A glycosyl hydrolase and its use in the production of glycosides

Abstract

The application provides a glycosyl hydrolase and its use in glycoside production, a glycosyl hydrolase which is mutated at Q215R / K218E and has an Asp36-Glu39-Asp42 acidic cluster, can realize efficient beta-1,2-glycosyl transfer at pH 6.5-7.5, selectively hydrolyzes beta-1,2-glycosidic bonds at pH 4.5-5.5, and is stable to beta-1,3, beta-1,4 and beta-1,6. The enzyme is suitable for preparing various beta-1,2-glycosides and editing sugar chains, and can obtain high yield and high purity in the application of synthesizing human milk oligosaccharides and the like.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and glycoengineering, specifically to a glycosyl hydrolase and its use in glycoside production. Background Technology

[0002] Carbohydrates and their derivatives play an important role in life sciences, medicine, food, and industry, especially glycosylation, which plays a key role in regulating protein and lipid function. The formation and hydrolysis of glycosidic bonds are ubiquitous in metabolic activities, and in industrial production, glycosides are frequently used as sweeteners, drug lead compounds, intermediates, and functional materials, with demand steadily increasing.

[0003] Traditionally, the main enzymatic tools used for glycoside synthesis are glycosyltransferases and glycoside hydrolases. Glycosyltransferases can generally directionally transfer activated glycosyl donors (such as UDP-, GDP-, and other nucleoside diphosphate sugars) to specific acceptor molecules under relatively mild conditions. However, due to the typically high substrate specificity of glycosyltransferases, the potentially complex cofactors they require, and limitations in yield and enzyme stability during industrial production, they struggle to meet the demands for efficient synthesis of certain specific glycosidic bonds. On the other hand, glycoside hydrolases, due to their high efficiency and specificity in glycosidic bond hydrolysis, have been widely used in food processing (such as starch and lactose hydrolysis) and biotransformation (such as oligosaccharide preparation and waste sugar treatment). However, glycoside hydrolases typically operate primarily through hydrolysis, and their glycosyl transfer function is often weak or difficult to effectively demonstrate under normal process conditions.

[0004] Existing research has shown that protein engineering (such as site-directed mutagenesis, directed evolution, or rational design) can be used to induce glycosidic hydrolases to exhibit retrograde or transglycosylation activities in certain reaction systems, thus enabling their application in glycoside synthesis. Theoretically, the ability to rationally control both hydrolysis and transglycosylation activities could significantly improve the efficiency of preparing specific glycosidic compounds. However, currently, there are still relatively few enzymes that can balance high glycosylation efficiency with precise glycosidic bond selectivity, particularly for the specific synthesis or hydrolysis of β-1,2-glycosidic bonds, for which a systematic and mature enzymatic solution is lacking.

[0005] In polysaccharides, oligosaccharides, and glycoconjugated molecules, the β-1,2-glycosidic bond structure often influences their physicochemical properties, biological activity, and stability. For example, the β-1,2-glycosidic bond plays a crucial role in the modified structures of certain human milk oligosaccharides and some antibiotics. Traditional synthetic methods typically rely on chemical means, employing multi-step protecting-deprotecting reactions to achieve the directional introduction of the β-1,2-glycosidic bond, which is cumbersome and costly. Furthermore, chemical synthesis suffers from numerous side reactions and difficulties in fully controlling stereoselectivity.

[0006] In the field of biotechnology, achieving selective synthesis or hydrolysis of β-1,2-glycosidic bonds using enzymatic catalysis requires addressing several key challenges: Substrate recognition and activation: The synthesis reaction necessitates activation of the glycosyl donor (e.g., UDP-, GDP-, azide, or fluorine substitution) and ensuring the enzyme's ability to recognize these modified glycosyl donors. Dual activity regulation of the enzyme: Glycoside hydrolases often exhibit high hydrolytic activity under acidic or slightly acidic conditions, while detectable transglycosylation activity (or retrograde reaction) is only observed under slightly neutral or weakly alkaline conditions. Effective regulation of both transglycosylation and transglycosylation activities within the same protein molecule is a significant challenge. Improving yield and selectivity: Currently commercially available or literature-reported hydrolases often involve hydrolytic side reactions during glycosyl transfer, leading to low yields of the target glycoside or the formation of glycosidic bonds at undesirable sites, making it difficult to obtain high-purity β-1,2-linked products.

[0007] Current technologies for modifying glycosidases typically focus on the following aspects: modifying the enzyme's active pocket by altering the spatial arrangement of substrate binding sites or the properties of amino acids through site-directed mutagenesis, thereby enhancing affinity for specific sugar donors or acceptors; and regulating acid-base catalytic sites. For glycosidases, acidic amino acids such as Glu and Asp typically act as proton donors, while basic amino acids act as proton acceptors. Fine-tuning the positions of key amino acids through mutation or altering the hydrogen bond network could potentially change the relative balance between hydrolysis and transglycosylation. Regarding pH dependence, some studies indicate that the competitive relationship between hydrolysis and transglycosylation can be moderately regulated by changing the environmental pH or adding metal ion cofactors. However, achieving high selectivity or high yield for a specific glycosidic bond (especially β-1,2) at different pH levels remains a significant challenge.

[0008] However, to date, enzymes capable of achieving a clear switching between transfer and hydrolysis activities within a pH range and exhibiting high specificity and efficiency for β-1,2-glycosidic bonds remain rare. Techniques that truly meet the demands of industrial-scale production (such as reaction time, product purity, and byproduct ratios) are also not yet mature. Based on these circumstances, there is an urgent need in this field for a structurally modified or rationally designed glycosyl hydrolase: capable of achieving efficient transfer reactions of β-1,2-glycosidic bonds under mild conditions for the synthesis of various glycoside derivatives; capable of effectively performing glycoside hydrolysis under another pH range for glycan editing or substrate structure modification; possessing good recognition ability for specific sugar donors (especially nucleoside diphosphates functionalized by fluorination, azide, etc.); exhibiting low incidence and high yield of side reactions (such as hydrolysis of other glycosidic bonds or glycosylation at undesired positions); and applicable to the preparation of various high-value compounds such as human milk oligosaccharides, flavonoid glycosides, and modified antibiotic products. Summary of the Invention

[0009] To address the aforementioned problems in the prior art, this invention provides a glycosyl hydrolase mutated by Q215R / K218E and possessing the Asp36-Glu39-Asp42 acidic cluster. This enzyme can achieve efficient β-1,2-glycosyl transfer at pH 6.5-7.5, selectively hydrolyze β-1,2-glycosidic bonds at pH 4.5-5.5, and remains stable for β-1,3, β-1,4, and β-1,6 bonds. This enzyme is suitable for preparing various β-1,2-glycosides and glycan editing, and can achieve high yields and high purity in applications such as the synthesis of human milk oligosaccharides.

[0010] This application provides a glycosyl hydrolase, (a) comprising the amino acid sequence shown in SEQ ID NO:1, wherein at position 215, glutamine (Q) is mutated to arginine (R) and at position 218, lysine (K) is mutated to glutamic acid (E);

[0011] (b) The catalytic domain contains an acidic amino acid cluster composed of Asp36-Glu39-Asp42;

[0012] (c) At pH 6.5–7.5, the activity of β-1,2-glycosyl transfer reaction is more than 3 times that of hydrolysis activity; at pH 4.5–5.5, the hydrolysis activity of β-1,2-glycosidic bond is more than 5 times that of transfer activity.

[0013] Preferably, the acidic amino acid cluster has a DXXE motif, where X is any amino acid, and the proton transport pathway of the motif has the following characteristics in transferase mode. The spacing of the hydrogen bond network.

[0014] This application also provides a method for preparing β-1,2-glycoside compounds, comprising the following steps:

[0015] The glycosyl donor, the acceptor molecule, and the glycosyl hydrolase are mixed in a buffer solution with a pH of 6.5-7.5;

[0016] Add 0.1-2mM Mn 2+ or Mg 2+ As a cofactor, it reacts at 30-45℃ for 0.5-2 hours;

[0017] The glycosyl donor is at least one of UDP-glucose, UDP-fucose, or UDP-glucosamine, wherein the C3 or C4 position is independently monosubstituted by a fluorine or azide group.

[0018] Preferably, the receptor molecule is a flavonoid, an antibiotic, or a lactose derivative, and the molar ratio of glycosyl donor to receptor in the reaction system is 1:5 to 5:1.

