oligosaccharide to the four probiotics are respectively 0.182, 0.320, 0.290 and 0.333, which shows that the proliferation effect of the 3' -fucosyllactose on the four probiotics is better than that of the fructo-oligosaccharide; in addition, the 3' -fucosyllactose ferments four harmful bacteria (including escherichia coli ATCC 11775, salmonella typhimurium AS1.1552, listeria monocytogenes CICC 21635 and staphylococcus aureus AS 1.1861), the growth amounts (OD595) after 24h of fermentation are 0.091, 0.074, 0.086 and 0.095 respectively, which are close to the sugar-free group; and the growth amounts (OD595) of the four harmful bacteria fermented by the fructo-oligosaccharide under the same condition for 24 hours are respectively 0.179, 0.186, 0.199 and 0.109, which shows that the proliferation effect of the 3' -fucosyllactose on the four harmful bacteria is lower than that of the fructo-oligosaccharide, and the fructo-oligosaccharide shows excellent probiotic activity.

The alpha-L-fucosidase (PbFuc) provided by the invention 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 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 the 29 family 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 citric acid-trisodium citrate (■) has a pH of 3.0-6.0; PB buffer (□) pH 6.0-8.0; Tris-HCl buffer (. diamond-solid.) pH 6.0-9.0; CHES buffer pH (. DELTA.) 8.0-10.0, CAPS buffer pH 10.0-11.0, 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 the TAIL I to be used as a template for the second round (TAIL II) of PCR amplification reaction, properly diluting the PCR product of the 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 compared and analyzed in the BLAST project, and one gene sequence which has higher homology with the reported alpha-L-fucosidase and better novelty is selected for cloning and expression.

5. Designing a primer P1 according to the coding sequence of the amplified hitAIL-PCR fragment: 5' -CCGGAATTCCAGGATTACACACCTACAGCCGC-3' (underlined restriction site for restriction enzyme EcoRI) 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 alpha 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, the DNA molecule shown in the 10 th to 1278 th sites of SEQ ID No.1 is fused with the DNA molecule containing the coding sequence (1 st to 108 th sites of SEQ ID No. 2) of His-tag label (composed of 6 histidine residues) on the vector to form the DNA molecule of SEQ ID No.2, and the finally expressed protein is the fusion protein of SEQ ID No. 4.

Further, the protein of SEQ ID No.3 is compared with the known structural protein sequences in the PDB database to obtain a multiple sequence alignment chart of the protein of SEQ ID No.3 and alpha-L-fucosidase of GH29 family. As a result, it was found that the protein of SEQ ID No.3 has the highest sequence similarity (36.41%) with the GH29 family α -L-fucosidase derived from Paenibacillus thiolyticus (PDB accession No.: 6GN6), and secondly with the GH29 family α -L-fucosidase derived from Bacteroides thetaiotaomicron (BtFuc2970, PDB: 2WVT, 29.6%). In addition, the protein of SEQ ID No.3 was aligned with the UniProtKB/Swiss-prot (swissprot) database protein sequence, and the similarity of the protein of SEQ ID No.3 to GH29 family α -L-fucosidase (accession number: Q9BTY2) derived from human (Homo sapiens) was found to be 30.54%. Followed by another human (Homo sapiens) derived GH29 family alpha-L-fucosidase (31.28%, accession number P04066) and one cynomolgus Macaca fascicularis (Macaca fascicularis) derived alpha-L-fucosidase (31.08%, accession number Q60HF 8). But less similar to other proteins (see figure 3).

The protein of SEQ ID No.3 is compared and analyzed through BLAST project, and the result shows that the protein has the highest homology with the reported alpha-L-fucosidase of GH29 family, so the protein is preliminarily assumed to be the alpha-L-fucosidase of GH29 family, namely the protein of SEQ ID No.3 is named as alpha-L-fucosidase (PbFuc), the coding gene thereof is shown as the 10 th to 1278 th positions of SEQ ID No.1 and is named as alpha-L-fucosidase gene (PbFuc gene); the N end of PbFuc of SEQ ID No.3 is connected with MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEF to obtain the fusion protein (named as recombinant alpha-L-fucosidase, namely PbFuc-His) of SEQ ID No.4, and the coding gene of the fusion protein is shown in SEQ ID No.2 and named as recombinant alpha-L-fucosidase gene, namely PbFuc-His gene.

