JPWO2020040257A1 - Enzyme synthesis method of β-glycoside of lacto-N-biose I or galacto-N-biose using modified phosphorylase - Google Patents

Abstract

In the present application, a modified GLNBP protein in which a mutation is introduced into a predetermined region of a non-mutated GLNBP, and the 1-position of galactose-1-phosphate using the same, and an N-acetylglucosamine residue or β-bond bound to a non-reducing terminal. A method for β-1,3 binding of the N-acetylglucosamine residue or the 3-position of the N-acetylgalactosamine residue in a sugar having an N-acetylgalactosamine residue and one or more sugar residues. I will provide a.

Description

The present disclosure relates to a method for synthesizing a β-glycoside of lacto-N-biose I or galacto-N-biose and a modified enzyme used therein.

Bifidobacterium-dominant bacterial flora is formed in the intestinal tract of breast-fed infants, which involves oligosaccharide components contained in human milk. Among the oligosaccharide components, lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc) is known to be a true bifidobacteria.
It is known that the formation of the intestinal flora in infancy has a great effect on obesity and the development of allergies in adulthood, and WHO recommends breastfeeding, but the rate of breastfeeding continues to decline. Also, the weaning time is earlier. In addition, the caesarean section rate exceeds 25% worldwide, and it is known that the intestinal bacterial flora of the delivered baby is significantly different from that of the vaginal delivered baby. For these reasons, there is an urgent need to develop prepared milk that is closer to human milk. However, although a chemical synthesis method and a fermentation method using metabolic engineering (for example, Non-Patent Document 1) have been developed as existing lacto-N-tetraose synthesis methods, none of them have been put into practical use.

β-1,3-galactosyl-N-acetylhexosamine phosphorylase (EC 2.4.1.211; also known as: galacto-N-biose / lacto-N-bios I phosphorylase) (hereinafter, may be abbreviated as GLNBP) is lacto-. N-Biose I (Galβ1-3GlcNAc) to galactose-1-phosphate (Gal-1-P) and N-acetylglucosamine (GlcNAc), and galacto-N-Biose (Galβ1-3GalNAc) to galactose-1-phosphate It is known as an enzyme that has an activity of phosphorylase decomposition and its reverse reaction catalytic activity on acid (Gal-1-P) and N-acetylgalactosamine (GalNAc), and its existence is present in many bacteria such as Bifidobacterium spp. Widely found in fungi. However, since GLNBP does not act on β-glycosides of lacto-N-biose I such as lacto-N-tetraose, these compounds cannot be prepared using the reverse reaction.

Much research has been done on the structure of GLNBP. For example, based on the results of Bifidobacterium longum subsp. Longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b) (hereinafter sometimes abbreviated as Bifidobacterium longum JCM1217 strain) and its storage stability, 27 in the genus Bifidobacterium. It is suggested that it has a helix structure of (Non-Patent Document 2). In addition, many research results have been reported on the active center in the three-dimensional structure (for example, Non-Patent Document 3).

Baumgaertner, F. et al., (2014), Synthesis of the Human Milk Oligosaccharide Lacto-N-Tetraose in Metabolically Engineered, plasmid-Free E. coli. ChemBioChem, 15 (13), 1896-1900. RCSB PDB ID 2ZUV, Crystal structure of Galacto-N-biose / Lacto-N-biose I phosphorylase in complex with GlcNAc, Ethylene glycol, and nitrate, Released: 2008-12-30 [Search August 10, 2018], URL: http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=2ZUV Hidaka, M., Nishimoto et al., The crystal structure of galacto-N-biose / lacto-N-biose I phosphorylase: A large deformation of a tim barrel scaffold, J.Biol.Chem., 284: 7273-7283, 2009

All disclosures of the prior art documents cited herein are incorporated herein by reference.

An object of the present application is to provide a method for producing a β-glycoside of lacto-N-biose I or galacto-N-biose, such as a novel lacto-N-tetraose.

As a result of diligent studies to solve the above problems, the present inventors have found that the modified GLNBP in which amino acid deletion / substitution / insertion is introduced into a predetermined region of GLNBP (SEQ ID NO: 1) of the Bifidobacterium longum JCM1217 strain is found. Reversibly catalyzes the synthesis of galactose-1-phosphate (Gal-1-P) and lacto-N-triose II (GlcNAcβ1-3Galβ1-4Glc) into lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc) We found out that we could do it and came to this application. Bifidobacterium longum JCM1217 strain GLNBP (SEQ ID NO: 1) has a loop region located near the active center in the tertiary structure between the 19th helix structure (448-451) and the 20th helix structure (468-478). (Positions 452 to 467) are present, and the region into which the mutation has been introduced is included in this loop region. Furthermore, in addition to lacto-N-triose II, the modified GLNBP has galactose-1-phosphate (Gal-) for a wide range of sugars having a β-linked N-acetylglucosamine (GlcNAc) residue at the non-reducing end. We have found that the formation of β-1,3 bonds with 1-P) is reversibly catalyzed, and have reached the present invention.
Aspects of the invention of the present application will be described in detail below.

[1] When the amino acid sequence of the non-mutant GLNBP is aligned with the amino acid sequence shown in SEQ ID NO: 1, one or more amino acids are deleted in the region corresponding to positions 452 to 467 in SEQ ID NO: 1. Substituted and / or inserted; and has a β-linked N-acetylglucosamine residue or a β-linked N-acetylgalactosamine residue at the 1-position of galactose-1-phosphate and 1 or A modified GLNBP protein having an enzymatic activity that binds β-1,3 to the N-acetylglucosamine residue or the 3-position of the N-acetylgalactosamine residue in a sugar having a plurality of sugar residues.

[2] One or more amino acids have been deleted, substituted and / or inserted in the loop region located in the three-dimensional vicinity of the active center of non-variant GLNBP; and one of galactose-1-phosphate. The N-acetylglucosamine residue or the N-acetylglucosamine residue in a sugar having a β-linked N-acetylglucosamine residue or a β-linked N-acetylgalactosamine residue and one or more sugar residues at the position and the non-reducing end. A modified GLNBP protein having an enzymatic activity that binds β-1,3 to the 3-position of N-acetylgalactosamine residue.

[3] When the loop region located in the three-dimensional vicinity of the active center of the non-mutant GLNBP aligns the amino acid sequence of the non-mutant GLNBP with the amino acid sequence shown in SEQ ID NO: 1, 452 to 467 in SEQ ID NO: 1. The modified GLNBP protein according to [2], which is a region of a position corresponding to a position.

[4] Lacto-N-triose II is a sugar having a β-linked N-acetylglucosamine residue or a β-linked N-acetylgalactosamine residue at the non-reducing end and having one or more sugar residues. , The modified GLNBP protein according to any one of [1] to [3], which has an enzymatic activity for synthesizing lacto-N-tetraose.

[5] In the loop region located three-dimensionally near the active center of the non-mutant GLNBP, 1 to 16 amino acids were deleted, 1 to 16 amino acids were replaced by 1 to 16 amino acids, and / Alternatively, the modified GLNBP protein according to any one of [2] to [4], wherein 1 to 10 amino acids are inserted.

[6] When the amino acid sequence of the non-variant GLNBP was aligned with the amino acid sequence shown in SEQ ID NO: 1, 1 to 16 amino acids were deleted in the region corresponding to positions 452 to 467 in SEQ ID NO: 1. The modified GLNBP protein according to any one of [1] to [5], wherein 1 to 16 amino acids are replaced by 1 to 16 amino acids and / or 1 to 10 amino acids are inserted.

