Fucoside hydrolase and application thereof

Abstract

The invention discloses a fucoside hydrolase. The hydrolase is as follows: a) a fucoside hydrolase containing a sequence as shown in SEQ ID NO: 2 or a sequence as shown in SEQ ID NO: 2; or b) a mutant based on SEQ ID NO: 2 containing one or two or more mutations. The fucoside hydrolase is used as a catalyst in synthesis of L-fucose, a substrate is low in price and easy to obtain, the industrial cost of industrial production of L-fucose is greatly reduced, meanwhile, reaction conditions are mild, the method is environmentally friendly, the operation difficulty of synthesis of L-fucose by using the fucoside hydrolase is further simplified, and the method is suitable for industrial production of L-fucose. Good industrial development prospects are realized.

Description

Technical Field

[0001] The present application relates to the field of bioengineering technology, and in particular to a fucoside hydrolase and its application. Background Art

[0002] Fucose is a hexose sugar, also known as 6-deoxygalactose and methylpentose. The vast majority of fucose found in nature is L-fucose, with D-fucose found only as a rare sugar in some glycoside compounds. L-fucose is abundant in seaweed and gums, and is also found in the polysaccharides of certain bacteria. Fucose, as a component of the sugar chains in glycoproteins, is widely present on the plasma membranes of various cell surfaces. Fucose has one less hydroxyl group on the sixth carbon atom than other hexose sugars, making it less hydrophilic and more hydrophobic than other monosaccharides. In certain blood type molecules, fucose can serve as a specific blood type marker. Fucose is the terminal sugar of many cell surface polysaccharides and plays an important role in many physiological and pathological processes. For example, sialylated Lewis X, with its terminal L-fucose, is a tetrasaccharide expressed in white blood cells such as granulocytes and monocytes. This glycan is closely associated with a variety of tumors, including pancreatic, breast, colon, and lung cancers. However, the mechanisms of action of these glycans remain unclear due to the complexity of their structure. Modification of their terminal sugars, such as L-fucose, by adding probes, can track the action of this sugar and further investigate its mechanism of action. Furthermore, studies have shown that fucosylated substances play a crucial role in embryonic development and are involved in immune responses. In particular, L-fucose has been found to be a key component of human breast milk. As a major component of human milk oligosaccharides (HMOs), it plays a crucial role in healthy childhood development. L-fucose is an important starting material in the chemical and pharmaceutical industries and is also used in the production of cosmetics and nutritional supplements. 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 seaweeds, including the Fucaceae and Laminariaceae families. Currently, L-fucose is primarily obtained by harvesting brown seaweeds from the Fucaceae family, which can be found worldwide and are abundant along the Atlantic coast of Europe. For example, Japanese patent JP2000351790 discloses methods for extracting fucoidan and for obtaining and separating fucose oligosaccharides from the extracted fucoidan. However, large-scale harvesting of brown seaweed from coastal areas raises environmental concerns and is subject to certain environmental regulations. Furthermore, purification of monosaccharides from complex oligosaccharide hydrolysates often requires the use of toxic chemicals, such as lead acetate and excess organic solvents. Furthermore, the composition of oligosaccharides in seaweed is significantly affected by seasonal variations, making L-fucose yields difficult to predict. In addition to hydrolyzing fucoidan from brown seaweed, recent studies have shown that L-fucose can also be obtained through bacterial polysaccharide hydrolysis containing naturally occurring L-fucose. European Patent WO2012 / 034996A1 discloses strains belonging to the Enterobacteriaceae family that are capable of producing extracellular polysaccharides containing L-fucose. To produce L-fucose, the polysaccharide produced by the above-mentioned strain is recovered and hydrolyzed, for example, by treatment with sulfuric acid or hydrochloric acid, which requires cumbersome purification and separation steps. In addition to extracting L-fucose from polysaccharide or oligosaccharide hydrolysates, several synthetic pathways for L-fucose have been developed starting from other monosaccharides, such as L-arabinose, D-galactose, L-rhamnose, D-mannose, and D-glucose. Generally, these raw materials are expensive, and the synthesis reaction involves several protection and deprotection steps, resulting in a low overall yield of the final product. Chinese patents such as CN112813118A report a method for synthesizing L-fucose using L-galactonic acid-1,4-lactone as a raw material, but the raw material price remains high. U.S. Patent No. 8642297B2 specifically proposes a universal fermentation method that uses recombinant mannitol-1-dehydrogenase from celery (Apium graveolens) to produce L-fucose. However, no specific operational examples are disclosed, nor are alternative enzymes for the reaction. BASF of Germany has disclosed a biocatalytic method for synthesizing L-fucose from L-fucose (CN107454915A). L-fucose is first chemically synthesized from galactose, followed by the conversion of L-fucose to L-fucose using galactose oxidase and catalase. Although the reaction has high substrate tolerance, the conversion rate is only 62%, and the raw material cost is high. Summary of the Invention