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

Construction of recombinant strain and expression of recombinant alpha-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, adding IPTG (with IPTG as experimental group and without IPTG as control group) toFinal concentration 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, whereas 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 solutions of the experimental groups were experimentally determined to have the ability to hydrolyze pNP-FUC, indicating that the experimental groups had higher α -L-fucosidase activity (the crude enzyme solutions shown in Table 3 had α -L-fucosidase activity), but the control group had no activity at all.

Purification of di, recombinant alpha-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)^-1pH8.0Tris-HCl buffer, 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，100mmol L^-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 proteins and the assembly of the head of bacterial proteins T4.Nature 227: 680-685), and the results are shown in FIG. 5, in which M is a low molecular weight standard protein; 1 is crude enzyme solution; 2 is pure enzyme solution. The result shows that the pure enzyme solution obtains an obvious single band, the molecular weight is 50kDa, and the specific enzyme activity is 26.3U/mL. The results of purification of the recombinant α -L-fucosidase are shown in table 3. The protein content was determined by Lowry et AL (Lowry OH, Rosebrough NJ, Farr AL, Randall RJ.1951.protein measurement with the folin phenol reagent. J Biol Chem 193: 265) -275) using bovine serum albumin as the standard protein and the enzyme activity was determined as in the first step of example 3 below.

TABLE 3 purification Table of Deuterobacter alpha-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

A method for measuring the activity of alpha-L-fucosidase in reference to Janet et al (Janet et al, alpha-Fucosidases with differential functionalities from 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 the alpha-L-fucosidase at 35 ℃, and drawing by taking the highest point of the enzyme activity as 100%.

PbFuc pH stability assay: using the above different pH valuesThe buffer solution of (A) and two other 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^-1Diluting pure enzyme solution by proper times with citric acid-trisodium citrate with pH of 5.0, treating at different temperatures for 30min, cooling in ice water bath for 30min, and measuring residual enzyme activity according to standard method with untreated enzyme activity as control.

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 was used as a fucosyl donor, lactose was used as an acceptor, and an appropriate amount of recombinant α -L-fucosidase was added to react in a buffer solution of an appropriate pH for an appropriate time. The reaction solution was boiled for 10min for inactivation, filtered through a 0.22 μm microporous membrane, and analyzed by HPLC. The quantitative method is referred to the method of Birgite et al (Birgite et al, Substrate specificity and transfection activity of GH29 a-L-polynucleotides for enzymatic procedures of human milk oligosaccharides [ J ] New Biotechnology, 2018, 41: 34-45.). And (3) quantifying the product by taking the purified 3 '-fucosyllactose as a standard substance and establishing a relation curve between the concentration of the 3' -fucosyllactose and the peak area of a corresponding HPLC signal. The concentration of 3' -fucosyllactose in the product was calculated according to a standard curve.

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 3h of adding the recombinant alpha-L-fucosidase (PbFuc) into the reaction system, HPLC analysis shows that saccharides with higher polymerization degrees than fucose (monosaccharide) and lactose (disaccharide) are generated in the product (FIG. 8), which shows that PbFuc can synthesize a transglycosylation product with higher polymerization degrees by using pNP-FUC and lactose as substrates, and the enzyme is proved to have transglycosylation activity, and further performs separation and purification, molecular weight determination and structure identification on the transglycosylation product.

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^TM Q 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]⁺A single ion peak at 511.1611 (fig. 9) was present, indicating that the molecular weight of the product was 488.1611, which is consistent with the 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 data were compared and the compound was consistent with the Alcaligenes sp. derived α -L-fucosidase transglycosidation product (Murata et al.,. enzymic synthesis of alpha-L-fucosyl-N-acetyl-lipids and3 '-O-alpha-L-fucosylating alpha-L-fucosidases. carbohydrate research.1999, 320: 192. sup. 199.) indicating that the compound was 3' -fucosyllactose (Fuc α 1, 3-Gal β 1, 4-Glu).

