Mutant milk-N-diglycosidase

Abstract

The mutant milk-N-diglycosidase has improved properties as compared to the wild type milk-N-diglycosidase isolated from Bifidobacterium longum.

Description

Technical Field

[0001] The present invention relates to a lacto-N-biosidase having enhanced transglycosidase synthesis activity and an enzymatic reaction for preparing oligosaccharides having a lacto-N-biose terminus at the non-reducing end in one enzymatic step. Background Art

[0002] Over the past few decades, interest in the preparation and commercialization of human milk oligosaccharides (HMOs) has steadily increased. The importance of HMOs is directly related to their unique biological activities, making them important potential products in the fields of nutrition and therapy. Consequently, low-cost, industrially scalable production methods for HMOs have been sought.

[0003] To date, the structures of over 140 HMOs have been determined, and a significant number of them may be present in human milk. HMOs consist of a lactose (Galβ1-4Glc) moiety at the reducing end, which may be extended by N-acetylglucosamine, one or more N-acetyllactosamine moieties (Galβ1-4GlcNAc), and / or a lacto-N-biose moiety (Galβ1-3GlcNAc). Lactose and N-acetyllactosaminated or lacto-N-biosylated lactose derivatives can be further substituted with one or more fucose and / or sialic acid residues, or lactose can be substituted with additional galactose, forming the currently known HMOs. In human milk / colostrum, type I oligosaccharides containing lacto-N-biose predominate over type II oligosaccharides containing N-acetyllactosamine. This is a characteristic characteristic of humans and not found in other mammals. The predominance of type I oligosaccharides has been of particular interest in the development of bifidobacterial flora in the infant colon and may have other benefits. N-acetyllactosamine can serve as an internal, non-reducing terminal disaccharide unit, while lacto-N-biose can only serve as a non-reducing terminal disaccharide unit (Urashima et al.: Milkoligosaccharides, Nova Biomedical Books, 2011; Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). The core HMO structure with a terminal lacto-N-biose is shown in Table 1 below.

[0004]

[0005] Table 1.

[0006] Over the past decade, efforts to develop HMO synthesis processes have increased significantly. Numerous processes have been developed for their production through microbial fermentation, enzymatic reactions, chemical synthesis, or a combination of these techniques. While several manufacturers have recently achieved industrial-scale synthesis and purification of simpler HMOs, such as trisaccharide and tetrasaccharide HMOs, using biotechnology approaches, including the use of genetically modified microorganisms, the same task remains challenging for more complex HMOs.

[0007] In biological systems, Leloir-type glycosyltransferases (GTs, EC 2.4.1.-) and glycosidases (also known as glycoside hydrolases: GHs, EC 3.2.1.-) constitute the two main classes of carbohydrate-processing enzymes used to produce HMOs. Both classes of enzymes transfer a glycosyl group from a donor to an acceptor, thereby generating oligosaccharides. The application of glycosyltransferases in industrial in vitro synthesis processes is primarily limited by the high cost of activating the donor sugar. Compared to glycosyltransferases, glycosidases have a broader substrate spectrum, typically using monosaccharides, oligosaccharides, and / or engineered substrates (i.e., substrates bearing various functional groups). They typically exhibit activity towards a wide range of carbohydrate and non-carbohydrate acceptors. Another advantage of glycosidases over glycosyltransferases in in vitro applications is their robustness and accessibility.

[0008] Lacto-N-biosidases (EC 3.2.1.140) are primarily classified in the GH20 family. Lacto-N-biosidases typically function through a retaining mechanism and specifically hydrolyze terminal lacto-N-biosyl residues at the non-reducing ends of oligosaccharides. Lacto-N-biosidase (LnbB) from Bifidobacterium bifidum JCM1254 has been shown to have low transglycosylation activity to prepare LNT from suitable precursors (Wada et al. Appl. Environ. Microbiol. 74, 3996 (2008)), and other lacto-N-biosidases have also been suggested for enzymatic HMO synthesis (WO 2012 / 156897, WO 2012 / 156898). Recently, specific mutants of the lacto-N-biosidase LnbB with reduced hydrolytic activity have been disclosed, which are able to produce LNT from a suitable lacto-N-biose donor and lactose (WO 2020 / 126613, Castejón-Vilatersana et al. Int. J. Mol. Sci. 22, 3230 (2021), Vuillemin et al. Appl. Sci. 11, 11493 (2021)).

[0009] Sakamura et al. (J. Biol. Chem. 288, 25194 (2013)) discovered a lacto-N-biosidase (LnbX) from Bifidobacterium longum JCM1217 that is completely different from the GH20 lacto-N-biosidases identified to date in terms of amino acid sequence, substrate specificity, structure, and catalytic mechanism. LnbX is a founding member of the GH136 family.

[0010] New approaches to the enzymatic synthesis of type I oligosaccharides, preferably type I HMOs, and especially type I HMOs other than LNT, have been sought. Summary of the Invention

[0011] The present invention relates to a mutant lacto-N-biosidase having:

[0012] - an amino acid sequence that is substantially identical to the sequence of amino acids 45 to 625 in SEQ ID No. 1, i.e., has at least 70% sequence identity therewith; and

[0013] - a mutation at one or more amino acid positions selected from the group consisting of 410, 416, 439 and 442, the amino acid numbering following SEQ ID No. 1.

[0014] Preferably, the mutant lacto-N-biosidase comprises one or more of the following mutations:

[0015] - at position 410, Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or

[0016] - at position 416, Asp(D) is substituted by Asn or Gln, preferably by Asn; and / or

[0017] - at position 439, Met (M) is substituted by Leu, Val or Ile, preferably by Leu; and / or

[0018] - At position 442, Asn(N) is substituted by Trp, Tyr, Phe or His, preferably by Trp.

[0019] In one embodiment, the amino acid sequence of the mutant lacto-N-biosidase is substantially identical to the sequence of amino acids 31 to 625 in SEQ ID No. 1, and has a mutation at at least one or more amino acid positions selected from 410, 416, 439 and 442, wherein the amino acid numbering follows SEQ ID No. 1.

[0020] According to another aspect, the present invention relates to a method for preparing the mutant lacto-N-biosidase, comprising the following steps:

[0021] (a) providing a DNA sequence encoding a mutant lacto-N-biosidase, and then

[0022] (b) expressing the mutant lacto-N-biosidase in a host cell transformed with the DNA sequence obtained in step (a).

[0023] In addition, a method for synthesizing carbohydrates containing lacto-N-biose is provided, the method comprising the steps of reacting a lacto-N-biosyl donor with a carbohydrate acceptor in the presence of lacto-N-biosidase to transfer the lacto-N-biosyl residue of the donor to the carbohydrate acceptor, wherein the lacto-N-biosidase comprises an amino acid sequence that is substantially identical to the sequence of amino acids 31 or 45 to 625 in SEQ ID No. 1, i.e., has at least 70% sequence identity therewith.

[0024] In another aspect of the present invention, there is provided a use of lacto-N-biosidase for preparing carbohydrates containing lacto-N-biose, wherein the lacto-N-biosidase comprises an amino acid sequence that is substantially identical to the sequence of amino acids 31 or 45 to 625 in SEQ ID No. 1, i.e., has at least 70% sequence identity therewith.

