JP5917913B2 - Improved methods for carbohydrate-related enzymes - Google Patents

Description

  The present invention relates to a method for improving the function of a carbohydrate-related enzyme or its catalytic domain. Specifically, a carbohydrate-binding enzyme (CBM) region that binds to a glycoprotein sugar chain is fused to another carbohydrate-related enzyme or a catalytic region thereof to form a chimeric polypeptide, or the carbohydrate-related enzyme or The present invention relates to a method for improving the function of the catalytic region or imparting a new function, and a chimeric polypeptide thereof.

  About half of the world's population is infected with Helicobacter pylori, and it is still regarded as a causative agent for gastric cancer, gastric malignant tumors and gastric ulcers. At present, the inspection system for Helicobacter pylori is almost satisfied, and the effect of reducing the risk of infection with Helicobacter pylori is increasing. However, in the sterilization treatment, the use of antibiotics induces the risk of developing new resistant bacteria. In fact, even in recent years when antibiotics are used in combination with two or three antibiotics, The incidence of resistant bacteria tends to increase (Non-Patent Document 1). Furthermore, such heavy use of antibiotics may damage the human body and kill enteric bacteria, resulting in adverse effects on carriers. On the other hand, the risk of Helicobacter pylori to humans is thought to vary depending on the number of Helicobacter pylori colonization in the stomach, but in animal experiments, when the average number of Helicobacter pylori in the stomach of mice is about 1/10, It has been reported that gastric ulcer does not develop (Non-patent Document 2). This means that the risk can be greatly reduced if the number of H. pylori is limited to a certain extent. From this situation, several dairy manufacturers currently sell lactic acid bacteria and yogurt containing bifidobacteria that are effective against H. pylori and explain the risk reduction effect. However, since lactic acid bacteria and bifidobacteria are difficult to survive in a severe environment (strongly acidic condition) in the stomach, their effects are limited.

  H. pylori inhabits the surface mucus secreted from the surface layer of the gastric mucosa, but not in the gland mucus secreted from the mucosa or the deep mucosa. This gland mucus is composed of gastric mucin with a specific structure, and gastric mucin (mucin-type glycoprotein) binds α-linked N-acetylglucosamine residue (αGlcNAc residue) and galactose residue (Gal residue). O-glycoprotein sugar chain (O-glycan) having the GlcNAcα1 → 4Galβ residue at the non-reducing end is characteristically included. In recent years, it has also been reported that these sugar chains appear with the appearance of gallbladder cancer, pancreatic cancer, cervical cancer and other neoplasms (Non-patent Document 3).

Nakayama et al. Reported that the core di-branched O-glycan having an αGlcNAc residue at the non-reducing end (mainly Gal β1 → 4GlcNAcβ1 → 6 ( Gal β1 → 3) Gal terminal of the sugar chain having the structure of GalNAcα1 → R (underlined part) Glycoprotein sugar chain in which GlcNAc is bound to α) is bound to the growth of Helicobacter (including H. pylori). It has been clarified that it is an enzyme activity inhibition of glucosylcholesterol synthase (CHLαGcT) (Non-Patent Document 4, Non-Patent Document 5) possessed only by (Non-Patent Document 4). Helicobacter pylori requires a glycosyl cholesterol component (CGL) for its own growth, but it cannot synthesize CGL itself, so it takes cholesterol from the outside and builds a cell wall by adding glucose near the cell membrane of the fungus. It is thought that Therefore, it is speculated that glycoprotein sugar chains having O-glycans containing αGlcNAc residues have the property of inhibiting the construction of the cell wall of H. pylori, and are expected to be applied as growth inhibitors specific to H. pylori. it can. Since gastric mucin containing αGlcNAc also has livestock such as pigs, preparation by extraction from them is also conceivable. However, the separation and extraction requires a complicated multi-stage extraction process, and there is a problem in the supply amount of animal gastric mucin as a raw material. On the other hand, a method using a separating agent (antibody or lectin) specific for αGlcNAc is also conceivable. For proteins that specifically bind to gastric mucins including αGlcNAc, monoclonal antibodies previously found by Horita et al. (HIK1083 (Non-patent Document 6); IgM and HGM504 (Kanto Chemical); IgM, HGM507 (Kanto Chemical) ); IgM). HIK1083 is a monoclonal antibody recognizing αGlcNAc residue, HGM504 is a monoclonal antibody recognizing GlcNAcα1 → 4Galβ1 → 4GlcNAc residue, and HGM507 is a monoclonal antibody recognizing GlcNAcα1 → 4Gal residue. Has been used. However, considering the preparation of gastric mucin as a functional substance, the use of expensive antibodies is impractical (and the above-mentioned antibodies are IgM, so the cost effectiveness is quite low). Moreover, although there is a report example (nonpatent literature 7) about development of the lectin with respect to (alpha) GlcNAc, there are many problems regarding genetic engineering application and mass productivity.

  On the other hand, preparing such glycoprotein sugar chains by a multi-step synthetic chemical method is not a solution in productivity. Patent Document 1 discloses a Helicobacter pylori-binding substance that is an oligosaccharide having a Galβ1 → 3GlcNAcGalβ1 → 4GlcNAc structure. However, since this substance also has a complicated structure, it must be prepared through a multi-step process and cannot be prepared easily in large quantities.

On the other hand, the inventors have found that a derivative in which an alkyl group such as an ethyl group is introduced into αGlcNAc also has an effect of suppressing the growth of H. pylori (Patent Document 1). However, although the compound having this simple structure is advantageous in preparation, the effect is several tens to several hundreds of less than the mucin-type glycoprotein having αGlcNAc described above. It is difficult to expect the effect (Non-patent Document 5).
Therefore, a new method for preparing αGlcNAc-containing mucin using inexpensive mucin-type glycoprotein as a raw material has been desired.

  By the way, the inventors have found an enzyme (AgnC) that specifically releases GlcNAc in the intestinal bacteria from the above-mentioned gastric mucin containing αGlcNAc. This enzyme consists of a region having a catalytic function for hydrolysis and a region that specifically binds to a sugar residue of O-glycan in gastric mucin. The latter is composed of multiple sugar-binding modules (CBM), which binds particularly strongly to Muc6-type O-glycans. Among the multiple CBM regions, one CBM is αGlcNAc-specific and some others are non-reducing. It is suggested that the terminal β-binding galactose (Galβ) specifically binds (see Non-Patent Document 8 and Japanese Patent Application No. 2011-167900 claiming priority from Japanese Patent Application No. 2010-173229). Therefore, the inventors have investigated whether AgnC transfers to galactose (Gal) using paranitrophenyl αN acetylglucosamine (GlcNAcαpNP) or a compound as shown below as a sugar donor. It was found that it has no function of transferring (transfer function).

