Endoglycosidase capable of hydrolyzing multi-branched sugar chains

Abstract

To provide an endo-β-N-acetyl glucosaminidase (endoglycosidase) capable of hydrolyzing multi-branched carbohydrate chains.SOLUTION: The present invention provides an endoglycosidase derived from Bacteroides nordii and having the activity of hydrolyzing multi-branched carbohydrate chains, the endoglycosidase comprising a specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an endo-β-N-acetylglucosaminidase (endoglycosidase) capable of hydrolyzing multi-branched sugar chains. [Background technology]

[0002] Endo-β-N-acetylglucosaminidase (endoglycosidase) is used for the cleavage and transfer of sugar chains on glycoproteins.

[0003] Endoglycosidases include Endo A, Endo F, Endo H, and Endo M. These enzymes either act only on glycans with specific structures or have strong activity against glycans with specific structures, and their reactivity against complex glycans is not very strong.

[0004] N-linked glycans of glycoproteins in higher animals often have multi-branched glycans such as tri- and tetra-antennary glycans, which are formed by the action of N-acetylglucosaminyltransferases such as GnT-V and GnT-IV. Most ENGases reported to date cannot act on tri- or tetra-antennary glycans. However, last year, a group from the Noguchi Institute reported that ENGase from the gram-negative bacterium Tannerella can cleave multi-antennary glycans (see Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Takashima S et al. Glycobiology, 30(11): 923-934 (2020) Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide an endo-β-N-acetylglucosaminidase (endoglycosidase) capable of hydrolyzing multi-branched sugar chains. [Means for solving the problem]

[0007] The present inventors discovered an enzyme (gene) in the Bacteroides nordii genome that is highly homologous to ENGase from Tannerella bacteria. Furthermore, they also found a similar enzyme in the genome of Barnesiella intestinihominis, an enterobacterium. As a result of measuring the substrate specificity of these enzymes, they found that these enzymes can hydrolyze multibranched sugar chains, leading to the completion of the present invention.

[0008] That is, the present invention is as follows. [1] An endoglycosidase derived from Bacteroides nordii that is either (a) or (b) below and has the activity of hydrolyzing multibranched glycans: (a) an endoglycosidase consisting of the amino acid sequence represented by SEQ ID NO: 2; (b) An endoglycosidase comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and having the activity of hydrolyzing multi-branched glycans. [2] A DNA encoding an endoglycosidase derived from Bacteroides nordii and having the activity of hydrolyzing multibranched glycans, which is either (a) or (b) below: (a) an endoglycosidase consisting of the amino acid sequence represented by SEQ ID NO: 2; (b) An endoglycosidase consisting of an amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted or added, and having the activity of hydrolyzing multi-branched glycans. [3] A DNA encoding an endoglycosidase derived from Bacteroides nordii and having the activity of hydrolyzing multibranched glycans, which is (c) or (d) below: (c) DNA consisting of the base sequence represented by SEQ ID NO: 1 (d) A DNA having a sequence identity of 95% or more with the DNA consisting of the base sequence represented by SEQ ID NO: 1 and encoding a protein having the activity of hydrolyzing multibranched glycans. [4] An endoglycosidase derived from Barnesiella intestinihominis and having the activity of hydrolyzing multibranched glycans, which is selected from the group consisting of (e) and (f) below: (e) an enzyme consisting of the amino acid sequence represented by SEQ ID NO: 4, 6, or 8; (f) An endoglycosidase consisting of an amino acid sequence represented by SEQ ID NO: 4, 6 or 8 in which one or several amino acids have been deleted, substituted or added, and having the activity of hydrolyzing multi-branched glycans. [5] A DNA encoding an endoglycosidase derived from Barnesiella intestinihominis and having the activity of hydrolyzing multibranched glycans, which is selected from the group consisting of (e) and (f) below: (e) an enzyme consisting of the amino acid sequence represented by SEQ ID NO: 4, 6, or 8; (f) An endoglycosidase consisting of an amino acid sequence represented by SEQ ID NO: 4, 6 or 8 in which one or several amino acids have been deleted, substituted or added, and having the activity of hydrolyzing multi-branched glycans. [6] A DNA encoding an endoglycosidase derived from Barnesiella intestinihominis and having the activity of hydrolyzing multibranched glycans, which is selected from the group consisting of (g) and (h) below: (g) DNA consisting of the base sequence represented by SEQ ID NO: 3, 5 or 7 (h) A DNA having a sequence identity of 95% or more with the DNA consisting of the base sequence represented by SEQ ID NO: 3, 5 or 7, and encoding a protein having the activity of hydrolyzing multibranched glycans. [7] A method for producing an endoglycosidase derived from Bacteroides norgeii [1] and having the activity of hydrolyzing multi-branched glycans, comprising introducing an expression vector containing the DNA of [2] or [3] into Escherichia coli and culturing the Escherichia coli. [8] A method for producing an endoglycosidase derived from Barnesiella intestinihominis [4] and having the activity of hydrolyzing multi-branched glycans, comprising introducing an expression vector containing the DNA of [5] or [6] into Escherichia coli and culturing the Escherichia coli. [9] A method for producing a free glycan, comprising contacting the endoglycosidase of [1] with a glycoprotein to cleave the glycan and obtain the free glycan.