[0004] In the prior art, 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. The present application proposes a method for preparing L-fucose by hydrolysis of relatively inexpensive fucosyllactose as a substrate under the action of a fucoside hydrolase. The present invention provides an efficient fucoside hydrolase and its gene, as well as a recombinant expression vector and recombinant expression transformant containing the gene, as well as a recombinant enzyme and its preparation method, and the use of the hydrolase and its mutants in the synthesis of L-fucose.

[0005] The specific technical solutions of this application are as follows:

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

[0007] a) comprises the sequence shown in SEQ ID NO: 2 or the sequence shown in SEQ ID NO: 2; or

[0008] b) a mutant based on SEQ ID NO: 2 comprising one or two or more mutations.

[0009] 2. The hydrolase according to item 1, wherein the amino acid sequence of the mutant comprises amino acid mutations at at least one, two, three or four positions corresponding to F103, E283, G293 and D400 of SEQ ID NO: 2.

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

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

[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 according to item 4 or 5, wherein the expression vector is preferably a plasmid, cosmid, phage or animal vector.

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

[0015] 8. Use of the fucoside hydrolase according to any one of Items 1 to 3, the nucleic acid molecule according to Item 4 or 5, the expression vector according to Item 6, or the host cell according to Item 7 for producing L-fucose.

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

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

[0018] 10. The method according to item 0, wherein the hydrolysis reaction temperature is 20 to 60°C, or

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

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

[0021] Effects of the Invention

[0022] The fucoside hydrolase of the present application is used as a catalyst in the synthesis of L-fucose. The substrate is cheap and easily available, which greatly reduces the industrial cost of industrial production of L-fucose. At the same time, the reaction conditions are mild and environmentally friendly, and the operational difficulty of synthesizing L-fucose using the fucoside hydrolase is further simplified, which has good industrial development prospects. At the same time, the fucoside hydrolase of the present application has good tolerance to the substrate, improves the conversion rate of the substrate, and greatly shortens the production time of industrial L-fucose. DETAILED DESCRIPTION

[0023] The present application is described in detail below. Although 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 by 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 words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of this application shall be as defined by the attached claims.

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

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

[0027] Chinese name Alphabet abbreviations Chinese name Alphabet 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 glutamate E Tryptophan W Lysine K Methionine (methionine) M Arginine R Tyrosine Y Histidine H

[0028] The present application provides a fucoside hydrolase, wherein the hydrolase is:

[0029] a) comprises the sequence shown in SEQ ID NO: 2 or the sequence shown in SEQ ID NO: 2; or

[0030] b) a mutant based on SEQ ID NO: 2 comprising one or two 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 of SEQ ID NO: 2.

[0043] Based on the amino acid sequence of the present invention, the present invention may contain changes at one or more positions, i.e., substitutions, insertions and / or deletions, and still retain its activity.

[0044] The fucoside hydrolase described in the present application can be a fucoside hydrolase ancestral enzyme constructed by an ancestral sequence reconstruction method, a fucoside hydrolase obtained by artificially synthesizing the entire amino acid sequence, or a fucoside hydrolase obtained by cloning and expressing through a genetic engineering method.