  Based on the above situation, the inventors investigated whether or not other host-derived enzymes have a transfer function, and as a result, some of the enterobacterial enzymes efficiently converted GlcNAc into α-linked Gal. (Patent document 2). The technical feature of the present invention is that a sugar donor (N acetyl α-glucosaminyl α-dimethoxytriazole, GlcNAcα-DMT) that can be prepared in one step in an aqueous solution is converted to αN acetylglucosaminidase derived from Bacteroides thetaiotaomicron ( (AgnBT) transfers GlcNAc to a sugar receptor containing Gal. It was shown that an oligosaccharide chain containing GlcNAcα1 → 4Gal can be prepared by this method. Moreover, it has been suggested that the oligosaccharides obtained by these methods already have anti-pylori activity (Non-patent Document 9). However, the degree of the effect of these oligosaccharides is only a few hundred μM level, and in the preparation of the oligosaccharide, the preparation process of the oligosaccharide (compound having Gal at the terminal) serving as a sugar receptor becomes complicated. Disadvantages are an issue.

  Therefore, the inventors tried the above transfer method (method described in Patent Document 2) in order to introduce α-linked GlcNAc into a mucin-type glycoprotein sugar chain that is α-linked and does not have GlcNAc at the terminal. Almost no introduced product could be obtained. This was thought to be due to the fact that the catalytic region of the enzyme was buried in the mucin-type glycoprotein sugar chain in which the target reaction sites (ie, galactose) were densely placed and could not be approached. Therefore, in the glycosyltransferase reaction of the above AgnBT that uses a mucin-type glycoprotein sugar chain as a sugar acceptor, it has become necessary to improve the translocation activity by devising an enzyme close to the reaction point.

  In view of the above situation, the present inventors examined the use of CBM having affinity for glycoprotein. This is because many CBMs are derived from microorganisms and are easy to use in genetic engineering. That is, since the CBM of AgnC described above has an affinity for glycoprotein sugar chains, it was considered whether such CBM could be used for enzyme transfer reactions.

  CBMs are currently classified into 64 groups based on amino acid sequence homology (Non-patent Document 10), and are found accompanying carbohydrate-related enzymes. In each family, amino acid residues essential for binding to a sugar substrate are defined from three-dimensional structural analysis data such as X-ray crystal structure analysis. Many CBMs having affinity for polysaccharides such as cellulose and amylose have been reported so far, but it has recently been found that many families have CBMs having affinity for glycoprotein sugar chains. .

  So far, several fusion enzymes with CBM have been reported. For example, attempts have been reported for a long time to introduce CBM (having affinity for β-linked glucose, which is a structural unit of cellulose) into cellulase, and to make cellulosic sugar from cellulose powder. In addition, by introducing CBM that binds to cellulose into an enzyme that phosphorolyzes cellodextrin, the activity of the enzyme is improved 2-3 times (Non-patent Document 11). On the other hand, with regard to glycoprotein sugar chains, in recent years, CBM (Non-patent Document 12) having affinity for cellulose is introduced into an enzyme that hydrolyzes an N-linked glycoprotein sugar chain, and CBM is introduced into the N-terminal region of the enzyme. Activity has been reported to be maintained in some fusions, but no improvement in activity has been reported.

  As described above, there are many examples of applications to sugar chains (polysaccharides) such as cellulose and chitin that do not exist in the human body as described above, but sugar chains (glycoprotein sugar chains) in the human body. There are few examples that target it. In addition, the CBM having an affinity for polysaccharides such as cellulose and chitin and the CBM having an affinity for sugar chains frequently found in the living body such as O-linked glycans and N-linked glycans have amino acid sequences. Belongs to another group (Non-patent Document 13), and the properties are considered to be quite different.

  From the above facts, by fusing / introducing a CBM having an affinity for a glycoprotein sugar chain (for example, the above-mentioned αGlcNAc-containing mucin-type glycoprotein sugar chain) to a protein having another function via an appropriate linker, The inventors have come up with the idea that if the function before fusion is maintained, the method can be a new and effective method that can efficiently introduce the target sugar (glycosyl transfer) into the target glycoprotein.

  Carbohydrate hydrolases (derived from microorganisms) are highly productive at the industrial level, but at present, most of the target products produced by the transglycosylation reaction are still at the oligosaccharide synthesis level. Therefore, the present invention has been considered to be an important means for preparing “glycoprotein sugar chains” useful in the medical field at an industrial level.

International Publication No. 2008/084561 Pamphlet JP 2011-244482 A

Pandey R et al., Asian Pacific Journal of Cancer Prevention, 2010, Vol. 11, p. 583-588. Kimura K, Probiotics and Biogenics, 2005, p. 345-356. Mikami Y et al., Modern Pathology, 2004, Vol. 17, p. 962-972. Hirai Y et al., Journal of Bacteriology, 1995, 177, p. 5327-5333. Kawakubo M et al., Science, 2004, 305, p. 1003-1006. Ishihara K, et al., Biochemistry Journal, 1966, 318, p. 409-416. Molchanova V, et al., Biochimica et Biophysica Acta, 2005, 1723, p. 82-90. Fujita M et al., Journal of Biological Chemistry, 2011, Vol. 286, p. 6479-6489. Lee H et al., Biochemical and Biophysics Research Communication, 2006, 349, p. 1235-1241. CAZy site, Internet (URL: http://www.cazy.org) Xinhao Y et al., Applied Microbiology and Biotechnology, 2011, Vol. 92, p. 551-560. Kwan EM et al., Protein Engineering Design and Selection, 2005, Vol. 18, p. 497-501. Guillen D et al., Applied Microbiology and Biotechnology, 2010, vol. 85, p. 1241-9.

  The present invention has been made to solve the above-mentioned problems. In order to efficiently introduce a target sugar into a glycoprotein sugar chain, a CBM having an affinity for the glycoprotein sugar chain, a sugar-related enzyme, This fusion protein maintains a function before fusion and newly imparts a function useful for glycoprotein preparation. Specifically, a CBM region polypeptide that specifically binds to a mucin-type glycoprotein sugar chain including GlcNAcα1 → 4Gal is fused to a polypeptide having an enzyme function, and the enzyme function is improved compared to the enzyme before the fusion. . That is, as a result, GlcNAc is efficiently and selectively introduced into mucin-type glycoprotein sugar chains.