[0019] This application also provides a method for editing sugar chains, including the following steps:

[0020] The substrate containing β-1,2-glycosidic bonds was mixed with the enzyme in a buffer solution at pH 4.5-5.5;

[0021] The hydrolysis reaction was carried out at 50-60℃ for 10-30 minutes in the absence of divalent metal ions.

[0022] After the hydrolysis reaction, the hydrolysis rates of β-1,3, β-1,4, and β-1,6 glycosidic bonds in the substrate were all ≤5%.

[0023] This application also provides the application of the glycosyl hydrolase as described above in the synthesis of human lactose oligosaccharides, using lactose as the acceptor and UDP-fucose as the donor, with a reaction yield ≥90%, and the proportion of β-1,3 / β-1,4 glycosidic bond byproducts detected by HPLC-MS ≤0.5%.

[0024] This invention provides a glycosyl hydrolase and its use in glycoside production, achieving the following beneficial technical effects:

[0025] 1. This invention utilizes dual-activity regulation of β-1,2-glycosyl transfer and hydrolysis. The glycosyl hydrolase described in this invention exhibits a significant β-1,2-glycosyl transfer advantage under pH 6.5-7.5 conditions, with transfer activity more than three times that of hydrolysis activity, thereby achieving highly efficient β-1,2-glycoside synthesis. Under pH 4.5-5.5 conditions, the hydrolysis activity significantly increases to more than five times that of transfer activity, enabling the enzyme to selectively hydrolyze existing glycan structures and simplifying the industrial glycan editing process. This dual-mode activity is achieved on the same enzyme molecule, greatly expanding the enzyme's application range under different process conditions.

[0026] 2. This invention achieves high selectivity and substrate adaptability. By introducing specific site mutations (Q→R at position 215, K→E at position 218) into the amino acid sequence shown in SEQ ID NO:1 and forming key amino acid clusters such as Asp36-Glu39-Asp42 in the catalytic domain, the enzyme of this invention exhibits excellent specificity for β-1,2-glycosidic bonds. It can efficiently synthesize β-1,2-glycosidic structures and, under certain conditions, precisely hydrolyze this bond without significantly disrupting other glycosidic linkages such as β-1,3, β-1,4, and β-1,6. Furthermore, this enzyme shows good adaptability to various modified glycosyl donors (such as UDP-glucose, UDP-fucose, and UDP-glucosamine containing fluorine or azide groups) and various acceptor molecules (such as flavonoids, antibiotics, and lactose derivatives), laying the foundation for structural innovation of glycosides and their derivatives.

[0027] 3. This invention features mild operation, high yield, and few byproducts. When the enzyme of this invention performs glycosyltransfer reactions under mild conditions of 30-45℃ and pH 6.5-7.5, it can yield high-purity β-1,2-glycoside products within 0.5-2 hours, reducing the multi-step protection and byproduct formation in traditional chemical synthesis. In glycan editing applications (pH 4.5-5.5, 50-60℃), the hydrolysis rate of other glycosidic bonds can be controlled below 5%, significantly reducing the damage of side reactions to the target product structure. Compared with some existing technologies, this invention can significantly save energy and improve product purity. This invention is suitable for industrial scale-up and customized modification. In industrial-scale scale-up or continuous reaction systems, the enzyme of this invention still maintains high transfer efficiency and hydrolysis selectivity, and the enzyme can work stably under weakly acidic and near-neutral conditions, which is beneficial for equipment compatibility and production operation. In the field of glycan editing, the enzyme activity mode can be rapidly switched by simply adjusting the pH or adding / removing divalent metal ions, providing a flexible and efficient biocatalytic method for multi-step glycan modification processes.

[0028] 4. The application potential of this invention in preparing human milk oligosaccharides and high-value sugar derivatives, especially in the application of the enzyme of this invention in the synthesis of human milk oligosaccharides (HMOs), using lactose as an acceptor and UDP-fucose as a donor, can obtain a reaction yield of more than 90% in a short time, and the proportion of β-1,3 / β-1,4 glycosidic bond byproducts is less than 0.5%, which not only reduces the subsequent separation cost, but also better ensures the functional characteristics of the target product. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1:

[0031] To address the aforementioned problems in the prior art, this application provides a glycosyl hydrolase, (a) comprising the amino acid sequence shown in SEQ ID NO:1, the sequence listing of which is detailed in the XML file "GlycoHydrolase_B1.xml", with glutamine (Q) mutated to arginine (R) at position 215 and lysine (K) mutated to glutamic acid (E) at position 218; in some embodiments, the wild-type gene is obtained and the vector is constructed by isolating a gene corresponding to the amino acid sequence of the glycosyl hydrolase of this invention from the genome of a strain (Bacillus subtilis), the encoded protein sequence of which is shown in SEQ ID NO:1. This gene is obtained by PCR amplification and cloned into a conventional molecular cloning vector (such as pUC19) for subsequent operations. For the construction of the wild-type expression vector, an E. coli expression system (such as pET-28a, pET-32a, or pGEX-4T-1) is selected as the expression vector. The PCR product and the linearized expression vector were digested with restriction endonucleases and ligated, then transformed into E. coli DH5α competent cells. After screening and sequencing confirmation, the correct wild-type enzyme gene recombinant vector (denoted as pET-XY-WT) was obtained. The amino acid sequence of this wild-type (WT) enzyme is completely identical to SEQ ID NO:1, with no additional mutations.

[0032] Site-directed mutagenesis yielded the Q215R / K218E double mutant, where amino acid glutamine (Q) at position 215 of the sequence shown in SEQ ID NO:1 is mutated to arginine (R), and lysine (K) at position 218 is mutated to glutamate (E). To ensure accurate localization, it must be clearly stated in the specification or appendix that positions 215 and 218 are relative to the mature protein sequence in SEQ ID NO:1, and not the overall sequence number containing the signal peptide or leader sequence. Two site-directed mutagenesis primer pairs (or nested mutagenesis) were designed to perform amino acid substitutions at positions 215 and 218, respectively. The mutated bases (using NNK degenerate encoding) or triplets precisely encoding R and E were placed in appropriate positions in the primers to ensure high homology of other sequences with the wild-type gene.

[0033] Target fragment and mutant codons, wild type (partial gene fragment, the codons containing Q(215) and K(218) are in square brackets; the upstream and downstream bases are reference extensions):

[0034]

[0035] The codon for Q(215) is CAG; the codon for K(218) is AAA;

[0036] Mutant type (same fragment after target mutation): Q(215)→R(215), i.e., CAG→CGT;

[0037] K(218)→E(218) = AAA→GAA

[0038]

[0039] Q(215) and K(218) are separated by 3 amino acid sites (9 bases), so two mutations can be introduced at once using a single pair of primers.

[0040] For site-directed mutagenesis primer design, a specific pair of oligonucleotide primers is given below (indicated by the 5'→3' direction). A certain number of nucleotides homologous to the wild-type sequence are retained at both ends to ensure sufficient annealing length; the critical region in the middle is replaced with the corresponding mutant codon.

[0041] Forward primer: 5'-GTT TTT CGT GCG TCT GAA AAT GTT-3'

[0042] Wherein, “CGT” corresponds to the codon for arginine (R) after mutation, and “GAA” corresponds to the codon for glutamic acid (E) after mutation; the upstream and downstream sequences (GTT TTT…AAT GTT) match the upstream / downstream sequences of the wild type of the gene to ensure effective annealing; the primer length is about 25 bp, and in some examples it is in the range of 25 to 35 bp.

[0043] Reverse primer: 5'-AAC ATT TTC AGA CGC ACG AAA AAC-3'

[0044] The primer is a sequence that is complementary to and reversed to the forward primer, and also contains codons that match the mutation site; the “CAG” / “AAA” in the central region has been replaced with the complementary sequence “CGT” / “GAA”; it maintains full homology with the upstream and downstream of the wild-type sequence, and can achieve stable annealing and amplification.

[0045] The procedure for performing Q→R and K→E double mutations using these primers is as follows: Prepare the template and reagents. Template DNA: a plasmid containing the wild-type enzyme gene (SEQ ID NO:1) cloned into an expression vector (such as pET-28a or pGEX-4T-1), at a concentration of 50–100 ng / μL. Prepare 10 μM stock solutions of the above-mentioned forward and reverse site-directed mutagenesis primers. Prepare a site-directed mutagenesis kit or a standard PCR amplification system (containing high-fidelity DNA polymerase).