[0045] In this application, ancestral sequence reconstruction (ASR) 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 the ancestral sequence at a specific node in the tree." Ancestral sequence reconstruction (ASR) is used in molecular evolution research. Unlike traditional evolutionary methods that study proteins by horizontally comparing related protein homologs at the ends of different branches of a phylogenetic tree, ASR vertically detects statistically inferred ancestral proteins within the tree nodes. A phylogenetic tree is a branching diagram that shows the evolutionary relationships between multiple biological species or other entities based on the similarities and differences in their physical or genetic characteristics. In a rooted phylogenetic tree, each node with descendants represents the inferred most recent common ancestor of these descendants. In ASR, multiple related homologs of the target protein are selected and aligned using a multiple sequence alignment (MSA) to construct a phylogenetic tree with statistically inferred sequences at the nodes of the branches. These sequences are called "ancestors," and the process of synthesizing the corresponding DNA, translating it into cells, and producing proteins is called "reconstruction."

[0046] Ancestral sequences are usually calculated by maximum likelihood, but Bayesian methods can also be performed. Since ancestors are inferred from phylogeny, the topology and composition of phylogeny play a major role in outputting ASR sequences. ASR does not claim to reconstruct the actual sequence of ancient proteins / DNA, but rather a sequence that may be similar to the sequence at the node. The maximum likelihood (ML) method works by generating such a sequence, in which the residue at each position is predicted to be most likely to occupy the position by the inference method used. Generally speaking, this is a score matrix (similar to those used in BLAST or MSA) calculated from existing sequences. Alternative methods include maximum parsimony (MP), which constructs sequences based on sequence evolution models, and the concept of the minimum number of nucleotide sequence changes usually represents the most effective evolutionary pathway and the most likely to occur. MP is often considered to be the most unreliable reconstruction method because it may oversimplify evolution to a degree that is not suitable for a billion-year scale. Other methods include Bayesian methods, which involve considerations of residue uncertainty. Such methods are sometimes used to supplement ML methods, but typically produce more ambiguous sequences (i.e., sequences containing residue positions where unambiguous substitutions cannot be predicted). Typically in such cases, multiple ASR sequences covering the majority of ambiguities are generated and compared to each other. In some embodiments, ancestral sequence reconstruction is performed using the online software FireProt-ASR (FireProt-ASR (muni.cz)).

[0047] The fucoside hydrolase of the present application is derived from Xanthomonas sp. The fucoside hydrolase is obtained by ancestral sequence reconstruction, constructing the ancestral enzyme of the fucoside hydrolase, artificially synthesizing the full amino acid sequence of the fucoside hydrolase, and cloning and expressing the obtained fucoside hydrolase through genetic engineering.

[0048] Among them, when 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 the hydrolase with the best catalytic performance was obtained, whose amino acid sequence is shown in SEQ NO: 2.

[0049] After obtaining the optimal hydrolase progenitor enzyme, codon optimization was performed based on the corresponding amino acid sequence to obtain the full-length gene sequence. The gene sequence was then delivered to a gene synthesis company for artificial synthesis. After obtaining the corresponding gene, the gene was amplified by PCR and the sequence was ligated into pET28a. The primers described 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] Wherein, the underlined portion of the upstream primer nucleotide sequence is the NdeI restriction enzyme site, and the underlined portion of the downstream primer is the BamHI restriction enzyme site.Then with the artificially synthesized gene as template, polymerase chain reaction (PCR) is utilized to carry out gene amplification, and complete hydrolase full-length DNA fragment is obtained.Wherein said hydrolase full-length gene (nucleotide sequence is as shown in SEQ NO:1 in the sequence table), named after lfh, total length is 1380 nucleotide bases.Its coding sequence stops from the 1st base to 1380 bases, and the start codon is ATG, and the stop codon is TAA.This sequence has no introns, and the amino acid sequence of the protein of this gene encoding is as shown in SEQ NO:2 in the sequence table.