[7] When the amino acid sequence of the non-variant GLNBP is aligned with the amino acid sequence shown in SEQ ID NO: 1, the region at the position corresponding to positions 452 to 467 (for example, positions 454 to 465 and 457 to 463) in SEQ ID NO: 1 In, 3 to 10 consecutive amino acids (for example, 4 to 8, 5 to 7, and 5 (for example, the region corresponding to the 458 to 462 position)) are deleted or 3 to consecutive amino acids are deleted. 10 consecutive amino acids (eg 4-8, 5-7, 7 (eg region corresponding to positions 457-463)) containing at least one glycine in a row of 2-10 (eg 2-7) The modified GLNBP protein according to any one of [1] to [6], which is substituted with 3) amino acids.

[8] When the amino acid sequence of the non-variant GLNBP is aligned with the amino acid sequence shown in SEQ ID NO: 1, the position corresponding to positions 452 to 467 (for example, positions 454 to 465, for example 457 to 463) in SEQ ID NO: 1 In the region, 3 to 10 consecutive amino acids (for example, 4 to 8, 5 to 7, and 7 (for example, the region corresponding to the 457 to 463 position)) of 2 to 10 consecutive amino acids (for example, 2 to 2) 7. The modified GLNBP protein according to any one of [1] to [7], which is substituted with glycine (7).

[9] When the amino acid sequence of the non-variant GLNBP is aligned with the amino acid sequence shown in SEQ ID NO: 1, the region at the position corresponding to positions 452 to 467 (for example, positions 454 to 465 and 457 to 463) in SEQ ID NO: 1 In, 3 to 10 consecutive amino acids (for example, 4 to 8, 5 to 7, and 7 (for example, the region corresponding to the position 457 to 463)) are composed of X ¹ X ² G and X ¹ GX ^2. , X ¹ GG, GX ¹ G, or GGG (X ¹ and X ² indicate amino acids other than the same or different glycine), substituted with three consecutive amino acids selected from [1]-[7]. ] The modified GLNBP protein according to any one of the items.

[10] When the amino acid sequence of the non-variant GLNBP is aligned with the amino acid sequence shown in SEQ ID NO: 1, the region at the position corresponding to positions 452 to 467 (for example, positions 454 to 465 and 457 to 463) in SEQ ID NO: 1 In, 3 to 10 consecutive amino acids (for example, 4 to 8, 5 to 7, and 7 (for example, the region corresponding to the 457 to 463 position)) are composed of AGG, CGG, EGG, FGG, HGG, and From the group consisting of IGG, KGG, LGG, MGG, NGG, PGG, QGG, SGG, TGG, WGG, YGG, GPG, GGC, GGL, GGM, GGP, GGGQ, GGS, GGGY, MGL, MGS, LGL, and LGG. The modified GLNBP protein according to any one of [1] to [7], which is substituted with three consecutive amino acids selected.

[11] The non-mutant GLNBP is derived from the genera Bifidobacterium, Anaerococcus, Clostridium, Cutibacterium, Erysipelothrix, Lachnoclostridium, Streptobacillus, or Vibrio, according to any one of [1] to [10]. The modified GLNBP protein described.

[12] The non-mutant GLNBP is
(I) Amino acid sequence selected from SEQ ID NOs: 1 to 14 and 100; or (ii) Having 75% or more sequence identity with the amino acid sequence selected from SEQ ID NOs: 1 to 14 and 100, and GLNBP activity. Have;
The modified GLNBP protein according to any one of [1] to [11].

[13] The modified GLNBP protein according to any one of [1] to [12], which has an amino acid sequence selected from the group consisting of the following:
(I) Amino acid sequence selected from SEQ ID NOs: 15-48, 98 and 99;
(Ii) An amino acid sequence having 80% or more sequence identity with the amino acid sequence selected from SEQ ID NOs: 15 to 48, 98 and 99.

[14] The N-acetylglucosamine residue in a sugar having the 1-position of galactose-1-phosphate and an N-acetylglucosamine residue β-bonded to a non-reducing terminal and having one or more sugar residues. The modified GLNBP protein according to any one of [1] to [13], which has an enzymatic activity of binding β-1,3 to the 3-position of.

[15] A polynucleotide encoding the modified GLNBP protein according to any one of [1] to [14].

[16] A vector containing the polynucleotide according to [15].

[17] A host cell transformed with the vector according to [16].

[18] The method for producing a modified GLNBP protein according to any one of [1] to [14], which comprises culturing the host cell according to [17] and recovering the modified GLNBP protein from the culture.

[19] In the presence of the modified GLNBP protein according to any one of [1] to [14], an N-acetylglucosamine residue or β-bonded to galactose-1-phosphate at a non-reducing terminal was β-bonded. The 1-position of the galactose-1-phosphate and the N-acetylglucosamine residue or N- A method of β-1,3 binding with the 3-position of an acetylgalactosamine residue.

[20] A sugar having a β-linked N-acetylglucosamine residue or a β-linked N-acetylgalactosamine residue at the non-reducing end and having one or more sugar residues is lacto-N-triose II. , [19].

[21] Lacto-N comprising a step of reacting galactose-1-phosphate with lacto-N-triose II in the presence of the modified GLNBP protein according to any one of [1] to [14]. -How to synthesize tetraose.

The modified GLNBP protein of the present application has galactose-1-phosphate and a β-linked N-acetylglucosamine residue or a β-linked N-acetylgalactosamine residue at the non-reducing end, and has one or more sugar residues. It has an enzymatic activity that binds β-1,3 to the sugar it has. The modified GLNBP protein of the present application can synthesize lacto-N-tetraose by using galactose-1-phosphate and lacto-N-triose II as substrates.