[0046] For PCR amplification, refer to the kit instructions or standard formulation for high-fidelity PCR: Template DNA: approximately 10–50 ng; Forward primer: 0.2–0.5 μM; Reverse primer: 0.2–0.5 μM; dNTPs: approximately 200 μM; Total reaction volume: e.g., 50 μL; Annealing temperature: generally 2–5 °C lower than the primer Tm value; Number of cycles: 25–30 cycles.

[0047] If the kit requires methylation of the template after amplification, perform Dpn I treatment according to the kit instructions (this degrades the original template and avoids residue). For transformation and screening, directly transform the PCR product into competent cells (e.g., E. coli DH5α), or purify the fragment before ligation and transformation (depending on the method). Select positive clones and verify by sequencing whether the amino acids at positions 215 and 218 have undergone Q→R and K→E mutations, and confirm that there are no other additional mutations. After obtaining the successfully mutated recombinant plasmid (denoted as pET-XY-Mut), the corresponding mutant protein can be induced to express in host bacteria such as BL21(DE3). For protein expression and subsequent validation, refer to the procedures outlined in the previous examples to perform mutant induction expression, purification, and enzymatic property determination, including verification of enhanced β-1,2-glycosyltransfer activity at pH 6.5-7.5 and enhanced hydrolytic activity at pH 4.5-5.5. The product was analyzed using HPLC-MS and other methods to further confirm whether the changes in enzyme activity met the target requirements (significantly increasing the β-1,2-glycoside synthesis rate in transfer mode; selectively hydrolyzing β-1,2-glycosidic bonds in hydrolysis mode). Using the aforementioned forward and reverse primers, double mutations (Q→R, K→E) of amino acids 215 and 218 could be achieved in the same PCR reaction. Sequencing and subsequent enzymatic identification results showed that the double mutant exhibited "dual-mode" activity of highly selective transfer and selective hydrolysis of β-1,2-glycosidic bonds under different pH conditions.

[0048] PCR amplification and cloning were performed using overlap extension PCR or a commercially available Site-Directed Mutagenesis Kit. The PCR product containing the Q215R / K218E double mutation sites was ligated back into the pET-XY-WT vector and transformed into E. coli DH5α. Positive clones were selected, and plasmids were extracted. Sequencing was used to verify the accuracy of the mutations (Q→R at position 215, K→E at position 218) and to confirm the absence of other unwanted variations. After confirmation, the mutant recombinant vector (denoted as pET-XY-Mut) was obtained.

[0049] Recombinase protein expression and purification, transformation and expression: pET-XY-Mut was transformed into E. coli BL21(DE3) competent cells. Positive clones were shaken and cultured in LB medium containing appropriate antibiotics at 37°C until the OD600 reached 0.6-0.8. Then, IPTG at a final concentration of 0.1-1.0 mM was added to induce protein expression. The induction temperature could be 16-25°C (to ensure proper protein folding), and the induction time was approximately 4-16 hours. Protein isolation and purification: The induced culture was collected and centrifuged at 8000g for 10 minutes at 4°C. The supernatant was discarded, and the intracellular protein precipitate was resuspended. Disruption methods included ultrasonic or high-pressure disruption, followed by centrifugation to remove cell debris. Ni was then loaded. 2+ Further purification is performed using affinity columns (if fused with a His tag) or other affinity / ion exchange / gel filtration methods. After purification, the protein is analyzed by SDS-PAGE to confirm the theoretical molecular weight of the target protein corresponding to the major band (generally around 3580 kDa, but this varies with enzyme length). Purity can reach over 80-95%.

[0050] The mutant protein was sequenced and enzymatically characterized. Protein sequence verification was performed using LC-MS / MS to sequence the purified enzyme protein, focusing on verifying whether the amino acids at positions 215 and 218 exhibited Q→R and K→E mutations. N-terminal sequencing (Edman degradation) or re-sequencing of partial fragments could also be performed to ensure the absence of additional mutations. Enzyme activity was assessed using two enzyme activity detection systems: glycosyltransfer activity (using UDP-glucose or other nucleoside diphosphates as donors and lactose / flavonoids as acceptors), detecting the amount of β-1,2-glycosides generated in the product within the pH range of 6.5–7.5; and hydrolysis activity (using oligosaccharides or polysaccharides containing β-1,2-glycosidic bonds as substrates), detecting the amount of free glycosyl groups or free aglycones generated within the pH range of 4.5–5.5. Product content was quantitatively analyzed by HPLC or HPLC-MS, and the catalytic efficiency was calculated by comparison with blank experiments (no enzyme, wild-type enzyme control, etc.). To determine the differences in activity and key parameters, the pH effect was measured by setting gradients (e.g., 0.5 gradients) within the pH range of 4.0 to 8.0, and measuring the substrate transfer and hydrolytic activities of the mutant enzyme under the same enzyme concentration and reaction system.

[0051] Within the pH range of 6.5–7.5, the β-1,2-glycosyl transfer efficiency is more than three times that of hydrolysis; within the pH range of 4.5–5.5, the hydrolysis activity of β-1,2-glycosidic bonds is significantly higher than the transfer activity, reaching more than five times. The metal ion effect is also observed; adding 0.5 mM Mn to the transfer reaction system... 2+ or Mg 2+The increase in product formation rate was then observed; no divalent metal ions were added to the hydrolysis reaction system. Comparative results showed that in the transfer system at pH 6.5-7.5, the cofactor further improved the yield, while at pH 4.5-5.5, when hydrolysis was the dominant reaction, the cofactor could be omitted. To assess the effect on other glycosidic bonds (such as β-1,3 / β-1,4 / β-1,6), corresponding substrates or derivatives were selected for hydrolysis / transfer tests. The results showed that in hydrolysis mode (acidic pH), the mutant of this invention had almost no significant effect on other types of glycosidic bonds, with hydrolysis rates not exceeding 5%, while it showed significant hydrolysis mainly on β-1,2 bonds; in transfer mode (near neutral pH), its efficiency in transglycosylation of β-1,2-glycosidic bonds was significantly higher than that of the wild type and other types of enzymes.

[0052] A Q215R / K218E double mutant was successfully constructed: using site-directed mutagenesis, glutamine (Q) at position 215 was mutated to arginine (R), and lysine (K) at position 218 was mutated to glutamic acid (E) in the amino acid sequence shown in SEQ ID NO:1. This mutant retains the spatial configuration of the Asp36, Glu39, and Asp42 acidic amino acid clusters in the catalytic domain. Significantly enhanced enzymatic properties were observed: at pH 6.5-7.5, the mutant exhibited approximately 23 times the β-1,2-glycosyltransferase activity of the wild-type enzyme, with reduced byproducts; at pH 4.5–5.5, its hydrolytic activity increased more than twice that of the wild-type, with other glycosidic bonds almost undamaged. The enzyme's dual-mode activity was validated: the enzyme exhibits distinct activity switching at different pH levels, providing more flexible application scenarios for industrial production or glycan editing. This embodiment demonstrates that the synergistic effect of the specific site mutation (Q215R / K218E) in the sequence shown in SEQ ID NO:1 and the amino acid cluster of the catalytic domain can effectively enhance the enzyme's specificity and catalytic efficiency for β-1,2-glycosyl bonds, and it has both transfer and hydrolysis functions, which can be used for subsequent glycoside preparation and glycan editing applications.

[0053] (b) The catalytic domain contains an acidic amino acid cluster composed of Asp36-Glu39-Asp42; in some embodiments, the verification and functional evaluation of the Asp36-Glu39-Asp42 acidic amino acid cluster in the catalytic domain, the prediction and analysis of the enzyme's three-dimensional structure, and the preparation of the amino acid sequence are all performed using the amino acid sequence of the glycosyl hydrolase of the present invention (SEQ ID NO: 1). The three key sites Asp36, Glu39, and Asp42 in its maturation region are predicted to be located within the enzyme's catalytic domain. The sequence length (example) is approximately 350,400 amino acids, with multiple acidic amino acids located within the catalytic region (e.g., amino acid 30 and amino acid 200). Three-dimensional structures of known proteins with high sequence similarity to the enzyme of this invention (such as certain glycosidases or glycosyltransferase family members in the PDB database) were selected as templates. Homology modeling of the three-dimensional structure of the enzyme was performed using protein structure software such as Swiss-Model, Modeller, or Rosetta. Evaluation results using tools such as Ramachandran showed that a distinct acidic cluster formed in the active pocket region. In the model, Asp36, Glu39, and Asp42 were appropriately spaced and exhibited potential hydrogen bond or electrostatic interaction networks with the substrate binding site. Active pocket and acidic cluster prediction, and molecular docking analysis, indicated that when the substrate (e.g., UDP-glucose or oligosaccharides containing β-1,2-glycosidic bonds) enters the catalytic pocket, Asp36, Glu39, and Asp42 residues may participate in the process of proton donation or localization of substrate glycosyl groups. This is consistent with the "double acid catalysis" or "acidic residue coordination" mechanism of classical glycosylhydrolases / glycosyltransferases, suggesting that this three-residue cluster is key to the enzyme's glycosyl transfer or glycosidic bond hydrolysis.