  In the present invention, a carbohydrate-related enzyme itself, or a chimera in which the N- or C-terminus of its catalytic region is fused with the CBM region of a carbohydrate-related enzyme related to another glycoprotein sugar chain (ie, CBM that binds to a glycoprotein). Type polypeptide (Example 1) is allowed to act on a glycoprotein sugar chain serving as a sugar acceptor in the presence of a sugar donor in a buffer solution, thereby improving the transglycosylation activity and introducing the target sugar into the sugar You can get protein. The linker between the CBM and the catalytic region only needs to be 10 amino acid residues or more, but by using the sequence of 10 amino acid residues upstream from the N-terminus of the CBM of the original enzyme having CBM, Increases the possibility of maintaining each of its enzyme functions.

  Specifically, the region of one or more CBMs in the region from CBM 2 to 6 of AgnC belonging to GH family 89 (FIG. 1) is the number of amino acid residues at the C-terminus of AgnBT belonging to the same GH family 89. By allowing a polypeptide (chimeric protein) fused via a 25-residue polypeptide to act on a sugar acceptor (mucin-type glycoprotein) in a buffer solution in the presence of a sugar donor (GlcNAc-αDMT), An αGlcNAc-containing mucin-type glycoprotein is efficiently obtained (Example 7, Example 9). At this time, if there is a polypeptide site having a length of 10 or more amino acid residues, the enzyme function is maintained. Moreover, the polypeptide of the present invention brings about the improvement of hydrolysis activity (activity for releasing GlcNAc) on not only oligosaccharides but also glycoprotein sugar chains (Example 2, Example 3 and Example 6). By the above method, the target sugar (GlcNAc) can be introduced in the target binding mode (stereoselectivity 100%). This is because the enzyme (catalyst region) has strict stereoselectivity.

That is, the present invention relates to a method of using the following polypeptide (chimeric polypeptide) and the chimeric polypeptide.
(Claim 1) A carbohydrate-binding enzyme (CBM) region that binds to a glycoprotein sugar chain is fused to another carbohydrate-related enzyme or a catalytic region thereof to form a chimeric polypeptide. A method of improving the function of the catalyst region or adding a new function.
(Claim 2) The function or new function of the carbohydrate-related enzyme or the catalytic domain thereof is a transglycosylation activity for introducing a sugar into a target oligosaccharide, polysaccharide or glycoprotein sugar chain using a sugar donor. The method of claim 1.
(Claim 3) The method according to claim 1, wherein the function or new function of the carbohydrate-related enzyme or the catalytic region thereof is hydrolytic activity for oligosaccharide, polysaccharide or glycoprotein sugar chain.
(Claim 4) The method according to claim 1, wherein the function or new function of the carbohydrate-related enzyme or the catalytic domain thereof is resistant to organic solvents.
(Claim 5) The method according to claim 1, wherein the sugar binding module (CBM) region that binds to a glycoprotein sugar chain is a CBM region belonging to the CBM family 32.
(Claim 6) The sugar binding module (CBM) region that binds to a glycoprotein sugar chain is a CBM region associated with α-N-acetylglucosaminidase belonging to glycosyl hydrolase family 89 (GH89). Method.
(Claim 7) The method according to claim 1, wherein the other carbohydrate-related enzyme is α-N-acetylglucosaminidase belonging to glycosyl hydrolase family 89 (GH89).
(Claim 8) It has an amino acid sequence of 10 amino acid residues or more adjacent to the N-terminus of the CBM region in the original enzyme of the sugar binding module (CBM) region that binds to a glycoprotein sugar chain as a spacer during fusion. The method according to claim 1, wherein a polypeptide is used.

(Claim 9) A chimeric polypeptide in which a sugar-binding module (CBM) region that binds to a glycoprotein sugar chain is fused to another carbohydrate-related enzyme or a catalytic region thereof.
(Claim 10) The chimeric polypeptide according to claim 9, wherein the sugar binding module (CBM) region that binds to a glycoprotein sugar chain is a CBM region belonging to the CBM family 32.
(Claim 11) The sugar binding module (CBM) region that binds to a glycoprotein sugar chain is a CBM region associated with α-N-acetylglucosaminidase belonging to glycosyl hydrolase family 89 (GH89). Chimeric polypeptide.
(Claim 12) The chimeric polypeptide according to claim 9, wherein the other carbohydrate-related enzyme is α-N-acetylglucosaminidase belonging to glycosyl hydrolase family 89 (GH89).
(Claim 13) A polypeptide having an amino acid sequence of 10 amino acid residues or more adjacent to the N-terminus of the CBM region in the original enzyme of the sugar binding module (CBM) region that binds to a glycoprotein sugar chain as a fusion spacer The chimeric polypeptide according to claim 9, comprising:

  With the chimeric polypeptides having various CBMs of the present invention, the target sugar can be introduced into the non-reducing end of the target glycoprotein. That is, in the introduction of a new sugar residue in vitro, it was difficult to introduce a sugar by enzymatic transglycosylation reaction for a glycoprotein having a dense sugar chain. Thus, the intended sugar residue can be introduced.

  By the method according to claims 5 to 7 of the present invention, α-linked GlcNAc can be efficiently introduced into a sugar chain (O-glycan) in which galactose is concentrated (Example 9). In addition, after preparation, α-linked GlcNAc is used as a non-reducing end using, for example, a CBM (CBM4, described in Patent Document 3) having affinity for GlcNAcα1 → 4Galβ residue-containing O-glycan. The O-glycan possessed can be purified (highly purified).

  O-glycans containing GlcNAcα1 → 4Galβ residues are present only in certain mucin-type glycoproteins (Muc6). However, if the polypeptide of the present invention is a glycoprotein sugar chain having galactose at its end, GlcNAc is transferred to the galactose, so that various glycoproteins (sugars having glycans having galactose at the non-reducing end) are used. Protein) can be used as a sugar receptor, and α-linked GlcNAc residues can be introduced into, for example, inexpensive egg white mucins. In addition, many other types of glycoprotein sugar chains can be prepared by the same method as the polypeptide of the present invention. This is because sugar donors (such as sialic acid, galactose, fucose, N-acetylgalactosamine, etc.) can be obtained by introducing a leaving group into sugars constituting glycoprotein sugar chains or oligosaccharide chains containing them. Synthetic methods have been established that lead to a substrate for hydrolase and glycosyltransferase and to a substrate (sugar donor) for transglycosylation. This is because the type has been found and expected (such as the CAZy site described above).