FIG. 1 shows the mutation sites of GLNBP mutants (TP, 2G, 3G, 4G, 5G, 6G, 7G). FIG. 2 shows a thin-layer chromatogram of a cell-free extract of GLNBP mutants (TP, 2G, 3G, 4G, 5G, 6G, 7G). triose II: Lacto-N-triose II. FIG. 3 shows the results of SDS-polyacrylamide gel electrophoresis (Coomassie staining) showing the degree of purification of GLNBP mutant (3G, 4G, 5G, 6G, 7G) proteins. FIG. 4 shows a thin layer chromatogram in the measurement of LNT (lacto-N-tetraose) synthetic activity of purified GLNBP-MGG. Deployed twice with BuOH: AcOH: H ₂ O = 2: 1: 1. Lane 1: 1 mM Lacto-N-triose ll Lane 2: 1 mM LNT Lane 3: 1 mM Sucrose Lane 4: 1 mM Fructose Lane 5: Reaction solution (810 min) _20-fold dilution FIG. 5 shows the results of HPAEC-PAD method analysis in the measurement of LNT synthetic activity of purified GLNBP-MGG. FIG. 6 shows the results of HPAEC-PAD analysis of synthetic lacto-N-tetraose and lacto-N-tetraose standards. ^{FIG. 7 shows the 1} H-NMR spectra of lacto-N-tetraose standard and synthetic lacto-N-tetraose. FIG. 8 shows a thin layer chromatogram in the measurement of Gal / GlcNAc residue binding activity of purified GLNBP-MGG. Deployed twice with 75% propanol. Lane 1: 1 mM N, N'-diacetylchitobiose Lane 2: 1 mM GlcNAc-β-pNP Lane 3: 1 mM Gal-1-P Lane 4: 1 mM Sucrose Lane 5: 1 mM Fructose Lane 6: GlcNAc β1-4 GlcNAc reaction Liquid 10-fold dilution lane 7: GlcNAcβ1-4 GlcNAc enzyme-free control 10-fold dilution lane 8: GlcNAc-β-pNP reaction solution 10-fold dilution lane 9: GlcNAc-β-pNP enzyme-free control 10-fold dilution FIG. 9-1 shows the alignment result of the GLNBP homolog of the genus Bifidobacterium (following FIG. 9-2). FIG. 9-2 shows the alignment result of the GLNBP homolog of the genus Bifidobacterium. Figure 10 shows RCSB PDB ID 2ZUV, Crystal structure of Galacto-N-biose / Lacto-N-biose I phosphorylase in complex with GlcNAc, Ethylene glycol, and nitrate, Released: 2008-12-30 [August 10, 2018] Search], URL: http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=2 Quoted from ZUV, Bifidobacterium longum subsp. Longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b ) Shows a schematic diagram showing the secondary structure. FIG. 11 corresponds to the 19th helix structure (positions 448 to 451), the loop region (positions 452 to 467), and the 20th helix structure (positions 468 to 478) of GLNBP (SEQ ID NO: 1) of the Bifidobacterium longum JCM1217 strain. The figure which shows the part is shown. FIG. 12 shows the amino acid sequence identity of each Bifidobacterium genus GLNBP homolog to GLNBP (SEQ ID NO: 1) of the Bifidobacterium longum JCM1217 strain. FIG. 13-1 shows the alignment result of the GLNBP homolog (continued from FIG. 13-2). FIG. 13-2 shows the alignment result of the GLNBP homolog. FIG. 14 corresponds to the 19th helix structure (positions 448 to 451), the loop region (positions 452 to 467), and the 20th helix structure (positions 468 to 478) of GLNBP (SEQ ID NO: 1) of the Bifidobacterium longum JCM1217 strain. The figure which shows the part is shown. FIG. 15 shows a model diagram in which LNT is incorporated into the active center of wild-type BlGLNBP. White: entire GLNBP, gray: LNT, black: loop area

In the present application, when the amino acid sequence of non-mutant GLNBP is aligned with the amino acid sequence shown in SEQ ID NO: 1, one or more amino acids are deleted in the region corresponding to positions 452 to 467 in SEQ ID NO: 1. Substituted and / or inserted; and having a β-linked N-acetylglucosamine residue or a β-linked N-acetylglucosamine residue at the 1-position of galactose-1-phosphate and 1 or Provided is a modified GLNBP protein having an enzymatic activity of β-1,3 binding to the N-acetylglucosamine residue or the 3-position of the N-acetylgalactosamine residue in a sugar having a plurality of sugar residues.

In addition, the present application has deleted, substituted and / or inserted one or more amino acids in a loop region located three-dimensionally near the active center of non-mutated GLNBP; and of galactose-1-phosphate. The N-acetylglucosamine residue in a sugar having a β-linked N-acetylglucosamine residue or a β-linked N-acetylgalactosamine residue at the 1-position and one or more sugar residues. Alternatively, a modified GLNBP protein having an enzymatic activity that binds β-1,3 to the 3-position of the N-acetylgalactosamine residue is provided.

As shown in FIG. 15, the inventors of the present application created a model diagram in which lacto-N-tetraose was incorporated into the active center of GLNBP of Bifidobacterium longum. According to this figure, it exists between the 19th and 20th helix structures in the secondary structure and is located near the active center in the tertiary structure (forming a part of the + subsite of the active center). ) The loop region collides with the lacto-N-tetraose. It is speculated that in the modified GLNBP protein of the present application, the structure of the loop region is changed by introducing a mutation into the loop region, the collision with sugars such as lacto-N-tetraose is eliminated, and the enzyme activity is exhibited. Will be done. The speculation of the mechanism does not limit the invention of the present application at all.

In the present application, the modified GLNBP protein means a protein prepared based on the non-mutated GLNBP. The modified GLNBP protein is the N-acetylglucosamine residue of a sugar having one position of galactose-1-phosphate and an N-acetylglucosamine residue β-linked to the non-reducing end and having one or more sugar residues. Enzymatic activity that binds β-1,3 to the 3-position of the group (hereinafter, may be abbreviated as Gal / GlcNAc residue-binding activity), and / or 1-position of galactose-1-phosphate and a non-reducing terminal. Enzymatic activity that binds β-1,3 to the 3-position of the N-acetylgalactosamine residue of a sugar having a β-linked N-acetylgalactosamine residue and one or more sugar residues (hereinafter, It may have Gal · GalNAc residue binding activity). Since non-mutant GLNBP is an enzyme having reversible GLNBP activity as described below, the Gal / GlcNAc / Gal / GalNAc residue binding activity of the modified GLNBP protein is also reversible. Therefore, the activity of phosphoric acid-degrading the β-1,3 bond can also be regarded as the Gal / GlcNAc / Gal / GalNAc residue binding activity of the modified GLNBP protein of the present application. The assay method for Gal / GlcNAc / Gal / GalNAc residue binding activity is not particularly limited and can be carried out by a method known to those skilled in the art, for example, by the method described in Examples of the present application. The modified GLNBP protein may or may not have the GLNBP activity described below.

In one embodiment of the present application, the modified GLNBP protein has one or more sugar residues having a β-linked N-acetylglucosamine residue at the 1-position of galactose-1-phosphate and a non-reducing end. It has an enzymatic activity that binds β-1,3 to the 3-position of the N-acetylglucosamine residue of the sugar.

In the present application, the "non-mutant GLNBP" has an enzymatic activity that catalyzes the following reaction (hereinafter, may be abbreviated as GLNBP enzyme activity), and the amino acid sequence of the non-mutant GLNBP is represented by SEQ ID NO: 1. When aligned with the amino acid sequence shown in, it means that there is no artificial mutation in the region corresponding to the position 452 to 467 in SEQ ID NO: 1 or in the loop region located in the three-dimensional vicinity of the active center. do.

The "non-mutant GLNBP" may be a wild-type GLNBP that is of biological origin and does not contain an artificial mutation, or as long as it has GLNBP enzyme activity, the amino acid sequence of the non-mutant GLNBP is SEQ ID NO: 1. When aligned with the amino acid sequence shown in, artificial mutation occurs in a location other than the region corresponding to positions 452 to 467 in SEQ ID NO: 1 or a location other than the loop region located in the three-dimensional vicinity of the active center. May have.

The "non-mutant GLNBP" may be of any origin, and may be any enzyme derived from prokaryotes such as bacteria, yeast, fungi, animals or the like, or is a recombinant enzyme. You may. Such enzymes can be commercially available, or can be purified by methods well known to those of skill in the art, such as from nature, or can be obtained by genetic recombination.