[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 the degeneracy of codons, the nucleic acid molecule encoding the above hydrolase (amino acid sequence as shown in SEQ ID NO: 2) is not limited to the nucleic acid molecule with the sequence as shown in SEQ ID NO: 1. A homolog of a polynucleotide can also be provided by appropriately introducing substitutions, deletions, changes, insertions, or additions of nucleotides.

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

[0087] The above-mentioned "corresponding" has the meaning generally understood by those skilled in the art. Specifically, "corresponding" means that after the two sequences are aligned for homology or sequence identity, a position in one sequence corresponds to a specified position in the other sequence.

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

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

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

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

[0092] In a specific embodiment, the amino acid sequence of the mutant is that only mutations at F103 and E283 exist relative to the amino acid sequence shown in SEQ ID NO: 2.

[0093] In a specific embodiment, the amino acid sequence of the mutant is that only mutations at F103 and E283 exist relative to the amino acid sequence shown in SEQ ID NO: 2.

[0094] In a specific embodiment, the amino acid sequence of the mutant is that only mutations at F103 and G293 exist relative to the amino acid sequence shown in SEQ ID NO: 2.

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

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

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

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

[0099] In a specific embodiment, the amino acid sequence of the mutant is that only mutations at F103, E283 and G293 exist relative to the amino acid sequence shown in SEQ ID NO: 2.

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

[0101] In a specific embodiment, the amino acid sequence of the mutant is that only mutations at F103, G293 and D400 exist relative to the amino acid sequence shown in SEQ ID NO: 2.

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

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

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

[0105] In a specific embodiment, the amino acid sequence of the mutant is a mutation in which the G293 position is mutated to alanine A relative to the amino acid sequence shown in SEQ ID NO: 2.

[0106] In a specific embodiment, the amino acid sequence of the mutant is a mutation in which the D400 position is mutated to alanine A relative to the amino acid sequence shown in SEQ ID NO: 2.

[0107] In a specific embodiment, the amino acid sequence of the mutant is the only mutation that exists in the amino acid sequence shown in SEQ ID NO: 2, where the F103 site is mutated to threonine T and the E283 site is mutated to serine S.

[0108] In a specific embodiment, the amino acid sequence of the mutant is the only mutation that exists in the amino acid sequence shown in SEQ ID NO: 2, where the F103 site is mutated to threonine T and the G293 site is mutated to alanine A.

[0109] In a specific embodiment, the amino acid sequence of the mutant is the amino acid sequence shown in SEQ ID NO: 2, except that the mutations at position F103 are mutated to threonine T and the mutation at position D400 is mutated to alanine A.

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

[0111] In a specific embodiment, the amino acid sequence of the mutant is that, relative to the amino acid sequence shown in SEQ ID NO: 2, only the mutation of the F103 position to threonine T, the mutation of the G293 position to alanine A, and the mutation of the D400 position to alanine A exist.

[0112] In a specific embodiment, the amino acid sequence of the mutant is that, relative to the amino acid sequence shown in SEQ ID NO: 2, only the mutation of the G283 site to serine S, the mutation of the G293 site to alanine A, and the mutation of the D400 site to alanine A exist.

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

[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. The mutation can be carried out according to conventional methods in the art, for example, by using directed mutagenesis or construction of synthetic oligonucleotides, and then expressing the mutated DNA sequence in a host cell to obtain a mutant with substitution, insertion and / or deletion of the amino acid sequence.

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

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

[0129] The present application provides a nucleic acid molecule encoding the above-mentioned hydrolase. In a specific embodiment, 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. 3 or the sequence 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] CAGCAAGCAGGTACAACGTCCGCTACGGATGTGCGTTTCACGCAAAG

[0155] GATGATGGGCTTTACGCAATCGTCTTGATTGGCCTGGGGACGCAATTA

[0156] TCTTATTGGCTTCCGATGTGACGAAAGGTACCAGTGTATCTTGTTGGG

[0157] CTCCGATGGGCCGCTTAAATGGAAGCAAACTGGAGGAGGACTGGTTAT

[0158] CAATCTTCCAGCCGCAAAGCCCTCAGACCACGCCTGCGCCTACGTGTT

[0159] CAAAATTGAGTTGAAGGGTAAGCCCGCCTAA.

