Fucosidase mutant and application thereof

Abstract

The present disclosure relates to a novel mutant form of an alpha-fucosidase (alpha-L-fucosidase) that exhibits enhanced alpha-(1, 6) fucosidase activity. The present disclosure also relates to compositions comprising the novel mutated forms of alpha-fucosidase, and methods of cleaving alpha-(1, 6) linked fucose in glycoconjugates using the novel mutated forms of alpha-fucosidase.

Description

Technical Field

[0001] The present disclosure relates to a novel mutant form of α-fucosidase (α-L-fucosidase) that exhibits enhanced α-(1,6) fucosidase activity. The present disclosure also relates to a composition comprising the novel mutant form of α-fucosidase, and a method for using the novel mutant form of α-fucosidase to cleave α-(1,6)-linked fucose in a glycoconjugate. Background Art

[0002] N-glycosylation is one of the most common post-translational modifications observed in mammalian proteins and plays a crucial role in regulating the intrinsic properties and biological functions of these proteins. For example, the attachment of N-glycans can significantly affect protein folding, stability, antigenicity, and immunogenicity. In addition, N-glycans can be directly involved in a wide range of biological recognition processes, including cell adhesion, host-pathogen interactions, cancer metastasis, and immune responses. Although mammalian N-glycans share a basic oligosaccharide core structure, the introduction of additional modifications to the core structure (such as sialylation and fucosylation) has helped to increase the level of structural diversity that affects biological function.

[0003] The physiological activity of therapeutic antibodies is mediated by two different mechanisms. The first mechanism involves neutralization or apoptosis of the target antigen, which helps to improve the therapeutic efficacy. The second mechanism involves antibody effector functions, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activated by the formation of immune complexes. The significance of ADCC in the clinical effectiveness of therapeutic antibodies, especially anti-cancer antibodies, has been elucidated through genetic analysis of patient leukocyte receptor (FcγR) polymorphisms. Therefore, the bioindustry has been vigorously developing ADCC enhancement technologies, including modification of N-glycans attached to the constant region (Fc) of antibodies. It is known that the amount of α-(1,6)-linked fucose (core fucose) attached to the innermost GlcNAc of the N-glycan on the Fc region of the antibody affects ADCC activity. Removing core fucose from the innermost GlcNAc of the N-glycan on the Fc region significantly enhances the ADCC activity of IgG because the binding affinity of non-fucosylated antibodies to the FcγRIIIα receptor is increased.

[0004] Many approaches have been developed to enhance FcγRIIIα binding and ADCC by reducing fucosylation of IgG. Some strategies involve the development of production cell lines that eliminate or reduce the expression of α-(1,6)-fucosyltransferase. Alternative strategies to reduce fucosylation include silencing the α-(1,6)-fucosyltransferase gene by using RNAi. However, enzymatic defucosylation of N-glycans remains challenging in vitro because N-glycans are buried between the two Fc domains. The presence of the Fc domain limits the access of α-fucosidase to fucose residues, creating steric hindrance and resulting in lower efficiency of enzymatic defucosylation.

[0005] Only a few α-L-fucosidases such as α-L-fucosidase from bovine kidney, α-L-fucosidase from human (FucA1), α-L-fucosidase from Bacteroides fragilis (BfFuc), and α-L-fucosidase C (AlfC) from Lactobacillus casei are disclosed to be able to remove core fucose from the innermost GlcNAc residue after removing the outer N-glycans on the Fc region. Since the intrinsic function and activity of the wild-type enzyme are limited and low, there is a need to develop a modified enzyme to efficiently cleave core fucose from N-glycans. Summary of the invention

[0006] In one embodiment, the present disclosure provides a mutant form of α-L-fucosidase for improving enzymatic hydrolysis of fucose in vitro.

[0007] In one embodiment, the present disclosure provides a mutant α-L-fucosidase comprising a polypeptide having at least 95% sequence identity to SEQ ID NO: 1 and having a substitution mutation at amino acid position 247 (K247).

[0008] In one embodiment, the present disclosure provides a mutant α-L-fucosidase comprising a polypeptide having at least 97% sequence identity to SEQ ID NO: 1 and having a substitution mutation at amino acid position 247 (K247).

