Fucosidase and use thereof

Abstract

This application discloses a fucoside hydrolase, which is: a) containing the sequence shown in SEQ ID NO:2; or b) containing one or more mutants based on SEQ ID NO:2. The fucoside hydrolase of this application, as a catalyst in the synthesis of L-fucose, offers advantages such as inexpensive and readily available substrates, significantly reducing the industrial cost of L-fucose production. Furthermore, the reaction conditions are mild and environmentally friendly, and the operation of synthesizing L-fucose using the fucoside hydrolase is further simplified, demonstrating excellent prospects for industrial development.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, and in particular to a fucoside hydrolase and its applications. Background Technology

[0002] Fucose is a hexacarbon sugar, also known as 6-deoxygalactose or methylpentose. The vast majority of fucose found in nature is L-fucose; D-fucose is found only as a rare sugar in some glycosides. L-fucose is abundant in seaweed and gums, and also found in polysaccharides of some bacteria. As a component of the glycan chains in glycoproteins, fucose is widely present on the plasma membranes of various cell surfaces. Fucose has one less hydroxyl group on its sixth carbon atom than most hexacarbon sugars, making it less hydrophilic and more hydrophobic than other monosaccharides. In some blood group molecules, fucose can serve as a blood type marker. Fucose is the terminal sugar of many cell surface glycans and plays an important role in many physiological and pathological processes. For example, sialylated Lewis X has an L-fucose terminus, a tetrasaccharide expressed in leukocytes such as granulocytes and monocytes. This glycan is closely associated with tissue origins in various tumors, including pancreatic cancer, breast cancer, colon cancer, and lung cancer. However, the mechanisms of action of these glycans are not yet clear due to the complexity of their structures. Structural modifications to their terminal sugars (such as L-fucose), such as adding probes, can track the sugar's action process and thus study its mechanism of action. Furthermore, studies have shown that fucosylated substances play a crucial role in embryonic development and are involved in the organism's immune response. In particular, L-fucose has been found to be an important component of human breast milk, serving as a major component of human milk oligosaccharides (HMOs) and playing a vital role in the development of healthy children. L-fucose is an important starting material for the chemical and pharmaceutical industries and can also be used in the production of cosmetics and nutritional products. Therefore, developing green and efficient methods for the large-scale production of L-fucose is of great significance.

[0003] L-fucose is primarily derived from the polysaccharide fucoidan, a fucoidan monosulfate found in all common brown seagrasses, including those from the families Fucaceae and Laminariaceae. Currently, large-scale acquisition of L-fucose mainly relies on harvesting brown seagrass from the Fucaceae family, which is found globally and abundantly along the European coast of the Atlantic Ocean. For example, Japanese Patent JP2000351790 discloses methods for extracting fucoidan and for obtaining and separating oligosaccharides from the extracted fucoidan. However, large-scale harvesting of brown seagrass from coastlines raises environmental concerns and is subject to certain environmental regulations. Furthermore, purifying monosaccharides from complex oligosaccharide hydrolysis products often requires the use of toxic chemicals such as lead acetate and excessive organic solvents. In addition, the composition of oligosaccharides in seaweed is highly susceptible to seasonal variations, making the yield of L-fucose difficult to predict. Besides hydrolyzing fucoidan from brown seaweed, recent studies have shown that L-fucose can also be obtained via the hydrolysis of bacterial polysaccharides containing naturally occurring L-fucose. European Patent WO2012 / 034996A1 discloses a strain belonging to the Enterobacteriaceae family that can produce extracellular polysaccharides containing L-fucose. To produce L-fucose, the polysaccharide produced by the aforementioned strain is recovered and hydrolyzed, for example, by treatment with sulfuric acid or hydrochloric acid; however, this requires cumbersome purification and separation steps. In addition to extracting L-fucose from polysaccharide or oligosaccharide hydrolysates, several synthetic routes for L-fucose have been developed starting with other monosaccharides, such as L-arabinose, D-galactose, L-rhamnose, D-mannose, and D-glucose. Typically, these raw materials are expensive, and the synthetic reactions involve several protection and deprotection steps, resulting in low overall yields of the final product. Chinese patent CN112813118A and others report a method for synthesizing L-fucose using L-galactonide-1,4-lactone as a raw material, but the raw material price remains high. US patent US8642297B2 specifically presupposes a general fermentation method that anticipates using recombinant mannitol-1-dehydrogenase from celery (Apium graveolens) to produce L-fucose; however, it does not disclose specific operational examples or alternative enzymes for the reaction. BASF AG of Germany disclosed a biocatalytic method for synthesizing L-fucose from L-fucoitol (CN107454915A), first synthesizing L-fucoitol chemically from galactose, then catalyzing the synthesis of L-fucose with galactose oxidase and catalase. Although this reaction exhibits high substrate tolerance, the conversion rate is only 62%, and the raw material cost is high. Summary of the Invention

[0004] Currently, there is a lack of L-fucose synthesis methods with high industrial production feasibility. Therefore, it is necessary to develop an efficient L-fucose synthase and establish a green enzymatic synthesis process for L-fucose. This application proposes a method for preparing L-fucose by hydrolyzing relatively inexpensive fucoidan-lactose as a substrate under the action of a fucoside hydrolase. This invention provides an efficient fucoside hydrolase and its gene, as well as a recombinant expression vector and recombinant expression transformant containing the gene, the recombinant enzyme and its preparation method, and the application of the hydrolase and its mutants in the synthesis of L-fucose.

[0005] The specific technical solution of this application is as follows:

[0006] 1. A fucoside hydrolase, wherein the hydrolase is:

[0007] a) Contains a sequence as shown in SEQ ID NO:2 or a sequence as shown in SEQ ID NO:2; or

[0008] b) A mutant based on SEQ ID NO:2 containing one or more mutations.

[0009] 2. The hydrolase according to claim 1, wherein the amino acid sequence of the mutant contains an amino acid mutation at at least one, two, three, or four sites corresponding to F103, E283, G293, and D400 of SEQ ID NO:2.

[0010] 3. A fucoside hydrolase comprising the sequence as described in SEQ ID NO:4 or the sequence as described in SEQ ID NO:4.

[0011] 4. A nucleic acid molecule encoding any one of items 1 to 3 of the above.