[0160] The present application provides an expression vector comprising the above-mentioned nucleic acid molecule.

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

[0162] In this application, the above-mentioned cosmid refers to the cosmid

[0163] For example, the expression vector can be prepared by the following method:

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

[0165] The present application provides a host cell comprising the above-mentioned expression vector.

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

[0167] The bacterium is preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3) or Escherichia coli DH5α.

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

[0169] The present application provides a method for preparing fucoside hydrolase, which comprises inoculating the above-mentioned host cells into a culture medium for fermentation to obtain a fermentation broth, and centrifuging the fermentation broth to collect bacterial cells, and crushing the bacterial cells to obtain the fucoside hydrolase.

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

[0171] In this application, there are no particular limitations on the culture method and conditions. They can be appropriately selected based on factors such as the host cell type and culture method, as long as the transformants can grow and produce fucoside hydrolase. Other specific operations for culturing the transformants can be performed according to conventional procedures in the art.

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

[0173] The present 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 producing L-fucose.

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

[0175] The fucoside hydrolase is used to hydrolyze the fucosyllactose to obtain L-fucose. In a specific embodiment, the fucosyllactose is 2-fucosyllactose.

[0176] In one embodiment, the temperature of the hydrolysis reaction 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 embodiment, the pH of the hydrolysis reaction is 6.0-10.0, 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, 10.0, preferably 7.0-8.0.

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

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

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

[0181] Using the above-mentioned fucoside hydrolase for hydrolysis reaction can obtain L-fucose with a purity of more than 99%, and the reaction conditions are mild, the operation is relatively gradual, and it has good industrial application prospects. At the same time, the above-mentioned fucoside hydrolase has good tolerance to the substrate, which increases the conversion rate of the substrate to as high as 99%, greatly shortening the production time of L-fucose in industrial production.

[0182] Example

[0183] This application provides general and / or specific descriptions of the materials and experimental methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., percentage by weight. All reagents or instruments used without manufacturer indication are commercially available conventional reagents.

[0184] Example 1 Fucoside hydrolase

[0185] The amino acid sequence of the fucoside hydrolase ancestral enzyme constructed according to conventional ancestral sequence reconstruction methods in the art is shown in SEQ ID NO: 2. The encoding gene was obtained by codon optimization, and the full-length sequence was further artificially synthesized 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] Among them, the underlined part of the upstream primer is the NdeI restriction site, and the underlined part of the downstream primer is the BamHI restriction site.

[0191] PCR amplification was performed using the artificially synthesized fucoside hydrolase ancestral enzyme DNA as a template. The PCR system was: 2×Taq PCR MasterMix 10μL, upstream primer and downstream primer 1μL (0.3μmol / L), DNA template 1μL (0.1μg) and ddH2O 7μL. The PCR amplification program was as follows: (1) 95℃, pre-denaturation for 3min; (2) 94℃, denaturation for 30s; (3) 55℃ annealing for 30s; (4) 72℃ extension for 2min; steps (2) to (4) were repeated for 30 cycles; (5) 72℃ extension for 10min, and then cooled 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 1380bp in length after DNA sequencing and named lfh. The nucleotide sequence of the gene is shown in SEQ ID NO.1 in the sequence table.

[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 double-digested with restriction endonucleases NdeI and BamHI at 37°C for 2 hours, purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit. The target fragments were ligated overnight at 4°C using T4 DNA ligase to obtain the expression plasmid pET28a-lfh.