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 NRRL B-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 > 0.1, 3' -fucosyllactose is consideredHas proliferation effect on the bacterium when 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 AS 1.2435(L.casei subsp. casei AS 1.2435, Zhang et al, D-galactose production Lactobacillus NZ9000 Cells Harboring Lactobacillus strain L-enamel L-associated strain L-Lactobacillus strain I.P. J.E.P.J.P.J.P.L.J.P.P.J.P.P.P.E.M.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.S.P.P.S.P.S.P.S.S.P.S. NRRL No. 7, NRRL No.2, NRRL No. 7, NRrl-D-beta.S. 3, D-beta.S. 3, Lactobacillus strain, D. 3, D-strain, Lactobacillus strain, D-strain, Lactobacillus strain, D-strain, D, S. S.S.S. S. S.S.S. S. S.S.S.S.S. S. S.S. S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.P.S.S.S.S.S.S.S.S.P.P.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.P.S.S.P.P.S.S.S.S.S.S.S.S.S.S.S.P.P.S.S.S.S.P.P.P.P.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.P.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.P.P.P.S.P.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.P.S.S.S.S., mani-

et al, biological availability and storage stability of yogurts and transformed mills prepared with a local 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). Simultaneously, fermenting four strains of harmful bacteria (including Escherichia coli ATCC 11775, Salmonella typhimurium AS1.1552, Listeria monocytogenes CICC 21635 and Staphylococcus aureus AS 1.1861, see figure 13) with 3' -fucosyllactoseThe growth after 24h (OD595) (see table 5) was 0.091, 0.074, 0.086 and 0.095, respectively, close to the sugarless 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> alpha-L-fucosidase and related biological material and application thereof

<130> GNCFY191297

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1294

<212> DNA

<213> Geobacillus (Pedobacter sp.)