[0009] In one embodiment, the present disclosure provides a mutant α-L-fucosidase comprising a polypeptide having at least 99% sequence identity to SEQ ID NO: 1 and having a substitution mutation at amino acid position 247 (K247).

[0010] In one embodiment, the present disclosure provides a mutant α-L-fucosidase, which includes a polypeptide having a sequence of SEQ ID NO: 1 and having a substitution mutation at amino acid position 247 (K247).

[0011] In some embodiments, the substitution mutation is selected from the group consisting of K247A, K247C, K247D, K247E, K247F, K247G, K247I, K247L, K247M, K247N, K247P, K247Q, K247S, K247T, K247V, K247W, and K247Y.

[0012] In some embodiments, the mutant α-L-fucosidase has the sequence of any one of SEQ ID NOs: 2-18.

[0013] In some embodiments, the mutant α-L-fucosidase of the present disclosure can be used to efficiently cleave core fucose in native glycoproteins without denaturing or degrading the function of the glycoproteins.

[0014] In some embodiments, the fucosidase described herein can hydrolyze one or more α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linked fucose. The fucose can be present in N- and / or O-linked glycans in the glycoconjugate.

[0015] In some embodiments, the α-fucosidase is a recombinant Bacteroides α-fucosidase.

[0016] In some embodiments, the α-fucosidase exhibits a pH optimum of 6.5-7.5.

[0017] In some embodiments, the present disclosure provides a composition comprising a mutant α-L-fucosidase as described above to facilitate Fc glycoengineering of an antibody or Fc fusion protein (eg, a therapeutic antibody).

[0018] In some embodiments, the composition further comprises at least one glycosidase.

[0019] In some embodiments, the glycosidase can be an exoglycosidase.

[0020] In some embodiments, the glycosidase may be an endoglycosidase. The endoglycosidase includes but is not limited to endo-β-N-acetylglucosaminidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2 and variants thereof.

[0021] In some embodiments, the compositions are useful and efficient for defucosylation of glycoconjugates in vitro. Specifically, the compositions described herein can be used for core defucosylation of glycoproteins in vitro.

[0022] In some embodiments, the core defucosylation is core α-(1,6) defucosylation.

[0023] In some embodiments, the core defucosylation is core α-(1,3) defucosylation.

[0024] In some embodiments, the defucosylation can be performed without denaturing or degrading the function of the glycoprotein.

[0025] In one embodiment, the present disclosure provides a method for preparing a defucosylated glycoconjugate in vitro, the method comprising contacting a glycoconjugate comprising one or more fucoses with an endoglycosidase and a mutant α-L-fucosidase as described above sequentially or simultaneously.

[0026] In some embodiments, the method comprises the following sequential steps: (a) contacting the glycoconjugate with the endoglycosidase; and (b) contacting the glycoconjugate with a mutant α-L-fucosidase as described above.

[0027] In some embodiments, the method further comprises step (c) terminating the reaction.

[0028] In some embodiments, step (c) is performed at about 65° C. for 15-25 minutes.

[0029] In some embodiments, step (a) and / or step (b) is performed at about 37° C. for 0.5-2 hours.

[0030] In some embodiments, the method comprises the following sequential steps: (a) contacting the glycoconjugate with the endoglycosidase, the endoglycosidase including but not limited to endo-β-N-acetylglucosaminidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2 and variants thereof; (b) contacting the glycoconjugate with the mutant α-fucosidase of the present invention described above. The glycoconjugate comprises one or more fucose selected from α-(1,2), α-(1,3), α-(1,4) and α-(1,6) linked fucose. The fucose may be present in N- and / or O-linked glycans in the glycoconjugate.

[0031] In some embodiments, the method comprises contacting a composition comprising the mutant α-L-fucosidase described above and at least one glycosidase. The endoglycosidase includes but is not limited to endo-β-N-acetylglucosaminidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2 and variants thereof. The glycoconjugate comprises one or more fucose selected from α-(1,2), α-(1,3), α-(1,4) and α-(1,6) linked fucose. The fucose may be present in N- and / or O-linked glycans in the glycoconjugate. DETAILED DESCRIPTION

[0032] For convenience, certain terms used in the context of the present disclosure are collected here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0033] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0034] When referring to polypeptides, the term "substantially identical" or "substantially the same" means that at least 95%, and more preferably at least 96%, 97%, 98% or 99% of the amino acid residues have polypeptide sequence identity with the entire sequence of a reference polypeptide sequence when optimally aligned with another amino acid residue by appropriate polypeptide insertion or deletion, as measured by any known sequence identity algorithm (such as BLAST, ALIGN or Megalign (DNASTAR) software). A polypeptide sequence having substantial identity to a reference polypeptide sequence can exhibit the same function.