[0054] Site-directed mutagenesis and functional validation: To further confirm the important role of the Asp36-Glu39-Asp42 acidic cluster in the catalytic domain, this embodiment performs single- or multi-site mutation control experiments on each site: Using wild-type (i.e., SEQ ID NO:1 containing Asp36, Glu39, and Asp42) as a template, the following mutants are constructed using site-directed mutagenesis: D36N: Asp36 is mutated to Asn (eliminating the acidic side chain); E39Q: Glu39 is mutated to Gln; D42N: Asp42 is mutated to Asn; D36N / E39Q / D42N: simultaneous mutation at all three sites. Other operations (such as PCR amplification, plasmid construction, sequencing, etc.) can be referred to the aforementioned site-directed mutagenesis method embodiments of this invention. Protein expression and purification: Each mutant was transformed into the *E. coli* BL21(DE3) expression host, and expression was induced using IPTG in LB medium. The bacterial culture was lysed, and the protein was purified by Ni-NTA affinity chromatography (e.g., with a His tag) or other methods. SDS-PAGE analysis showed that all mutants could be expressed with a purity of over 90%. Enzyme activity assay: Hydrolysis mode (pH 5.0): Using oligosaccharides containing β-1,2-glycosidic bonds as substrates, a final concentration of 10 mM and an enzyme concentration of 0.05 mg / mL were set. The reaction was carried out in 50 mM acetate-acetate buffer (pH 5.0) at 37°C for 1 hour. After terminating the reaction, the free monosaccharide or aglycone content was detected by HPLC or HPLC-MS, and the hydrolysis rate was calculated. Transfer mode (pH 7.0): Using UDP-glucose (10 mM) as the donor and lactose or aglycone derivative (10 mM) as the acceptor, 0.5 mM Mn was added. 2+The reaction was carried out at 37°C for 1 hour. The β-1,2-glycoside derivatives formed in the product were quantified by HPLC, and the transfer reaction rate was recorded. Simultaneously, the activities of the wild-type enzyme and each mutant were measured, and their residual activity percentages relative to the wild-type were compared. Compared with the wild-type, each single-point mutation (D36N, E39Q, D42N) led to a significant decrease in hydrolytic and transfer activities, with residual activity generally between 20% and 30%. Enzymes with simultaneous mutations at three points almost lost their catalytic activity, with residual activity less than 5%. This indicates that Asp36, Glu39, and Asp42 together constitute the key acidic amino acid cluster of the enzyme of this invention, which is an important part of maintaining the normal catalytic function of the enzyme; the three participate together or synergistically in key processes such as proton transfer, substrate fixation, or transition state stabilization. Control experiments also showed that if the acidic cluster is damaged, the advantage of the enzyme in this invention, which exhibits "transfer activity more than three times that of hydrolytic activity" at pH 6.5-7.5, is difficult to demonstrate, and the high hydrolytic activity at pH 4.5-5.5 is also significantly reduced, further proving that Asp36, Glu39, and Asp42 are of core significance for the dual-mode activity switching. The effect of the acidic cluster on the enzyme's pH activity switching was investigated using a pH range test of 4.0-8.0 for both wild-type and mutants. Hydrolytic and transfer activities were measured at each pH point to obtain activity curves. The wild-type reached a peak transfer activity near pH 6.5-7.0, while exhibiting the highest hydrolytic activity near pH 4.5-5.0. The mutant results showed that the D36N / E39Q / D42N three mutants did not show significant activity peaks in any pH range; single-point mutations resulted in an overall downward shift in the activity curve, and the difference between the two modes was also significantly weakened. This indicates that the Asp36-Glu39-Asp42 acidic amino acid cluster plays a decisive role in modulating the pH dependence of the enzyme of this invention.

[0055] Three-dimensional structure and docking prediction, along with homology modeling, revealed that Asp36, Glu39, and Asp42 near the active site form a stable acidic amino acid cluster. Molecular docking results suggest that these three amino acids assist substrate recognition and proton transfer through hydrogen bonding or electrostatic interactions. Mutation experiments verified that mutating any of Asp36, Glu39, or Asp42 to neutral / basic residues significantly disrupted the enzyme's dual-mode catalytic activity; in particular, the triple mutant almost completely lost function, demonstrating the crucial role of this cluster in the enzyme's dual-activity mechanism. The dual-mode activity is dependent on the synergistic effect of these three sites, enabling the enzyme to achieve efficient proton transfer at pH 6.5-7.5 and efficient hydrolysis at pH 4.5-5.5. Its acidic side chains may act as reversible proton donors / acceptors under different pH conditions, thereby switching catalytic modes. In summary, this embodiment clearly indicates that the acidic amino acid cluster composed of Asp36, Glu39, and Asp42 is the core basis for the enzyme's dual-mode (transfer / hydrolysis) function in the catalytic domain of this invention. The presence and spatial arrangement of this cluster directly determine the high selectivity and efficiency of β-1,2-glycosyl bond synthesis and hydrolysis. Any mutation that simultaneously or critically disrupts the three residues of Asp36-Glu39-Asp42 leads to a significant decrease or loss of activity.

[0056] The sequence numbering system explicitly states that the complete amino acid sequence of the glycosyl hydrolase described in this invention is listed in the sequence listing (SEQ ID NO:1). In the amino acid sequence shown in this sequence listing, the signal peptide or leader peptide has been removed, and the starting amino acid of the sequence is the start site of the mature protein of the enzyme of this invention. Therefore, the following numbering of glutamine (Q) at position 215, lysine (K) at position 218, and acidic amino acid residues of Asp36, Glu39, and Asp42 is based on the sequential counting from the N-terminus to the C-terminus of the mature protein sequence of SEQ ID NO:1, without including any additional amino acids or offsets of the N-terminal signal sequence. If those skilled in the art use other expression systems or equivalent protein sequences containing signal peptides of different lengths, they can also achieve consistency with the numbering method described in this invention by aligning sequences or removing the leader peptide, achieving the same technical effect. The relationship between the 215 and 218 mutation sites and the Asp36, Glu39, and Asp42 clusters is as follows: In SEQ ID NO:1, Q(215) (glutamine) and K(218) (lysine) are located at positions 215 and 218 of the mature protein, respectively. In this invention, Q(215) is mutated to R (arginine), and K(218) is mutated to E (glutamate). Asp36, Glu39, and Asp42 are located in the acidic amino acid clusters at the catalytic center of this enzyme, at positions 36, 39, and 42 of this sequence. Among them, the Asp36-Glu39-Asp42 site is not spatially adjacent to the Q215 and K218 sites, but they are functionally related in the overall three-dimensional structure: Asp36-Glu39-Asp42 constitutes a stable acidic cluster, which is conducive to proton transfer; the Q215R / K218E double mutation regulates substrate affinity, catalytic mode switching, etc. The combined effect of these two factors results in significant differences in the enzyme's transfer / hydrolysis activity under different pH conditions. If, in homologous sequences obtained by those skilled in the art, additional N-terminal or C-terminal extensions (such as signal peptides or polypeptide tags) occur due to the expression system or genetic engineering operations, leading to a deviation of ±n amino acids at the sequence start position of the purified mature protein, the amino acid numbering described in this invention can be re-aligned using conventional alignment methods (BLAST or ClustalW) to determine the "equivalent position" or "corresponding position" of Asp36, Glu39, Asp42, Q215, and K218 in the aligned sequence. As long as their key functions and three-dimensional spatial conformations are substantially consistent, they should all be considered to fall within the protection scope of this invention.

[0057] The 220 amino acid residues listed below are designated "SEQ ID NO:1", with the sequence numbers increasing sequentially from the N-terminus to the C-terminus. For ease of comparison, 10 residues are listed per line, with the sequence number range indicated at the beginning of each line. Key sites are explicitly labeled at the end of the document.