[0012] 5. A nucleic acid molecule, wherein the nucleic acid molecule comprises the sequence shown in SEQ ID NO.1 or SEQ ID NO.3 or the sequence shown in SEQ ID NO.1 or SEQ ID NO.3.

[0013] 6. An expression vector comprising the nucleic acid molecule described in claim 4 or 5, wherein the expression vector is preferably a plasmid, granule, bacteriophage, or animal vector.

[0014] 7. A host cell comprising the expression vector described in item 6 or 7, wherein the host cell is preferably a bacterial, fungal, plant, or animal cell.

[0015] 8. The use of any one of items 1 to 3, the nucleic acid molecule described in item 4 or 5, the expression vector described in item 6, or the host cell described in item 7 in the production of L-fucose.

[0016] 9. A method for producing L-fucose, comprising:

[0017] L-fucose is obtained by hydrolyzing fucoidan lactose using any one of items 1 to 3.

[0018] 10. According to the method described in item 0, the hydrolysis reaction temperature is 20–60°C, or

[0019] The hydrolysis reaction occurs at pH 6.0-10.0, or

[0020] The concentration of fucose-lactose is 50mM-1000mM.

[0021] The effects of the invention

[0022] The fucoidan hydrolase of this application, used as a catalyst in the synthesis of L-fucose, offers advantages such as inexpensive and readily available substrates, significantly reducing the industrial cost of L-fucose production. Furthermore, the reaction conditions are mild and environmentally friendly, further simplifying the operation of synthesizing L-fucose using the fucoidan hydrolase, thus demonstrating excellent industrial development prospects. Additionally, the fucoidan hydrolase exhibits good substrate tolerance, improving substrate conversion rates and greatly shortening the production time of L-fucose in industrial production. Detailed Implementation

[0023] The present application will now be described in detail. While specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0024] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0025] In the following amino acid and polypeptide sequences, the uppercase letters I, P, V, Q, E, S, T, and W each represent an amino acid or its amino acid residues. The correspondence between the uppercase letters and amino acids is shown in Table 1.

[0026] Table 1. Amino acids and their corresponding capital letters.

[0027] Chinese name abbreviations Chinese name abbreviations glycine G Serine S alanine A threonine T Valine V Cysteine C Leucine L Asparagine N Isoleucine I glutamine Q proline P Aspartic acid D Phenylalanine F glutamic acid E Tryptophan W Lysine K Methionine M Arginine R Tyrosine Y Histidine H

[0028] This application provides a fucoidan hydrolase, wherein the hydrolase is:

[0029] a) Contains a sequence as shown in SEQ ID NO:2 or a sequence as shown in SEQ ID NO:2; or

[0030] b) A mutant based on SEQ ID NO:2 containing one or more mutations.

[0031] The amino acid sequence of SEQ ID NO:2 is as follows:

[0032] MKSTVILLLLLLAKAAVATAPAAGPYQPTWESLDQHPLPEWFDDAKF

[0033] GIFIHWGVYSVPAWAPRGGYAEWYWYNMQTPGSPVYEYHRKTYGEDFS

[0034] YKDFIPQFKAEKWDPEEWAELFKEAGAKYVVLTAEHHDGFALWDSKVT

[0035] DWNAVEMGPKRDIVGELAEAVRKRGMKFGLSYHGLLNWFNPDYPGNKS

[0036] DEPSEEYYAEYMLPQIKELIDKYQPDLLWFDGGWDNPAEYWRSKEILAY

[0037] YYNQAPKRPKEVVVNDRLGKGERKKHGDFYTPEYGTLADTSREHKWET

[0038] TRGIGHSFGYNRNEDEEDYMSAEELIHMLVDIVSKNGNLLLNVGPRADG

[0039] TIPEVQQERLREIGEWLKVNGEAIYGTRPWTSQQAGTTSATDVRFTAKDD

[0040] GLYAIVLDWPGDDIILLASDVTKGTSVSLLGSDGPLKWKQTGGGLVINLP

[0041] AAKPSDHACAYVFKIELKGKPA

[0042] The mutant refers to the amino acid sequence relative to SEQ ID NO:2, in SEQ ID NO:2

[0043] Based on the amino acid sequence, it contains changes at one or more positions, namely substitution, insertion and / or deletion, and still retains its activity.

[0044] The fucoside hydrolase described in this application can be a fucoside hydrolase ancestor enzyme constructed by ancestor sequence reconstruction, a fucoside hydrolase obtained by artificially synthesizing the full amino acid sequence, or a fucoside hydrolase obtained by cloning and expressing it through genetic engineering methods.

[0045] In this application, the ancestral sequence reconstruction (ASR) described is discussed in Randall et al. (Nat. Commun. 7: 12847 doi: 10.1038 / ncomms 12847 (2016)). The authors define ASR as "the process of analyzing modern sequences in an evolutionary / phylogenetic context to infer ancestral sequences at specific nodes in a tree." Ancestor sequence reconstruction (ASR) is used in molecular evolutionary studies. Unlike traditional methods of studying protein evolution by horizontally comparing related protein homologs at the ends of different branches of a phylogenetic tree, ASR probes statistically inferred ancestral proteins within tree nodes in a vertical manner. A phylogenetic tree is a branching graph showing evolutionary relationships between multiple biological species or other entities based on similarities and differences in their physical or genetic characteristics. In a rooted phylogenetic tree, each node with offspring represents the inferred most recent common ancestor of those offspring. In ASR, multiple related homologs of the target protein are selected and aligned using multiple sequence alignment (MSA) to construct a phylogenetic tree with statistically inferred sequences at the nodes of the branches. These sequences are known as "ancestors." The process of synthesizing the corresponding DNA, converting it into cells, and producing proteins is called "reconstruction."

[0046] Ancestor sequences are typically calculated using maximum likelihood, although the Bayesian method can also be performed. Since ancestors are inferred from phylogeny, the topology and composition of the phylogeny play a major role in the output ASR sequence. An ASR does not claim to reconstruct the actual sequence of an ancient protein / DNA, but rather a sequence likely similar to the sequence at that node. Maximum likelihood (ML) works by generating sequences where residues at each position are predicted to be most likely to occupy that position using the inference method used. Typically, this is a score matrix calculated from existing sequences (similar to those used in BLAST or MSA). Alternative methods include maximum parsimony (MP), which constructs sequences based on sequence evolution models, where the concept of the minimum number of nucleotide sequence changes typically represents the most efficient and most probable evolutionary pathway. MP is often considered the least reliable reconstruction method because it can oversimplify evolution to a degree unsuitable for a billion-year scale. Other methods include the Bayesian method, which involves considering residue uncertainties. Such methods are sometimes used to supplement ML methods, but they typically produce more ambiguous sequences (i.e., sequences containing residue positions for which definitive substitutions cannot be predicted). In such cases, multiple ASR sequences covering most of the ambiguity are usually generated and compared with each other. In some implementations, ancestor sequence reconstruction is performed using the online software FireProt-ASR (FireProt-ASR(muni.cz)).