[0035] Ranges are expressed herein as from "about" one particular value and / or to "about" another particular value. When such ranges are expressed, embodiments include ranges from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation by using the term "about", it is understood that the particular value forms another embodiment. It is further understood that the endpoints of each of the ranges are valid relative to and independent of the other endpoint. As used herein, the term "about" refers to ± 20%, preferably ± 10%, and more preferably ± 5%.

[0036] As used herein, the term "glycan" refers to a polysaccharide or an oligosaccharide. A glycan may be a homopolymer or heteropolymer of monosaccharide residues and may be linear or branched. Glycan may also be used to refer to the carbohydrate portion of a glycoconjugate such as a glycoprotein, glycolipid, or proteoglycan.

[0037] As used herein, the term "glycosidase" refers to an enzyme that catalyzes the hydrolysis of glycosidic linkages or glycosidic bonds.

[0038] As used herein, the term "glycoconjugate" refers to all molecules in which at least one sugar moiety is covalently linked to at least one other moiety, including but not limited to N-linked glycoproteins, O-linked glycoproteins, glycolipids, proteoglycans, and the like.

[0039] As used herein, the term "N-linked glycoprotein" refers to a glycoprotein in which the glycan is attached to the nitrogen of the normal asparagine or arginine side chain.

[0040] As used herein, the term "O-linked glycoprotein" refers to a glycoprotein in which the glycan is attached to the hydroxyl oxygen of a normal serine, threonine, tyrosine, hydroxylysine or hydroxyproline side chain or to the oxygen of a lipid such as ceramide.

[0041] The first aspect of the present invention relates to a polypeptide fragment, comprising a mutant form of α-L-fucosidase from Lactobacillus casei, wherein the mutant form has a sequence substantially identical to polypeptide SEQ ID NO: 1 and has a substitution mutation at position 247.

[0042] SEQ ID NO: 1 refers to the sequence of α-fucosidase from Lactobacillus casei, with GeneBank accession number CAQ67984.1.

[0043] MNDNVAWFKQAKYGMMIHWGLYSLLAGEYRGESSSAYAEWIQSKFQIPNAEYGNLAT

[0044] AFNPLYFDAKKIVALAKQCGMQYLVVTTKHHDGFAMYHSKVDAYNVYDATPFHRDII

[0045] GELAEACQKAGLKFGLYYSQDLDWHDPNGGGYKSNDVETAGTTWDNSWDFPDEDQ

[0046] KNFDLLCFDNKILPQIKEIMSNYGDIATAWFDVPMTLSEAQSQTIYDTVRELQPNCLINSR

[0047] LGNGKYDFVSLGDNEIPKNKEDMNKTDVDYNEITGFKPSPLGLYETAGTINDSWGFSY

[0048] HDQNWKTPRTLYRYKQHLNDFGINYLLNVGLDPLGRVPMMAEENLLAAKALEDEANR (SEQ ID NO: 1).

[0049] In some embodiments, the substitution mutations described above are selected from the group consisting of K247A, K247C, K247D, K247E, K247F, K247G, K247I, K247L, K247M, K247N, K247P, K247Q, K247S, K247T, K247V, K247W and K247Y, preferably K247D and K247E.