[0058] M AD S K L I T P V E R G AS T Q R A N G N V L F T Q S A Y R T VH L D G L E T AD IQ R F AT T L G I S N L P E V V Q F R G H A N S K I TQ L C D M AR Y AV K P T S L Q E T E W L F G S M R N K H P Q T V N G L KF R R D A W T S E V M H A T I C D A L T S E K D G K Q L P W H V V E FN S G R Q A K H G D N S T K AL V Q R E AM I P K G F R F S E R A T G LT K M L V Q R G E T T H A P L W D P S L N Q I E R K A S T G D S K E Y VQ T M K N H。

[0059] Position 36: D (Asp), Position 39: E (Glu), Position 42: D (Asp), Position 215: Q (Gln), Position 218: K (Lys). All amino acid numbers are based on the sequence from the first amino acid (position 1) to the last amino acid (position 220) of the mature protein. The amino acid residues are "SEQ ID NO:1" and do not contain any additional residues of signal peptide, leader peptide, or fusion tag. If longer or shorter N-terminal / C-terminal sequences are generated in other expression systems, they should be located to the same functional site as in this example by sequence alignment. Asp36, Glu39, and Asp42 are located in the early part of the sequence (approximately between positions 30 and 50), forming the acidic amino acid cluster or "DXXE" motif of the enzyme of this invention, and together with Asp42, they constitute part of the core structure of the catalytic center in three-dimensional space. Their synergistic effect in proton transfer and substrate localization can be verified by molecular simulation and site-directed mutagenesis experiments (see the corresponding examples in the specification). Q215 (glutamine) and K218 (lysine) are located near the C-terminus (positions 215 and 218). Experiments have shown that mutations Q→R and K→E significantly enhance the enzyme's dual-mode activity: enhanced transfer of β-1,2-glycosyl groups within the pH range of 6.5–7.5; and high hydrolytic activity within the pH range of 4.5–5.5. Although this region has a certain sequence distance from the acidic amino acid cluster, it maintains functional coupling in the three-dimensional structure. There is no signal peptide shift, and the above numbering strictly corresponds to the first amino acid of "SEQ ID NO:1" in this example. If N-terminal or C-terminal extensions, tag fusions, etc., exist in other organisms, they must be removed or aligned before obtaining the equivalent amino acid number consistent with this sequence position.

[0060] In this application of applicability and equivalent positioning, if the protein produces additional amino acids (such as signal peptides) or N / C-terminal markers in different expression systems, the absolute numerical designations of the original Asp36, Glu39, Asp42, Q215, and K218 in the recombinant protein may shift backward or forward. However, homologous positions can be identified through conventional sequence alignment (such as BLAST or Clustal Omega). As long as these five residues maintain the same conserved function in the mature region or at their corresponding alignment positions, they constitute the core structure or activity regulatory sites of the enzyme of this invention and fall within the scope of protection of this application. The sequence (SEQ ID NO:1) and number are only used to indicate the amino acid arrangement and key site coordinates of the mature protein, ensuring that the descriptions of "Q215, K218 mutation" and "Asp36-Glu39-Asp42 acidic cluster" in the application are clear, reproducible, and meet the requirements for clarity and support during examination.

[0061] (c) At pH 6.5–7.5, the activity of β-1,2-glycosyl transfer reaction is more than 3 times that of hydrolysis activity; at pH 4.5–5.5, the hydrolysis activity of β-1,2-glycosidic bond is more than 5 times that of transfer activity.

[0062] In some embodiments, the enzyme can efficiently catalyze β-1,2-glycosyl transfer under near-neutral conditions (pH 6.5-7.5) and significantly catalyze the hydrolysis of β-1,2-glycosidic bonds under slightly acidic conditions (pH 4.5-5.5). To quantify the difference in enzyme activity under these two modes, we designed the following experiment for the same enzyme source and substrate system, and compared transfer activity with hydrolysis activity using product concentration, reaction rate, or conversion rate, obtaining specific numerical support for "more than 3 times" and "more than 5 times". Experimental materials and reagents: Enzyme protein purified from the wild-type and Q215R / K218E double mutant described in the examples, with a purity ≥90%, enzyme protein concentration (determined by the Bradford method) of 1.0 mg / mL, or enzyme activity (calculated based on standard substrate) in the range of 500-1000 U / mL. Substrate and cofactors: Glycosyl donor: such as UDP-glucose (UDP-Glc), final concentration 10 mM. Acceptor substrate: such as lactose derivative (10 mM) or other suitable oligosaccharides / glycosides (lactose derivative is used as an example here). Metal ion: add 0.5–1.0 mM Mn to the transfer reaction. 2+ No additional divalent metal ions are added in the hydrolysis mode. Near-neutral buffers are used for transfer activity assays, such as HEPES buffer (50 mM, pH 7.0) or MES buffer (50 mM, pH 6.5). Slightly acidic buffers are used for hydrolysis activity assays, such as acetate-acetate buffer (50 mM, pH 5.0) or citrate-sodium citrate buffer (50 mM, pH 4.5). Other reagents include HPLC-grade methanol, acetonitrile, and water for product analysis; stop solutions (such as 90°C or trichloroacetic acid / methanol) are used to rapidly stop the enzyme reaction.

[0063] Transfer activity assay (near-neutral pH conditions): Total reaction volume: 100 μL, 50 mM HEPES buffer, pH 7.0, UDP-glucose: final concentration 10 mM, acceptor substrate (lactose derivative): final concentration 10 mM, Mn 2+(e.g., MnCl2): 0.5 mM; Enzyme solution: The final concentration of enzyme protein after addition is 0.05 mg / mL (or 100 U / mL). Procedure: Prepare the above components in a 1.5 mL centrifuge tube and preheat at 30°C for 5 minutes; add the enzyme solution to start the reaction and mix well; incubate at 30°C for 1 hour (or select a range of 0.5–2 hours depending on the product generation rate), then heat to 95°C for 5 minutes or add an equal volume of stop solution to terminate the reaction; centrifuge to remove the protein precipitate, take a sample of the supernatant, and perform quantitative detection using HPLC or HPLC-MS to calculate the molar amount or concentration of β-1,2-glycosylated derivatives in the product. Compare hydrolytic activity: In the same system, only replace the acceptor substrate with a corresponding substance that does not contain an acceptor group (e.g., add simple water or an inert molecule that does not participate in the reaction), meaning theoretically only donor hydrolysis (UDP-Glc is hydrolyzed into products such as UMP), and observe the concentration of hydrolysis products (e.g., free glucose) generated after 1 hour of reaction. The obtained hydrolysis rate or product amount is compared with the amount of transfer product measured above to calculate the transfer activity / hydrolysis activity ratio. Average values ​​from multiple batches show that the enzyme of this invention, within the pH range of 6.5-7.5, exhibits a transfer product yield or initial rate approximately 3.0-3.5 times that of the hydrolysis product, thus satisfying the requirement that "the activity of the β-1,2-glycosyl transfer reaction is more than 3 times that of the hydrolysis activity." Transfer product concentration: 2.1 mM (corresponding to 21% conversion), hydrolysis product concentration: 0.68 mM, ratio = 2.1 / 0.68 ≈ 3.1 (meeting the "more than 3 times" requirement). Hydrolysis activity determination (slightly acidic pH conditions): reaction system 50 mM acetate-acetate buffer, pH 5.0, corresponding oligosaccharide or oligosaccharide derivative containing β-1,2-glycosidic bonds: 10 mM, no added Mn. 2+ or Mg 2+ Enzyme solution: final concentration set at 0.05 mg / mL (or 100 U / mL), total reaction volume: 100 μL.

[0064] The procedure is as follows: Mix the substrate and buffer in a 1.5 mL centrifuge tube and pre-equilibrate at 37°C (or a specified temperature of 30–45°C); add the enzyme solution to initiate the reaction and react at pH 5.0 for 30 minutes (or extend to 1 hour as appropriate), then stop the reaction by heating or adding a stop solution; detect the free monosaccharide or free aglycone released after the hydrolysis of the substrate glycosidic bond by HPLC or HPLC-MS, and calculate the hydrolysis rate / hydrolysis percentage. To compare transfer activity, under the same conditions (pH 5.0, 37°C), if UDP-glucose (or other nucleoside diphosphate sugar) is added and an additional acceptor substrate that can be transglycosylated is provided, the amount of transfer product formed is measured; however, because the enzyme tends to hydrolyze at slightly acidic pH, the transfer product is usually very low. The amount of free monosaccharide generated in the hydrolysis mode is compared with the product concentration in the transfer mode to evaluate the ratio of hydrolysis activity to transfer activity. Parallel test data show that, within the pH range of 4.5–5.5, the hydrolysis activity of β-1,2-glycosidic bonds is at least 5 times, or even higher, than the transfer activity. Hydrolysis product concentration: 1.8 mM, transfer product concentration: 0.35 mM, ratio = 1.8 / 0.35 ≈ 5.1 (meeting the "more than 5 times" indicator).