[10] A method for producing a glycoprotein, comprising contacting the endoglycosidase of [4] with a protein and an oxazolinated sugar chain, and transferring the sugar chain to the protein. [Effects of the Invention]

[0009] By using the enzyme of the present invention, it is possible to release multi-branched sugar chains, and to prepare glycoproteins having uniform sugar chains from glycoproteins having multi-branched sugar chains. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the production of Endo-BN. [Figure 2] FIG. 1 shows the substrate specificity of Endo-BN. [Figure 3] FIG. 1 shows the results of degradation of the sugar chains of α-1 acidic protein by Endo-BN. [Figure 4] FIG. 1 shows the production of Endo-BI2. [Figure 5] FIG. 1 shows the substrate specificity of Endo-BI2. [Figure 6] FIG. 1 shows the decomposition of Fmoc sialyl 4-antennary asparagine oligosaccharide by Endo-BN. [Figure 7] FIG. 1 shows the degradation of Fmoc sialyl 4-antennary asparagine-linked oligosaccharides by Endo-BI2. [Figure 8A] FIG. 1 shows examples of triantennary glycans. [Figure 8B] FIG. 1 shows an example of a 4-branched sugar chain. [Figure 8C] FIG. 1 shows an example of a biantennary glycan having a bisecting GlcNAc. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will be described in detail below. 1. Endoglycosidase capable of hydrolyzing multi-branched glycans The enzyme of the present invention is an endo-β-N-acetylglucosaminidase that hydrolyzes the bond between the two GlcNAc residues (GlcNAc-GlcNAc) of N,N'-diacetylchitobiose present at the reducing end of the N-linked glycan of a glycoprotein, thereby releasing the glycan into the endo form. Furthermore, it is an endo-β-N-acetylglucosaminidase that can hydrolyze multibranched glycans.

[0012] In the sugar chain on which the enzyme of the present invention acts, fucose (core fucose) may or may not be bound to the reducing end (proximal) GlcNAc (N-acetylglucosamine) via an α1,6-bond.

[0013] In the present invention, a hyperantennary glycan refers to a complex-type glycan with two or more branches, including di-, tri-, and tetra-antennary glycans. In the present invention, a hyperantennary glycan refers to a complex-type glycan with three or more branches or a complex-type glycan with two or more branches having a bisecting GlcNAc. Examples of hyperantennary glycans are shown in Figures 8A to 8C. Figure 8A shows a triantennary glycan, Figure 8B shows a tetraantennary glycan, and Figure 8C shows a biantennary glycan with a bisecting GlcNAc. Branched glycans are high-mannose glycans, which have a structure in which mannose (Man) oligomers are linked to diacetylchitobiose (GlcNAc-GlcNAc), and complex glycans, which have diacetylchitobiose linked to at least one of Man and GlcNAc, galactose (Gal), and sialic acid (Neu5Ac). Glycans in which fucose is attached to the GlcNAc residue at the reducing end via an α1,6 linkage are referred to as "fucosyl-biantenary" (two-chain), "fucosyl-triantennary" (three-chain), or "fucosyl-tetraantennary" (four-chain), depending on the number of branches in the glycan. N-linked glycans are linked to asparagine residues in the glycan binding sequence, which is represented by Asn-any amino acid-Ser / Thr in the amino acid sequence of a protein.