As an example of "non-mutant GLNBP"
Bifidobacterium genus: For example
Bifidobacterium longum (eg, Bifidobacterium longum subsp. Infantis ATCC 15697 (eg, Blon_2174: SEQ ID NO: 2); Bifidobacterium longum subsp. Longum JCM 1217 (eg, BLLJ_1623: SEQ ID NO: 1));
Bifidobacterium breve (eg, Bifidobacterium breve DSM 20213 = JCM 1192 (eg, BBBR_1586: SEQ ID NO: 3));
Bifidobacterium scardovii (eg BBSC_1789: SEQ ID NO: 4);
Bifidobacterium bifidum PRL2010 (eg BBPR_1055: SEQ ID NO: 5; BBPR_0233: SEQ ID NO: 6);
Bifidobacterium bifidum JCM1254 (eg, SEQ ID NO: 100);
Anaerococcus genus: eg Anaerococcus prevotii (eg SEQ ID NO: 7);
Genus Clostridium: eg Clostridium perfringens (eg SEQ ID NO: 8);
Genus Cutibacterium: for example, Cutibacterium acnes (eg, SEQ ID NO: 9);
Genus Erysipelothrix: eg, Erysipelothrix rhusiopathiae (eg SEQ ID NO: 10);
Genus Lachnoclostridium: eg, Lachnoclostridium phytofermentans (eg, SEQ ID NOs: 11 and 12);
Genus Streptobacillus: for example, Streptobacillus moniliformis (eg SEQ ID NO: 13) or
Genus Vibrio: eg Vibrio vulnificus (eg SEQ ID NO: 14)
The origin GLNBP can be mentioned.
In one embodiment, GLNBP is from the genus Bifidobacterium.

SEQ ID NOs: 1 to 14 and 100 shown below are amino acid sequences of proteins already known as GLNBP homologs having or highly likely to have GLNBP activity, and these GLNBP homologs are examples of the "non-mutant GLNBP" of the present application. Can be mentioned.

Further, in the example of "non-mutant GLNBP", the amino acid sequence selected from SEQ ID NOs: 1 to 14 and 100 and 75% or more, for example 80% or more, preferably 90% or more, more preferably 95% or more, for example. Those having 97% or more or 98% or more amino acid sequence identity and having GLNBP enzyme activity are included. The assay method for GLNBP enzyme activity is not particularly limited, and can be carried out by a method known to those skilled in the art, for example, according to the method described in Examples of the present application.

In one embodiment, the "non-mutant GLNBP" has SEQ ID NO: 1; or 75% or more, such as 80% or more, preferably 90% or more, more preferably 95% or more, for example 97% with SEQ ID NO: 1. It has the above or 98% or more amino acid sequence identity and has GLNBP enzyme activity.

As used herein, the percentage of "sequence identity" is determined by comparing sequences juxtaposed optimally in a comparison window. For example, when the optimum alignment of two or more sequences is obtained, a gap may occur in the reference sequence (if other sequences contain additions, a gap may occur, but the reference sequence here is deleted with additions for convenience. The portion of the subject sequence within the comparison window may contain additions or deletions (ie, gaps) as compared to (assuming no). Here, by specifying the number of positions where the "same" character string is recognized in both the reference array and the comparison target array, the number of their associated positions is obtained, and the number is calculated in the comparison window. The percentage of "sequence identity" can be calculated by dividing by the total number of positions of and multiplying the obtained result by 100. By using sequence analysis software known in the art, sequence identity can be easily measured. In addition, there are variations in the specific calculation procedure, discrimination criteria, how to handle the reference sequence, selection of the alignment method that is the premise, etc., depending on the software used, but those skilled in the art will appropriately select according to the purpose of use. It is possible to use it.

In the present application, "a region at a position corresponding to positions 452 to 467 in SEQ ID NO: 1 when the amino acid sequence of non-mutant GLNBP is aligned with the amino acid sequence shown in SEQ ID NO: 1" is based on SEQ ID NO: 1. Position 452-467 in SEQ ID NO: 1 when alignment was performed in the usual manner in the art (eg, using a program such as ClustalW (https://www.genome.jp/tools-bin/clustalw)). Means the area corresponding to and the determined area.

As used herein, "alignment" compares two or more sequence groups and constitutes a string (eg, a polypeptide sequence) that constitutes each sequence (eg, a polypeptide sequence). It refers to a process for appropriately associating (a character string indicating an amino acid residue to be used), and also refers to a comparison result of sequences after the association. Here, the appropriate association is evaluated based on "identity", "similarity", etc. between the sequences to be compared. The "identity" of a sequence (eg, the primary sequence of a protein) refers to the degree to which two or more contrastable sequences have the same string (eg, individual amino acids) relative to each other. Further, the "similarity" of a sequence (for example, an amino acid sequence) refers to the degree of a character string in which two or more contrastable sequences have the same or (if not the same) specific properties with respect to each other. As an example, in the evaluation of similarity of "amino acids", the properties of amino acids forming so-called conservative substitutions are evaluated as "common". A BLOSUM score matrix or the like is known as an exemplary index for quantitatively evaluating such "similarity", and those skilled in the art will appropriately select such an index according to the object to be compared and have similarities. It can be used for sexual evaluation.

In the present application, "alignment" may be performed by structural alignment using a method commonly used in the art. "Structural alignment" takes into account not only (primary) sequence information between molecules (eg, proteins) to be compared, but also structural information of the molecule (eg, information on secondary, tertiary, etc. of proteins). , This is a process of associating the sequences constituting the molecule. In general, since the three-dimensional structure of a protein is evolutionarily highly conserved, structural alignment results in poor similarity on the primary sequence (evolutionarily, when the sequences are more distantly related to each other). Also, it is possible to specify the region corresponding to a predetermined region of the reference sequence in (the sequence of) the molecule to be compared.

In the present application, the "loop region located in the three-dimensional vicinity of the active center of the non-mutant GLNBP" means a loop region forming a part of the + subsite of the active center of the non-mutant GLNBP. The loop region can be confirmed by analyzing the tertiary structure of non-mutant GLNBP by methods commonly used in the art. Examples of the method include an X-ray crystal structure analysis method, a nuclear magnetic resonance method, and an electron microscopy method as experimental methods, and can be confirmed by predicting the three-dimensional structure of the non-mutant GLNBP by a bioinformatics method.

In the wild-type GLNBP (SEQ ID NO: 1) of the Bifidobacterium longum JCM1217 strain, the loop region is located between the 19th helix structure (positions 448 to 451) and the 20th helix structure (positions 468 to 478) (452 to 467). Position). When aligned between homologs based on the amino acid sequence of an enzyme whose tertiary structure is already known, the corresponding region (particularly the active center that has a profound effect on enzyme activity and the region corresponding to its three-dimensional neighborhood) has the tertiary structure. Among them, it is presumed to be present at the same position, and it can be presumed that it has a similar effect on GLNBP activity. In the present application, the "loop region located in the three-dimensional vicinity of the active center of the non-mutant GLNBP" refers to 452 to 452 in SEQ ID NO: 1 when the amino acid sequence of the non-mutant GLNBP is aligned with the amino acid sequence shown in SEQ ID NO: 1. The region at the position corresponding to the position 467 may or may not form the loop region, although the region corresponding to the position 452 to 467 in the SEQ ID NO: 1 in the non-mutant GLNBP may or may not form the loop region. May be good.

When the amino acid sequence of the non-mutant GLNBP is aligned with the amino acid sequence shown in SEQ ID NO: 1, the region at the position corresponding to positions 452 to 467 in SEQ ID NO: 1 is
Blon_2174: Positions 452 to 467 in SEQ ID NO: 2 (Bifidobacterium longum subsp. Infantis ATCC 15697);
BBBR_1586: Positions 451 to 466 in SEQ ID NO: 3 (Bifidobacterium breve DSM 20213 = JCM 1192);
BBSC_1789: Positions 452 to 467 in SEQ ID NO: 4 (Bifidobacterium scardovii);
BBPR_1055: Position 452-467 in SEQ ID NO: 5 (Bifidobacterium bifidum PRL2010);
BBPR_0233: Position 452-467 in SEQ ID NO: 6 (Bifidobacterium bifidum PRL2010);
In SEQ ID NO: 100 (Bifidobacterium bifidum JCM1254), positions 452 to 467;
In SEQ ID NO: 7 (Anaerococcus prevotii), positions 454 to 469;
Positions 454 to 469 in SEQ ID NO: 8 (Clostridium perfringens);
In SEQ ID NO: 9 (Cutibacterium acnes), positions 465 to 480;
In SEQ ID NO: 10 (Erysipelothrix rhusiopathiae), positions 455-470;
In SEQ ID NO: 11 (Lachnoclos tridium phytofermentans), positions 455-470;
Positions 456 to 471 in SEQ ID NO: 12 (Lachnoclos tridium phytofermentans);
In SEQ ID NO: 13 (Streptobacillus moniliformis), positions 445-460;
In SEQ ID NO: 14 (Vibrio vulnificus), positions 456 to 477;
Is.