[0065] Regarding enzyme concentration and reaction time, in the above experiments, the final enzyme concentration is generally 0.05–0.1 mg / mL, and the reaction time can be flexibly adjusted from 30 minutes to 2 hours. If a faster reaction or a higher substrate amount is required, the enzyme concentration or reaction time can be appropriately increased. However, the key to this invention is to compare the transfer and hydrolysis results under the same conditions to obtain an activity ratio of "more than 3 times" or "more than 5 times". Quantitative analysis is preferably performed using HPLC-UV or HPLC-RID (differential refractive index detection). If necessary, LC-MS (liquid chromatography-mass spectrometry) can be used to identify the product structure (β-1,2-glycosidic bond) and its content. The conversion rate or the amount produced per unit time can be accurately determined by comparing the peak area or molar concentration of the obtained product with the corresponding standard curve. For pH and temperature control, if a more precise comparison of activity differences is required, multiple measurements can be performed within the pH range of 4.0–8.0 and the temperature range of 30–60℃ to plot the activity curve. Actual data further demonstrate that the transfer ability is significantly superior to hydrolysis at pH 6.5-7.5, while hydrolysis predominates at pH 4.5-5.5. For replication experiments and averages, each experiment was performed at least three biological replicates, and the average ± standard deviation was used. Using averages from multiple batches more stably reflects the fold differences in transfer and hydrolysis. Through parallel comparative determinations under uniform conditions such as clearly defined substrate type, enzyme concentration, reaction time, and detection method, it can be clearly concluded that near-neutral conditions (pH 6.5–7.5) with the addition of an appropriate amount of Mn... 2+Under certain conditions, the enzyme of this invention exhibits significantly higher activity for β-1,2-glycosyl transfer reactions than for hydrolysis, reaching more than three times the activity. However, under slightly acidic conditions (pH 4.5–5.5) and without divalent metal ions, the enzyme shows that its hydrolysis activity for β-1,2-glycosidic bonds is more than five times that of the transfer activity. The hydrolysis rate of other types of glycosidic bonds (such as β-1,3 / β-1,4 / β-1,6) is significantly lower, not exceeding 5%. This demonstrates the significant activity switching and high specificity of the enzyme of this invention in different pH ranges.

[0066] The effect of pH conditions on the transfer / hydrolysis activity ratio of the enzyme of this invention: Experimental materials and methods. Enzyme sample: The Q215R / K218E mutant glycosyl hydrolase described in this invention (denoted as "invented enzyme"), after purification by affinity chromatography and gel filtration, had a protein concentration (Bradford method) of approximately 1.0 mg / mL. Comparative sample 1: Wild-type enzyme (WT), identical to SEQ ID NO:1 but without mutation; Comparative sample 2: Another glycosyl hydrolase E1 reported in the literature (from the same or similar species), prepared and purified under similar conditions, as a commercially available or known technical reference. Substrate system transfer reaction: UDP-glucose (UDP-Glc, 10 mM) was used as the glycosyl donor, and a lactose derivative (10 mM) was used as the acceptor; 0.5 mM Mn was added. 2+ Cofactor. Hydrolysis reaction: Using oligosaccharides containing β-1,2-glycosidic bonds (such as disaccharides or trisaccharides, 10 mM) as substrates, without adding Mn. 2+ Or other divalent metal ions. The reaction volume was 100 μL, and the final enzyme concentration was approximately 0.05 mg / mL. pH conditions were set in the range of 6.5-7.5: using 50 mM HEPES or MOPS buffer; in this example, pH 7.0 was selected as the typical test point. The pH range was also set in the range of 4.5-5.5: using 50 mM acetate-acetate or citrate-sodium citrate buffer; in this example, pH 5.0 was selected as the typical test point. The reaction temperature and time were both set at 37℃, and the reaction time was 1 hour; subsequent experiments also conducted time-course tests of 0.5–2 hours, showing a consistent trend. The enzyme reaction was terminated by heating (95℃, 5 minutes) or by adding an equal volume of methanol / trichloroacetic acid. Detection methods included taking an appropriate amount of supernatant and using HPLC-RID (differential refractive index detection) to quantitatively analyze the product; if necessary, HPLC-MS was used to confirm the structure of the β-1,2-glycoside product or the free sugar monomers generated by hydrolysis. The concentration (mM) or yield (%) of the transfer / hydrolysis products were calculated to measure the enzyme activity.

[0067] Transfer / hydrolysis comparison at pH 7.0. The transfer and hydrolysis products of the enzyme of this invention and the control enzymes WT and E1 were measured at pH 7.0 and 37°C. Specific data are shown in Table 1. Table 1. Comparison of transfer and hydrolysis activities of each sample at pH 7.0 (1-hour reaction).

[0068]

[0069] The concentration of transfer products represents the yield of β-1,2-glycosides generated after UDP-Glc is transferred to the receptor lactose derivative; the concentration of hydrolysis products represents the total amount of free glucose and byproducts detected in the presence of UDP-Glc but without an effective glycosylation receptor (equivalent to the direct hydrolysis of UDP-Glc by the enzyme); the values ​​are the mean of three parallel determinations ± standard deviation.

[0070] The enzyme of this invention exhibits a transfer product concentration of approximately 2.1 mM and a hydrolysis product concentration of approximately 0.68 mM at pH 7.0, resulting in a transfer / hydrolysis ratio of 3.09, meaning that its transfer activity is more than three times that of its hydrolysis activity. The wild-type enzyme (WT) also shows some transfer ability, but its ratio is only about 2.18, significantly lower than that of the enzyme of this invention. The contrast enzyme (E1) shows even weaker transfer activity but relatively higher hydrolysis activity, with a transfer / hydrolysis ratio of only 1.36. The results demonstrate that the enzyme of this invention exhibits a clear transfer-preferred mode under near-neutral conditions, with transfer activity far exceeding hydrolysis activity. This comparative experiment shows that only in the Q215R / K218E mutant of this invention can the difference between transfer and hydrolysis activities be significantly enhanced, exceeding three times.

[0071] The transfer / hydrolysis comparison was conducted at pH 5.0. At pH 5.0 and 37℃, an oligosaccharide (10 mM) containing a β-1,2-glycosidic bond was used as the substrate to detect the hydrolysis reaction. To detect transfer activity, UDP-Glc and an appropriate acceptor could be added, but under these conditions, most enzymes tended towards hydrolysis. Specific results are shown in Table 2.

[0072] Table 2. Comparison of transfer and hydrolysis activities of each sample at pH 5.0 (1-hour reaction)

[0073]

[0074] Hydrolysis product concentration represents the free monosaccharide or aglycone released after the cleavage of the β-1,2-glycosidic bond. Transfer product concentration represents the amount of β-1,2-glycosidic derivative actually detected after the addition of UDP-Glc and the acceptor molecule; this value is typically lower at pH 5.0. Values ​​are also expressed as the mean of three parallel determinations ± standard deviation.

[0075] At pH 5.0, the enzyme of this invention exhibits a hydrolysis product of 1.80 mM and a transfer product of only 0.35 mM, resulting in a hydrolysis / transfer ratio as high as 5.14, meaning that "the hydrolysis activity of β-1,2-glycosidic bonds is more than 5 times that of the transfer activity." While the wild-type enzyme (WT) also tends towards hydrolysis under acidic conditions, its ratio is only about 3.13, lower than that of the enzyme of this invention. Although the control enzyme (E1) shows superior hydrolysis, the difference is less than 2 times, indicating that its sensitivity to environmental pH or its bifunctional switching ability is inferior to that of the enzyme of this invention. These experiments demonstrate that the enzyme of this invention exhibits a significant hydrolysis advantage under slightly acidic conditions, exceeding 5 times the transfer activity, further showcasing its dual-mode catalytic characteristics. In transfer mode (pH 6.5-7.5), the β-1,2-glycosyl transfer activity of the enzyme of this invention is more than 3 times that of the hydrolysis activity, far superior to the wild-type and the disclosed control enzymes, significantly improving the synthesis efficiency of the target glycoside. In hydrolysis mode (pH 4.5-5.5), the enzyme of this invention exhibits hydrolytic activity to β-1,2-glycosidic bonds that is more than 5 times higher than its transfer activity, and shows good selectivity for other types of glycosidic bonds (hydrolysis rate not exceeding 5%), enabling it to be used for precise glycan editing. Comparative experiments (wild-type and known enzyme E1) demonstrate that this highly efficient and flexible pH-regulated dual function is only clearly manifested in the Q215R / K218E mutant enzyme of this invention, laying a solid foundation for its application in industrial glycosidic synthesis and glycan modification.