[0194] Transform the above expression plasmid into E. coli DH5α competent cells. Screen positive clones on a plate containing kanamycin, pick single colonies, and verify positive clones by colony PCR. Cultivate these cells, amplify the plasmid, and retransform them into E. coli BL21(DE3) competent cells. Plate the transformation solution onto an LB plate containing kanamycin and incubate inverted at 37°C overnight to obtain positive transformants (E. coli BL21(DE3) / pET28a-lfh). Verify positive clones by colony PCR and sequencing. Inoculate a positive colony into LB medium and incubate for 12 hours as a seed culture. Transfer the culture to 100 mL of fresh LB medium and continue incubating until the OD600 reaches 0.6-0.8. Add the inducer lactose or IPTG to a final concentration of 0.2 mM and continue incubating at 25°C for 12 hours. The culture medium was removed by centrifugation to obtain cells expressing recombinant fucoside hydrolase.

[0195] Add 10 mM phosphate buffer (100 mM, pH 7.0) to cells expressing recombinant fucoside hydrolase, disrupt the cells by ultrasound, and release the intracellular fucoside hydrolase. Centrifuge at 4°C and 8000 rpm for 10 minutes to obtain the supernatant, which is the crude fucoside hydrolase enzyme solution. Freeze-dry the recombinant fucoside hydrolase cells to obtain lyophilized cells.

[0196] Example 3 Determination of Fucoside Hydrolase Activity

[0197] The activity assay was performed as follows: 10 mmol / L 2-fucosyllactose was added to a 200 μL reaction system (100 mmol / L sodium phosphate buffer, pH 8.0). The mixture was incubated at 30°C for 2 minutes, followed by addition of an appropriate amount of the crude enzyme solution prepared in Example 2. The mixture was rapidly mixed, allowed to react for 10 minutes, and then heated to terminate the reaction. The reduction in substrate was measured by HPLC, and the enzyme activity was calculated. Enzyme activity (U) is defined as the amount of enzyme required to catalyze the hydrolysis of 1 μmol of 2-fucosyllactose per minute under the above conditions. The specific activity of fucoside hydrolase LFH on 2-fucosyllactose was determined to be 165 U / mg.

[0198] Example 5 Fucoside hydrolase catalyzes the hydrolysis of 2-fucosyllactose

[0199] First, the reaction pH was fixed at 8.0 and the substrate concentration was 50 mM. The effects of catalytic reaction at 20°C, 30°C, 40°C, 50°C, and 60°C were examined. The results, shown in Table 1, show that after 12 hours of reaction, the conversion rate was high at temperatures between 30°C and 50°C. The highest conversion rate, reaching 96%, was achieved at a reaction temperature of 40°C and a pH of 8.0. Furthermore, the reaction temperature was fixed at 30°C, and the effects of catalytic reaction at pH 6.0, 7.0, 8.0, 9.0, and 10.0 were examined. See Table 1 for details.

[0200] Table 1 Effects of different temperatures and pH on fucoside 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 reaction of 2-fucosyllactose. Reactions 1-5 show that the conversion rate first increases and then decreases with the increase in the temperature of the reaction system. When the temperature reaches 40°C, the conversion rate can reach a maximum of 96%. Reactions 2 and 6-9 show that the conversion rate first increases and then decreases with the increase in the temperature of the reaction system. When the pH is 8.0 and 7.0, the conversion rate can reach a maximum of 95%. Therefore, the pH of 7.0-8.0 and the temperature of 40°C are the optimal reaction conditions for the hydrolysis reaction of 2-fucosyllactose catalyzed by the fucoside hydrolase.

[0203] Example 6 Preparation of Fucoside Hydrolase Mutants

[0204] A structural model of fucoside hydrolase lfh was constructed, and the interaction between the fucoside hydrolase LFH and the substrate was analyzed after the substrate was docked into the active center. The interaction between the enzyme and the substrate was strengthened by introducing mutations. The full-length gene sequence of the hydrolase obtained in Example 1 (nucleotide sequence as shown in SEQ ID NO.1) was mutated by 4 bases. The mutation positions of the mutant were respectively mutating F at position 103 of the fucoside hydrolase gene coding sequence to T, E at position 283 to S, G at position 293 to A, and D at position 400 to A. The sequence of the obtained mutant gene 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. PCR amplification was performed using the fucoside hydrolase expression vector pET28a-lfh prepared in Example 2 as a template. 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 10 μL Upstream primer 1 μL Downstream primer 1 μL DNA template 1 μL <![CDATA[ddH2O]]> 7μL