  Further, the fusion of CBM as described above results in a significant increase in the original function of the enzyme (for example, hydrolysis activity) (actual example 2, example 3, and example 6). Therefore, for an enzyme having specificity for a certain oligosaccharide molecule, when it is desired to increase the function of the glycoprotein sugar chain having the oligosaccharide, the sugar residue located at the non-reducing end of the glycoprotein sugar chain The purpose can be achieved by introducing (fused) a CBM having an affinity for a CBM, particularly a tandem CBM, by the method described above. For example, an enzyme (endo αN-acetylgalactosaminidase) that hydrolyzes oligosaccharides (Galβ1 → 3GalNAcα-Ser / Thr) containing N-acetylgalactosamine bound to serine or threonine in an endo form (Ashida H, et al. , Glycobiology, 2008, Vol. 18, p.727-734)), a tandem CBM having an affinity for galactose or sialic acid located at the non-reducing end of a mucin-type glycoprotein sugar chain ( The enzyme activity can be increased predominately by introducing a non-hydrophobic enzyme (not limited to that of the present invention) into the enzyme. As a result, various O-glycan libraries can be obtained from glycoprotein sugar chains that are difficult to hydrolyze. It can be prepared.

  Furthermore, the chimeric polypeptide (chimeric enzyme) of the present invention has an advantageous effect in enzyme activity (glycosylation reaction) even in a mixed solvent with various organic solvents (Example 8). That is, it is considered that introduction of CBM into a protein having an enzyme function as in the present invention brings resistance of the enzyme to an organic solvent, resulting in an improvement in hydrolyzing activity of glycolipids and a sugar donor. In the presence, glycosylation to lipids is also possible.

  The glycoprotein sugar chain containing α-linked O-glycan obtained by the chimeric enzyme of the present invention is used as a food or drink to reduce, cure or prevent gastric diseases. Useful. Since the substance exhibits a strong H. pylori growth inhibitory action, an excellent anti-H. Pylori action can be obtained just by adding a small amount of H. pylori growth inhibitor to food and drink.

  The glycoprotein sugar chain containing α-linked O-glycan obtained by the chimeric enzyme of the present invention can be used as a pharmaceutical preparation or in combination with antibiotics (effective against H. pylori). It is used for the treatment, symptom relief and prevention of gastric diseases such as chronic gastritis and gastric ulcers derived from bacteria. In addition, since the sugar chain-containing substance exhibits a strong H. pylori-specific growth-inhibiting action, it is possible to produce an excellent anti-H. Pylori action just by taking a small amount of this pharmaceutical preparation, and no side effects occur. Useful for healing.

Primary structure of amino acid sequence of α-N-acetylglucosaminidase homolog. CBM: sugar binding module, FIVAR: found in variety mechanism domain, TM: transmembrane domain Detection by HPLC of hydrolysis activity against GlcNAc-α-Gal-β-pMP substrate Release of GlcNAc from Class III Mucin containing GlcNAc-α Hydrolysis activity of AgnBT1 chimeric enzyme against Class III Mucin ΑGlcNAc transfer activity of AgnBT1-chimeric enzyme (examination of enzyme concentration) ΑGlcNAc transfer activity of AgnBT1-chimeric enzyme (influence by organic solvent (5% v / v)) ΑGlcNAc transfer activity of AgnBT1 chimeric enzyme against mucin

  CBM is widely found in saccharide-related enzymes hosted by bacteria and higher organisms such as mammals, and is particularly prominent in saccharide-hydrolyzing enzymes of bacteria found in intestinal bacteria and soil. In claim 1, it is described that fusion of CBM having an affinity for glycoprotein sugar chain and a sugar-related enzyme improves enzyme activity and imparts a new function. If there is a target glycoprotein sugar chain and the target sugar site has been determined, an enzyme that acts on the oligosaccharide site containing the sugar site is selected and has an affinity for the glycoprotein sugar chain. A CBM having a property may be selected. Here, CBM includes galactose (Gal), fucose (Fuc), sialic acid (Sia, (N acetylneuraminic acid (NeuAc), and N acetylglycolic acid (GlcAc)), which are sugars constituting the glycoprotein sugar chain. ), Those having affinity for N acetylgalactosamine (GalNAc) can be selected by the above-mentioned CAZy site, etc., among those having an affinity for glycoprotein sugar chains. (CBM families 13, 32, 40, 47, (50), (51), 57) are particularly desirable.) Types of glycoprotein sugar chains having affinity even within the same family In addition, there is no particular limitation as long as the enzyme into which CBM is introduced is a carbohydrate-related enzyme. As shown below, fusion between the same GH (or glycosyltransferase (GT)) family (that is, the enzyme family before being fused and the same family of enzymes having the CBM to be introduced) Is particularly preferred.

  In the second to fourth aspects, as in the case of the method using the polypeptide according to the first aspect described above, it is used for glycosyl transfer, sugar release (hydrolysis), and resistance to organic solvents. It is described that it is a method. Moreover, the polypeptide used at that time is described in Claim 9 and Claim 10, respectively. The glycosyl transfer according to claim 2 may be a combination of a sugar donor and a sugar acceptor, and a glycoprotein having a non-reducing terminal as a sugar acceptor. The method can be used for any glycoprotein. In addition, both sugar donor and sugar acceptor can be used at any concentration as long as they are dissolved in the buffer. Similarly, the hydrolysis according to claim 3 can also be used as a method for hydrolyzing the glycoprotein itself in the absence of a sugar donor. In this case as well, any concentration can be used as long as the glycoprotein is dissolved in the buffer. In claim 4, it is described that the method according to claim 1 can be used for obtaining the organic solvent resistance of the target enzyme. The organic solvent that can be used here is an aprotic solvent such as tetrahydrofuran (THF), acetonitrile, and acetone as in Example 8, and any other solvent that is miscible with the buffer solution. Things can also be used. The concentration of the solvent used depends on the sugar-related enzyme to be introduced and the CBM to be introduced, but it can be used from 0 to 80% / Vol with respect to the buffer solution. For enzymes, it has significant activity in 5-20% / Vol organic solvents. On the other hand, protic solvents (alcohols) can result in inhibition of the intended reaction because the solvent itself can be a sugar acceptor. In addition, when a solvent that is not miscible with the buffer such as chloroform, hexane, ether, and toluene is used, the transglycosylation activity is maintained, but the reaction efficiency may be lowered.

  The CBM32 family described in claim 5 is a CBM belonging to the CBM32 family described in the above CAZy site, and among them, there is a report (or a presumed one) that particularly binds to a glycoprotein sugar chain. Is preferably used.