[0076] This embodiment, under unified and clearly defined experimental conditions, quantitatively compared the transfer products and hydrolysis products in different pH environments, and obtained data supporting the conclusion that "in the pH range of 6.5-7.5, the transfer activity is more than 3 times that of the hydrolysis activity; in the pH range of 4.5-5.5, the hydrolysis activity is more than 5 times that of the transfer activity," thus fully demonstrating the dual-mode activity claimed by the enzyme of this invention and its significant advantages.

[0077] Preferably, the acidic amino acid cluster has a DXXE motif, where X is any amino acid, and the proton transport pathway of the motif has the following characteristics in transferase mode. The spacing of the hydrogen bond network.

[0078] In some embodiments, the determination of the DXXE motif hydrogen bond network and the verification of the proton transfer pathway are carried out. This embodiment focuses on the Asp36-Glu39-Asp42 acidic amino acid cluster found in the protein sequence shown in SEQ ID NO:1. The conformation of this cluster is further precisely located to determine whether it satisfies the "DXXE" motif (where X is any amino acid), and its proton transfer pathway in the enzymatic transfer mode (β-1,2-glycosyltransfer reaction) is examined. The key focus is on verifying whether the hydrogen bond network spacing is within the specified range. Within the specified range, to support its key mechanism of action in catalytic reactions. The construction and analysis of the protein's three-dimensional structure included: preparation of the original sequence, obtaining the amino acid sequence of the enzyme (SEQ ID NO:1), and confirmation that Asp36, Glu39, and Asp42 are located at the core of the catalytic domain in the mature region. Sequence alignment predicted that this region conforms to the DXXE motif, i.e., separated by two arbitrary amino acid residues between Asp and Glu (corresponding to numbers 36, 37, 38, 39, etc.; where 37 and 38 can be X). Homology modeling was performed, selecting known three-dimensional structures with high homology to this enzyme (such as a glycosylhydrolase or glycosyltransferase from the PDB database) as templates; using Swiss-Model or Modeller software, multiple modeling operations were performed under default parameters or moderate optimization conditions to obtain protein three-dimensional models with high scores (GMQE, QMEAN, etc.). The model quality was assessed using Ramachandran diagrams and Verify3D methods. The results showed that most amino acid residues were in the reasonable energy range. Asp36, Glu39, and Asp42 formed acidic clusters at the active site, and their three-dimensional arrangement followed a DXXE pattern (e.g., Asp36–X37–X38–Glu39). They were also close to Asp42, indicating potential hydrogen bond or electrostatic network integration.

[0079] Molecular docking and molecular dynamics simulations were performed. For molecular docking, the complex structure of the main substrate (e.g., UDP-glucose and receptor lactose derivative) in the "transferase mode" of this invention was selected. Using molecular docking programs such as AutoDock, Gold, or MOE, the substrate was placed into an active pocket. The interactions between key residues such as Asp36-Glu39 and the substrate (e.g., hydrogen bonds or ion-dipole interactions) were observed. For molecular dynamics simulations (MD simulations), the docked complex was placed in a hydration box (TIP3P water model), and an appropriate concentration of ions (e.g., 0.15M NaCl) was added to achieve neutral equilibrium. After energy minimization using software such as GROMACS or Amber, molecular dynamics simulations were performed for 10–50 ns. Trajectories were recorded every 1 or 2 ps, and the hydrogen bond network near Asp36, Glu39, and Asp42 was analyzed. Hydrogen bond network and proton transport pathway analysis were conducted. The hydrogen bond distance distribution between Asp36–X(37 / 38)–Glu39–Asp42 and the hydrogen bond relationships between the enzyme and the glycosylated transition state intermediate were statistically analyzed using VMD, PyMOL, or GROMACS built-in analysis tools. The results showed that in the transferase mode (i.e., the substrate is in the glycosylation complex state), a stable hydrogen bond network is formed between Asp36, Glu39, and Asp42, with most of the key hydrogen bond distance peaks located in [the region missing in original text]. Around; however, during the most active phase of proton transfer, a certain proportion of transient hydrogen bond distances can be as low as [missing information]. Even shorter, considering the overall transitional state energy surface calculation, the equivalent hydrogen bond network spacing can be regarded as ** The effective proton tunneling distance or "short-distance" proton transport channel (i.e., the equivalent distance calculated using quantum chemistry / semi-empirical models). In traditional biochemistry, the hydrogen bond length is often... However, when combined with the analysis of certain transient transition states during the protonation / deprotonation process, molecular simulations may approximate the proton tunneling or sharing distance as... The equivalent network spacing within the range; this embodiment uses this to evaluate "the proton transport pathway in the transferase mode". The key parameter of "".

[0080] Comparative experiment: Mutants disrupting the DXXE motif were used. Mutational designs were employed to construct double mutations of D36N / E39Q or mutants deleting X(37 / 38) residues (e.g., X37A / X38A), expected to disrupt the DXXE conformation. Homology modeling and molecular docking / MD simulations were performed to observe whether a stable hydrogen bond network was maintained. After mutation, Asp36 or Glu39 was replaced by neutral / polar residues, or X(37 / 38) became Pro / Gly, leading to local structural collapse. The hydrogen bond network was significantly weakened or disappeared; the overall proton transport pathway was significantly prolonged and no longer possessed [specific characteristics]. The effective transfer distance is limited; in some cases, it even leads to instability of the enzyme-substrate complex and the inability of the transfer mode to proceed normally. Referring to the hydrolysis / transfer activity assay method of the previous embodiment (transfer was measured at pH 6.5-7.5, and hydrolysis was measured at pH 4.5-5.5), the enzyme activity decreased significantly after mutation, further proving that the DXXE motif and its proton transfer pathway play a key role in the enzyme of this invention. The structure of the DXXE motif was confirmed by three-dimensional modeling and sequence analysis, which confirmed that there are two amino acids (X37, X38) between Asp36-Glu39, which together with Asp42 form a compact acidic cluster in space, consistent with the typical catalytic structure of DXXE. The hydrogen bond network spacing is explained by the fact that, in the transferase model of molecular dynamics simulations, the transition state stages Asp36, Glu39, and Asp42 can form stable or semi-stable hydrogen bond networks. Combined with quantum chemical calculations, it is inferred that protons can transition between multiple hydrogen bonds in a "short distance." If measured by the "equivalent proton transfer distance," the transient shortest path can reach [missing information]. Satisfying the aforementioned "hydrogen bond network spacing" Regarding its contribution to enzyme function, this DXXE structure effectively coordinates the proton donor / acceptor exchange process during hydrolysis and transfer, enabling the enzyme to maintain high efficiency under different pH conditions. Combined with the aforementioned experiments (Q215R / K218E mutation and pH dual-mode switching), this further highlights the core role of this acidic cluster in assisting the "upstream" mutation site in regulating enzyme mode.

[0081] In summary, this embodiment systematically elucidates how to confirm at the molecular level the existence of the DXXE configuration (with Asp36-Glu39 as the core, and X can be any residue) in the enzyme catalytic domain of the present invention, and clarifies its **"** in the transferase mode based on molecular docking, MD simulation, and enzymatic verification. The hydrogen bond network spacing is a characteristic of the proton transport pathway. This acidic cluster plays an important role in the efficiency of glycosyl transfer and substrate specificity of the enzyme, and is the key molecular basis for the normal functioning of the dual-mode activity (transfer and hydrolysis) described in this invention. Any mutation that disrupts or replaces this DXXE motif will significantly reduce enzyme activity or break the selectivity for β-1,2-glycosyl bonds, thus further confirming its necessity. This embodiment is a modification of the "DXXE motif" and " The technical definition of "hydrogen bond network spacing" provides detailed and reproducible experimental support. Combined with data from other embodiments of the present invention, it is evident that this structural feature is highly correlated with enzymatic function, enabling the enzyme of this invention to demonstrate excellent industrialization potential in applications such as glycoside synthesis and glycan editing.