[0047] The fucoside hydrolase of this application is derived from Xanthomonas. This fucoside hydrolase was obtained through ancestral sequence reconstruction, constructing an ancestral fucoside hydrolase, synthesizing the complete amino acid sequence of the fucoside hydrolase artificially, and cloning and expressing the resulting fucoside hydrolase using genetic engineering.

[0048] In constructing the ancestral enzyme of fucoside hydrolase, the cloned enzymes were repeatedly compared and screened by comparing the activities of the hydrolases, and finally the ancestral enzyme of hydrolase with the best catalytic performance was obtained, and its amino acid sequence is shown in SEQ NO:2.

[0049] After obtaining the optimal progenitor enzyme for the hydrolase, the full-length gene sequence was obtained through codon optimization based on the corresponding amino acid sequence. This gene sequence was then delivered to a gene synthesis company for artificial synthesis. After obtaining the gene, it was amplified using PCR, and the sequence was ligated into pET28a. The primers used are as follows:

[0050] Upstream primer:

[0051] 5'-gtgccgcgcggcagc catatgATGACTACAGATTCAAGGCAACTAGC-3'(SE Q ID NO:5)

[0052] Downstream primer:

[0053] 5'-acggagctcgaattc ggatcc TTACGGAGTCGGGGAAGACA-3'(SEQ ID NO:6)

[0054] In this sequence, the underlined portion of the upstream primer's nucleotide sequence represents the NdeI restriction site, and the underlined portion of the downstream primer represents the BamHI restriction site. Then, using the artificially synthesized gene as a template, polymerase chain reaction (PCR) was employed to amplify the gene, obtaining the complete full-length DNA fragment of the hydrolase. The full-length hydrolase gene (nucleotide sequence shown in SEQ NO:1 of the sequence listing) is named lfh and is 1380 nucleotides in length. Its coding sequence, from the first base to the 1380th base, has a start codon of ATG and a stop codon of TAA. This sequence contains no introns, and the amino acid sequence of the protein encoded by this gene is shown in SEQ NO:2 of the sequence listing.

[0055] The sequence of SEQ ID NO:1 is as follows:

[0056] ATGAAAAGCACCGTTATCCTGTTGCTGCTGTTGCTTGCTAAAGCCG

[0057] CCGTGGCGACTGCTCCAGCCGCAGGTCCTTACCAACCGACGTGGGAG

[0058] AGCCTGGATCAGCATCCTCTGCCGGAGTGGTTTGATGATGCTAAGTTTG

[0059] GTATCTTTATTCATTGGGGGGTCTACTCAGTACCAGCCTGGGCGCCGCG

[0060] TGGCGGTTACGCAGAATGGTACTGGTACAACATGCAGACACCGGGTAG

[0061] TCCCGTCTATGAATATCACCGCAAAACGTATGGGGAGGACTTCTCTTAC

[0062] AAAGACTTTATTCCGCAGTTTAAGGCCGAAAAAATGGGACCCTGAGGAA

[0063] TGGGCGGAGTTATTCAAAGAGGCCGGCGCCAAATACGTTGTTTTAACG

[0064] GCCGAACATCATGACGGCTTCGCCCTGTGGGACAGTAAGGTCACGGAC

[0065] TGGAATGCGGTCGAAATGGGGCCCAAGCGTGATATCGTGGGAGAGCTG

[0066] GCGGAGGCCGTACGCAAACGCGGTATGAAATTTGGATTGAGCTACCAC

[0067] GGCTTATTAAATTGGTTCAACCCCGATTATCCAGGCAATAAGTCGGACG

[0068] AGCCATCAGAAGAATATTACGCTGAATACATGCTTCCTCAGATTAAGGA

[0069] ATTGATCGACAAATACCAACCTGACTTGTTGTGGTTCGATGGTGGATGG

[0070] GATAATCCCGCAGAATACTGGCGTTCAAAGGAAATCCTGGCATACTACT

[0071] ACAATCAGGCGCCAAAGCGTCCCAAAGAAGTTGTCGTAAACGATCGCT

[0072] TAGGCAAGGGTGAACGTAAGAAGCACGGCGACTTTTATACTCCTGAGT

[0073] ACGGGACATTAGCTGATACGTCTCGCGAACACAAGTGGGAGACTACGC

[0074] GCGGGATTGGCCACTCTTTCGGCTACAACCGCAATGAGGACGAAGAG

[0075] GACTACATGAGCGCGGAGGAACTTATCCATATGCTGGTTGACATCGTGT

[0076] CAAAAAATGGTAACTTACTTCTGAACGTAGGTCCACGCGCAGATGGAA

[0077] CTATTCCCGAAGTGCAACAGGAGCGCTTACGCGAGATCGGTGAGTGGT

[0078] TGAAAGTGAACGGAGAGGCCATCTACGGGACACGCCCGTGGACAAGC

[0079] CAGCAAGCAGGTACAACGTCCGCTACGGATGTGCGTTTCACCGAAAG

[0080] GATGATGGGCTTTACGCAATCGTCTTGGATTGGCCTGGGGACGACATTA

[0081] TCTTATTGGCTTCCGATGTGACGAAAGGTACCAGTGTATCTTTGTTGGG

[0082] CTCCGATGGGCCGCTTAAATGGAAGCAAACTGGAGGAGGACTGGTTAT

[0083] CAATCTTCCAGCCGCAAAGCCCTCAGACCACGCCTGCGCCTACGTGTT

[0084] CAAAATTGAGTTGAAGGGTAAGCCCGCCTAA.