As an example of where amino acid deletions, substitutions and / or insertions are introduced with respect to the modified GLNBP protein of the present application, SEQ ID NO: 1 when the amino acid sequence of non-mutant GLNBP is aligned with the amino acid sequence shown in SEQ ID NO: 1. Within the range of positions corresponding to positions 452 to 467, within the range of positions corresponding to positions 454 to 465 in SEQ ID NO: 1, within the range of positions corresponding to positions 456 to 464, and positions corresponding to positions 457 to 463. Within the range of.

In the modified GLNBP protein of the present application, the total number of amino acids that can be deleted is not particularly limited as long as it is 1 or more and equal to or less than the number of amino acids in the amino acid sequence of the region in which the mutation can be introduced. Examples of the range of total number of amino acids that can be deleted include 1-16, 3-10, 4-8, 5-7, and 5 (eg, the region corresponding to positions 458-462). Can be mentioned. Also, examples of the range of total number of amino acids that can be deleted include a lower limit selected from 1, 2, 3, 4, and 5 and selected from 16, 13, 10, 9, 8, and 7. It can be indicated by a combination with the upper limit to be determined. The total number of amino acids may be consecutively deleted, or one and / or two or more consecutive amino acids may be deleted discontinuously.

In the modified GLNBP protein of the present application, the total number of amino acids that can be substituted is not particularly limited as long as it is 1 or more and equal to or less than the number of amino acids in the amino acid sequence of the region in which the mutation can be introduced. Examples of the total number of amino acids that can be substituted include 1-16, 3-10, 4-8, 5-7, and 7 (eg, the region corresponding to positions 457-463). Further, examples of the range of the total number of amino acids that can be substituted are selected from the lower limit value selected from 1, 2, 3, 4, and 5, and 16, 13, 10, 9, 8, and 7. It can be indicated by a combination with the upper limit value. The total number of amino acids may be continuously substituted, or one and / or two or more consecutive amino acids may be substituted discontinuously.

When an amino acid is substituted, it can be substituted by as many amino acids as the number of amino acids to be substituted, or by more / or less amino acids than the number of amino acids to be substituted. The total number of amino acids used for the substitution is 1 or more and is not particularly limited, and examples thereof include 1 to 16, 1 to 10, 2 to 7, and 3. Examples of the range of total number of amino acids used for the substitution are the lower limit selected from 1, 2, 3 and the upper limit selected from 16, 13, 10, 9, 8, 7, 5, 3. Can be indicated in combination with.

The amino acid used for the substitution may contain at least one glycine from the viewpoint of giving structural flexibility. Examples of amino acids used for the substitution include 2 to 10 consecutive (eg, 2-7, 3) amino acids containing at least one glycine. As an example, ^{three consecutive pieces selected from X 1} X ² G, X ¹ GX ² , X ¹ GG, GX ¹ G, or GGG (X ¹ and X ² indicate amino acids other than the same or different glycine). Amino acids can be mentioned. Further examples of amino acids used for the substitution are GG, GGG, GGGGG, GGGGG, GGGGGGG, GGGGGGG, AGG, CGG, EGG, FGG, HGG, IGG, KGG, LGG, MGG, NGG, PGG, QGG, SGG, TGG. , WGG, YGG, GPG, GGC, GGL, GGM, GGP, GGQ, GGS, GGY, MGL, MGS, LGL, and LGS. Further examples include FGG, LGG, and MGG.

In the present application, the amino acid means the following 20 natural amino acids, and the "amino acid other than glycine" specifically means alanine, leucine, arginine, lysine, aspartic acid, methionine, aspartic acid, phenylalanine, and cysteine. , Proline, glutamine, serine, glutamic acid, threonine, tryptophan, histidine, tyrosine, isoleucine, and valine.

In the modified GLNBP protein of the present application, the total number of amino acids that can be inserted is 1 or more, and is not particularly limited. Examples of the total number of amino acids that can be inserted include 1-10 and 2-7. In addition, an example of the range of the total number of inserted amino acids is selected from the lower limit value selected from 1, 2, 3, 4, and 5, and 10, 9, 8, 7, 6, and 5. It can be indicated by a combination with the upper limit value. The total number of amino acids may be inserted consecutively, or one and / or two or more consecutive amino acids may be inserted discontinuously. The amino acid that can be inserted may contain at least one glycine from the viewpoint of giving structural flexibility.

One embodiment includes a modified GLNBP protein having an amino acid sequence selected from SEQ ID NOs: 15-48, 98 and 99 below.

One embodiment of the modified GLNBP protein of the present application includes a modified GLNBP protein having an amino acid sequence selected from the group consisting of:
(I) Amino acid sequence selected from SEQ ID NOs: 15-48, 98 and 99;
(Ii) Has 80% or more, preferably 90% or more, more preferably 95% or more, for example 97% or more or 98% or more sequence identity with the amino acid sequence selected from SEQ ID NOs: 15 to 48, 98 and 99. It has a β-linked N-acetylglucosamine residue or a β-linked N-acetylgalactosamine residue at the 1-position of galactose-1-phosphate and one or more sugar residues at the non-reducing end. An amino acid sequence having an enzymatic activity that binds β-1,3 to the N-acetylglucosamine residue or the 3-position of the N-acetylgalactosamine residue in the sugar having.

The modified GLNBP protein of the present application can be prepared using a general method known to those skilled in the art. For example, a site-specific mutagenesis method such as a combination of the Kunkel method and the PCR method (for example, Inverse PCR, QuikChange method) is used to obtain a polynucleotide encoding a modified GLNBP protein from a polynucleotide encoding a non-mutated GLNBP. Is incorporated into a vector such as a plasmid, bacteriophage, or cosmid by a conventional method, the host cell is transformed with the vector, the culture is cultured, and the modified GLNBP can be obtained by appropriately purifying the culture.

In the present application, "a sugar having a β-linked N-acetylglucosamine residue or a β-linked N-acetylgalactosamine residue and having one or more sugar residues" is a non-reducing terminal (sugar). It has an N-acetylglucosamine residue or an N-acetylgalactosamine residue and one or more sugar residues on the leftmost side when the reducing end of the chain is on the right), and the N-acetylglucosamine residue. Alternatively, the N-acetylgalactosamine residue is not particularly limited as long as the hydroxy group at its 3-position is unmodified and its reducing end is β-bonded. The sugar may have sugar residues linked linearly, branched chains, or cyclically. In one embodiment, the sugar is linearly linked. The number of sugar residues is not particularly limited as long as it is one or more, and examples thereof include 2 to 6, 2 to 4, and 2 to 3. The type of sugar residue is not particularly limited, and examples thereof include fucose, galactose, N-acetylgalactosamine, glucose, N-acetylglucosamine, and sialic acid found in components of breast milk oligosaccharides and / or mucin-type sugar chains. .. The sugar may have a phosphate group, UDP, pNP (p-nitrophenyl group) or the like bonded to a hydroxy group other than the 3-position of the sugar residue at the non-reducing end.
Examples of the sugar include lacto-N-triose II (GlcNAcβ1-3Galβ1-4Glc), N, N'-diacetylchitobiose (GlcNAcβ1-4GlcNAc), GlcNAc-β-pNP, and GalNAc-β-pNP.