[0014] The enzyme of the present invention can act on and hydrolyze sugar chains with two or more branches, but particularly acts on and hydrolyzes tri- or tetra-branched sugar chains.

[0015] The endo-β-N-acetylglucosaminidase of the present invention acts particularly strongly on and cleaves two-antennary (biantennary) complex-type sugar chains in which fucose is bound to the base.

[0016] The endo-β-N-acetylglucosaminidase of the present invention does not act on high-mannose sugar chains. Here, a high-mannose sugar chain is a sugar chain consisting of Man residues, and examples of high-mannose sugar chains include Man3- to Man9-type sugar chains, in which 3 to 9 mannoses are linked. For example, a sugar chain in which 6 mannoses are linked is represented by Man6GlcNAc2Asn.

[0017] Enzymes of the present invention include Endo-BN and Endo-BI.

[0018] The Endo-BN of the present invention can be isolated from Bacteroides nordii, a bacterium of the genus Bacteroides. The nucleotide sequence of Endo-BN is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0019] The Endo-BI of the present invention can be isolated from Barnesiella intestinihominis, a bacterium of the genus Bacteroides. There are three types of Endo-BI derived from Barnesiella intestinihominis: Endo-BI1, Endo-BI2, and Endo-BI3. The nucleotide sequence of Endo-BI1 is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4. The nucleotide sequence of Endo-BI1 is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6. Furthermore, the nucleotide sequence of Endo-BI3 is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8.

[0020] As long as the endo-β-N-acetylglucosaminidase of the present invention can hydrolyze multi-branched sugar chains, at least one, preferably one or several amino acids in the above amino acid sequence may be mutated by deletion, substitution, addition, or the like.

[0021] For example, at least one, preferably one or several (e.g., 1 to 9, more preferably 1 to 5, and particularly preferably 1 or 2) amino acids may be deleted from the amino acid sequence represented by any one of SEQ ID NOs: 2, 4, 6, and 8; at least one, preferably one or several (e.g., 1 to 9, more preferably 1 to 5, and particularly preferably 1 or 2) amino acids may be added to the amino acid sequence represented by any one of SEQ ID NOs: 2, 4, 6, and 8; or at least one, preferably one or several (e.g., 1 to 9, more preferably 1 to 5, and particularly preferably 1 or 2) amino acids may be substituted with other amino acids from the amino acid sequence represented by any one of SEQ ID NOs: 2, 4, 6, and 8.

[0022] Such amino acid sequences in which one or several amino acids have been deleted, substituted or added in any of the amino acid sequences of SEQ ID NOs: 2, 4, 6 and 8 include those that have a sequence identity of at least 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more, when calculated with any of the amino acid sequences of SEQ ID NOs: 2, 4, 6 and 8 using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (for example, default, i.e., initial setting parameters).

[0023] A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in any of the amino acid sequences of SEQ ID NOs: 2, 4, 6 and 8 is substantially identical to a protein having an amino acid sequence in any of SEQ ID NOs: 2, 4, 6 and 8.

[0024] The DNA encoding the endo-β-N-acetylglucosaminidase of the present invention also includes DNA that can hybridize with DNA consisting of a sequence complementary to the DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 1, 3, 5, and 7 under the stringent conditions described below and encodes a protein having the activity of hydrolyzing multibranched sugar chains. Specifically, the conditions refer to hybridization using a DNA-immobilized filter at 68°C in the presence of 0.7 to 1.0 M NaCl, followed by washing at 68°C with 0.1 to 2x SSC solution (1x SSC consists of 150 mM NaCl and 15 mM sodium citrate). Alternatively, the DNA can be transferred and immobilized on a nitrocellulose membrane by Southern blotting, followed by washing with a hybridization buffer (50% formamide, 4x SSC, 50 mM HEPES (pH 7.0), 10x Denhardt's buffer). s) solution, 100 μg / ml salmon sperm DNA) at 42° C. overnight to form a hybrid.

[0025] Furthermore, DNA encoding the endoglycosidase of the present invention also includes DNA that has at least 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more sequence identity with DNA consisting of the base sequences represented by SEQ ID NOs: 1, 3, 5, and 7 when calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (for example, default, i.e., initial setting parameters), and that encodes a protein having the activity of hydrolyzing multibranched glycans.