[0025] In yet another aspect of the present invention, there is provided a mixture consisting of, or consisting essentially of, LNT, LNnT, pLNH, and optionally lactose.

[0026] In a further aspect of the present invention, a method for obtaining the mixture according to the fifth aspect of the present invention is provided, wherein the method comprises reacting LNT and LNnT in the presence of an enzyme selected from LnbX, a truncated functional analogue thereof and a mutant lacto-N-biosidase according to the first aspect of the present invention to produce a reaction mixture, and then removing the enzyme and optionally lactose from the reaction mixture. DETAILED DESCRIPTION

[0027] The present inventors have unexpectedly discovered that lacto-N-biosidase (LnbX) from Bifidobacterium longum JCM1217 (Sakamura et al., J. Biol. Chem. 288, 25194 (2013), GenBank no. DAA64542, SEQ ID No. 1) and its truncated functional analogs can be used as translacto-N-biosidases to produce oligosaccharides containing linear lacto-N-biose. Furthermore, it was discovered that mutations in LnbX or its truncated functional analogs at certain positions can increase the yield of enzymatic synthesis.

[0028] A "truncated functional analog" of LnbX refers to a truncated LnbX protein that substantially retains the desired activity. Examples of truncated functional analogs of LnbX include polypeptide fragments of amino acids 31 to 1573, 31 to 1431, 38 to 1431, 45 to 1431, 31 to 1005, 31 to 904, 31 to 639, 31 to 625, or 45 to 625 of SEQ ID No. 1, preferably polypeptide fragments of amino acids 31 to 625 or 45 to 625.

[0029] The first aspect of the present invention relates to a mutant lacto-N-biosidase, which comprises a polypeptide fragment having:

[0030] - has substantial identity (i.e. at least 70% sequence identity) to the polypeptide fragment of amino acids 45 to 625 of SEQ ID No. 1, and

[0031] - a mutation at one or more amino acid positions selected from 410, 416, 439 and 442 (ie, an amino acid in the wild-type sequence is substituted by another amino acid), the amino acid numbering following SEQ ID No. 1.

[0032] Preferably, the one or more amino acid positions are:

[0033] - Position 410, wherein Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or

[0034] - Position 416, wherein Asp (D) is substituted by Asn or Gln, preferably by Asn; and / or

[0035] - Position 439, wherein Met (M) is substituted by Leu, Val or Ile, preferably by Leu; and / or

[0036] - Position 442, wherein Asn(N) is substituted by Trp, Tyr, Phe or His, preferably by Trp.

[0037] Thus, a mutant lacto-N-biosidase can be obtained, which, compared with the wild-type lacto-N-biosidase of SEQ ID No. 1 or a truncated functional analog thereof (e.g., an enzyme comprising a polypeptide fragment of amino acids 31 or 45 to 625 of SEQ ID No. 1):

[0038] - providing improved lacto-N-biosidase synthesis performance in a reaction between a lacto-N-biosyl donor and an acceptor to produce a lacto-N-biose-containing product; and / or

[0039] - providing a reduced hydrolysis of lacto-N-biose-syl donors and / or lacto-N-biose-containing products.

[0040] Therefore, the present invention provides a mutant lacto-N-biosidase comprising a polypeptide fragment having at least 70% sequence identity with the polypeptide fragment of amino acids 45 to 625 in SEQ ID No. 1, and

[0041] a) has a mutation at one or more amino acid positions selected from the group consisting of 410, 416, 439 and 442, preferably wherein at least one mutation is selected from the group consisting of:

[0042] - at position 410, Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or

[0043] - at position 416, Asp(D) is substituted by Asn or Gln, preferably by Asn; and / or

[0044] - at position 439, Met (M) is substituted by Leu, Val or Ile, preferably by Leu, and / or

[0045] - at position 442, Asn(N) is substituted by Trp, Tyr, Phe or His, preferably by Trp,

[0046] and / or

[0047] b) providing improved translacto-N-biosidase synthetic performance in a reaction between a lacto-N-biosyl donor and an acceptor to produce a lacto-N-biose-containing product, and / or reduced hydrolysis activity towards a lacto-N-biose-containing product in such a reaction, compared to the wild-type lacto-N-biosidase of SEQ ID No. 1 or a truncated functional analog thereof (e.g., an enzyme comprising a polypeptide fragment from amino acids 31 or 45 to 625 of SEQ ID No. 1).

[0048] The polypeptide fragment from amino acids 31 to 625 in SEQ ID No. 1 has been identified as the core portion of LnbX, responsible for the complete catalytic hydrolysis activity (Yamada et al., Cell Chem. Biol. 24, 515 (2017)). In addition, the polypeptide fragment from amino acids 45 to 625 in SEQ ID No. 1 has been identified as the related domain of LnbX classified in the GH136 family.

[0049] According to the present invention, the terms "substantial identity" and "substantially identical" in the context of two or more nucleic acid or amino acid sequences preferably mean that the two or more sequences are identical, or have at least about 70% of their amino acid residues identical (i.e., the sequences have at least about 70% identity), when compared and aligned for maximum correspondence over a comparison window or designated nucleic acid or amino acid sequence. The percent identity of nucleic acid or amino acid sequences can be measured using the BLAST 2.0 (or higher) sequence alignment algorithm under default parameters, or by manual alignment and visual inspection (e.g., see http: / / www.ncbi.nlm.nih.gov / BLAST / ). According to the present invention, the percentage identity of a polypeptide fragment substantially identical to amino acids 31 or 45 to 625 of SEQ ID No. 1, or a substantially identical amino acid sequence of SEQ ID No. 1, is preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 92%, especially at least 93%, more especially at least 94%, even more especially at least 95%, even more especially at least 96%, especially at least 97%, more especially at least 98%, and most especially at least 99%. Suitably, this definition preferably excludes 100% sequence identity, for example, imposes a maximum limit of 99.9%, 99.8% or 99.7% on sequence identity, or requires that there is at least one amino acid difference between the compared sequences. This definition also applies to the complementary sequences of the test sequences and sequences with deletions and / or additions and substitutions. An example of an algorithm suitable for determining percent identity and sequence similarity is the BLAST+2.13.0 algorithm, which is described in Altschul et al., Nucl. Acids Res. 25, 3389 (1997). BLAST+2.13.0 is used to determine the percent sequence identity of the proteins of the present invention. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0050] LnbX transfers lacto-N-biosyl residues from donor substrates to acceptors. If the acceptor is a carbohydrate (monosaccharide, disaccharide, or oligosaccharide), lacto-N-biosidase acts as a translacto-N-biosidase (capable of generating carbohydrate products containing lacto-N-biose). On the other hand, the same lacto-N-biosidase can transfer the same lacto-N-biosyl residue previously added to the carbohydrate acceptor from the product to a water molecule, thereby acting as a hydrolase. These two processes occur simultaneously. Overall synthetic performance is the ratio of translacto-N-biosidase activity to hydrolytic activity.