  The CBM described in claims 6 (method) and 11 (thing used in claim 6) is described as being a CBM associated with the GH89 family. In the following examples, a tandem type CBM belonging to the C-terminus of α-N-acetylglucosaminidase (AgnC) belonging to GH89 family derived from Clostridium perfringens strain 13 is effective. It is described. Here, with respect to such tandem CBMs, if one or more of them are introduced into the target enzyme, an improvement in function or the like can be expected. Similar to AgnC, tandem CBMs are also found in Bifidobacterium bifidum, Eubacterium terricum and Collinella stercoris (FIG. 1). Can be used. At this time, as the number of CBMs to be introduced increases, an improvement in affinity for glycoprotein can be expected, so that an improvement in hydrolysis activity, transglycosylation activity, and the like can be further expected.

  Claims 7 (method) and 12 (the product used in claim 7) include a catalytic region of α-N-acetylglucosaminidase belonging to glycosyl hydrolase family 89 (GH89) as the sugar-related enzyme to be introduced. It is described that. Note that the GH89, A Sid Agrobacterium Kapusuratamu (Acidobacterium capsulatum), Akarumanshia Mushinifiria (Akkermansia muciniphila), Arisuchipesu Shahi (Alistipes shahii), Asuchikakorisu EXEN tri Kas (Asticcacaulis excentricus), Bacteroides carboxymethyl Rani Sol Benz (Bacteroides xylanisolvents), Bacteroides fragilis ( Bacteroides fragilis, Bacteroides hercogenes, Bacteroides salanitronis, Bactero Death Seta thetaiotaomicron (Bacteroides thetaiotaomicron), Bacteroides Barugatasu (Bacteroides vulgatus), view Themba Luzia Kabarunae (Beutenbergia cavernae), Bifidobacterium bifidum (Bifidobacterium bifidum), Bra key Mycobacterium faecium (Brachybacterium faecium), Candidatus Arusuromitasu (Candidatus Arthromitus) , Caulobacter crescentus, Caulobacter segnis, Chitinophaga pinensis a pinensis), Clostridium perfringens, Clostridium spiroforme, Clostridium spiroforme, Flavobacterium johnisoniae, Glanuricera marsiensis Palidibacterium propionicigenes), Pedobacter heparinus, Pedobacter sultans, Prevotella Lumi Nicola (Prevotella ruminicola), Salmonella enterica (Salmonella enterica), stacker Blanc Zia Nassoenshisu (Stackebrandtia nassauensis), Streptomyces ambofaciens (Streptomyces ambofaciens), Streptomyces avermitilis (Streptomyces avermitilis), Streptomyces bottle Ken Jen cis (Streptomyces binchenggenis) , Streptomyces scabiei, Streptomyces venezuelae, Streptomyces violaceus niger ( treptomyces violaceusniger), Terigurobasu Sanenshisu (Terriglobus saanensis), Xanthomonas Akusonopodisu (Xanthomonas axonopodis), Xanthomonas oryzae (Xanthomonas oryzae), Zoberia junk Nibora Nsu (Zobellia galactanivorans), Zunonwanjia professional Fanda (Zunongwangia profunda), Anopheles Gambia (Anopheles gambiae), Arabidopsis Thaliana (Arabidopsis thaliana), Aspergillus oryzae, Bos taurus, Mosquito Roh Love di Sevilla Burigusae (Caenorhabditis briggsae), mosquito Enola Budi Sevilla elegans (Caenorhabditis elegans), Cheronasu Inanitasu (Chelonus inanitus), Doromaiusu Nova ho Randy (Dromaius Novaehollandiae), Drosophila melanogaster (Drosophila melanogaster), Homo sapiens (Homo sapiens), Micromonas, Mus musculus, Nicotiana tabacum, Oryza sativa Indica, Oryza sativa japonica Oryza sativa Japonica), Bitisu Binifera (Vitis vinifera), indicating a di-Mace (Zea mays), α-N- acetylglucosaminidase derived (see CAZy site).

  In addition, as a polypeptide for a linker between the CBM to be fused and the catalytic site of the carbohydrate-related enzyme to be fused according to claim 8 (method) and 13 (the product used in claim 8), About 10-25 residues upstream from the N-terminal side of the CBM (in the enzyme associated with the CBM, a polypeptide site on the N-terminal side about 10-25 residues from the N-terminal amino acid site of the CBM) By using it, the fused polypeptide can easily maintain or improve the enzyme activity. The AgnC CBM of the present invention introduces a 20-residue region from the N-terminus to the N-terminus of the second CBM from the N-terminus of AgnC (the first CBM to the C-terminus of the catalytic region). The enzyme activity is maintained as shown in the following examples by the amino acid sequence of 2 residues by adding the restriction enzyme site.

  A chimeric protein of AgnBT and a second to sixth polypeptide from the N-terminal side of a plurality of CBM regions associated with the C-terminus of AgnC and AgnBT is prepared as in Example 1 below. be able to. In addition, combinations of CBMs from other GH89 homologs and catalytic regions of other GH89 can be similarly prepared as chimera types.

  Furthermore, these chimeric polypeptides can be used by immobilizing them on a carrier. For immobilization on a carrier, a tag such as histidine tag (His) or glutathione-S-transferase (GST) can be introduced into the chimeric polypeptide in either the N-terminal or C-terminal direction.

  Affinity of the polypeptide prepared in the present invention (described in claims 6 and 11) to a Galβ residue-containing O-glycan in the case of a glycosyltransferase reaction and a GlcNAcα1 → 4Galβ residue-containing O-glycan in a sugar hydrolysis reaction The conditions for exhibiting the properties can be used as long as they are in a buffer solution having a pH of 4 to 9, but the specific conditions are as follows.

  Examples of the solution include a phosphate buffer solution, a carbonate buffer solution, and a Tris buffer solution, and phosphate buffered saline (PBS) is particularly preferable. The pH of the solution is preferably near neutral, and particularly preferably from pH 6.0 to pH 8.0. The reaction temperature is preferably 15 to 40 ° C, particularly preferably 25 ° C. The concentration of the oligosaccharide serving as the substrate can be used up to about several tens of millimetres, as long as the substrate is dissolved in the buffer solution under the conditions for reaction in both hydrolysis and transglycosylation reactions. . In the glycosyltransferase reaction, the concentration of glycoprotein serving as a sugar acceptor and the concentration of a sugar donor need only be dissolved, and can be used from several tens μM to several hundred μM. Any sugar donor may be used as long as it is a substrate for the transglycosylation reaction. In sugar donors, O-linked glycans transfer sugars to many non-reducing ends of glycoprotein sugar chains, so the sugar transfer yield is 10 to several hundred times higher than the sugar acceptor. improves. The reaction time is preferably from several tens of minutes to several hundreds of μM to several tens of minutes to 10 hours, particularly about 30 minutes to 5 hours in both the hydrolysis reaction and the transglycosylation reaction. preferable. However, in the glycosyltransferase reaction, the reaction rate tends to be relatively high in 5 hours or more when the polypeptide concentration is several tens of nM or less. Any reaction vessel may be used as long as it can control the temperature in the system and does not particularly adsorb dissolved proteins and carbohydrate compounds. In the reaction, the reaction time can be shortened by stirring and shaking.