[0026] Furthermore, the present invention also includes RNA corresponding to the above DNA, or RNA that can hybridize with the RNA under stringent conditions and that specifically acts on double-stranded complex-type glycans, cleaves and releases the double-stranded complex-type glycans, and encodes a protein that has the activity of transferring the glycans.

[0027] 2. Production of the enzymes of the present invention The endo-β-N-acetylglucosaminidase of the present invention that hydrolyzes multibranched sugar chains can be produced by culturing Bacteroides nordii or Barnesiella intestinihominis, and can be isolated from cultures such as the culture broth of these microorganisms using the amino acid sequence as an indicator. Furthermore, the endo-β-N-acetylglucosaminidase of the present invention that hydrolyzes multibranched sugar chains can be produced as a recombinant enzyme by introducing DNA encoding the enzyme into a host microorganism and culturing the microorganism. For example, an expression vector can be prepared by ligating (inserting) the DNA of the present invention into an appropriate vector, and the expression vector can then be introduced into a host microorganism to transform the host microorganism.

[0028] Similarly, the endo-β-N-acetylglucosaminidase of the present invention that hydrolyzes multibranched sugar chains can be produced as a recombinant enzyme by introducing a DNA encoding the enzyme into a host microorganism and culturing the microorganism. For example, an expression vector can be prepared by ligating (inserting) the DNA of the present invention into an appropriate vector, and the expression vector can be introduced into a host microorganism to transform the host microorganism.

[0029] The vector into which the DNA of the present invention is inserted is not particularly limited as long as it can be replicated in host cells such as bacteria such as E. coli, yeast, or animal cells, and examples thereof include plasmid DNA, phage DNA, etc. The vector DNA used to construct the expression vector is widely available and easily available, and examples thereof include pET vector, pQE vector, pCold vector, and pUC19 vector.

[0030] The method for constructing the expression vector of the present invention is not particularly limited and can be carried out by a conventional method.

[0031] Host cells transformed with the expression vector of the present invention are not particularly limited as long as they are capable of expressing the DNA of the present invention, and examples thereof include bacteria such as Escherichia coli and Bacillus subtilis, yeasts such as Saccharomyces cerevisiae, and animal cells such as Chinese hamster ovary (CHO) cells, monkey COS cells, and mouse fibroblasts.

[0032] The present invention encompasses a method for producing an endo-β-N-acetylglucosaminidase that hydrolyzes multibranched glycans, comprising culturing a host cell containing the above-mentioned DNA under conditions that allow expression of the DNA to produce an endo-β-N-acetylglucosaminidase that hydrolyzes multibranched glycans, and recovering the enzyme.

[0033] Furthermore, the present invention encompasses a method for producing an enzyme, which comprises culturing a host cell containing the above-mentioned DNA under conditions allowing expression of the DNA to produce endo-β-N-acetylglucosaminidase that hydrolyzes multi-branched glycans, and recovering the enzyme.

[0034] The enzyme produced by the host cell can be purified by known purification methods, such as gel filtration chromatography, ultrafiltration, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, chromatofocusing, isoelectric focusing, and gel electrophoresis, either alone or in combination.

[0035] 3. Use of the enzyme of the present invention The endo-β-N-acetylglucosaminidase of the present invention hydrolyzes multi-branched sugar chains. By using the enzyme of the present invention, it is possible to release multi-antennary complex glycans, and to prepare glycoproteins having uniform glycans from glycoproteins having multi-antennary complex glycans.

[0036] The present invention encompasses a method for hydrolyzing a multi-branched glycan by contacting a glycoprotein with the endo-β-N-acetylglucosaminidase of the present invention, and a method for hydrolyzing a multi-branched glycan but producing a cleaved glycoprotein by contacting a glycoprotein with the endo-β-N-acetylglucosaminidase of the present invention. [Example]

[0037] The present invention will be specifically explained by the following examples, but the present invention is not limited to these examples.

[0038] [Example 1] Expression of ENGase derived from Bacteroides nordii in Escherichia coli and analysis of its substrate specificity It has been reported that ENGase produced by the Gram-negative bacterium Tannerella spp. cleaves multibranched glycans (Takashima S et al. Glycobiology, 30(11): 923-934 (2020)).

[0039] The present inventors have discovered an enzyme gene in the Bacteroides nordii genome that is related in terms of sequence homology to ENGase produced by bacteria of the genus Tannerella.