[0050] In some embodiments, the substitution mutation as described above is K247D, and the mutant α-L-fucosidase has the sequence

[0051] In some embodiments, the substitution mutation as described above is K247E, and the mutant α-L-fucosidase has the sequence

[0052] In some embodiments, the substitution mutation as described above is K247F, and the mutant α-L-fucosidase has the sequence

[0053]

[0054] In some embodiments, the substitution mutation as described above is K247A, and the mutant α-L-fucosidase has the sequence

[0055]

[0056] In some embodiments, the substitution mutation as described above is K247C, and the mutant α-L-fucosidase has the sequence

[0057]

[0058] In some embodiments, the substitution mutation as described above is K247G, and the mutant α-L-fucosidase has the sequence

[0059]

[0060] In some embodiments, the substitution mutation as described above is K247I, and the mutant α-L-fucosidase has the sequence

[0061]

[0062]

[0063] In some embodiments, the substitution mutation as described above is K247L, and the mutant α-L-fucosidase has the sequence

[0064]

[0065] In some embodiments, the substitution mutation as described above is K247M, and the mutant α-L-fucosidase has the sequence

[0066]

[0067] In some embodiments, the substitution mutation as described above is K247N, and the mutant α-L-fucosidase has the sequence

[0068]

[0069] In some embodiments, the substitution mutation as described above is K247P, and the mutant α-L-fucosidase has the sequence

[0070]

[0071]

[0072] In some embodiments, the substitution mutation as described above is K247Q, and the mutant α-L-fucosidase has the sequence

[0073]

[0074] In some embodiments, the substitution mutation as described above is K247S, and the mutant α-L-fucosidase has the sequence

[0075]

[0076] In some embodiments, the substitution mutation as described above is K247T, and the mutant α-L-fucosidase has the sequence

[0077]

[0078] In some embodiments, the substitution mutation as described above is K247V, and the mutant α-L-fucosidase has the sequence

[0079]

[0080]

[0081] In some embodiments, the substitution mutation as described above is K247W, and the mutant α-L-fucosidase has the sequence

[0082]

[0083] In some embodiments, the substitution mutation as described above is K247Y, and the mutant α-L-fucosidase has the sequence

[0084]

[0085] The mutant α-fucosidase can hydrolyze one or more α-(1,2), α-(1,3), α-(1,4) and α-(1,6) linked fucose. Fucose can be present in N- and / or O-linked glycans in glycoconjugates. Fucose can be core α-(1,3)-fucose or core α-(1,6)-fucose.

[0086] The second aspect of the present invention relates to a composition comprising the mutant α-fucosidase described above and at least one glycosidase.

[0087] In some embodiments, the glycosidase is an exoglycosidase.

[0088] In some embodiments, the glycosidase is an endoglycosidase. The endoglycosidase includes but is not limited to endo-β-N-acetylglucosaminidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2 and variants thereof.

[0089] In some embodiments, the endoglycosidase is EndoS comprising a mutation at position D233.

[0090] In some embodiments, the endoglycosidase is EndoS2 comprising mutations at positions T138, D226, T227, and / or T228.

[0091] In some embodiments, the endoglycosidase is EndoS2 comprising a mutation selected from the group consisting of T138D, T138E, T138F, T138H, T138K, T138L, T138M, T138N, T138Q, T138R, T138V, T138W, D182Q, D226Q, T227Q, and T228Q.

[0092] In some embodiments, the compositions are useful and efficient for defucosylation of glycoconjugates in vitro. Specifically, the compositions described herein can be used for defucosylation of the core of glycoproteins in vitro. Defucosylation can be performed without denaturing or degrading the function of the glycoprotein.

[0093] In some embodiments, the compositions of the present invention relate to the efficient hydrolysis of N-glycans on the Fc region of antibodies. As used herein, the term "N-glycan" refers to N-linked oligosaccharides attached via N-acetylglucosamine (GlcNAc) linked to the amide nitrogen of asparagine residues in the Fc region of antibodies or fragments thereof.

[0094] In some embodiments, the N-glycans are of the high mannose, hybrid or complex type with or without core fucose.

[0095] The third aspect of the present invention relates to a method for preparing defucosylated glycoconjugates in vitro. The glycoconjugates can be treated with the endoglycosidase and mutant α-fucosidase as described above sequentially or simultaneously.

[0096] In some embodiments, the method comprises the following steps: (a) contacting the glycoconjugate with an endoglycosidase, including but not limited to endo-β-N-acetylglucosaminidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2 and variants thereof; (b) contacting the glycoconjugate with the mutant α-fucosidase of the present invention described above. The glycoconjugate includes one or more fucose selected from α-(1,2), α-(1,3), α-(1,4) and α-(1,6) linked fucose. Fucose can be present in N- and / or O-linked glycans in the glycoconjugate.