[0051] By comparison, the mutant lacto-N-biosidases of the present invention exhibit higher overall synthetic performance, meaning that their translacto-N-biosidase activity is higher relative to their hydrolytic activity. In this regard, the relatively low translacto-N-biosidase synthetic activity of the mutants of the present invention can be compensated by a significant reduction in the mutant's hydrolytic activity, resulting in improved synthetic performance. Similarly, the relatively high hydrolytic activity of the mutants can be overcome by a significant increase in their translacto-N-biosidase synthetic activity. The translacto-N-biosidase synthetic performance of the mutant lacto-N-biosidases of the present invention is improved compared to wild-type LnbX or its truncated functional analogs.

[0052] Suitably, the mutant lacto-N-biosidases of the present invention are non-natural lacto-N-biosidases, that is, they are not produced or naturally occurring in nature, but are prepared by chemical synthesis, genetic engineering or similar laboratory methods to produce synthetic mutant lacto-N-biosidases.

[0053] Preferably, the lacto-N-biosidase of the first aspect comprises a polypeptide sequence having at least 70% sequence identity with the fragment of amino acids 45 to 625 in SEQ ID No. 1 above, and the following amino acid mutations, wherein:

[0054] - at position 410, Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or

[0055] - At position 416, Asp(D) is substituted by Asn or Gln, preferably by Asn.

[0056] Also preferably, the lacto-N-biosidase comprises a polypeptide sequence having at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even even more preferably at least 95% sequence identity to the fragment of amino acids 45 to 625 of SEQ ID No. 1, and the following mutations:

[0057] - at position 410, wherein Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or

[0058] - at position 416, wherein Asp(D) is substituted by Asn or Gln, preferably by Asn; and / or

[0059] - at position 439, wherein Met (M) is substituted by Leu, Val or Ile, preferably by Leu; and / or

[0060] - at position 442, wherein Asn(N) is substituted by Trp, Tyr, Phe or His, preferably by Trp.

[0061] Preferably, the lacto-N-biosidase of the first aspect comprises a polypeptide sequence having at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even even more preferably at least 95% sequence identity to the fragment of amino acids 45 to 625 of SEQ ID No. 1, and the following amino acid mutations, wherein:

[0062] - at position 410, Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or

[0063] - At position 416, Asp(D) is substituted by Asn or Gln, preferably by Asn.

[0064] More preferably, the lacto-N-biosidase comprises a polypeptide sequence identical to the fragment of amino acids 45 to 625 in SEQ ID No. 1, and the following mutations:

[0065] - at position 410, wherein Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or

[0066] - at position 416, wherein Asp(D) is substituted by Asn or Gln, preferably by Asn; and / or

[0067] - at position 439, wherein Met (M) is substituted by Leu, Val or Ile, preferably by Leu; and / or

[0068] - at position 442, wherein Asn(N) is substituted by Trp, Tyr, Phe or His, preferably by Trp.

[0069] Even more preferably, the lacto-N-biosidase of the first aspect comprises a polypeptide sequence identical to the fragment of amino acids 45 to 625 in SEQ ID No. 1, and the following amino acid mutations, wherein:

[0070] - at position 410, Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or

[0071] - At position 416, Asp(D) is substituted by Asn or Gln, preferably by Asn.

[0072] In other preferred embodiments of the first aspect of the present invention, the lacto-N-biosidase comprises a polypeptide sequence having at least 70% sequence identity to a fragment of amino acids 31 to 625 of SEQ ID No. 1, and the following mutations:

[0073] - at position 410, wherein Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or

[0074] - at position 416, wherein Asp(D) is substituted by Asn or Gln, preferably by Asn; and / or

[0075] - at position 439, wherein Met (M) is substituted by Leu, Val or Ile, preferably by Leu; and / or

[0076] - at position 442, wherein Asn(N) is substituted by Trp, Tyr, Phe or His, preferably by Trp.

[0077] Preferably, the lacto-N-biosidase comprises a polypeptide sequence having at least 70% sequence identity to a fragment of amino acids 31 to 625 of SEQ ID No. 1, and the following amino acid mutations, wherein:

[0078] - at position 410, Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or

[0079] - At position 416, Asp(D) is substituted by Asn or Gln, preferably by Asn.

[0080] Also preferably, the lacto-N-biosidase comprises a polypeptide sequence having at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even even more preferably at least 95% sequence identity to the fragment of amino acids 31 to 625 of SEQ ID No. 1, and the following mutations:

[0081] - at position 410, wherein Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or

[0082] - at position 416, wherein Asp(D) is substituted by Asn or Gln, preferably by Asn; and / or

[0083] - at position 439, wherein Met (M) is substituted by Leu, Val or Ile, preferably by Leu; and / or

[0084] - at position 442, wherein Asn(N) is substituted by Trp, Tyr, Phe or His, preferably by Trp.

[0085] Preferably, the lacto-N-biosidase of the first aspect comprises a polypeptide sequence having at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even even more preferably at least 95% sequence identity to the fragment of amino acids 31 to 625 of SEQ ID No. 1, and the following amino acid mutations, wherein:

[0086] - at position 410, Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or

[0087] - At position 416, Asp(D) is substituted by Asn or Gln, preferably by Asn.

[0088] More preferably, the lacto-N-biosidase comprises a polypeptide sequence identical to the fragment of amino acids 31 to 625 in SEQ ID No. 1, and the following mutations:

[0089] - at position 410, wherein Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or

[0090] - at position 416, wherein Asp(D) is substituted by Asn or Gln, preferably by Asn; and / or

[0091] - at position 439, wherein Met (M) is substituted by Leu, Val or Ile, preferably by Leu; and / or

[0092] - at position 442, wherein Asn(N) is substituted by Trp, Tyr, Phe or His, preferably by Trp.

[0093] Even more preferably, the lacto-N-biosidase comprises a polypeptide sequence identical to the fragment of amino acids 31 to 625 of SEQ ID No. 1, and the following amino acid mutations, wherein:

[0094] - at position 410, Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or

[0095] - At position 416, Asp(D) is substituted by Asn or Gln, preferably by Asn.

[0096] Embodiments of the first aspect, including preferred and more preferred embodiments, may be histidine-tagged at either the N-terminus or the C-terminus. A His tag is a short DNA sequence encoding a specific polypeptide, typically inserted into the coding site of a target gene for expression at the N-terminus or C-terminus of the desired protein. The His tagging method is particularly useful because it allows for convenient purification and detection of recombinant proteins. In one embodiment, the His tag is attached to the N-terminus. In another embodiment, the His tag is attached to the C-terminus. The presence of the His tag does not significantly affect the essential characteristics of the claimed artificial lacto-N-biosidase mutant.

[0097] According to a second aspect of the present invention, there is provided a method for preparing the mutant lacto-N-biosidase according to the first aspect of the present invention, comprising the following steps:

[0098] (a) providing a DNA sequence encoding a mutant lacto-N-biosidase; then

[0099] (b) expressing the mutant lacto-N-biosidase in a host cell transformed with the DNA sequence obtained in step (a).