[0207] Table 3 PCR process

[0208]

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

[0210] Table 4 Comparison of hydrolysis performance of different fucoside hydrolase mutants

[0211]

[0212] As shown in Table 4, when the substrate concentration was 50 mM, mutants F103T and E283S were able to completely convert the substrate after a reaction time of 12 h, and their catalytic performance was superior to that of the crude fucoside hydrolase solution prepared in Example 3. Mutants G293A and D400A were able to achieve complete substrate conversion in 8 h and 6 h, respectively, and their catalytic performance was significantly superior to that of the crude fucoside hydrolase solution prepared in Example 3. The combined mutants F103T / E283S / G293A / D400A achieved a conversion rate of >99% after only 6 h of reaction, so the combined mutants F103T / E283S / G293A / D400A were preferred.

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

[0214] Freeze-dried cells of the fucoside hydrolase combination mutant F103T / E283S / G293A / D400A were prepared by following the method for preparing freeze-dried cells in Example 2. The freeze-dried cells of the fucoside hydrolase combination mutant F103T / E283S / G293A / D400A were dissolved in water to obtain catalysts at different concentrations. The conversion reaction was investigated at substrate concentrations of 50 mM, 100 mM, 200 mM, 400 mM, 1000 mM, and 1500 mM 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 Combination 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 when the ratio of substrate concentration to catalyst amount remains constant, the mutant combination F103T / E283S / G293A / D400A can completely convert up to 1000 mM fucosyllactose. At a substrate concentration of 1500 mM and a catalyst level of 30 g / L, the conversion rate was only 89% after a 24-hour reaction. Furthermore, when the catalyst level was increased to 50 g / L at this substrate concentration and the reaction time was 24 hours, the conversion rate reached 98%. This demonstrates that this fucosyllactose hydrolase mutant has high substrate tolerance.

[0217] Example 8 Preparation of L-fucose by catalysis of the fucoside 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), and 2-fucosyllactose was added to a final concentration of 1 mol / L. The reaction was continued until the substrate was completely converted. After the reaction, cells were removed by centrifugation, and then L-fucose was separated from lactose by molecular sieve resin. L-fucose was obtained by decolorization with activated carbon and recrystallization. The product yield was 87% and the chemical purity was >99%.

[0219] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application in any other manner. Any person skilled in the art may utilize the above disclosed technical content to modify or modify the present application into equivalent embodiments with equivalent variations. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present application and are based on the technical essence of the present application shall still fall within the scope of protection of the present application.

Claims

1. A fucoside hydrolase, wherein the hydrolase is: a) comprises the sequence shown in SEQ ID NO: 2 or the sequence shown in SEQ ID NO: 2; or b) a mutant based on SEQ ID NO: 2 comprising one or two or more mutations.

2. The hydrolase according to claim 1, wherein The amino acid sequence of the mutant comprises amino acid mutations at at least one, two, three or four positions corresponding to F103, E283, G293 and D400 of SEQ ID NO:

2.

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

4. A nucleic acid molecule encoding the fucoside hydrolase according to any one of claims 1 to 3.

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.

6. An expression vector comprising the nucleic acid molecule according to claim 4 or 5, wherein the expression vector is preferably a plasmid, cosmid, phage or animal vector.

7. A host cell comprising the expression vector according to claim 6, wherein the host cell is preferably a bacterial, fungal, plant or animal cell.

8. Use of the fucoside hydrolase according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4 or 5, the expression vector according to claim 6, or the host cell according to claim 7 in producing L-fucose.

9. A method for producing L-fucose, comprising: L-fucose is obtained by hydrolyzing fucose lactose using the fucoside hydrolase according to any one of claims 1 to 3.

10. The method according to claim 0, wherein the hydrolysis reaction temperature is 20 to 60°C, or The hydrolysis reaction is pH 6.0-10.0, or The concentration of fucose lactose is 50mM-1000mM.

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