The present application has one or more N-acetylglucosamine residues or β-linked N-acetylgalactosamine residues β-linked to galactose-1-phosphate at the non-reducing end in the presence of the modified GLNBP protein of the present application. By reacting with a sugar having a sugar residue, the 1-position of the galactose-1-phosphate and the 3-position of the N-acetylglucosamine residue or the N-acetylgalactosamine residue are β-1,3. Provide a method of combining. Lacto-N-tetraose can be synthesized by using galactose-1-phosphate and lacto-N-triose II as substrates. The modified GLNBP proteins of the present application reversibly catalyze the reaction, but bias the equilibrium towards the product by removing the product out of the system and / or by overusing one of the substrates. obtain.

Hereinafter, the present invention will be described in more detail with reference to test examples, but the present invention is not limited to these examples.

Test Example 1: Glycine substitution mutant

<Preparation of TP, 2G, 3G, 4G, 5G, 6G, 7G mutants of GLNBP (SEQ ID NO: 1) derived from Bifidobacterium longum JCM1217 strain>
The mutants shown in FIG. 1 were prepared by Inverse PCR using pET28a-GLNBP (Kitaoka et al. AEM 71: 3158-3162, 2005) as a template and the primers shown in Table 1. The amplified fragment was phosphorylated using T4 polynucleotide kinase (Takara Bio), purified by agarose gel electrophoresis, and self-ligated using a DNA ligation kit (Takara Bio). Mutation was confirmed by a sequence using the primers shown in Table 2.

<Expression of each mutant and preparation of cell-free extract>
The prepared plasmid was introduced into BL21 (DE3) ΔlacZ / pRARE2 strain (Yoshida et al. Glycobiol. 22: 361-368, 2012), and the obtained transformant was used in 50 mL LB medium (30 mg / L Km, 15). Inoculated into (including mg / mL Cm). When the OD600 of the culture solution reached 0.5, 0.1 mM IPTG was added to induce protein expression, and the cells were cultured at 18 ° C. for 48 hours. After that, the cells were collected by centrifugation, suspended in 3 mL of Lysis Buffer (50 mM HEPES (pH 8.0), 300 mM NaCl, 10 mM imidazole), and the cells were disrupted by ultrasonic waves (Qsonica Q125). ). The supernatant obtained by centrifugation was used as a cell-free extract. Protein quantification was performed by the Bradford method (Bio-Rad), and bovine serum albumin was used as a standard product.

<Enzyme reaction (cell-free extract)>
Add a cell-free extract with a final concentration of 0.1 mg / mL to 20 mM sodium phosphate buffer (pH 7) containing 4 mM lacto-N-tetraose, and 30 for lacto-N-biose I (LNB). After incubating at ° C for 10 minutes and in the case of lacto-N-tetraose (LNT) overnight, it was subjected to TLC. Butanol: AcOH: water (2/1/1) was used as the developing solution, and the sugar was visualized according to the method of Anderson et al. (Anal Biochem. 287: 337-339, 2000).
The results are shown in FIG.

As shown in FIG. 2, GLNBP mutants (TP, 2G, 3G, 4G, 5G, 6G, 7G) showed the activity of degrading lacto-N-tetraose into lacto-N-triose II.

<Protein purification>
The cell-free extract prepared as described above was subjected to Ni-NTA Spin Columns (QIAGEN) to purify the mutant enzyme. The purification was performed according to the protocol of QIAGEN, but HEPES buffer was used instead of the phosphate buffer. The active fraction was collected and the buffer was exchanged for 50 mM MOPS-NaOH (pH 7) using Amicon Ultra 50K (Millipore) and the protein was concentrated. Protein quantification was calculated by the extinction coefficient based on amino acid composition (https://web.expasy.org/protparam/). The degree of purification was confirmed by SDS-PAGE as shown in FIG.

<Enzyme activity (purified protein)>
The activity of phosphoric acid decomposition and its reverse reaction (synthetic reaction) was carried out according to the method of Nishimoto M and Kitaoka M (BBB 71: 2101-2104, 2007). The phosphoric acid decomposition reaction was carried out using 10 mM lacto-N-biose I (LNB) or 10 mM lacto-N-tetraose (LNT) as a substrate in the presence of 10 mM sodium phosphate. The synthetic reaction was carried out using 1 mM galactose-1-phosphate and 10 mM GlcNAc or 10 mM lacto-N-triose II (LNTri). In the phosphoric acid decomposition reaction, 1 micromol of galactose-1-phosphate was defined as 1 minute, and in the synthetic reaction, the amount of enzyme producing 1 micromol of phosphoric acid per minute was defined as 1 U.
The results are shown in the table below.

Test Example 2: Amino acid substitutions of GLNBP mutant (3G) (XGG, GXG, GGX)

<Preparation of each amino acid substitution (XGG, GXG, GGX) of GLNBP mutant (3G)>
The mutants shown in the table below were prepared by the QuikChange method or the Inverse PCR method using pET28a-GLNBP (3G) as a template and the primers listed in the table below. Mutation was confirmed by a sequence using the same primers as in Table 2.

<Expression of each mutant and preparation of cell-free extract>
As mentioned above, I went.

<LNT-added phosphoric acid decomposition activity: thin layer chromatography>
Cell-free extract having a final concentration of 0.5 mg / mL was added to 10 mM sodium phosphate buffer (pH 7) containing 4 mM lacto-N-tetraose, incubated overnight, and then subjected to TLC. Butanol: AcOH: water (2/1/1) was used as the developing solution for TLC, and sugar was visualized according to the method of Anderson et al. (Anal Biochem. 287: 337-339, 2000). TLC was visually judged and evaluated as high: (++); same: (+); low: (-); compared to the original 3G mutant (Δ457-463 :: GGG). nd indicates no detection, and NA indicates not applicable (because no mutant was obtained). The results are shown in the table below.

Test Example 3: Purified GLNBP-3G, AGG, FGG, LGG, MGG, MGS, MGL, LGS, LNT synthetic activity of LGL mutants

<Expression and purification of mutants>
The GLNBP -3G, AGG, FGG, LGG, and MGG mutants obtained as described above were purified and LNT synthetic activity was measured. Purification was performed using Ni-NTA Spin Columns (QIAGEN) as described above. In addition, the MGS, MGL, LGS, and LGL mutants shown in Table 7 were also prepared and used for activity measurement. These mutants were subjected to the QuikChange method using pET28a-GLNBP (MGG) and pET28a-GLNBP (LGG) as templates. The primers used are as shown in Table 8.

<Measurement of LNT synthetic activity (purified protein)>
The activity was carried out according to the method of Nishimoto M and Kitaoka M (BBB 71: 2101-2104, 2007) using 1 mM galactose-1-phosphate and 10 mM lacto-N-triose II as substrates. The amount of enzyme that produces 1 micromol of phosphoric acid per minute was defined as 1 U.
The results are shown in the table below.