[0097] In some embodiments, the method comprises contacting a composition comprising the mutant α-L-fucosidase described above and at least one glycosidase. Endoglycosidases include but are not limited to endo-β-N-acetylglucosaminidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2, and variants thereof. The glycoconjugate comprises one or more fucose selected from α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linked fucose. Fucose can be present in N- and / or O-linked glycans in the glycoconjugate.

[0098] In some embodiments, the endoglycosidase is Streptococcus pyogenes endoglycosidase S2, Streptococcus pyogenes endoglycosidase S has a mutation at amino acid position D233, Streptococcus pyogenes endoglycosidase S2 has a mutation at amino acid position T138, Streptococcus pyogenes endoglycosidase S2 has a mutation at amino acid position D182, Streptococcus pyogenes endoglycosidase S2 has a mutation at amino acid position D184, Streptococcus pyogenes endoglycosidase S2 has a mutation at amino acid position D186, Streptococcus pyogenes endoglycosidase S2 has a mutation at amino acid position D226, or Streptococcus pyogenes endoglycosidase S2 has a mutation at amino acid position T227.

[0099] In some embodiments, the endoglycosidase is EndoS comprising a mutation at position D233.

[0100] In some embodiments, the endoglycosidase is EndoS2 comprising mutations at positions T138, D226, T227, and / or T228.

[0101] In some embodiments, the endoglycosidase is EndoS2 comprising a mutation selected from the group consisting of T138D, T138E, T138F, T138H, T138K, T138L, T138M, T138N, T138Q, T138R, T138V, T138W, D182Q, D226Q, T227Q, and T228Q.

[0102] In some embodiments, the duration of the reaction mixture is set to at least 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes or 100 minutes, preferably less than 60 minutes. The reaction temperature is preferably room temperature, more preferably about 20°C, 25°C, 30°C, 35°C, 40°C or 45°C, and most preferably about 37°C.

[0103] Examples

[0104] Example 1. Protein expression constructs

[0105] Wild-type α-fucosidase C (AlfC) was amplified from Lactobacillus casei BL23 genomic DNA (ATCC 393) by polymerase chain reaction (PCR) and cloned into pET47N with a His tag fusion at the N-terminus. Substitution of the wild-type lysine at position 247 with each of the other 19 natural amino acids was generated by saturation mutagenesis using the following forward primers and plasmid-encoded wild-type AlfC as template.

[0106] Table 1. Forward primers used for site-directed mutagenesis.

[0107]

[0108] All substitutions used the following as reverse primer: 5'-CGG GAT CTC GTT ATC GCC CAG-3' (SEQ ID NO:38). The PCR program for saturation mutagenesis was as follows: 95°C for 3 minutes; 16 cycles of 95°C for 45 seconds, 58°C for 50 seconds, 68°C for 8 minutes; and a final step of 72°C for 10 minutes. Mutations were confirmed by DNA sequencing. Other enzymes used in the study, such as EndoS or EndoS2, were cloned into pET28a with a His tag fusion at the N-terminus.

[0109] Example 2. Protein expression and purification

[0110] The protein expression construct was transformed into BL21 (DE3) (EMD Biosciences, San Diego, CA). The protein was overexpressed at 16°C for 20 hours by induction with 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG). The cells were disrupted by a high pressure homogenizer and the supernatant was collected after centrifugation. The collected supernatant was loaded into a Ni-NTA agarose column and washed with ten column volumes of wash buffer (Tris-HCl, pH 7.4, 200 mM sodium chloride and 40 mM imidazole). Elution was performed by two column volumes of elution buffer (Tris-HCl, pH 7.4, 200 mM sodium chloride and 300 mM imidazole). The protein purity was checked by SDS-PAGE and the protein was purified by The protein buffer was exchanged into reaction buffer (50 mM Tris-HCl, 50 mM mannitol, 50 mM sorbitol, pH 7.0) using Ultra-0.5 centrifugal filter devices (UFC5010BK, 10 kDa cutoff).