[0100] Step (a) can be carried out in a conventional manner by preparing a mutant DNA sequence encoding the mutant lacto-N-biosidase of the first aspect of the present invention. Then, in step (b), such a mutant DNA sequence is introduced at the gene level by conventional molecular biological methods. The DNA sequence of the enzyme variant can be cloned into an expression vector, which can be imported into a suitable host expression strain, such as Escherichia coli, containing a DNA plasmid with the information required for regulating and controlling the expression of the enzyme variant. The sequence encoding the enzyme variant can be placed under the control of an inducible promoter. As a result, the expression of the enzyme variant can be controlled by adding an inducing agent (usually using isopropyl-β-D-thiogalactoside (IPTG)). The host cells thus transformed are then cultured in a conventional nutrient medium (such as Lennox broth, M9 minimal medium) and induced with IPTG. After expression, biomass can be harvested by centrifugation. After appropriate cell lysis and purification, the mutant enzyme can be separated from the biomass. In this process, conventional centrifugation, precipitation, ultrafiltration and / or chromatography methods can be used.

[0101] According to a third aspect of the present invention, there is provided a method for synthesizing carbohydrates containing lacto-N-biose, comprising reacting a lacto-N-biosyl donor and a carbohydrate acceptor in the presence of LnbX, a truncated functional analogue thereof, or a mutant lacto-N-biosidase according to the first aspect of the present invention, thereby transferring the lacto-N-biosyl residue of the lacto-N-biosyl donor to the carbohydrate acceptor.

[0102] The carbohydrate acceptor used in the third aspect of the present invention can be any monosaccharide, disaccharide or oligosaccharide, preferably an oligosaccharide of 3-10 monosaccharide units, the lacto-N-biosyl moiety on its terminal monosaccharide unit can be transferred by LnbX, its truncated functional analogs or the mutant lacto-N-biosidase according to the first aspect of the present invention. The oligosaccharide acceptor preferably contains a galactose unit at the non-reducing end. In a preferred embodiment, the galactose-containing acceptor is lactose. In other preferred embodiments, the acceptor includes an N-acetylglucosamine unit adjacent to the galactose unit, thereby forming an N-acetyllactosamine (Galpβ1-4GlcNAcp) or lacto-N-biosyl (Galpβ1-3GlcNAcp) moiety, preferably an N-acetyllactosamine moiety. Examples of such receptors include N-acetyllactosamine, LNnT (Galβ1-4GlcNAcβ1-3Galβ1-4Glc), or Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc.

[0103] LnbX, its truncated functional analogs, or the mutant lacto-N-biosidase according to the first aspect of the present invention exhibit strong β1-3 selectivity when performing the method of the third aspect of the present invention. As a result, the reaction product is a β1-3-lacto-N-biosyl monosaccharide, disaccharide, or oligosaccharide, preferably an oligosaccharide of 3-10 monomer units. Preferably, LnbX, its truncated functional analogs, or the mutant lacto-N-biosidase according to the first aspect of the present invention brings a lacto-N-biosyl residue from a suitable donor to the 3-position of the terminal galactose of the acceptor. Therefore, LnbX, its truncated functional analogs, or the mutant lacto-N-biosidase according to the first aspect of the present invention are preferably used to synthesize oligosaccharides containing lacto-N-biose, wherein lacto-N-biose is linked to galactose, preferably a linear core HMO containing lacto-N-biose, such as those listed in Table 2 below.

[0104] receptors product lactose Lacto-N-tetraose (LNT) LNJ p-Lacto-N-hexaose (pLNH) Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc p-Lacto-N-octaose (pLNO)

[0105] Table 2.

[0106] The lacto-N-biosyl donor used in the third aspect of the invention may be LnbX, a truncated functional analogue thereof, or any lacto-N-biosyl compound from which the mutant lacto-N-biosidase according to the first aspect of the invention is capable of transferring a lacto-N-biosyl residue to a carbohydrate acceptor as described above. Suitably, the lacto-N-biosyl donor may be a compound of formula 1 or formula 2:

[0107]

[0108] wherein X is selected from the group consisting of a monosaccharide, a disaccharide or an oligosaccharide, an azide, a fluorine, an optionally substituted phenoxy group, an optionally substituted pyridyloxy group, a group A, a group B, a group C and a group D,

[0109]

[0110] where R a are independently H or alkyl, or two adjacent R a Group representation =C(R b )2 group, wherein R b are independently H or alkyl, R c R is independently selected from alkoxy, amino, alkylamino and dialkylamino, d Selected from H, alkyl and -C(=O)R e , where R e is OH, alkoxy, amino, alkylamino, dialkylamino, hydrazine, alkylhydrazine, dialkylhydrazine or trialkylhydrazine,

[0111] Preferably, X in Formula 1 is selected from monosaccharides, disaccharides or oligosaccharides, phenoxy, p-nitrophenoxy, 2,4-dinitrophenoxy and 2-chloro-4-nitrophenoxy. Advantageously, X in Formula 1 is lactose, which means that the lacto-N-disaccharyl donor is LNT.

[0112] It is advantageous if the process according to the third aspect of the invention is carried out at an acceptor / donor molar ratio of 1 :1 to 5:1 , preferably 3:1 to 5:1 , at a pH in the range of 4.5 to 6.5 and a temperature of room temperature to 60°C, preferably 35-55°C.

[0113] According to a fourth aspect of the present invention, there is provided the use of LnbX, a truncated functional analogue thereof or a mutant lacto-N-biosidase according to the first aspect of the present invention for the synthesis of carbohydrates containing lacto-N-biose, preferably wherein the lacto-N-biose is linked to a terminal galactose, more preferably linked to the galactose via a β1-3 bond, and even more preferably for the synthesis of a linear core HMO containing lacto-N-biose, in particular pLNH.

[0114] According to a fifth aspect of the present invention there is provided a mixture consisting of, or consisting essentially of, LNT, LNnT, pLNH and optionally lactose.

[0115] According to a sixth aspect of the present invention, there is provided a method for obtaining a mixture according to the fifth aspect of the present invention, wherein the method comprises reacting LNT and LNnT in the presence of an enzyme selected from LnbX, a truncated functional analogue thereof and a mutant lacto-N-biosidase according to the first aspect of the present invention to produce a reaction mixture, and then removing the enzyme and optionally lactose from the reaction mixture.

[0116] A mixture consisting of, or consisting essentially of, LNT, LNnT, pLNH, and optionally lactose can be formulated as a pharmaceutical, cosmetic, and / or nutritional composition containing a pharmaceutically, cosmetically, and / or nutritionally acceptable carrier, such as a phosphate-buffered saline solution, an unbuffered saline solution, a mixture of ethanol in water, water, and an emulsion (e.g., an oil / water or water / oil emulsion), as well as various wetting agents and / or excipients. The pharmaceutical, cosmetic, and / or nutritional composition can also contain other substances that do not produce adverse, allergic, or other undesirable reactions when administered to a patient.

[0117] The carriers and other substances included in the pharmaceutical composition, cosmetic composition and / or nutritional composition may include one or more of the following: solvents, dispersants, coatings, absorption enhancers, controlled release agents and one or more inert excipients (such as starch), polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, binders and disintegrants. If desired, the tablet dosage form of the anti-infective mixture can be coated by aqueous or non-aqueous techniques known to those skilled in the art.

[0118] The pharmaceutical, cosmetic, and / or nutritional compositions according to the present invention can be administered orally, buccally, sublingually, topically, and / or rectally. They can be formulated as tablets, capsules, suppositories, effervescent tablets, pellets, lozenges, troches, gels, pastes, solutions, suspensions, emulsions, syrups, boluses, electuaries, slurries, powders, or granules in aqueous or non-aqueous liquids containing a predetermined concentration of the HMO mixture.