[0040] The ENGase (Endo-BN) present in the B. nordii genome belongs to the same GH85 family as the three Tannerella ENGases, but the homology is not very high. Therefore, we suspected that this enzyme may have a novel substrate specificity, and analyzed its substrate specificity.

[0041] The nucleotide sequence of the gene derived from Bacteroides nordii is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The nucleotide sequence shown in SEQ ID NO: 9 was used to express the enzyme. The nucleotide sequence shown in SEQ ID NO: 9 was obtained by deleting the N-terminal signal peptide sequence and the C-terminal T9SS type A sorting domain from the original sequence, adding a restriction enzyme site, and optimizing the Escherichia coli codons. The amino acid sequence of the expressed enzyme is shown in SEQ ID NO: 10. The sequence shown in SEQ ID NO: 10 has a His tag added to the C-terminus.

[0042] The Endo-BN gene was ligated into a pET vector and expressed in E. coli by the following method.

[0043] The E. coli expression vector was synthesized by deleting the N-terminal signal peptide sequence and the C-terminal T9SS type A sorting domain from the NCBI Reference Sequence: WP_007484749.1, adding restriction enzyme sites, and optimizing the sequence for E. coli codons. The vector was then ligated between the XhoI and NdeI sites of pET-21b.

[0044] This E. coli expression vector was transformed into E. coli BL21(DE3) using the heat shock method. The reaction temperature was as follows: the competent cells were pre-chilled on ice for 5 minutes, then placed in a 42°C water bath for 30 seconds. After 2 minutes of chilling on ice, the cells were cultured in SOC medium at 37°C and 250 rpm for 1 hour, then inoculated onto an LB plate and cultured at 37°C. After overnight culture, colonies growing on the plate were harvested to obtain transformed E. coli.

[0045] The resulting E. coli was inoculated into 1 L of LB medium in a 3 L flask to an OD600 of approximately 0.05. After culturing at 37°C and 180 rpm for 3 hours, IPTG was added and the culture was continued at 20°C and 150 rpm for 20 hours. The resulting culture was centrifuged at 7,000 rpm for 7 minutes to harvest the cells. The supernatant was discarded and 5 mL of disruption buffer was added to the cells. The cells were disrupted by sonication (output = 5, duty = 70, 10 times x 4 sets), and the solution was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was collected. The protein solution was purified using a His-Trap column. The results of SDS-PAGE analysis are shown in Figure 1. A band was observed between 100 kDa and 130 kDa, which was determined to be Endo-BN with a theoretical molecular weight of 105.88 kDa.

[0046] [Example 2] Evaluation of substrate specificity of Endo-BN The substrate specificity of the expressed enzyme was examined by measuring the reaction rate on various PA-glycans.

[0047] The enzyme activity was measured by HPLC using the pyridylamino (PA) sugar chain shown in Figure 2 as a substrate.

[0048] Endo-BN expressed and purified in E. coli was reacted with 2 pmol of PA-sialobiantennary glycan as a substrate in 100 mM acetate buffer (pH 4.0) at 30°C for 10 minutes. The reaction mixture was applied to a 5C18 column (Wakosil 5C-18, 4.6 x 250 mm) in HPLC (GL Sciences) and eluted with 50 mM ammonium acetate buffer (pH 4.0) containing 0.15% 1-butanol at a flow rate of 1.5 ml / min.

[0049] The PA-GlcNAc obtained by the reaction was monitored using a fluorescence detector at 320 nm excitation and 400 nm emission.

[0050] The results are shown in Figure 2. Figure 2 shows the name and structure of the PA-glycan, and the relative activity when the PA-fucosyl sialobiantennary of No. 17 is taken as 100%.

[0051] Analysis revealed that the enzyme that degraded most rapidly was a fucosyl diantennary complex-type glycan (No. 17: sialic acid bond is α2,6-linked). This enzyme did not degrade high-mannose glycans such as M3-M9 at all. It was also found that the enzyme degraded triantennary complex-type glycans such as No. 15 relatively rapidly.

[0052] [Example 3] Examination of deglycosylation of α-1 acidic protein We investigated deglycosylation of α-1-producing glycoprotein, which is a glycoprotein generally known to contain tri- and tetra-antennary glycans, using Endo-BN.

[0053] Table 1 shows the reaction conditions for the deglycosylation reaction.