<400> 1

ccggaattcc aggattacac acctacagcc gctaatttaa aacaaagagc atggtttaat 60

gatgcacggt ttgggttatt tatccactgg ggacctttta gcataccagg cagtggcgaa 120

tgggtaatga acgatagaaa gctgactgtt cataattata ccaaccttaa agattttttt 180

aaccctgttg agtttaatgc ggaacaatgg gtaactatgg caaagaatgc cggtatgaaa 240

tacattacgt taattaccag gcaccatgat ggctttagca tgtgggacac taaatattct 300

gatttcaaca ttatgaatac cccatataaa aaagatattg taaaaatgat ggctgaagaa 360

tgccataagc aaggtatacc attatactta tattactcct tactagattg gcgaagggaa 420

gattatccgc atgaaactgg ccgtactgga caacattcag gcaggacagg taaaggcgat 480

tatgccagtt atttgcagtt tatgaaaaac cagttgaccg agttattgac taattacggc 540

gaaataggcg gcatctggtt cgatggacat tgggatcaaa ctgaaccgga aggatcaaaa 600

gacagaacct cgcggataga ttggaaatat aatgagattt atggtttgat ccacaaattg 660

caaccacagt gtatgattgg taataaccac cacttaactc catttgcagg tgaagatttc 720

cagatgtttg aacgcgatct tcccggagaa aataagtcag gtttaagttt tcagaaagca 780

tcagataagc tcccattaga aacctgtgaa accatttcaa actcctgggg atataatctg 840

agtgatacct attacaaatc aaataaagaa ctggttcata tgctggttaa agcagcaagc 900

ttaggttcca atcttttact gaacattggt ccgatgccaa gtggtaaaat ccagccggag 960

tttcaggatc gtttggcggg tttgggcaat tggcttaaaa tttacggcga aagcatttac 1020

ggtacaaagg caggatttat taaaccacag gcctggggaa gcattaccca aaaagataat 1080

agaatttata tccatattgt agatggaaaa acgacatctc ttgagctgga gaatgtaccg 1140

gtaaagaaaa ttaaaaaggc atacttacta aaagataaaa gccaggtaaa ttttaccttt 1200

aaaaaagcaa aattatcaat taccagcgca gcaagcgctt cagaaccaga tcaggtgatt 1260

gttttggaga ttggataggc ggccgcattc ttat 1294

<210> 2

<211> 1377

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcca ggattacaca 120

cctacagccg ctaatttaaa acaaagagca tggtttaatg atgcacggtt tgggttattt 180

atccactggg gaccttttag cataccaggc agtggcgaat gggtaatgaa cgatagaaag 240

ctgactgttc ataattatac caaccttaaa gattttttta accctgttga gtttaatgcg 300

gaacaatggg taactatggc aaagaatgcc ggtatgaaat acattacgtt aattaccagg 360

caccatgatg gctttagcat gtgggacact aaatattctg atttcaacat tatgaatacc 420

ccatataaaa aagatattgt aaaaatgatg gctgaagaat gccataagca aggtatacca 480

ttatacttat attactcctt actagattgg cgaagggaag attatccgca tgaaactggc 540

cgtactggac aacattcagg caggacaggt aaaggcgatt atgccagtta tttgcagttt 600

atgaaaaacc agttgaccga gttattgact aattacggcg aaataggcgg catctggttc 660

gatggacatt gggatcaaac tgaaccggaa ggatcaaaag acagaacctc gcggatagat 720

tggaaatata atgagattta tggtttgatc cacaaattgc aaccacagtg tatgattggt 780

aataaccacc acttaactcc atttgcaggt gaagatttcc agatgtttga acgcgatctt 840

cccggagaaa ataagtcagg tttaagtttt cagaaagcat cagataagct cccattagaa 900

acctgtgaaa ccatttcaaa ctcctgggga tataatctga gtgataccta ttacaaatca 960

aataaagaac tggttcatat gctggttaaa gcagcaagct taggttccaa tcttttactg 1020

aacattggtc cgatgccaag tggtaaaatc cagccggagt ttcaggatcg tttggcgggt 1080

ttgggcaatt ggcttaaaat ttacggcgaa agcatttacg gtacaaaggc aggatttatt 1140

aaaccacagg cctggggaag cattacccaa aaagataata gaatttatat ccatattgta 1200

gatggaaaaa cgacatctct tgagctggag aatgtaccgg taaagaaaat taaaaaggca 1260

tacttactaa aagataaaag ccaggtaaat tttaccttta aaaaagcaaa attatcaatt 1320

accagcgcag caagcgcttc agaaccagat caggtgattg ttttggagat tggatag 1377

<210> 3

<211> 422

<212> PRT

<213> Geobacillus (Pedobacter sp.)

<400> 3

Gln Asp Tyr Thr Pro Thr Ala Ala Asn Leu Lys Gln Arg Ala Trp Phe

1 5 10 15

Asn Asp Ala Arg Phe Gly Leu Phe Ile His Trp Gly Pro Phe Ser Ile

20 25 30

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

35 40 45

Asn Tyr Thr Asn Leu Lys Asp Phe Phe Asn Pro Val Glu Phe Asn Ala

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

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

340 345 350

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

355 360 365

Thr Ser Leu Glu Leu Glu Asn Val Pro Val Lys Lys Ile Lys Lys Ala

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

<210> 4

<211> 458

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

20 25 30

Gly Ser Glu Phe Gln Asp Tyr Thr Pro Thr Ala Ala Asn Leu Lys Gln

35 40 45

Arg Ala Trp Phe Asn Asp Ala Arg Phe Gly Leu Phe Ile His Trp Gly

50 55 60

Pro Phe Ser Ile Pro Gly Ser Gly Glu Trp Val Met Asn Asp Arg Lys

65 70 75 80

Leu Thr Val His Asn Tyr Thr Asn Leu Lys Asp Phe Phe Asn Pro Val

85 90 95

Glu Phe Asn Ala Glu Gln Trp Val Thr Met Ala Lys Asn Ala Gly Met

100 105 110

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

115 120 125

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

130 135 140

Asp Ile Val Lys Met Met Ala Glu Glu Cys His Lys Gln Gly Ile Pro

145 150 155 160

Leu Tyr Leu Tyr Tyr Ser Leu Leu Asp Trp Arg Arg Glu Asp Tyr Pro

165 170 175

His Glu Thr Gly Arg Thr Gly Gln His Ser Gly Arg Thr Gly Lys Gly

180 185 190

Asp Tyr Ala Ser Tyr Leu Gln Phe Met Lys Asn Gln Leu Thr Glu Leu

195 200 205

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

210 215 220

Asp Gln Thr Glu Pro Glu Gly Ser Lys Asp Arg Thr Ser Arg Ile Asp

225 230 235 240

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

245 250 255

Cys Met Ile Gly Asn Asn His His Leu Thr Pro Phe Ala Gly Glu Asp

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

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

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) protein with an amino acid sequence of SEQ ID No. 3.

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:

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

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

D1) use of the protein of claim 1 as an 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 an 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.