[0085] Due to codon degeneracy, nucleic acid molecules encoding the aforementioned hydrolases (amino acid sequences as shown in SEQ ID NO:2) are not limited to those with sequences as shown in SEQ ID NO:1. A polynucleotide homologue can also be provided by appropriately introducing substitutions, deletions, alterations, insertions, or additions of nucleotides.

[0086] In one specific embodiment, the amino acid sequence of the mutant contains an amino acid mutation at at least one of the sites F103, E283, G293 and D400 corresponding to SEQ ID NO:2, preferably containing an amino acid mutation corresponding to the D400 site and an amino acid mutation contained at the sites F103, E283, G293 and D400 corresponding to SEQ ID NO:2.

[0087] The term "corresponds" has the meaning commonly understood by those skilled in the art. Specifically, "corresponds" means the position in one sequence that corresponds to a specified position in another sequence after homology or sequence identity alignment.

[0088] In one specific embodiment, the amino acid sequence of the mutant is a mutation at the F103 site relative to the amino acid sequence shown in SEQ ID NO:2.

[0089] In one specific embodiment, the amino acid sequence of the mutant is a mutation at the E283 site relative to the amino acid sequence shown in SEQ ID NO:2.

[0090] In one specific embodiment, the amino acid sequence of the mutant is a mutation at the G293 site relative to the amino acid sequence shown in SEQ ID NO:2.

[0091] In one specific embodiment, the amino acid sequence of the mutant is a mutation at the D400 site relative to the amino acid sequence shown in SEQ ID NO:2.

[0092] In one specific embodiment, the amino acid sequence of the mutant is a mutation with only F103 and E283 sites relative to the amino acid sequence shown in SEQ ID NO:2.

[0093] In one specific embodiment, the amino acid sequence of the mutant is a mutation with only F103 and E283 sites relative to the amino acid sequence shown in SEQ ID NO:2.

[0094] In one specific embodiment, the amino acid sequence of the mutant is a mutation with only F103 and G293 sites relative to the amino acid sequence shown in SEQ ID NO:2.

[0095] In one specific embodiment, the amino acid sequence of the mutant is a mutation at only the F103 and D400 sites relative to the amino acid sequence shown in SEQ ID NO:2.

[0096] In one specific embodiment, the amino acid sequence of the mutant is a sequence with mutations at only the E283 and G293 sites relative to the amino acid sequence shown in SEQ ID NO:2.

[0097] In one specific embodiment, the amino acid sequence of the mutant is a sequence with mutations at only the E283 and D400 sites relative to the amino acid sequence shown in SEQ ID NO:2.

[0098] In one specific embodiment, the amino acid sequence of the mutant is a mutation at only the G293 and D400 sites relative to the amino acid sequence shown in SEQ ID NO:2.

[0099] In one specific embodiment, the amino acid sequence of the mutant is a mutation with only F103, E283 and G293 sites relative to the amino acid sequence shown in SEQ ID NO:2.

[0100] In one specific embodiment, the amino acid sequence of the mutant is a sequence with mutations at only the F103, E283, and D400 sites relative to the amino acid sequence shown in SEQ ID NO:2.

[0101] In one specific embodiment, the amino acid sequence of the mutant is a mutation with only F103, G293 and D400 sites relative to the amino acid sequence shown in SEQ ID NO:2.

[0102] In one specific embodiment, the amino acid sequence of the mutant is a sequence with mutations at only the E283, G293, and D400 sites relative to the amino acid sequence shown in SEQ ID NO:2.

[0103] In one specific embodiment, the amino acid sequence of the mutant is a sequence in which mutations only occur at the F103, E283, G293 and D400 sites relative to the amino acid sequence shown in SEQ ID NO:2.

[0104] In one specific embodiment, the amino acid sequence of the mutant is such that, relative to the amino acid sequence shown in SEQ ID NO:2, there is only a mutation that changes the F103 position to threonine (T). In another specific embodiment, the amino acid sequence of the mutant is such that, relative to the amino acid sequence shown in SEQ ID NO:2, there is only a mutation that changes the E283 position to serine (S).

[0105] In one specific embodiment, the amino acid sequence of the mutant is such that, relative to the amino acid sequence shown in SEQ ID NO:2, there is only a mutation that mutates the G293 site to alanine A.

[0106] In one specific embodiment, the amino acid sequence of the mutant is such that, relative to the amino acid sequence shown in SEQ ID NO:2, there is only a mutation that mutates the D400 site to alanine A.

[0107] In one specific embodiment, the amino acid sequence of the mutant is such that, relative to the amino acid sequence shown in SEQ ID NO:2, there are only two mutations: one at position F103 is mutated to threonine T and the other at position E283 is mutated to serine S.

[0108] In one specific embodiment, the amino acid sequence of the mutant is such that, relative to the amino acid sequence shown in SEQ ID NO:2, there are only two mutations: one at position F103 is mutated to threonine T and the other at position G293 is mutated to alanine A.

[0109] In one specific embodiment, the amino acid sequence of the mutant is such that, relative to the amino acid sequence shown in SEQ ID NO:2, there are only two mutations: one that mutates the F103 site to threonine T and the other that mutates the D400 site to alanine A.

[0110] In one specific embodiment, the amino acid sequence of the mutant is such that, relative to the amino acid sequence shown in SEQ ID NO:2, it contains only the mutations of F103 to threonine T, G283 to serine S, and G293 to alanine A.

[0111] In one specific embodiment, the amino acid sequence of the mutant is such that, relative to the amino acid sequence shown in SEQ ID NO:2, it contains only the mutations of changing the F103 site to threonine T, changing the G293 site to alanine A, and changing the D400 site to alanine A.

[0112] In one specific embodiment, the amino acid sequence of the mutant is such that, relative to the amino acid sequence shown in SEQ ID NO:2, it contains only the mutations of G283 to serine S, G293 to alanine A, and D400 to alanine A.

[0113] In one specific embodiment, the amino acid sequence of the mutant is such that, relative to the amino acid sequence shown in SEQ ID NO:2, it contains only the following mutations: mutation of F103 to threonine T, mutation of G283 to serine S, mutation of G293 to alanine A, and mutation of D400 to alanine A.

[0114] Its amino acid sequence is shown in SEQ ID NO:4.