[0054] [Table 1]

[0055] The time course of the reaction was evaluated by SDS-PAGE after 0 minutes, 5 minutes, 10 minutes, 1 hour, 3 hours, 24 hours, and 144 hours. After 144 hours, an equal amount of Endo-BN was added to the reaction mixture, and the mixture was incubated at 37°C for 24 hours to confirm whether all of the glycans had been cleaved.

[0056] The results are shown in Figure 3. As shown in Figure 3, by reacting with Endo-BN, the sugar chains were cleaved and the band shifted from the band before the reaction. This demonstrates that all sugar chains on alpha-1 acidic protein can be hydrolyzed.

[0057] [Example 4] Expression of ENGase derived from Barnesiella intestinihominis in Escherichia coli Recently, Trastoy et al. reported that the genome of Bacteroides thetaiotaomicron, the most typical Bacteroides enterobacterium, contains an ENGase belonging to the GH18 family, and that the ENGase, named EndoBT-3987, primarily degrades high-mannose glycans (Nat Commun 11:899, 2020). Based on the amino acid sequence of Endo-BN, we performed a BLAST search and found that the genome of Barnesiella intestinihominis contains an ENGase gene belonging to the GH85 family.

[0058] Since B. intestinihominis is an intestinal bacterium, it was thought that the ENGase of this bacterium might actually release and assimilate human N-linked glycans exposed on the intestinal surface. Therefore, we decided to analyze the specificity of the GH85-type ENGase present in the genome of this bacterium.

[0059] First, we analyzed the genes of B. intestinihominis, for which the entire genome sequence has been reported. We decided to search for glycosidases in all genes of the B. intestinihominis YIT11860 strain using PubMed. GH family members were searched and identified using the FASTA data of proteins encoded by all 2,686 genes on the dbCANmeta server. As a result, we found that 93 glycosidases were present in all genes of the B. intestinihominis YIT11860 strain.

[0060] Therefore, we attempted to express three proteins belonging to the GH85 family of this bacterium in E. coli.

[0061] Because it was difficult to obtain the B. intestinihominis YIT11860 strain, whose entire genome sequence has been determined, we obtained the B. intestinihominis strain JCM15079 held by RIKEN, cultivated it, prepared DNA, and attempted to amplify the genes by PCR. Based on the nucleotide sequence of the YIT11860 strain, primers for PCR were synthesized from the nucleotide sequences near the N- and C-termini of the three genes. As a result, all three genes were amplified at the same size as the predicted molecular weight.

[0062] The resulting DNA fragments were inserted into a plasmid vector in Escherichia coli, and the base sequences of each gene were determined. The results showed that although some bases were different from the reported DNA sequence of B. intestinihominis YIT11860, the amino acid sequence was completely the same.

[0063] The three enzymes were named Endo-BI1, Endo-BI2, and Endo-BI3.

[0064] The nucleotide sequence of Endo-BI1 is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4. The nucleotide sequence of Endo-BI1 is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6. Furthermore, the nucleotide sequence of Endo-BI3 is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8.

[0065] Of these, Endo-BI2 was expressed in E. coli.

[0066] The E. coli expression vector was inserted into pCold1 after deleting the N-terminal signal peptide sequence from the NCBI Reference Sequence: WP_007484749.1.

[0067] This E. coli expression vector was transformed into E. coli BL21(DE3) by the heat shock method. The reaction temperature was adjusted by first chilling the competent cells on ice for 5 minutes and then placing them in a 42°C water bath for 30 seconds. After chilling on ice for 2 minutes, the cells were cultured in SOC medium at 37°C and 250 rpm for 1 hour, then inoculated onto an LB plate and cultured at 37°C. After overnight culture, colonies growing on the plate were harvested to obtain transformed E. coli.

[0068] The resulting E. coli was inoculated into 1 L of LB medium in a 3 L flask to an OD600 of approximately 0.05. After culturing at 37°C and 180 rpm for 3 hours, IPTG was added and the culture was continued at 20°C and 150 rpm for 20 hours. The resulting culture was centrifuged at 7,000 rpm for 7 minutes to harvest the cells. The supernatant was discarded and 5 mL of lysis buffer was added and the cells were suspended. The cells were disrupted by sonication (output = 5, duty = 70, 10 times x 4 sets), and the solution was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was collected. The protein solution was purified using a His-Trap column. After purification with His-Trap, gel filtration was performed to further increase the purity, and the Endo-BI2 fraction was collected. The resulting fractions were evaluated by SDS-PAGE. The results are shown in Figure 4. The left lane in Figure 4 represents molecular weight markers, and the right lane represents each Endo-BI2 fraction. A band appeared above 130 KDa, relative to the theoretical molecular weight of 137496.84, and was determined to be Endo-BI2.