Test Example 4: Measurement of LNT synthesis activity of purified GLNBP-MGG

(Test method)
Reaction solution consisting of the following in a total volume of 1 mL:
40 mM Sucrose (Suc),
30 mM Gal-1-P,
20 mM Lacto-N-triose II (LNTri),
0.2 μM Glc1,6bisP (GlcBP),
2 mM MgCl ₂ ,
1 U / mL sucrose phosphorylase (oriental yeast) (SucP),
5 U / mL phosphoglucomutase (sigma) (PGM), and
0.06 U / mg GLNBP-MGG (0.5 mg / mL);
Was incubated at 30 ° C., and the sample (20 μL) collected at 0, 10, 30, 90, 270, 810 minutes was analyzed by the HPAEC-PAD method and the TLC method.
The remaining reaction was used for purification of LNT from other components.

(TLC method)
The results are shown in FIG.
As shown in FIG. 4, in the reaction solution after 810 minutes, consumption of Lacto-N-triose II and production of lacto-N-tetraose and fructose were shown.

(HPAEC-PAD method)
Each sugar was analyzed by the HPAEC-PAD method (high performance anion exchange chromatography with pulsed amperometric detection) using the Dionex ICS 3000 system (with CarboPac PA-1 column (Dionex, Sunnyvale, CA)). A constant flow rate (0.25 ml / min) was eluted with a linear gradient of 1-330 mM sodium acetate / 125 mM NaOH for 20 minutes (30 ° C.) followed by an additional 10 minutes.
The results are shown in FIG.

As shown in FIG. 5, Gal-1-P, Lacto-N-triose II, and Sucrose were consumed over time to produce lacto-N-tetraose and fructose.

(LNT purification)
The reaction was deionized with Amberlite MB-4, lyophilized and subjected to HPLC on a Sugar-D column (20 x 250 mm, Nacalai Tesque, Kyoto, Japan). A constant flow rate (5.0 ml / min) of 72% acetonitrile was eluted at 40 ° C. and monitored with a refractive index detector (RID-10A, Shimadzu, Kyoto, Japan). Fractions of the corresponding peaks were collected, lyophilized and purified at room temperature using a gel filtration column Toyopearl HW-40C (20 mm X 500 mm) (manufactured by TOSOH). Elution was carried out with water at a flow rate of 1.0 ml / min. Then, the analysis was performed by the HPAEC-PAD method in the same manner as above. The results are shown in FIG.

Furthermore, the purified synthetic lacto-N-tetraose obtained above was analyzed at _{298 K, D 2} O using a nuclear magnetic resonance apparatus (Bruker Avance800) using 2-methyl-2-propanol as an internal standard. The obtained spectrum is shown in FIG.

As shown in FIGS. 6 and 7, it was confirmed that the lacto-N-tetraose was synthesized by the GLNBP-MGG mutant by comparison with the lacto-N-tetraose standard product.

Test Example 5: Gal / GlcNAc residue binding activity of purified GLNBP-MGG

A reaction solution was prepared in the same manner as in Test Example 4 except that GlcNAc-β-pNP or GlcNAcβ1-4GlcNAc was used instead of Lacto-N-triose II, and the sample was incubated overnight at 30 ° C. and collected (20 μL). ) Was analyzed by the TLC method. The results are shown in FIG.
As shown in FIG. 8, along with the consumption of pNP-beta-GlcNAc or N, N'-diacetylchitobiose, a new spot was confirmed in the portion having a lower Rf value than pNP-beta-GlcNAc or N, N'-diacetylchitobiose.

Among the mutants used in Test Examples 1 to 5 and 9, the sequence listing of the mutants in the table below is described above.

Test Example 6: Bifidobacterium GLNBP homolog alignment
Based on the GLNBP (SEQ ID NO: 1) of the Bifidobacterium longum JCM1217 strain, the GLNBP homolog (Table 10) of the genus Bifidobacterium registered in Complete Genomes of KEGG (Kyoto Encyclopedia of Genes and Genomes) was aligned. Clustal W (https://www.genome.jp/tools-bin/clustalw) and Boxshade (https://embnet.vital-it.ch/software/BOX_form.html) were used for alignment. The results are shown in FIG.
FIG. 10 shows a schematic diagram showing the secondary structure of GLNBP (SEQ ID NO: 1) of the Bifidobacterium longum JCM1217 strain.
From the above alignment results, FIG. 11 shows the 19th helix structure (positions 448 to 451) and loop region (positions 452 to 467) of the GLNBP (SEQ ID NO: 1) of the Bifidobacterium longum JCM1217 strain in each GLNBP homolog of the genus Bifidobacterium. The figure which shows the part corresponding to the 20th helix structure (positions 468 to 478) is shown.
FIG. 12 shows a table showing the amino acid sequence identity of each Bifidobacterium genus GLNBP homolog to GLNBP (SEQ ID NO: 1) of the Bifidobacterium longum JCM1217 strain.

Test Example 7: GLNBP homolog alignment
Based on the GLNBP (SEQ ID NO: 1) of the Bifidobacterium longum JCM1217 strain, the amino acid sequences of the GLNBP homologs in Table 11 whose GLNBP activity has been confirmed as a purified enzyme were aligned. Clustal W (https://www.genome.jp/tools-bin/clustalw) and Boxshade (https://embnet.vital-it.ch/software/BOX_form.html) were used for alignment. The results are shown in FIG.
In FIG. 14, from the above alignment results, the 19th helix structure (positions 448 to 451), the loop region (positions 452 to 467), and the 20th position of the GLNBP (SEQ ID NO: 1) of the Bifidobacterium longum JCM1217 strain in each GLNBP homolog. The figure which shows the part corresponding to the helix structure (positions 468 to 478) is shown.

Test Example 8: Model diagram in which LNT is incorporated into the active center of wild-type BlGLNBP. GlcNAc in the complex structure of wild-type GLNBP and GlcNAc (PDB ID: Chain D subunit of 2ZUW) is registered in PDB ID: 2Z8F. FIG. 15 shows a model when GlcNAc of LNT is superposed. It can be seen that the Lac structure (Gal β1-4 Glc) on the reducing end side of LNT collides with the loop existing between Helix-19 and Helix-20.
PDB ID: 2ZUW: Hidaka et al.JBC 284: 7273-7283 (2009)
PDB ID: 2Z8F: Suzuki et al. JBC 283: 13165-13173 (2008)

Test Example 9: BbXGLNBP2-3G mutant

<Preparation of BbXGLNBP2-3G mutant>
BbXGLNBP2-3G mutant (SEQ ID NO:) in which positions 457 to 463 of BbXGLNBP2 (SEQ ID NO: 100) derived from Bifidobacterium bifidum JCM1254 strain having an amino acid sequence that is 99% identical to BbGLNBP2 (SEQ ID NO: 6) of Bifidobacterium bifidum PRL2010 strain are replaced with GGG. 99) using pTN027 (Nishimoto M and Kitaoka M. Identification of the putative proton donor residue of lacto-N-biose phosphorylase (EC 2.4.1.211). Bioscience, Biotechnology, and Biochemistry 71: 1587-1591 (2007)) as a template. , Inverse PCR using the primers listed in Table 12. The amplified fragment was phosphorylated using T4 polynucleotide kinase (Takara Bio), purified by agarose gel electrophoresis, and self-ligated using a DNA ligation kit (Takara Bio). Mutation was confirmed by a sequence using the primers shown in Table 13.

<Expression of each mutant and preparation of cell-free extract>
This was done as in Test Example 1.