Hereinafter, the present invention will be specifically described with reference to examples in which the chimeric polypeptide of the present invention was prepared and its function was investigated. However, the present invention is not limited in any way.
[Example 1]
(1.1) Acquisition of His-tag fusion protein (AgnBT1) that serves as a catalytic site for CBM-binding chimeric protein Using Bacteroides thetaitaomicron VPI5482 genome as a template, sense primer BamHI-BT1Δ32 (underlined is restriction enzyme BamHI site) ( GCG GAT CCC GAT CCC CAA TCG ATT CAT TG) (SEQ ID NO: 1) and antisense primer BT1Δ32-SacI (underlined is a SacI site) ( GTG AGC TCC TTT GCT TCA ATC GC) (SEQ ID NO: 2), DNA polymerase PrimeStar (manufactured by Takara) is used to increase the gene with thermal cycler PTC100 (manufactured by MJ Research). (Reaction conditions (turn 98 ° C. 30 seconds, 63.5 ° C. 30 seconds, for 30 cycles at 72 ° C. 33 seconds)) is performed to obtain a post amplification AgnBT1-DNA fragment (2,149Bp). The obtained product was treated with restriction enzymes (BamHI and SacI) and then ligated to a protein expression vector pET32a (+) with restriction enzyme sites (BamHI on the 5 ′ end side and SacI on the 3 ′ end side). The reaction was carried out and transformed into Escherichia coli (E. coli (BL21 (DE3) strain)) as an expression host, and the transformed manipulation body was obtained on an LB agar medium containing an ampicillin concentration of 50 μg / ml. Is pre-cultured in LB medium (2 ml) containing an ampicillin concentration of 50 μg / ml, further inoculated to 400 ml of the same culture medium, and shake-cultured at 37 ° C. until OD600 reaches 0.7, and then IPTG is 0. E. coli expressing this protein was prepared by adding to 1 mM and shaking culture at room temperature for 24 hours. After that, 4 ml of PBS solution (50 mM PBS, 300 mM NaCl) was added to the collected cells and suspended, and then lysozyme (10 mg / ml) was added to 0.75 mg / ml, and further PBS solution was added. The total volume was 4 ml, and this suspension solution was sonicated on ice, centrifuged at 20000 × g for 10 minutes, and the supernatant was purified using His-tag affinity chromatography (manufactured by TALON Metal affinity resins, Clontech). By using an ultrafiltration membrane (excluded molecular size 30000), this solution was removed from the imidazole etc. used for the purification, and as a result, the target protein was obtained as a purified product of about 20 mg per 1000 ml culture. (Fig. 1) The protein obtained was His-. Leave ag fusion protein were. Also, AgnBT protein expression vectors prepared using the following analysis was used subsequently for the preparation of a chimeric protein expression vector.

(1.2) Acquisition of His-tag fusion CBM2-chimeric protein (AgnBT1-CBM2)
Using the genome of Clostridium perfringens strain 13 as a template, the gene sequence of CBM2 and its surrounding region (amino acids 912 to 1090 from the N-terminal side), sense primer SacI-CBM2 (underlined indicates the restriction enzyme SacI site) (CG G AGC TC A TGG ATA AAA TAT TAG G) (SEQ ID NO: 3) and antisense primer CBM2-XhoI (underlined XhoI site) (AA C TCG AG T TCC TTT TCT TTT CC) (Sequence Listing Sequence) No. 4), using DNA polymerase PrimeStar (manufactured by Takara) and gene amplification (reaction conditions (in order 98 ° C. 5 seconds, 58.1 ° C. 30) with thermal cycler PTC100 (manufactured by MJ Research). Second, 30 cycles at 72 ° C. for 1 minute))), and a CBM2-DNA fragment (487 bp) was obtained after amplification. After subjecting the obtained product to restriction enzyme (SacI and XhoI) treatment, the restriction enzyme sites (SacI on the 5 ′ end side and SacI on the 3 ′ end side) were transferred to the AgnBT1 protein expression pET32a (+) vector prepared above. XhoI) is used for ligation reaction, transformed into E. coli (BL21 (DE3) strain) as an expression host, and transformed on an LB agar medium containing an ampicillin concentration of 50 μg / ml. This was pre-cultured in LB medium (2 ml) containing an ampicillin concentration of 50 μg / ml, further inoculated to 400 ml of the same culture solution, and shake-cultured at 37 ° C. until OD600 reached 0.7. IPTG was added to a concentration of 0.1 mM, and E. coli expressing this protein was prepared by shaking culture at room temperature for 24 hours. After centrifugation, after removing the supernatant, 4 ml of PBS solution (50 mM PBS, 300 mM NaCl) was added to the collected cells and suspended, and then lysozyme (10 mg / ml) was added to 0.75 mg / ml. The suspension was further sonicated on ice and centrifuged at 20000 × g for 10 minutes, and the supernatant was purified using His-tag affinity chromatography (TALON Metal). The solution was purified by affinity resin, manufactured by Clontech Co., Ltd. By using an ultrafiltration membrane (excluded molecular size 30000), the imidazole used for the purification was removed, and as a result, the target protein was purified as a purified product. , Approximately 21 mg per 1000 ml culture (FIG. 1). Remains of ag fusion protein, was used for the analysis of after.