[0115] The sequence of SEQ ID NO:4 is as follows:

[0116] MKSTVILLLLLLAKAAVATAPAAGPYQPTWESLDQHPLPEWFDDAKF

[0117] GIFIHWGVYSVPAWAPRGGYAEWYWYNMQTPGSPVYEYHRKTYGEDFS

[0118] YKDFIPQTKAEKWDPEEWAELFKEAGAKYVVLTAEHHDGFALWDSKVT

[0119] DWNAVEMGPKRDIVGELAEAVRKRGMKFGLSYHGLLNWFNPDYPGNKS

[0120] DEPSEEYYAEYMLPQIKELIDKYQPDLLWFDGGWDNPAEYWRSKEILAY

[0121] YYNQAPKRPKEVVVNDRLGKGERKKHGDFYTPEYGTLADTSRSHKWET

[0122] TRGIAHSFGYNRNEDEEDYMSAEELIHMLVDIVSKNGNLLLNVGPRADG

[0123] TIPEVQQERLREIGEWLKVNGEAIYGTRPWTSQQAGTTSATDVRFTAKDD

[0124] GLYAIVLDWPGDAIILLASDVTKGTSVSLLGSDGPLKWKQTGGGLVINLP

[0125] AAKPSDHACAYVFKIELKGKPA.

[0126] This application does not impose any restrictions on the mutation method. Mutation can be carried out according to conventional methods in the art, such as directed mutagenesis or construction of synthetic oligonucleotides, and then the mutated DNA sequence can be expressed in the host cell to obtain mutants with amino acid sequence substitution, insertion and / or deletion.

[0127] The hydrolase of this application has high enzyme activity, which improves the industrial potential of the enzyme.

[0128] The mutant of this application has more than 90% homology with SEQ ID NO:2.

[0129] This application provides a nucleic acid molecule encoding the aforementioned hydrolase. In one specific embodiment, the nucleic acid molecule comprises a sequence as shown in SEQ ID NO:1 or SEQ ID NO:3, or a sequence as shown in SEQ ID NO:3, or a sequence as shown in SEQ ID NO:1 or SEQ ID NO:3.

[0130] The sequence of SEQ ID NO:3 is as follows:

[0131] ATGAAAAGCACCGTTATCCTGTTGCTGCTGTTGCTTGCTAAAGCCG

[0132] CCGTGGCGACTGCTCCAGCCGCAGGTCCTTACCAACCGACGTGGGAG

[0133] AGCCTGGATCAGCATCCTCTGCCGGAGTGGTTTGATGATGCTAAGTTTG

[0134] GTATCTTTATTCATTGGGGGGTCTACTCAGTACCAGCCTGGGCGCCGCG

[0135] TGGCGGTTACGCAGAATGGTACTGGTACAACATGCAGACACCGGGTAG

[0136] TCCCGTCTATGAATATCACCGCAAAACGTATGGGGAGGACTTCTCTTAC

[0137] AAAGACTTTATTCCGCAGACCAAGGCCGAAAAATGGGACCCTGAGGA

[0138] ATGGGCGGAGTTATTCAAAGAGGCCGGCGCCAAATACGTTGTTTTAAC

[0139] GGCCGAACATCATGACGGCTTCGCCCTGTGGGACAGTAAGGTCACGGA

[0140] CTGGAATGCGGTCGAAATGGGGCCCAAGCGTGATATCGTGGGAGAGCT

[0141] GGCGGAGGCCGTACGCAAACGCGGTATGAAATTTGGATTGAGCTACCA

[0142] CGGCTTATTAAATTGGTTCAACCCCGATTATCCAGGCAATAAGTCGGAC

[0143] GAGCCATCAGAAGAATATTACGCTGAATACATGCTTCCTCAGATTAAGG

[0144] AATTGATCGACAAATACCAACCTGACTTGTTGTGGTTCGATGGTGGATG

[0145] GGATAATCCCGCAGAATACTGGCGTTCAAAGGAAATCCTGGCATACTAC

[0146] TACAATCAGGCGCCAAAGCGTCCCAAAGAGTTGTCGTAAACGATCGC

[0147] TTAGGCAAGGGTGAACGTAAGAAGCACGGCGACTTTTATACTCCTGAG

[0148] TACGGGACATTAGCTGATACGTCTCGCAGCCACAAGTGGGAGACTACG

[0149] CGCGGGATTGCACACTCTTTCGGCTACAACCGCAATGAGGACGAAGAG

[0150] GACTACATGAGCGCGGAGGAACTTATCCATATGCTGGTTGACATCGTGT

[0151] CAAAAAATGGTAACTTACTTCTGAACGTAGGTCCACGCGCAGATGGAA

[0152] CTATTCCCGAAGTGCAACAGGAGCGCTTACGCGAGATCGGTGAGTGGT

[0153] TGAAAGTGAACGGAGAGGCCATCTACGGGACACGCCCGTGGACAAGC

[0154] CAGCAAGCAGGTACAACGTCCGCTACGGATGTGCGTTTCACCGCAAAG

[0155] GATGATGGGCTTTACGCAATCGTCTTGATTGGCCTGGGGACGCAATTA

[0156] TCTTATTGGCTTCCGATGTGACGAAAGGTACCAGTGTATCTTGTTGGG

[0157] CTCCGATGGGCCGCTTAAATGGAAGCAAACTGGAGGAGGACTGGTTAT

[0158] CAATCTTCCAGCCGCAAAGCCCTCAGACCACGCCTGCGCCTACGTGTT

[0159] CAAAATTGAGTTGAAGGGTAAGCCCGCCTAA.

[0160] This application provides an expression vector comprising the aforementioned nucleic acid molecules.

[0161] In this application, the expression vector is constructed by cloning the above-mentioned hydrolase gene into the expression vector using conventional methods in the art. The expression vector includes various conventional vectors in the art, such as commercially available plasmids, granules, bacteriophages or viral vectors, etc., preferably pET-28a plasmid.

[0162] In this application, the aforementioned clay particles refer to clay particles.

[0163] For example, expression vectors can be prepared in the following manner:

[0164] The hydrolase gene product obtained by PCR amplification was digested with restriction endonucleases NdeI and BamHI. At the same time, the expression vector, such as pET-28a, was also digested with restriction endonucleases NdeI and BamHI to form complementary sticky ends. The digested hydrolase gene product and the digested expression vector, such as pET-28a plasmid, were recovered and ligated using T4 DNA ligase to construct an expression vector containing the hydrolase gene, such as pET28a-lfh.