[0119] In addition, the pharmaceutical, cosmetic, and / or nutritional compositions of the present invention may also include binders, lubricants, inert diluents, flavoring agents, and wetting agents. Tablets, capsules, suppositories, or pills containing the pharmaceutical, cosmetic, and / or nutritional compositions of the present invention may optionally be coated or formulated to provide sustained, delayed, or controlled release of the anti-infective HMO mixture.

[0120] The HMO mixtures of the present invention may be supplemented with other pharmaceutical agents, active pharmaceutical ingredients, or agents that have an effect on the adverse health condition of the patient to whom the composition is administered.

[0121] In one embodiment, the composition can be in the form of a nutritional composition. For example, the nutritional composition can be a food composition, a rehydration solution, a medical food or a food for special medical purposes, a nutritional supplement, etc. The nutritional composition can contain a source of protein, lipid, and / or digestible carbohydrates and can be in powder or liquid form. The composition can be designed as a sole source of nutrition or a nutritional supplement.

[0122] Suitable protein sources include milk protein, soy protein, rice protein, pea protein and oat protein or mixtures thereof. The milk protein can be in the form of milk protein concentrate, milk protein isolate, whey protein or casein, or a mixture thereof. The protein can be whole protein or partially or deeply hydrolyzed protein. Hydrolyzed protein offers the advantage of being easier to digest, which is important for people with inflamed or damaged gastrointestinal tract (GI tract). Protein can also be provided in the form of free amino acids. Protein can comprise from about 5% to about 30% of the nutritional composition, typically from about 10% to 20%. Ideally, the protein source does not include excess lactose.

[0123] The protein source can be a source of glutamine, threonine, cysteine, serine, proline, or a combination of these amino acids. The glutamine source can be glutamine dipeptide and / or glutamine-rich proteins. Glutamine may be included because intestinal epithelial cells use glutamine as an energy source. Threonine, serine, and proline are important amino acids in the production of mucins. Mucins coat the gastrointestinal tract and can improve intestinal barrier function and mucosal healing. Cysteine is the primary precursor of glutathione, which is crucial for the body's antioxidant defenses.

[0124] Suitable digestible carbohydrates include maltodextrin, hydrolyzed or modified starch or corn starch, glucose polymers, corn syrup, corn syrup solids, high fructose corn syrup, carbohydrates derived from rice, carbohydrates derived from peas, carbohydrates derived from potatoes, tapioca, sucrose, glucose, fructose, sucrose, lactose, honey, sugar alcohols (e.g., maltitol, erythritol, sorbitol) or mixtures thereof. Preferably, the composition is reduced in amount with or without lactose or other FODMAP carbohydrates. Typically, digestible carbohydrates provide about 35% to about 55% of the energy of the nutritional composition. Particularly suitable digestible carbohydrates are low dextrose equivalent (DE) maltodextrins.

[0125] Suitable lipids include medium-chain triglycerides (MCT) and long-chain triglycerides (LCT). Preferably, lipid is a mixture of MCT and LCT. For example, MCT can include about 30% to about 70% by weight of lipid, more specifically about 50% to about 60% by weight. MCT provides the advantage of being more digestible, which can be important for people with inflamed or damaged gastrointestinal tract. Typically, lipid provides about 35% to about 50% of the energy of the nutritional composition. Lipid can contain essential fatty acids (ω-3 and ω-6 fatty acids). Preferably, these polyunsaturated fatty acids provide about 30% of the total energy of the lipid source.

[0126] Suitable sources of long-chain triglycerides are rapeseed oil, sunflower oil, palm oil, soybean oil, butterfat, corn oil, high oleic oil, and soy lecithin. Fractionated coconut oil is a suitable source of medium-chain triglycerides. The lipid profile of the nutritional composition is preferably designed to have a ratio of polyunsaturated fatty acids ω-6 (n-6) to ω-3 (n-3) of about 4:1 to about 10:1. For example, the ratio of n-6 fatty acids to n-3 fatty acids can be about 6:1 to about 9:1.

[0127] The nutritional composition can also include vitamins and minerals. If it is intended that the nutritional composition be used as a sole source of nutrition, it preferably includes a complete vitamin and mineral spectrum. Examples of vitamins include vitamin A, B-complex vitamins (such as B1, B2, B6, and B12), vitamins C, D, E, and K, niacin, and acid vitamins such as pantothenic acid, folic acid, and biotin. Examples of minerals include calcium, iron, zinc, magnesium, iodine, copper, phosphorus, manganese, potassium, chromium, molybdenum, selenium, nickel, tin, silicon, vanadium, and boron.

[0128] The nutritional composition may also include carotenoids, such as lutein, lycopene, zeaxanthin, and beta-carotene. The total amount of carotenoids included may vary from about 0.001 μg / ml to about 10 μg / ml. The amount of lutein included may be about 0.001 μg / ml to about 10 μg / ml, preferably about 0.044 μg / ml to about 5 μg / ml lutein. The amount of lycopene included may be about 0.001 μg / ml to about 10 μg / ml, preferably about 0.0185 μg / ml to about 5 μg / ml lycopene. The beta-carotene included may be about 0.001 μg / ml to about 10 mg / ml, for example, about 0.034 μg / ml to about 5 μg / ml beta-carotene.

[0129] The nutritional composition preferably also contains a reduced concentration of sodium, for example, from about 300 mg / l to about 400 mg / l. The remaining electrolytes may be present in concentrations sufficient to meet needs without providing an excessive renal solute burden on renal function. For example, potassium is preferably present in a range of from about 1180 to about 1300 mg / l; chloride is preferably present in a range of from about 680 to about 800 mg / l.

[0130] The nutritional composition may also contain various other conventional ingredients, such as preservatives, emulsifiers, thickeners, buffers, fibers and prebiotics (e.g., fructooligosaccharides, galacto-oligosaccharides), probiotics (e.g., Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium lactis HN019, Bifidobacterium lactis Bi07, Bifidobacterium infantis ATCC 15697, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus HNOO1, Lactobacillus acidophilus LA-5, Lactobacillus acidophilus NCFM, Lactobacillus fermentum CECT5716, Bifidobacterium longum BB536, Bifidobacterium longum AH1205, Bifidobacterium longum AH1206, Bifidobacterium breve M-16V, Lactobacillus reuteri ATCC 55730, Lactobacillus reuteri ATCC PTA-6485, Lactobacillus reuteri DSM 17938), antioxidant / anti-inflammatory compounds including tocopherols, carotenoids, ascorbic acid / vitamin C, ascorbyl palmitate, polyphenols, glutathione and superoxide dismutase (melon), other bioactive factors (e.g., growth hormones, cytokines, TFG-β), colorants, flavors, and stabilizers, lubricants, etc.

[0131] The nutritional composition can be formulated as a soluble powder, a liquid concentrate or a ready-to-use formulation. The composition can be supplied to a person in need via a nasogastric tube or orally. Various spices, fibers and other additives may also be present.