(1.3) Acquisition of His-tag fusion CBM2-6 chimeric protein (AgnBT1-CBM2-6)
Using the genome of Clostridium perfringens strain 13 as a template, the gene sequences of CBM2-6 and its front and back regions (amino acids 912 to 1640 from the N-terminal side), sense primer SacI-CBM2 (underlined indicate the restriction enzyme SacI site) (CGG AGC TC A TGG ATA AAA TAT TAG G) (Sequence Listing SEQ ID NO: 5) and antisense primer CBM26-XhoI (underlined XhoI site) (AT C TCG AG T GAA GTG GAA TCT C) (Sequence Listing SEQ ID NO: 6) and using DNA polymerase PrimeStar (manufactured by Takara), gene amplification (reaction conditions (in order 98 ° C. 5 seconds, 59.7 ° C. 30 seconds), thermal cycler PTC100 (manufactured by MJ Research), Performs 2 ℃ in 33 seconds is performed 28 cycle)), was obtained after amplification CBM2-6-DNA fragment (2,173bp). After subjecting the obtained product to restriction enzyme (SacI and XhoI) treatment, the restriction enzyme sites (SacI on the 5 ′ end side and SacI on the 3 ′ end side) were transferred to the AgnBT1 protein expression pET32a (+) vector prepared above. XhoI) is used for ligation reaction, transformed into E. coli (BL21 (DE3) strain) as an expression host, and transformed on an LB agar medium containing an ampicillin concentration of 50 μg / ml. This was pre-cultured in LB medium (2 ml) containing an ampicillin concentration of 50 μg / ml, further inoculated to 400 ml of the same culture solution, and shake-cultured at 37 ° C. until OD600 reached 0.7. IPTG was added to a concentration of 0.1 mM, and E. coli expressing this protein was prepared by shaking culture at room temperature for 24 hours. After centrifugation, after removing the supernatant, 4 ml of PBS solution (50 mM PBS, 300 mM NaCl) was added to the collected cells and suspended, and then lysozyme (10 mg / ml) was added to 0.75 mg / ml. The suspension was further sonicated on ice, centrifuged at 20000 × g for 10 minutes, and the supernatant was purified using His-tag affinity chromatography (TALON Metal). The solution was purified by affinity resin, manufactured by Clontech Co., Ltd. By using an ultrafiltration membrane (excluded molecular size 30000), the imidazole used for the purification was removed, and as a result, the target protein was purified as a purified product. It could be obtained at about 19 mg per 1000 ml culture (Fig. 1) His-ta. Remains of the fusion protein, was used for the analysis of after.

[Example 2]
Enzyme activity for 4-nitrophenyl derivative (GlcNAc-α-pNP) 3-5 μl of each protein solution (31-34 μg / μl) prepared above, 20 μl of 15 mM GlcNAc-α-pNP solution, 10-fold concentrated PBS (pH 7) .4) 10 μl of solution was added and the total volume was made up to 100 μl with sterilized water. This was incubated at 37 ° C. with agitation. After reacting for a certain period of time, the amount of pNP released was measured with an absorptiometer to calculate the specific activity and reaction rate, and the reactivity of each protein to GlcNAc-α-pNP substrate was compared. (Table 1) In the chimeric protein (AgnBT1-CBM2) to which CBM2 was imparted, the Vmax value was improved about 13 times and the specific activity was improved about 12 times compared to the wild type AgnBT1 protein. On the other hand, in the chimeric protein (AgnBT1-CBM2-6) provided with CBM2-6, no significant improvement in activity was observed.
<Table 1> Kinetic analysis of hydrolysis activity against GlcNAc-α-pNP

[Example 3]
Enzyme activity for 4-methoxyphenyl derivative (GlcNAc-α-Gal-β-pMP) 2 μl of 15 mM GlcNAc-α-Gal-β-pMP, each protein solution prepared above (3.1-3.4 μg / μl) 1 μl, 10 μl concentrated PBS (pH 7.4) solution 1 μl was added, and the total volume was adjusted to 10 μl with sterile water. After stirring the reaction solution at 37 ° C. for 3 hours with stirring, the amount of Gal-β-pMP that is considered to be released after cleavage of GlcNAc was determined by HPLC (HPLC apparatus; GL7400 (manufactured by Shimadzu), analytical column; ODS-3 (4 6 × 250 mm), detection; UV 283 nm). In all proteins, the peak of Gal-β-pMP was confirmed, and it was found that the hydrolysis activity was particularly high in the AgnBT1-CBM2-6 chimeric protein. (Figure 2)

GlcNAc-α-Gal-β-pMP is diluted to a 0.05-3 mM solution, and each protein solution (3.1-3.4 μg / μl) prepared above is 0.4-0.5 μl. 3 μl of 10-fold concentrated PBS (pH 7.4) solution was added, and the total volume was adjusted to 30 μl with sterilized water. After reacting for a certain period of time, the amount of Gal-β-pMP was measured by HPLC, the specific activity and the reaction rate were calculated, and the reactivity of each protein to GlcNAc-α-Gal-β-pMP substrate was compared. (Table 2) In the AgnBT1-CBM2-6 protein, the Vmax value was improved about 5 times and the kcat value was improved about 9 times compared to the wild type AgnBT1 protein. On the other hand, in the AgnBT1-CBM2 chimeric protein, both the Vmax value and the kcat value were improved by about 2-3 times.
<Table 2> Kinetic analysis of hydrolysis activity against GlcNAc-α-Gal-β-pMP substrate

[Example 4]
Evaluation of hydrolytic activity for glycoprotein having αGlcNAc residue at its terminal 50 μl of 2% solution of porcine gastric mucin glycoprotein Porcin Gastric Mucin (PGM) or ClassIII Mucin (manufactured by SIGMA-Aldrich), 10-fold concentrated PBS (pH 7.4) 10 μl of the solution and about 31-34 μg of each of the proteins prepared above were added to make a total volume of 500 μl, and incubated at 37 ° C. for 5 hours with stirring. After the reaction, the solution was subjected to centrifugal filtration, and the filtrate was subjected to centrifugal concentration, dissolved in a predetermined amount of distilled water, and measured by the following methods.

[Example 5]
The post-treated filtrate is subjected to HPLC (HPLC apparatus; GL7400 (manufactured by Shimadzu), analytical column; Asahipak NH2P-50 (4.6 × 250 mm), detection; UV 210 nm), and is considered to be released from PGM after the enzymatic reaction. GlcNAc was detected. An increase in a peak that seems to be GlcNAc was confirmed at an elution time of about 10 to 12 minutes. When the AgnBT1-CBM2-6 chimeric protein was used as the enzyme source, the amount of GlcNAc detected was the highest compared to the wild-type AgnBT1 protein or AgnBT1-CBM2 chimeric protein (FIG. 3).