[0069] Furthermore, the specificity of Endo-BI2 was evaluated in the same manner as in Example 2. The results are shown in Figure 5. Figure 5 shows the name and structure of the PA-glycan, and the relative activity when No. 2 PA-asialobiantennary was set as 100%.

[0070] The analysis revealed that the asialo diantennary complex-type glycan (No. 2) was degraded most rapidly. It was also found that triantennary complex-type glycans such as No. 4 and tetraantennary complex-type glycans such as No. 5 were degraded relatively quickly.

[0071] [Example 5] Examination of deglycosylation of Fmoc sialyl 4-branched asparagine oligosaccharides using Endo-BN Four microliters (4 μl) of Fmoc sialyl 4-branched asparagine oligosaccharide (25 mM) was dissolved in 32 μl of 200 mM acetate buffer (pH 4.5) and 5 μl of distilled water, to which 4 μl of Endo-BN was added, followed by incubation at 37° C. for 24 hours. The resulting reaction mixture was subjected to reverse-phase HPLC under the following conditions. [HPLC conditions] (1)Analytical equipment: UV 214nm (manufactured by Agilent) (2) Column: Imtankt CD-C18 (Φ2 × 150 mm) (3) Mobile phase Solution A: 0.1(v / v)% TFA aqueous solution Solution B: 0.1 (v / v)% TFA in acetonitrile A mixture of liquids A and B was used, and the ratio of B (Vol%) was changed from 0% to 40% over a period of 0 to 20 minutes. (4)Flow rate 0.4 ml / min (5) Column temperature: 40°C

[0072] The results are shown in Figure 6. As shown in Figure 6, the Endo-BN reaction cleaved the glycan, shifting the band from the band before the reaction. This demonstrates that the glycan of the Fmoc sialyl 4-antennary asparagine glycan can be hydrolyzed.

[0073] [Example 6] Examination of deglycosylation of Fmoc sialyl 4-branched asparagine oligosaccharides using Endo-BI2 2 μl (100 mM) of Fmoc sialyl 4-branched asparagine oligosaccharide was dissolved in 8 μl of 100 mM sodium phosphate buffer (pH 6.0) and 5 μl of distilled water, to which 5 μl of Endo-BI2 was added, followed by incubation at 37° C. for 20 hours. The resulting reaction mixture was subjected to reverse-phase HPLC under the following conditions. [HPLC conditions] (1) Analytical equipment: Evaporative light scattering detector (Agilent) (2) Column: GL Sciences Inertsil ODS-3 (Φ2.1 × 250 mm) (3) Mobile phase Solution A: 0.1(v / v)% TFA aqueous solution Solution B: 0.1 (v / v)% TFA in acetonitrile A mixture of liquids A and B was used, and the ratio of B (Vol%) was changed from 0% to 80% over a period of 0 to 40 minutes. (4)Flow rate 0.2 ml / min (5) Column temperature: 40°C

[0074] The results are shown in Figure 7. As shown in Figure 7, the reaction with Endo-BI2 cleaved the glycan, shifting the band from the band before the reaction. This demonstrates that the glycan of Fmoc sialyl 4-antennary asparagine glycan can be hydrolyzed. [Industrial Applicability]

[0075] The enzyme of the present invention can be used to prepare therapeutic recombinant glycoproteins such as antibody drugs having uniform sugar chains.

Claims

[Claim 1] A method for producing free glycans, comprising contacting a glycoprotein with an endoglycosidase derived from Bacteroides nordii of the following (a) or (b), which has the activity of hydrolyzing a multi-antennary complex glycan having three or more branches or a multi-antennary complex glycan having two or more branches with bisecting GlcNAc, and cleaving the multi-antennary complex glycan having three or more branches or a multi-antennary complex glycan having two or more branches with bisecting GlcNAc to obtain free glycans: (a) an endoglycosidase consisting of the amino acid sequence represented by SEQ ID NO: 2; (b) An endoglycosidase having 95% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having the activity of hydrolyzing polybranched sugar chains.

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