[0132] Nutritional composition can be prepared by any common manufacturing technology for preparing the nutritional composition of solid or liquid form. For example, composition can be prepared by combining various feed solutions (feed solution). Fat-in-protein (protein-in-fat) feed solution can be prepared by heating and mixing lipid sources, then adding emulsifier (such as lecithin), fat-soluble vitamins and at least a portion of protein source while heating and stirring. Then prepare carbohydrate feed solution by adding minerals, trace and ultra-trace minerals, thickener or suspending agent in water while heating and stirring. Before adding carbohydrates (such as HMO and digestible carbohydrate source), continue heating and stirring the resulting solution and keep 10 minutes. Then the feed solution of gained is mixed together while heating and stirring, and pH is adjusted to 6.6-7.0, then the composition is subjected to high temperature short-time processing, during which the composition is heat-treated, emulsified and homogenized, and then cooled. Add water-soluble vitamins and ascorbic acid, if necessary, pH is adjusted to the required range, add spices, and add water to reach the required total solid level.

[0133] For liquid products, the resulting solution can then be aseptically packaged to form an aseptically packaged nutritional composition. In this form, the nutritional composition can be in a ready-to-eat or concentrated liquid form. Alternatively, the composition can be spray-dried, processed, and packaged as a reconstitutable powder.

[0134] When the nutritional product is a ready-to-feed nutritional liquid, the total concentration of HMOs in the liquid may preferably be from about 0.1% to about 1.5%, including from about 0.2% to about 1.0%, such as from about 0.3% to about 0.7%, by weight of the liquid. When the nutritional product is a concentrated nutritional liquid, the total concentration of HMOs in the liquid may preferably be from about 0.2% to about 3.0%, including from about 0.4% to about 2.0%, such as from about 0.6% to about 1.5%, by weight of the liquid.

[0135] In another embodiment, the nutritional composition is a unit dosage form. The unit dosage form can contain an acceptable food grade carrier, for example, phosphate buffered saline, a mixture of ethanol in water, water and emulsions such as oil / water or water / oil emulsions, and various wetting agents or excipients. The unit dosage form can also contain other substances that do not produce adverse, allergic or other undesirable reactions when applied to a human. The carrier and other substances can include solvents, dispersants, coatings, absorption promoters, controlled release agents and one or more inert excipients (such as starch), polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, binding agents and disintegrants. The unit dosage form preferably mainly includes HMO and adds a minimum amount of binder and / or excipient. When the nutritional ingredients are incomplete or not intended to be the sole source of nutrition, the unit dosage form is particularly suitable.

[0136] Unit dosage forms can be administered orally, for example, as tablets, capsules, or pills containing a predetermined amount of the mixture, or as powders or granules containing a predetermined concentration of the mixture, or as gels, pastes, solutions, suspensions, emulsions, syrups, boluses, electuaries, or slurries containing a predetermined concentration of the mixture in an aqueous or non-aqueous liquid. Orally administrable compositions can include one or more binders, lubricants, inert diluents, flavorings, and wetting agents. Orally administrable compositions such as tablets can optionally be coated and can be formulated to provide sustained, delayed, or controlled release of the HMO mixture therein.

[0137] Unit dosage forms may also be administered by nasogastric tube or direct infusion into the gastrointestinal tract or stomach.

[0138] Unit dosage forms may also include therapeutic agents, such as antibiotics, probiotics, analgesics, and anti-inflammatory agents.

[0139] The appropriate dosage of a nutritional composition for a human can be determined in a conventional manner based on factors such as HMO concentration, human condition, immune status, weight, and age. The desired amount of HMO is generally from about 1 g to about 15 g per day, and in certain embodiments, from about 2 g to about 10 g per day, for example, from about 3 g to about 7 g per day. An appropriate dosage regimen can be determined by methods known to those skilled in the art.

[0140] In another embodiment, the HMO mixture can be formulated into a pharmaceutical composition. The pharmaceutical composition can contain a pharmaceutically acceptable carrier, such as phosphate-buffered saline, a mixture of ethanol in water, water, and an emulsion such as an oil / water or water / oil emulsion, as well as various wetting agents or excipients. The pharmaceutical composition can also contain other substances that do not produce adverse, allergic, or other undesirable reactions when administered to humans. The carrier and other substances can include solvents, dispersants, coatings, absorption enhancers, controlled release agents, and one or more inert excipients (such as starch), polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, binders, and disintegrants.

[0141] Pharmaceutical composition can be orally administered, for example as tablet, capsule or pill containing a predetermined amount, or as powder or granule containing a predetermined concentration, or gel, paste, solution, suspension, emulsion, syrup, bolus, lozenge or slurry containing a predetermined concentration in an aqueous or non-aqueous liquid. Orally administered compositions can include binding agents, lubricants, inert diluents, flavorings and wetting agents. Orally administered compositions such as tablets can optionally be coated, and can be formulated to provide a continuous, delayed or controlled release of a mixture thereof.

[0142] The pharmaceutical compositions may also be administered by rectal suppository, aerosol tube, nasogastric tube, or direct infusion into the gastrointestinal tract or stomach.

[0143] The pharmaceutical composition may also include therapeutic agents such as antibiotics, probiotics, analgesics, and anti-inflammatory agents. The appropriate dosage of the pharmaceutical composition can be determined in a conventional manner based on factors such as HMO concentration, patient condition, immune status, weight, and age. The desired amount of HMO is generally from about 1 g to about 15 g per day, and in certain embodiments, from about 2 g to about 10 g per day, for example, from about 3 g to about 7 g per day. Suitable dosage regimens can be determined by methods known to those skilled in the art.

[0144] The present invention also relates to the use of a mixture consisting of, or consisting essentially of, LNT, LNnT, pLNH and optionally lactose for medical purposes.

[0145] Individual HMOs are known to have beneficial health-related effects and are effective against bacterial and viral infections. While the individual HMOs alone are beneficial, the HMO combination mixtures described herein exhibit synergistic effects in treating infections compared to the individual components alone.

[0146] Another aspect of the present invention relates to a mixture consisting of or consisting essentially of LNT, LNnT, pLNH, and optionally lactose, or a composition comprising such a mixture consisting of or consisting essentially of LNT, LNnT, pLNH, and optionally lactose for use in treating, preventing, and / or ameliorating diseases and / or conditions associated with an imbalance in the microbiome. Alternatively, one aspect of the present invention relates to a method for treating, preventing, and / or ameliorating diseases and / or conditions associated with an imbalance in the microbiome in a human, comprising administering to the human a mixture consisting of or consisting essentially of LNT, LNnT, pLNH, and optionally lactose, or a composition comprising such a mixture consisting of or consisting essentially of LNT, LNnT, pLNH, and optionally lactose. In general, the mixture of LNT, LNnT, and pLNH according to the present invention can be used to modulate the human microbiome, for example, to increase the abundance of Bifidobacterium and Barnesiella, such modulation resulting in a decrease in the abundance of Firmicutes, particularly Clostridia.