[0165] This application provides a host cell that contains the above-described expression vector.

[0166] In this application, the host cell is a conventional host cell in the art, as long as the expression vector can stably replicate itself and the hydrolytic enzyme gene it carries can be effectively expressed. The host cell can be, for example, bacteria, fungi, plant cells, animal cells, etc.

[0167] The bacteria are preferably Escherichia coli, and more preferably Escherichia coli BL21(DE3) or Escherichia coli DH5α.

[0168] In this application, the expression vector, such as pET28a-lfh, can be transformed into a host cell, such as Escherichia coli BL21(DE3), to obtain the host cell, namely E.coli BL21(DE3) / pET28a-lfh.

[0169] This application provides a method for preparing fucoside hydrolase, which includes inoculating the above-mentioned host cells into a culture medium for fermentation to obtain a fermentation broth, centrifuging the fermentation broth to collect the cell bodies, and breaking the cell bodies to obtain fucoside hydrolase.

[0170] The culture medium can be any medium in the art that can grow the transformant and produce fucoside hydrolase. For example, the culture medium can be LB medium, preferably comprising: 5-15 g / L peptone, 1-10 g / L yeast extract, 5-15 g / L NaCl, and pH 6.0-8.0.

[0171] In this application, there are no special restrictions on the culture methods and conditions. Appropriate selections can be made based on factors such as host cell type and culture method, according to common knowledge in the art, as long as the transformant can grow and produce fucoside hydrolase. Other specific operations for culturing transformants can be performed in accordance with conventional operations in the art.

[0172] For example, the strain culture method includes: inoculating the host cells (e.g., E. coli BL21(DE3)) into LB medium containing kanamycin and culturing them. When the optical density OD600 of the culture medium reaches 0.6-0.8 (preferably 0.6), fucoside hydrolase can be efficiently expressed under the induction of isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.1-1.0 mmol / L (preferably 0.2 mmol / L).

[0173] This application provides the use of the above-mentioned fucoside hydrolase, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, or the above-mentioned host cell in the production of L-fucose.

[0174] This application provides a method for producing L-fucose, comprising:

[0175] L-fucose is obtained by hydrolyzing fucoidan-lactose using the aforementioned fucoside hydrolase. In one specific embodiment, the fucoidan-lactose is 2-fucolactose.

[0176] In one specific embodiment, the hydrolysis reaction temperature is 20–60°C, for example, 20°C, 23°C, 25°C, 28°C, 30°C, 33°C, 35°C, 38°C, 40°C, 43°C, 45°C, 48°C, 50°C, 53°C, 55°C, 58°C, or 60°C. Preferably, it is 30–50°C.

[0177] In one specific embodiment, the pH of the hydrolysis reaction is 6.0 to 10.0, for example, 6.0, 6.3, 6.5, 6.8, 7.0, 7.3, 7.5, 7.8, 8.0, 8.3, 8.5, 8.8, 9.0, 9.3, 9.5, 9.8, or 10.0. Preferably, it is 7.0 to 8.0.

[0178] In one specific embodiment, the concentration of fucose-lactose is 50mM-1000mM, for example, it can be 50mM, 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 550mM, 600mM, 650mM, 700mM, 750mM, 800mM, 850mM, 900mM, 950mM, or 1000mM.

[0179] In one specific embodiment, in a buffer system, the above-mentioned fucoside hydrolase is used to hydrolyze fucose lactose to obtain L-fucose. Preferably, the buffer is sodium phosphate buffer, Tris-HCl buffer, or glycine-NaOH buffer.

[0180] In one specific embodiment, the above-mentioned fucoidan hydrolase is dissolved in a buffer solution, and fucoidan-lactose is added to a final concentration of 50 mM-1000 mM. The reaction is carried out at 20-60°C with mechanical stirring until the conversion rate of the substrate fucoidan-lactose is close to 99%. After the reaction, L-fucose with a purity >99% is obtained by resin separation, activated carbon decolorization, and recrystallization.

[0181] Using the above-mentioned fucoside hydrolase for hydrolysis, L-fucose with a purity of over 99% can be obtained. The reaction conditions are mild and the operation is relatively gradual, showing good prospects for industrial application. At the same time, the above-mentioned fucoside hydrolase has good tolerance to the substrate, which improves the substrate conversion rate to up to 99%, greatly shortening the production time of L-fucose in industrial production.

[0182] Example

[0183] This application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0184] Example 1 Fucoside Hydrolase

[0185] The ancestral enzyme of fucoside hydrolase, constructed using conventional ancestral sequence reconstruction methods in this field, has the amino acid sequence shown in SEQ ID NO:2. Its encoding gene was obtained through codon optimization, and the full-length sequence was further synthesized artificially by a gene synthesis company. PCR primers were designed as follows:

[0186] Upstream primer:

[0187] 5'-gtgccgcgcggcagc catatg ATGACTACAGATTCAAGGCAACTAGC-3” (SEQ ID NO:5)

[0188] Downstream primer:

[0189] 5'-acggagctcgaattc ggatcc TTACGGAGTCGGGGAAGACA-3'(SEQ ID NO:6)

[0190] In this design, the underlined portion of the upstream primer represents the NdeI restriction site, and the underlined portion of the downstream primer represents the BamHI restriction site.

[0191] Using the synthetically produced fucoside hydrolase ancestor enzyme DNA as a template, PCR amplification was performed. The PCR system consisted of: 10 μL of 2×Taq PCR MasterMix, 1 μL each of upstream and downstream primers (0.3 μmol / L), 1 μL (0.1 μg) of DNA template, and 7 μL of ddH2O. The PCR amplification program was as follows: (1) 95℃ for 3 min pre-denaturation; (2) 94℃ for 30 s denaturation; (3) 55℃ for 30 s annealing; (4) 72℃ for 2 min extension; steps (2) to (4) were repeated for 30 cycles; (5) 72℃ for 10 min extension, followed by cooling to 4℃. The PCR product was purified by agarose gel electrophoresis and recovered using an agarose gel DNA recovery kit. A complete full-length gene sequence of fucoside hydrolase was obtained, which was sequenced to be 1380 bp in length and named lfh. The nucleotide sequence of the gene is shown in SEQ ID NO.1 in the sequence listing.