<Enzyme reaction (cell-free extract)>
_{Prepare a reaction solution containing 5 mM NaPO 4} and 2 mM lacto-N-biose I (LNB) or 5 mM NaPO ₄ and 2 mM lacto-N-tetraose (LNT) in 10 mM MOPS buffer (pH 7). Incubated overnight at 30 ° C. in the presence of a cell-free extract with a final concentration of 0.1 mg / mL, and then subjected to TLC. All were spotted by 1 μL. Butanol: AcOH: water (2/1/1) was used as the developing solution, and the sugar was visualized according to the method of Anderson et al. (Anal Biochem. 287: 337-339, 2000).
As a result, in the reaction solution containing lacto-N-tetraose, spots having the same Rf value as lacto-N-triose II were observed, indicating the decomposition activity of lacto-N-tetraose into lacto-N-triose II. Was done.

The present invention can be used for the production of oligosaccharides, particularly lacto-N-tetraose.

Claims (21)

When the amino acid sequence of non-mutant GLINBP is aligned with the amino acid sequence shown in SEQ ID NO: 1, one or more amino acids are deleted, substituted and / in the region corresponding to positions 452 to 467 in SEQ ID NO: 1. Or inserted; and having one or more β-linked N-acetylglucosamine residues or β-linked N-acetylgalactosamine residues at the non-reducing end with one position of galactose-1-phosphate. A modified GLNBP protein having an enzymatic activity that binds β-1,3 to the N-acetylglucosamine residue or the 3-position of the N-acetylgalactosamine residue in a sugar having a sugar residue. One or more amino acids have been deleted, substituted and / or inserted in the loop region located in the three-dimensional vicinity of the active center of non-mutant GLNBP; and at the 1-position of galactose-1-phosphate, The N-acetylglucosamine residue or N-acetyl in a sugar having a β-linked N-acetylglucosamine residue or a β-linked N-acetylgalactosamine residue at the non-reducing end and having one or more sugar residues. A modified GLNBP protein having an enzymatic activity that binds β-1,3 to the 3-position of a galactosamine residue. The loop region located in the three-dimensional vicinity of the active center of the non-mutant GLNBP corresponds to the 452 to 467 positions in SEQ ID NO: 1 when the amino acid sequence of the non-mutant GLNBP is aligned with the amino acid sequence shown in SEQ ID NO: 1. The modified GLNBP protein according to claim 2, which is a region of a position to be used. A sugar having a β-linked N-acetylglucosamine residue or a β-linked N-acetylgalactosamine residue at the non-reducing end and having one or more sugar residues is lacto-N-triose II, and lacto- The modified GLNBP protein according to any one of claims 1 to 3, which has an enzymatic activity for synthesizing N-tetraose. In the loop region located three-dimensionally near the active center of the non-mutant GLNBP, 1 to 16 amino acids are deleted, 1 to 16 amino acids are replaced by 1 to 16 amino acids, and / or 1 to 1 The modified GLNBP protein according to any one of claims 2 to 4, wherein 10 amino acids are inserted. When the amino acid sequence of the non-mutant GLNBP was aligned with the amino acid sequence shown in SEQ ID NO: 1, 1 to 16 amino acids were deleted in the region corresponding to positions 452 to 467 in SEQ ID NO: 1, 1 to 16 amino acids were deleted. The modified GLNBP protein according to any one of claims 1 to 5, wherein the amino acids are replaced by 1 to 16 amino acids and / or 1 to 10 amino acids are inserted. When the amino acid sequence of the non-mutated GLNBP was aligned with the amino acid sequence shown in SEQ ID NO: 1, 3 to 10 consecutive amino acids were deleted in the region corresponding to positions 452 to 467 in SEQ ID NO: 1. The modified GLNBP protein according to any one of claims 1 to 6, wherein 3 to 10 consecutive amino acids are replaced by 2 to 10 consecutive amino acids containing at least one glycine. When the amino acid sequence of the non-mutated GLNBP is aligned with the amino acid sequence shown in SEQ ID NO: 1, in the region corresponding to the position 452 to 467 in SEQ ID NO: 1, 3 to 10 consecutive amino acids are contiguous 2 to The modified GLNBP protein according to any one of claims 1 to 7, which is substituted with 10 glycines. When the amino acid sequence of the non-mutated GLNBP was aligned with the amino acid sequence shown in SEQ ID NO: 1, 3 to 10 consecutive amino acids were X ¹ X in the region corresponding to positions 452 to 467 in SEQ ID NO: 1. ^{Substituted by three consecutive amino acids selected from 2} G, X ¹ GX ² , X ¹ GG, GX ¹ G, or GGG (X ¹ and X ² indicate amino acids other than the same or different glycine) , The modified GLNBP protein according to any one of claims 1 to 7. When the amino acid sequence of the non-mutated GLNBP is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids at positions 452 to 467 in SEQ ID NO: 1 are AGG, CGG, EGG, FGG, HGG, IGG, KGG, LGG, MGG. , NGG, PGG, QGG, SGG, TGG, WGG, YGG, GPG, GGC, GGL, GGM, GGP, GGGQ, GGS, GGY, MGL, MGS, LGL, and LGS. The modified GLNBP protein according to any one of claims 1 to 7, which is substituted with the amino acid of. The modified GLNBP protein according to any one of claims 1 to 10, wherein the non-mutant GLNBP is derived from the genus Bifidobacterium, Anaerococcus, Clostridium, Cutibacterium, Erysipelothrix, Lachnoclostridium, Streptobacillus, or Vibrio. .. The non-mutant GLNBP
(I) Amino acid sequence selected from SEQ ID NOs: 1 to 14 and 100; or (ii) Having 75% or more sequence identity with the amino acid sequence selected from SEQ ID NOs: 1 to 14 and 100, and GLNBP activity. Have;
The modified GLNBP protein according to any one of claims 1 to 11. The modified GLNBP protein according to any one of claims 1 to 12, which has an amino acid sequence selected from the group consisting of the following:
(I) Amino acid sequence selected from SEQ ID NOs: 15-48, 98 and 99;
(Ii) An amino acid sequence having 80% or more sequence identity with the amino acid sequence selected from SEQ ID NOs: 15 to 48, 98 and 99. The 1-position of galactose-1-phosphate and the 3-position of the N-acetylglucosamine residue in a sugar having a β-bonded N-acetylglucosamine residue at the non-reducing end and having one or more sugar residues. The modified GLNBP protein according to any one of claims 1 to 13, which has an enzymatic activity of binding β-1,3 to and. A polynucleotide encoding the modified GLNBP protein according to any one of claims 1 to 14. A vector containing the polynucleotide according to claim 15. A host cell transformed with the vector according to claim 16. The method for producing a modified GLNBP protein according to any one of claims 1 to 14, which comprises culturing the host cell according to claim 17 and recovering the modified GLNBP protein from the culture. N-acetylglucosamine residue β-linked to galactose-1-phosphate and non-reducing terminal or β-linked N-acetylgalactosamine residue in the presence of the modified GLNBP protein according to any one of claims 1 to 14. The 1-position of the galactose-1-phosphate and the N-acetylglucosamine residue or the N-acetylgalactosamine residue, which comprises a step of reacting with a sugar having a group and having one or more sugar residues. A method of binding β-1,3 to the 3-position. Claim that the sugar having a β-linked N-acetylglucosamine residue or a β-linked N-acetylgalactosamine residue and having one or more sugar residues at the non-reducing end is lacto-N-triose II. 19. The method according to 19. Synthesize lacto-N-tetraose, which comprises the step of reacting galactose-1-phosphate with lacto-N-triose II in the presence of the modified GLNBP protein according to any one of claims 1 to 14. Method.

Images