[0147] In addition, another aspect of the present invention relates to a mixture consisting of or consisting essentially of LNT, LNnT, pLNH, and optionally lactose, or a composition comprising such a mixture consisting of or consisting essentially of LNT, LNnT, pLNH, and optionally lactose, for use in treating and / or reducing the risk of a broad range of bacterial or viral infections in humans. Alternatively, another aspect of the present invention relates to a method of treating and / or reducing the risk of a broad range of bacterial or viral infections in humans, comprising administering to a human a mixture consisting of or consisting essentially of LNT, LNnT, pLNH, and optionally lactose, or a composition comprising such a mixture consisting of or consisting essentially of LNT, LNnT, pLNH, and optionally lactose. Infections can occur in various body sites, not only in the intestinal microbiome, but also in other parts of the body exposed to the external environment, such as the skin, hair, ears, eyes, nose, and respiratory system. The one or more HMOs in the mixture according to the present invention can inhibit the adhesion of pathogenic bacteria such as Pseudomonas aeruginosa or Campylobacter jejuni, uropathogenic and enteropathogenic Escherichia coli, certain Salmonella and / or pneumonia-causing bacteria such as Pseudomonas aeruginosa, and viruses such as norovirus. The one or more HMOs in the mixture according to the present invention can also directly bind to pathogenic toxins such as those from Clostridium difficile. The one or more HMOs in the mixture according to the present invention can act as immune system modulators and influence intestinal health by stimulating bifidobacteria. The one or more HMOs in the mixture according to the present invention can inhibit the growth of group B streptococci in infants and breast milk. Group B streptococci are a major cause of neonatal sepsis, pneumonia, and meningitis. The one or more HMOs in the mixture according to the present invention can directly bind to the Shiga toxins Stx2 and Stx1B5 of Shigella dysenteriae. The one or more HMOs in the mixture according to the invention have the potential to reduce the risk of infectious diseases caused by bacterial or viral pathogens, most likely due to their binding to bacterial exotoxins.

[0148] In addition, another aspect of the present invention relates to the non-medical use of a mixture consisting of or essentially consisting of LNT, LNnT, pLNH and optionally lactose, or a composition comprising the mixture consisting of or essentially consisting of LNT, LNnT, pLNH and optionally lactose, for maintaining the symbiotic homeostasis of the intestinal flora in humans.

[0149] In addition, another aspect of the present invention relates to the non-medical use of a mixture consisting of or consisting essentially of LNT, LNnT, pLNH, and optionally lactose, or a composition comprising the mixture consisting of or consisting essentially of LNT, LNnT, pLNH, and optionally lactose, in the dietary management of a human. Dietary management refers to the complete or partial feeding of a patient suffering from a disease, disorder, or medical condition:

[0150] - limited, impaired, or disturbed ability to ingest, digest, absorb, metabolize, or excrete normal food or certain nutrients or metabolites contained therein, or

[0151] - Have other medically determined nutritional needs

[0152] (See: European Commission notification on the classification of foods for special medical purposes, Official Journal of the European Union C 401, 25 November 2017, pp. 10-11).

[0153] The present invention provides the following numbered aspects:

[0154] Aspect 1. A mutant lacto-N-biosidase having

[0155] - an amino acid sequence that is at least 70% identical to the sequence of amino acids 45 to 625 of SEQ ID No. 1; and

[0156] - a mutation at one or more amino acid positions selected from the group consisting of 410, 416, 439 and 442, the amino acid numbering following SEQ ID No. 1.

[0157] Aspect 2. The mutant lacto-N-biosidase according to aspect 1, comprising one or more of the following mutations:

[0158] - at position 410, Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or

[0159] - at position 416, Asp(D) is substituted by Asn or Gln, preferably by Asn; and / or

[0160] - at position 439, Met (M) is substituted by Leu, Val or Ile, preferably by Leu; and / or

[0161] - At position 442, Asn(N) is substituted by Trp, Tyr, Phe or His, preferably by Trp.

[0162] Aspect 3. The mutant lacto-N-biosidase according to aspect 2, comprising the following mutations:

[0163] - at position 410, Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or

[0164] - At position 416, Asp(D) is substituted by Asn or Gln, preferably by Asn.

[0165] Aspect 4. The mutant lacto-N-biosidase according to any one of the preceding aspects, which comprises or has a polypeptide sequence having a sequence identity of at least 75%, preferably at least 80%, more preferably at least 85%, still more preferably at least 90%, even more preferably at least 95% to the fragment of amino acids 45 to 625 in SEQ ID No. 1.

[0166] Aspect 5. The mutant lacto-N-biosidase according to any one of the preceding aspects, which comprises or has the same polypeptide sequence as the fragment of amino acids 45 to 625 in SEQ ID No. 1.

[0167] Aspect 6. The mutant lacto-N-biosidase according to any one of the preceding aspects, which comprises or has a polypeptide sequence having a sequence identity of at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even even more preferably at least 95% to a fragment of amino acids 31 to 625 of SEQ ID No. 1.

[0168] Aspect 7. A method for preparing the mutant lacto-N-biosidase according to any one of aspects 1 to 6, comprising the following steps:

[0169] (a) providing a DNA sequence encoding the mutant lacto-N-biosidase, and then

[0170] (b) expressing the mutant lacto-N-biosidase in a host cell transformed with the DNA sequence obtained in step (a).

[0171] Aspect 8. A method for synthesizing carbohydrates containing lacto-N-biose, the method comprising reacting a lacto-N-biosyl donor and a carbohydrate acceptor in the presence of an enzyme selected from LnbX, a truncated functional analog thereof, and the mutant lacto-N-biosidase according to any one of aspects 1 to 6, thereby transferring the lacto-N-biosyl residue of the lacto-N-biosyl donor to the carbohydrate acceptor, wherein the lacto-N-biosyl donor is preferably a compound of Formula 1 or Formula 2:

[0172]

[0173] wherein X is selected from the group consisting of a monosaccharide, a disaccharide or an oligosaccharide, an azide, a fluorine, an optionally substituted phenoxy group, an optionally substituted pyridyloxy group, a group A, a group B, a group C and a group D,

[0174]

[0175] where R a are independently H or alkyl, or two adjacent R a Group representation =C(R b )2 group, wherein R b are independently H or alkyl, R c R is independently selected from alkoxy, amino, alkylamino and dialkylamino, d Selected from H, alkyl and -C(=O)R e , where R e is OH, alkoxy, amino, alkylamino, dialkylamino, hydrazine, alkylhydrazine, dialkylhydrazine or trialkylhydrazine,

[0176] More preferably, X in Formula 1 is selected from monosaccharides, disaccharides or oligosaccharides, phenoxy, p-nitrophenoxy, 2,4-dinitrophenoxy and 2-chloro-4-nitrophenoxy, and advantageously the lacto-N-biosyl donor is LNT.

[0177] Aspect 9. The method according to aspect 8, wherein the donor is LNT, the acceptor is LNnT, and the lacto-N-biose-containing carbohydrate product is pLNH.

[0178] Aspect 10. A mixture consisting of, or consisting essentially of, LNT, LNnT, pLNH, and optionally lactose.

[0179] Example

[0180] In the following examples, a truncated version of lacto-N-biosidase from Bifidobacterium longum JCM1217 (LnbX, Sakamura et al., J. Biol. Chem. 288, 25194 (2013)), i.e., a fragment of amino acids 31 to 625 of SEQ ID No. 1, and mutants thereof were tested, with the positions of the mutations following SEQ ID No. 1. The truncated LnbX (WT, wild type) and mutants contained a His tag MGSSHHHHHHSSGLVPRGSHM at the N-terminus.