[0192] Example 2 Preparation of fucoside hydrolase expression vector and expression transformant

[0193] The fucoside hydrolase gene DNA fragment obtained in Example 1 and the empty pET-28a plasmid were digested with restriction endonucleases NdeI and BamHI at 37°C for 2 hours. The fragments were purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit. The target fragment was ligated overnight at 4°C using T4 DNA ligase to obtain the expression plasmid pET28a-lfh.

[0194] The expression plasmid was transformed into *Escherichia coli* DH5α competent cells. Positive clones were screened on kanamycin-containing resistant plates, and single clones were selected for colony PCR verification. The cells were cultured, and after plasmid amplification, the plasmid was extracted and re-transformed into *Escherichia coli* BL21(DE3) competent cells. The transformation solution was plated on LB agar plates containing kanamycin and incubated overnight at 37°C inverted mode to obtain the positive transformant *Escherichia coli* BL21(DE3) / pET28a-lfh. Colony PCR and gene sequencing verified the positive clones. Positive colonies were inoculated into LB medium and cultured for 12 h as a seed culture. The culture was then transferred to 100 mL of fresh LB medium and continued until the OD600 reached 0.6-0.8. Lactose or IPTG was added as an inducer to a final concentration of 0.2 mM, and the culture was continued at 25°C for another 12 h. Cells expressing recombinant fucoside hydrolase were obtained by centrifugation to remove the culture medium.

[0195] Cells expressing recombinant fucoside hydrolase were added to 10 mM phosphate buffer (100 mM, pH 7.0), and the cells were sonicated to release intracellular fucoside hydrolase. The supernatant obtained after centrifugation at 4°C and 8000 r / min for 10 min was the crude fucoside hydrolase solution. Cells expressing recombinant fucoside hydrolase were then freeze-dried to obtain frozen stem cells.

[0196] Example 3: Determination of Fucoside Hydrolase Activity

[0197] The activity assay was performed as follows: 10 mmol / L 2-fucolactose was added to a 200 μL reaction system (100 mmol / L sodium phosphate buffer, pH 8.0). After incubation at 30°C for 2 min, an appropriate amount of the crude enzyme solution prepared in Example 2 was added, and the mixture was rapidly mixed. The reaction was terminated by heating after 10 min. The reduction in substrate was determined by HPLC, and enzyme activity was calculated. Enzyme activity (U) was defined as the amount of enzyme required to catalyze the hydrolysis of 1 μmol of 2-fucolactose per minute under the above conditions. The specific activity of fucoside hydrolase LFH against 2-fucolactose was determined to be 165 U / mg.

[0198] Example 5: Fucoside hydrolase-catalyzed hydrolysis of 2-fucolactose

[0199] First, with the reaction pH fixed at 8.0 and substrate concentration at 50 mM, the effect of catalytic reaction on the reaction under conditions of 20°C, 30°C, 40°C, 50°C, and 60°C was investigated. The results are shown in Table 1. After 12 h of reaction, a high conversion rate was observed between 30-50°C, with the highest conversion rate (96%) achieved at 40°C and pH 8.0. Further, with the reaction temperature fixed at 30°C, the catalytic effect on the reaction was investigated under pH conditions of 6.0, 7.0, 8.0, 9.0, and 10.0. See Table 1 for details.

[0200] Table 1 Effects of different temperatures and pH on the fucoidan hydrolase reaction

[0201] reaction temperature pH Conversion rate 1 20℃ 8.0 67％ 2 30℃ 8.0 95％ 3 40℃ 8.0 96％ 4 50℃ 8.0 94％ 5 60℃ 8.0 84％ 6 30℃ 6.0 77％ 7 30℃ 7.0 95％ 8 30℃ 9.0 63％ 9 30℃ 10.0 32％

[0202] The fucoside hydrolase catalyzes the hydrolysis of 2-fucolactose. Reactions 1-5 show that the conversion rate initially increases and then decreases with increasing temperature. The conversion rate reaches a maximum of 96% when the temperature reaches 40℃. Reactions 2 and 6-9 show that the conversion rate initially increases and then decreases with increasing temperature. The conversion rate reaches a maximum of 95% when the pH is 8.0 and 7.0. Therefore, a pH of 7.0-8.0 and a temperature of 40℃ are the optimal reaction conditions for the fucoside hydrolase-catalyzed hydrolysis of 2-fucolactose.

[0203] Example 6: Preparation of Fucoside Hydrolase Mutant

[0204] A structural model of fucoside hydrolase lfh was constructed. The interaction between LFH and the substrate was analyzed after inserting the substrate pair into the active site. Mutations were introduced to enhance the interaction between the enzyme and the substrate. The full-length gene sequence of the hydrolase obtained in Example 1 (nucleotide sequence shown in SEQ ID NO. 1) was mutated by four bases. The mutation positions were: F at position 103 to T, E at position 283 to S, G at position 293 to A, and D at position 400 to A. The resulting mutant gene sequence is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4. The primers used for site-directed mutagenesis are shown in SEQ ID NO: 5 to NO. 12. The fucoside hydrolase expression vector pET28a-lfh prepared in Example 2 was used as a template for PCR amplification. The PCR system is shown in Table 2, and the PCR process is shown in Table 4.

[0205] Table 2 PCR System

[0206] system volume 2×KOD Plus Mix 10μL upstream primer 1μL Downstream primer 1μL DNA template 1μL <![CDATA[ddH2O]]> 7μL

[0207] Table 3 PCR process

[0208]

[0209] PCR products obtained using primers F103T-F (SEQ ID NO: 5) and F103T-R (SEQ ID NO: 6) were digested with restriction endonuclease DpnI to remove the template pET28a-lfh. The digested product was transformed into E. coli BL21(DE3), and sequencing confirmed the successful construction of mutant F103T. PCR products obtained using primers E283S-F (SEQ ID NO: 7) and E283S-R (SEQ ID NO: 8) were digested with restriction endonuclease DpnI to remove the template pET28a-lfh. The digested product was transformed into E. coli BL21(DE3), and sequencing confirmed the successful construction of mutant E283S. PCR products obtained using primers G293A-F (SEQ ID NO: 9) and G293A-R (SEQ ID NO: 6) were also digested with restriction endonuclease DpnI to remove the template pET28a-lfh. The digested product was transformed into E. coli BL21(DE3), and sequencing confirmed the successful construction of mutant E283S. PCR products obtained using primers (SEQ ID NO: 10) were digested with restriction endonuclease DpnI to remove the template pET28a-lfh. The digested product was transformed into E. coli BL21(DE3), and sequencing confirmed the successful construction of mutant G293A. PCR products obtained using primers D400A-F (SEQ ID NO: 11) and D400A-R (SEQ ID NO: 12) were digested with restriction endonuclease DpnI to remove the template pET28a-lfh. The digested product was transformed into E. coli BL21(DE3), and sequencing confirmed the successful construction of mutant D400A. Furthermore, a combined mutant F103T / E283S / G293A / D400A was constructed. After successful mutant construction, crude enzyme solution of the mutants was prepared according to the method described in Example 2. The crude enzyme solutions of fucoidan hydrolase prepared in Example 2, the constructed mutant F103T, E283S, G293A, D400A, and the combined mutant F103T / E283S / G293A / D400A were all taken at 50 mM and subjected to hydrolysis at 40 °C and pH 8.0. The results are shown in Table 4.