[Example 6]
Whether or not there was a change in the amount of αGlcNAc residue at the non-reducing end on Mucin was confirmed by dot blot using a HIK1083 antibody (Kanto Chemical) that recognizes GlcNAcα-residue. The mixture was treated with the primary antibody HIK1083 (diluted 500 times) at 37 ° C. for 1 hour, and anti-IgM-HRP (diluted 2000 times) (Santa Cruz Biotechnology, Inc.) was used as the secondary antibody. The amount of change in Galβ-residue newly appearing by cleaving αGlcNAc at the non-reducing end was detected by VECTASTAIN ABC systems (Vector Laboratories) using RCA ₁₂₀ lectin (Seikagaku Biobusiness Co., Ltd.). Mucin treated with the AgnBT1-CBM2-6 chimeric protein is less reactive to the HIK1083 antibody and more reactive to the RCA ₁₂₀ lectin than mucin treated with the wild-type AgnBT1 protein or the AgnBT1-CBM2 chimeric protein. It was. (Fig. 4)

[Example 7]
In order to confirm the presence or absence of αGlcNAc transfer activity of the AgnBT1 chimeric protein, a glycosyltransferase reaction was performed using Galβ-pMP as an acceptor and DMT-αGlcNAc as a sugar donor. Specifically, 12 μl of 250 mM Galβ-pMP, 4 μl of 250 mM DMT-αGlcNAc, and 2 μl of 10-fold concentrated PBS (pH 7.0) solution were added, and each protein solution prepared above was added to a final concentration of 0.4-1. The total volume was adjusted to 20 μl with sterilized water to 2 μl. The reaction solution was incubated at 37 ° C. for 30 min, 1 h, 5 h, and 16 hours with stirring. The reaction product is subjected to HPLC, and the peak of GlcNAc-α-Gal-β-pMP generated after the transfer of GlcNAc (HPLC apparatus; GL7400 (manufactured by Shimadzu), analytical column; ODS-3 (4.6 × 250 mm), detection UV 283 nm). It was confirmed that not only the wild-type AgnBT1 but also the AgnBT1 chimeric enzyme has an activity of transglycosylation, and the AgnBT1-CBM2 protein and the AgnBT1-CBM2-6 protein were at a lower concentration (0.4 μM) than the wild-type AgnBT1 protein. However, it has been found that it exhibits sufficient transglycosylation activity. (Fig. 5)

[Example 8]
The ability of transglycosylation activity under organic solvent-containing conditions was confirmed. Add 12 μl of 250 mM Galβ-pMP, 4 μl of 250 mM DMT-αGlcNAc, 2 μl of 10-fold concentrated PBS (pH 7.0) solution, and adjust each protein solution prepared above to a final concentration of 0.4-1.2 μl. In addition, 1 μl of acetonitrile was added to a final concentration of 5% (v / v), and the total volume was adjusted to 20 μl with sterilized water. In addition, acetone and tetrahydrofuran were reacted in the same manner. While stirring the reaction solution, it was incubated at 37 ° C. for 30 min, 1 h, 1.5 h, 16 hours, and the reaction product was subjected to HPLC after completion of the reaction. As a result, it was revealed that when acetonitrile or tetrahydrofuran was used, the activity of the wild-type AgnBT1 protein was lost and the transfer reaction did not proceed, but the activity was retained in the chimeric protein. In particular, the reactivity was kept high in acetonitrile. In acetone, it was found that particularly the AgnBT1-CBM2 protein retained high activity. (Fig. 6)

[Example 9]
The ability of AgnBT1 chimeric enzyme to transfer αGlcNAc to Gal residues on Mucin was examined. Mucin obtained by previously cleaving the terminal GlcNAcα-residue with the AgnC protein was prepared and used as an acceptor. Add 0.5 μl of 0.2% Mucin solution or 0.5% PGM solution treated with AgnC, 8 μl of 250 mM DMT-αGlcNAc, and 2 μl of 10-fold concentrated PBS (pH 7.0) solution. To a final concentration of 0.4-1.2 μl. The reaction solution was incubated at 37 ° C. for 1 h and 5 h with stirring. The reaction solution was spotted on a nitrocellulose membrane, and the above method was used to confirm the GlcNAcα-residue and Galβ-residue on Mucin by dot blot using HIK1083 antibody and RCA ₁₂₀ lectin, respectively. Mucin treated with the AgnBT1-CBM2-6 chimeric protein showed increased reactivity to the HIK1083 antibody compared to the mucin treated with the wild-type AgnBT1 protein or the AgnBT1-CBM2 chimeric protein, and conversely, the reactivity to the RCA ₁₂₀ lectin decreased. . (Fig. 7)

  The polypeptide provided by the present invention is a completely new method using a sugar chain binding tool possessed by enterobacteria, and the protein used is different from plant-derived lectins, and has a high binding force to sugar chains, genetic engineering. Therefore, the industrial applicability is high.

  The α-linked GlcNAc-containing O-glycan obtained by the polypeptide provided by the present invention is not only used as a pharmaceutical preparation for the treatment of gastric diseases, but also as a growth factor of good bacteria such as bifidobacteria, that is, as an intestinal agent. There is a possibility of use.

Claims (4)

A sugar-binding enzyme (CBM) region that binds to a glycoprotein sugar chain is fused to the C-terminus of another carbohydrate-related enzyme or its catalytic region to form a chimeric polypeptide, thereby the carbohydrate-related enzyme or its catalyst A method of improving the function of an area or adding a new function ,
A sugar binding module (CBM) region that binds to a glycoprotein sugar chain is a CBM region associated with α-N-acetylglucosaminidase belonging to glycosyl hydrolase family 89 (GH89);
Another carbohydrate-related enzyme is α-N-acetylglucosaminidase belonging to glycosyl hydrolase family 89 (GH89),
The function or new function of the carbohydrate-related enzyme or the catalytic domain thereof is (1) transglycosylation activity for introducing a sugar into a target oligosaccharide, polysaccharide or glycoprotein sugar chain using a sugar donor, (2) A method of hydrolyzing an oligosaccharide, polysaccharide or glycoprotein sugar chain, or (3) resistant to an organic solvent .   A polypeptide having an amino acid sequence of 10 amino acid residues or more adjacent to the N-terminus of the CBM region in the original enzyme of the sugar binding module (CBM) region that binds to a glycoprotein sugar chain is used as a spacer during fusion. Item 2. The method according to Item 1. A chimeric polypeptide in which a sugar-binding module (CBM) region that binds to a glycoprotein sugar chain is fused to the C-terminus of another carbohydrate-related enzyme or its catalytic region ,
A sugar binding module (CBM) region that binds to a glycoprotein sugar chain is a CBM region associated with α-N-acetylglucosaminidase belonging to glycosyl hydrolase family 89 (GH89);
A chimeric polypeptide in which the other carbohydrate-related enzyme is α-N-acetylglucosaminidase belonging to glycosyl hydrolase family 89 (GH89) . As a fusion of the spacer, claim 3 having the polypeptide having the amino acid sequence of more than 10 amino acid residues adjacent to the N-terminus of the CBM region in the original enzyme sugar binding modules (CBM) region that binds to glycoprotein sugar chains A chimeric polypeptide according to 1.

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