[0181] Example 1

[0182] exist The translacto-N-biosidase activity of truncated LnbX and its mutants was investigated, with the formation of pLNH monitored. Starting conditions: 10 mM LNT, 100 mM LNnT, 0.25 μM enzyme, 40°C, pH 6.0. The reaction was stopped by heating at 90°C for 10 minutes until the following time. Product formation was detected by HPLC-MS. The structure of pLNH was confirmed using a combination of HPLC-MS and various NMR techniques.

[0183] HPLC conditions:

[0184] Acetonitrile-water was used as the mobile phase in gradient elution mode at 25°C with a flow rate of 0.8 ml / min using an Accucore-150-Amide-HILIC 2.6 μm column (150 × 3 mm). The eluted substrate and product were detected by CAD. The concentration of each compound was calculated using a secondary calibration curve and its intercept was forced to zero.

[0185] The results are summarized in the table below.

[0186] Variants Conversion rate (time) WT 18％(1h) D416N 35％(1h) M439L 23％(1h) G410W 25％(3h) N442W 18％(3h)

[0187] Example 2:

[0188] The reaction described in Example 1 was performed with extended reaction times. For WT, D416N, N442W, and M439L, all pLNHs were hydrolyzed within 24 hours, while for G410W, 95% of the pLNHs formed were hydrolyzed within 24 hours. The WT product hydrolyzed the fastest. The product hydrolysis rates of all variants were lower than that of WT, in the following order (highest to lowest): D416N, N442W, M439L, G410W. The transglycosylation / hydrolysis rates of the variants were higher than that of WT, in the following order (highest to lowest): D416N, M439L, G410W, N442W.

[0189] Example 3

[0190] Mutant D416N was further tested to find the optimal reaction conditions. The following variables were tested:

[0191] - Temperature: 35-55℃,

[0192] -pH: 4.5-6.5,

[0193] -LNT concentration: 10-100 mM,

[0194] -Acceptor / donor molar ratio: 1-5,

[0195] - Enzyme concentration: 0.1-1 μM.

[0196] For each condition (28 in total), samples were collected at the following times: 15 min, 30 min, 1 h, 3 h, and 5 h.

[0197] The data showed that pH and temperature had minimal effects, with higher LNT concentrations and higher acceptor / donor ratios resulting in improved pLNH yield and product purity (expressed as the molar percentage of pLNH relative to the total amount of hexasaccharides and octasaccharides produced and detected). Based on the data, the optimal conditions were: an acceptor / donor ratio of 5:1, an enzyme concentration of 0.55 μM, a pH range of 4.5-6.5, and a temperature of 35-55°C.

[0198] The results for the optimal conditions are listed in the table below.

[0199] Temperature (℃) pH LNT (mM) LNnT (mM) Enzyme (μM) Conversion rate time pLNH purity 35 6.5 100 500 1 57％ 3h 82％ 55 4.5 100 500 0.55 52％ 1h 84％ 45 5.5 100 300 0.55 46％ 3h 78％ 55 6.5 100 500 0.1 30％ 5h 79％ 35 4.5 100 500 0.1 30％ 5h 81％

Claims

1. A mutant lacto-N-biosidase having - an amino acid sequence that is at least 70% identical to the sequence of amino acids 45 to 625 of SEQ ID No. 1; and - a mutation at at least one or more amino acid positions selected from the group consisting of 410, 416, 439 and 442, the amino acid numbering following SEQ ID No.

1.

2. The mutant lacto-N-biosidase according to claim 1, comprising one or more of the following mutations: - at position 410, Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or - at position 416, Asp(D) is substituted by Asn or Gln, preferably by Asn; and / or - at position 439, Met (M) is substituted by Leu, Val or Ile, preferably by Leu; and / or - At position 442, Asn(N) is substituted by Trp, Tyr, Phe or His, preferably by Trp.

3. The mutant lacto-N-biosidase according to claim 2, comprising the following mutation: - at position 410, Gly (G) is substituted by Trp, Tyr, Phe or His, preferably by Trp; and / or - At position 416, Asp(D) is substituted by Asn or Gln, preferably by Asn.

4. The mutant lacto-N-biosidase according to any one of the preceding claims, which has a polypeptide sequence having at least 80% sequence identity with a fragment of amino acids 45 to 625 of SEQ ID No.

1.

5. The mutant lacto-N-biosidase according to claim 4, which has a polypeptide sequence having at least 90% sequence identity with the fragment of amino acids 45 to 625 in SEQ ID No.

1.

6. The mutant lacto-N-biosidase according to any one of the preceding claims, which has a polypeptide sequence identical to a fragment of amino acids 45 to 625 of SEQ ID No.

1.

7. The mutant lacto-N-biosidase according to any one of claims 1 to 3, which has a polypeptide sequence having at least 70% sequence identity with a fragment of amino acids 31 to 625 in SEQ ID No.

1.

8. The mutant lacto-N-biosidase according to claim 7, which has a polypeptide sequence having at least 80% sequence identity with the fragment of amino acids 31 to 625 in SEQ ID No.

1.

9. The mutant lacto-N-biosidase according to claim 8, which has a polypeptide sequence having at least 90% sequence identity with the fragment of amino acids 31 to 625 in SEQ ID No.

1.

10. A method for preparing the mutant lacto-N-biosidase according to any one of claims 1 to 9, comprising the following steps: (a) providing a DNA sequence encoding the mutant lacto-N-biosidase, and then (b) expressing the mutant lacto-N-biosidase in a host cell transformed with the DNA sequence obtained in step (a).

11. A method for synthesizing carbohydrates containing lacto-N-biose, the method comprising reacting a lacto-N-biosyl donor and a carbohydrate acceptor in the presence of an enzyme selected from the group consisting of LnbX, a truncated functional analog thereof, and a mutant lacto-N-biosidase according to any one of claims 1 to 9, thereby transferring the lacto-N-biosyl residue of the lacto-N-biosyl donor to the carbohydrate acceptor.

12. The method according to claim 11, wherein the lacto-N-biosyl donor is a compound of Formula 1 or Formula 2: wherein X is selected from the group consisting of a monosaccharide, a disaccharide or an oligosaccharide, an azide, a fluorine, an optionally substituted phenoxy group, an optionally substituted pyridyloxy group, a group A, a group B, a group C and a group D, where R a are independently H or alkyl, or two adjacent R a Group representation =C(R b )2 group, wherein R b are independently H or alkyl, R c R is independently selected from alkoxy, amino, alkylamino and dialkylamino, d Selected from H, alkyl and -C(=O)R e , where R e is OH, alkoxy, amino, alkylamino, dialkylamino, hydrazine, alkylhydrazine, dialkylhydrazine or trialkylhydrazine.

13. The method according to claim 12, wherein X in Formula 1 is selected from monosaccharides, disaccharides or oligosaccharides, phenoxy, p-nitrophenoxy, 2,4-dinitrophenoxy and 2-chloro-4-nitrophenoxy.

14. The method according to claim 13, wherein X is lactose, and preferably the lacto-N-biosyl donor is LNT.

15. The method according to any one of claims 11 to 14, wherein the donor is LNT, the acceptor is LNnT, and the lacto-N-biose-containing carbohydrate product is pLNH.

16. A mixture consisting of or consisting essentially of LNT, LNnT, pLNH, and optionally lactose.

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