[0210] Table 4 Comparison of hydrolytic properties of different fucoside hydrolase mutants

[0211]

[0212] As shown in Table 4, when the substrate concentration is 50 mM, mutants F103T and E283S can completely convert the substrate after 12 hours of reaction, exhibiting better catalytic performance than the crude fucoidan hydrolase solution prepared in Example 3. Mutants G293A and D400A achieve complete substrate conversion in 8 hours and 6 hours, respectively, with significantly better catalytic performance than the crude fucoidan hydrolase solution prepared in Example 3. The combined mutant F103T / E283S / G293A / D400A achieves a conversion rate of >99% in just 6 hours. Therefore, the preferred combination is mutant D400A with mutant F103T / E283S / G293A / D400A.

[0213] Example 7: Fucoside hydrolase combination mutant F103T / E283S / G293A / D400A catalyzes the hydrolysis of fucose and lactose at different concentrations.

[0214] Frozen stem cells containing the fucoside hydrolase hybrid mutant F103T / E283S / G293A / D400A were prepared according to the method described in Example 2. Different concentrations of catalyst were obtained by dissolving these frozen stem cells in water. The effects of substrate concentrations of 50 mM, 100 mM, 200 mM, 400 mM, 1000 mM, and 1500 mM were investigated at 40 °C and pH 8.0. The results are detailed in Table 5. Table 5: Effect of different substrate concentrations on the hydrolysis reaction of the fucoside hydrolase hybrid mutant F103T / E283S / G293A / D400A.

[0215] reaction Substrate concentration Catalyst amount temperature pH reaction time Conversion rate 1 50mM 1g / L 40℃ 8.0 6h >99％ 2 100 mM 2g / L 40℃ 8.0 8h >99％ 3 200 mM 4g / L 40℃ 8.0 12h >99％ 4 400 mM 8g / L 40℃ 8.0 16h >99％ 5 1000mM 10g / L 40℃ 8.0 24h 90％ 6 1000mM 20g / L 40℃ 8.0 24h >99％ 7 1500mM 30g / L 40℃ 8.0 30h 89％ 8 1500mM 50g / L 40℃ 8.0 24h 98％

[0216] The above experiments show that, while maintaining a constant ratio of substrate concentration to catalyst amount, the combined mutant F103T / E283S / G293A / D400A can completely convert up to 1000 mM fucoidan. At a substrate concentration of 1500 mM and a catalyst concentration of 30 g / L, the conversion rate was only 89% after 24 hours of reaction. Further, at this substrate concentration, increasing the catalyst concentration to 50 g / L and reacting for 24 hours resulted in a conversion rate of 98%. This demonstrates that this fucoidan hydrolase mutant exhibits high substrate tolerance.

[0217] Example 8: Preparation of L-fucose catalyzed by the fucoidan hydrolase combination mutant F103T / E283S / G293A / D400A

[0218] The crude enzyme solution of the LFH mutant enzyme prepared in Example 6 was added to 100 mL of sodium phosphate buffer (100 mmol / L, pH 8.0), followed by the addition of 2-fuco-lactose to a final concentration of 1 mol / L. The reaction was allowed to proceed until the substrate was completely inverted. After the reaction, cells were removed by centrifugation, and L-fucose and lactose were separated by molecular sieve resin. L-fucose was obtained by decolorization with activated carbon and recrystallization, with a product yield of 87% and a chemical purity >99%.

[0219] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A fucoside hydrolase, wherein the hydrolase comprises: a) The sequence shown in SEQ ID NO:2; or b) Based on the mutant of SEQ ID NO:2; The mutant is, SEQ ID NO.2 has a phenylalanine mutation at position 103 to threonine; or SEQ ID NO.2 has a glutamic acid mutation at position 283 to a serine; or SEQ ID NO.2 has a glycine mutation at position 293 to alanine; or SEQ ID NO.2 has a mutation at position 400 where aspartic acid is replaced by alanine; or SEQ ID NO.2 has a mutation at position 103 where phenylalanine is replaced by threonine, at position 283 where glutamic acid is replaced by serine, at position 293 where glycine is replaced by alanine, and at position 400 where aspartic acid is replaced by alanine.

2. A fucoside hydrolase, the sequence of which is shown in SEQ ID NO:

4.

3. A nucleic acid encoding the fucoside hydrolase as described in claim 1 or 2.

4. A nucleic acid, wherein, The nucleic acid is shown in SEQ ID NO.1 or SEQ ID NO.

3.

5. An expression vector comprising the nucleic acid of claim 3 or 4.

6. The expression vector according to claim 5, wherein the expression vector is a plasmid or a bacteriophage.

7. A host cell comprising the expression vector of claim 5 or 6.

8. The host cell according to claim 7, wherein the host cell is a bacterial, fungal, plant or animal cell.

9. The use of the fucoside hydrolase of claim 1 or 2, the nucleic acid of claim 3 or 4, the expression vector of claim 5 or 6, or the host cell of claim 7 or 8 in the production of L-fucose.

10. A method for producing L-fucose, comprising: L-fucose is obtained by hydrolyzing fucoidan lactose using the fucoidan hydrolase described in claim 1 or 2.

11. The method according to claim 10, wherein the hydrolysis reaction temperature is 20~60℃, or The hydrolysis reaction occurs at pH 6.0-10.0, or The concentration of fucose-lactose is 50 mM-1000 mM.

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