[0111] Example 3. Defucosylation of the core of a glycoprotein

[0112] The core defucosylation activity of the mutant fucosidase was determined by analyzing the percentage of core fucose after enzyme treatment. ) (24 mg) was treated with EndoS2 (15.2 μg) at 37°C for 1 hour and then reacted with AlfC fucosidase mutant (2.8 μg) at 37°C for another 1 hour. The reaction was paused by heat inactivation at 65°C for 20 minutes and then incubated at 4 °C for 1 hour. Ultra filter device with ddH2O wash 3 times. Collect supernatant for mass analysis (Table 2). The percentage of core fucose on N-glycans in the blank group (no enzyme) was about 87%. The defucosylation activity of wild-type AlfC enzyme was about 19%. Mutation to a negatively charged amino acid residue at K247 residue significantly increased the defucosylation activity to about 80%, while other mutants at K247, in addition to the positively charged residue, also showed enhanced defucosylation activity. The defucosylation percentage was calculated by:

[0113]

[0114] Table 2. Analysis of defucosylation percentage.

[0115]

[0116]

[0117] "NF" indicates the structure of core GlcNAc-fucose, and "N" indicates the structure of core GlcNAc.

[0118] Although the present disclosure has been described in conjunction with the specific embodiments set forth above, many alternatives and modifications and variations thereof will be apparent to those skilled in the art. All such alternatives, modifications and variations are considered to fall within the scope of the present disclosure.

[0119] References:

[0120] US11193155B2

[0121] Klontz EH, Li C, Kihn K, Fields JK, Beckett D, Snyder GA, Wintrode PL, Deridge D, Wang LX, Sundberg EJ. Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation. Nat Commun. 2020 Dec 4;11(1):6204.

[0122] Li C, Zhu S, Ma C, Wang LX. Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins. J Am Chem Soc. 2017 Oct 25;139(42):15074-15087.

[0123] Osanjo G, Dion M, Drone J, Solleux C, Tran V, Rabiller C, Tellier C. Directed evolution of the alpha-L-fucosidase from Thermotoga maritima into an alpha-L-transfucosidase. Biochemistry. 2007 Jan 30;46(4):1022-33.

[0124] US10415021

[0125] Sequence Listing Translation

[0126] CHO PHARMA,INC.

[0127] FUCOSIDASE MUTANTS AND THE USE THEREOF Fucosidase mutants and their use.

Claims

1. A mutant α-L-fucosidase comprising a polypeptide having at least 95% sequence identity to SEQ ID NO: 1 and having a substitution mutation at amino acid position 247 (K247).

2. The mutant α-L-fucosidase according to claim 1, wherein the substitution mutation is selected from the group consisting of: K247A, K247C, K247D, K247E, K247F, K247G, K247I, K247L, K247M, K247N, K247P, K247Q, K247S, K247T, K247V, K247W and K247Y. 3 . The mutant α-L-fucosidase according to claim 1 , which has the sequence of any one of SEQ ID NOs: 2-18.

4. A composition comprising the mutant α-L-fucosidase according to any one of claims 1 to 3 and at least one glycosidase.

5. A method for preparing a defucosylated glycoconjugate in vitro, the method comprising contacting a glycoconjugate comprising one or more fucoses with an endoglycosidase and the mutant α-L-fucosidase according to any one of claims 1 to 3 sequentially or simultaneously.

6. The method according to claim 5, comprising the following steps in sequence: (a) contacting the glycoconjugate with the endoglycosidase; and (b) contacting the glycoconjugate with the mutant α-L-fucosidase according to any one of claims 1 to 3.

7. The method according to claim 5 or 6, further comprising (c) terminating the reaction.

8. The method of claim 7, wherein step (c) is performed at about 65°C for 15-25 minutes.

9. The method according to any one of claims 6 to 8, wherein the step (a) and / or the step (b) is performed at about 37°C for 0.5-2 hours.

10. The method according to any one of claims 6 to 9, wherein the endoglycosidase is endo-β-N-acetylglucosaminidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2 and variants thereof.

11. The method of claim 10, wherein EndoS2 comprises a substitution mutation selected from the group consisting of: T138D, T138E, T138F, T138H, T138K, T138L, T138M, T138N, T138Q, T138R, T138V, T138W, D182Q, D226Q, T227Q, and T228Q.

12. The method of any one of claims 5 to 11, wherein the fucose is selected from the group consisting of α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linked fucose.

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