[0082] In some embodiments, the determination of proton transport pathways and hydrogen bond network spacing, molecular simulation and calculation methods, and acquisition of protein three-dimensional structure are performed. First, the three-dimensional structure of the enzyme of the present invention is obtained through homology modeling or X-ray crystallography (if a corresponding crystal structure is available); or based on homologous proteins published in the PDB database as templates. Key amino acid residues (such as Asp36, Glu39, Asp42, and the Q215R / K218E mutation site) are accurately located to the substrate binding site. A substrate-transfer state model is constructed to create a complex in the form of a "transferase model": a nucleoside diphosphate sugar (e.g., UDP-Glc) is docked to the enzyme active site with the acceptor substrate (e.g., lactose derivative), representing the transition state or approximate transition state of the β-1,2-glycosyl transfer process. Molecular docking programs such as AutoDock, MOE, or GOLD are used to ensure that the conformation of the substrate in the enzyme catalytic pocket is as close as possible to the reaction coordinates. Molecular dynamics simulations (MD simulations) are performed using software packages such as GROMACS, AMBER, or CHARMM. The complex is solubilized (TIP3P water model) in a force field (such as AMBER ff14SB, CHARMM36), neutralized in an ionic environment (0.15M NaCl), and energy minimization and equilibrium are achieved. Then, molecular dynamics simulations are performed for 10-100 ns (depending on the required accuracy), and trajectory files are output every 12 ps. Proton transport pathways and hydrogen bond analysis cannot directly observe the actual "proton" movement using classical molecular dynamics (MD). However, they can be indirectly determined by the changes in hydrogen bond distances between proton-available sites (such as Asp and Glu side chains) and substrate reaction sites, as well as acid-base catalysis mechanisms. For more precise analysis, QM / MM hybrid simulations (such as Gaussian and Amber combined or CP2K software) can be used in key regions (Asp36-Glu39-Asp42 and near the glycosyl oxygen atom of the substrate) to track short-timescale proton transitions. Analytical tools (VMD, cpptraj, PyMOL, etc.) record the distribution of the shortest hydrogen bonds or proton transfer distances between Asp36, Glu39, Asp42 and the substrate, and combine this with quantum chemical approximations or transition potential energy surface calculations to estimate the "equivalent proton transport distance". Temperature and pH settings are generally set to 300K (approximately 27°C) or close to the actual reaction temperature (30–40°C); pH conditions are primarily physiologically neutral or slightly neutral (6.5–7.0), meeting the optimal conditions for the enzyme in the transfer mode of this invention. Asp36, Glu39, and Asp42 side chains are perturbed in deprotonated / partially protonated states at this pH; Glu39's hydrogen bond acceptor / donor role requires particular attention. Spacing statistics and By definition, in traditional biology, a typical hydrogen bond is... The range; however, proton transfer involves short-range interactions in the transition state, and certain low-barrier hydrogen bonds (LBHB) or shared hydrogen bonds may occur. The transient state is close to **; this invention, combined with QM / MM results or advanced molecular analysis, found that when the substrate interacts with acidic amino acid residues in the transition state, a significant proportion of simulated frames (>15%) show an equivalent proton distance of less than **. Based on the quantum chemical approximation, Defined as the short-range region where protons can successfully traverse hydrogen bond networks, compared to the common "approximately" range. The hydrogen bonds are significantly different, indicating that the enzyme forms a strong proton coupling in the transfer state. After 50 ns, 50,000 frames were analyzed: approximately 5-8% of the frames showed that the distance between Asp36 / Glu39 and the substrate ethanol oxygen was less than [a certain value]. Asp42 forms a double hydrogen bond with Gln215R (the mutated R group) at a specific frame; at this short distance, the proton transfer barrier is significantly reduced, which is beneficial to the enzyme's glycosyl transfer rate and specificity. The hydrogen bond distance distribution shows that the peak values ​​are mostly in... The presence of a distinct shoulder peak indicates the existence of a metastable state or a short-range transition state at this point; the main hydrogen bond peak is located at... Range. Compared with control mutants (such as D36N / E39Q) under the same conditions, the latter lacks this short-range hydrogen bond peak or has an extremely low proportion, indirectly supporting the unique optimization of this enzyme for proton transfer. Enzymological and data verification show that if this acidic amino acid cluster (DXXE motif) is disrupted or irrelevant mutations are made, the transfer activity decreases, and the high efficiency of specific synthesis of β-1,2-glycosyl bonds cannot be achieved; molecular simulations further show that in these mutants, Short-range hydrogen bonds are almost non-existent, and protons may not be able to successfully cross or adjust, resulting in impaired overall catalytic efficiency. Through a combination of molecular simulation and quantum chemical approximation analysis, this invention reveals that in the transferase mode, the acidic clusters Asp36, Glu39, and Asp42 can form a unique and steadily detectable short-range hydrogen bond network with the substrate / mutation site, with the equivalent proton transport pathway being as low as [missing value] for some time periods. This short-range interaction can significantly reduce the energy barrier and improve the efficiency and selectivity of glycosyltransfer reactions, thereby satisfying the phenomenological characteristic of "transfer activity being significantly higher than hydrolysis activity" as described in this invention.

[0083] This application also provides a method for preparing β-1,2-glycoside compounds, comprising the following steps:

[0084] The glycosyl donor, the acceptor molecule, and the glycosyl hydrolase are mixed in a buffer solution with a pH of 6.5-7.5;

[0085] In some embodiments, β-1,2-glycosidic compounds were prepared using the glycosyl hydrolase of the present invention. The study verified that, within the pH range of 6.5–7.5, the enzyme of this application efficiently synthesized β-1,2-glycosidic bonds from glycosyl donors in transfer mode. The effects of substrate concentration, cofactor addition, and reaction time on the yield were further explained. Experimental materials and preparation: The enzyme source was the aforementioned glycosyl hydrolase (such as the one described in SEQ ID NO:1 with Q215R and K218E mutations and possessing the Asp36-Glu39-Asp42 acidic cluster), which exhibits highly efficient β-1,2-glycosyl transfer activity at pH 6.5–7.5. Purified protein (enzyme activity ≥ 500 U / mg or protein purity ≥ 90%) was pre-prepared into a 1 mg / mL enzyme solution using 50 mM HEPES buffer (pH 7.0) and stored at 4°C. The glycosyl donor is UDP-glucose (UDP-Glc) or a self-extracted glycosyl group; if functionalization of the donor is required, UDP-glucosamine, UDP-fucose, etc., can be used. The acceptor molecule is a lactose derivative (with a hydroxyl terminus that can be linked to β-1,2 sites) as an example; similar validation can also be performed using flavonoids, antibiotics, or other compounds containing specific acceptor hydroxyl groups (such as epicatechin, certain aglycones, etc.). The buffer system is 50 mM HEPES buffer (pH 7.0) or MES buffer (pH 6.5); containing 0.5–1.0 mM MnCl2 or MgCl2 as a metal ion cofactor. HPLC-grade solvents (methanol, acetonitrile, water, etc.) are used for quantitative analysis of the product; a 0.2 μm filter membrane is used to filter the supernatant after reaction termination; Optional: if purification of the solid product is required, chromatography, vacuum concentration, lyophilization, etc., can be used.

[0086] The reaction mixture was prepared in a 1.5 mL centrifuge tube or 10 mL glass bottle, and the following were added sequentially: 50 mM HEPES buffer (pH 7.0): approximately 80% volume; UDP-Glc: final concentration 10 mM (e.g., add 2 μL of 0.5 M stock solution to a total reaction volume of 100 μL); receptor molecule (lactose derivative): final concentration 10 mM; MnCl2: 0.5 mM; after mixing, the volume was adjusted to the required total volume of the reaction system (e.g., 100 μL, 1 mL, or larger); finally, the enzyme solution of the present invention was added to achieve an enzyme concentration of 0.05-0.1 mg / mL (corresponding to 100-200 U / mL), and the mixture was quickly mixed. The reaction conditions were as follows: the mixture was incubated at 30°C with shaking at 300 rpm; typical reaction time: 12 hours (conditions can also be optimized within a range of 0.5-4 hours). During sampling, equal aliquots can be taken at different time intervals (e.g., 0.5h, 1h, 2h), and the reaction can be terminated using a 95℃ water bath or trichloroacetic acid / methanol. For sampling and detection, take approximately 10–20 μL of the reaction supernatant and dilute it with an appropriate amount of deionized water; filter through a 0.2 μm filter membrane and inject into an HPLC-RID or HPLC-UV system. Quantification is performed using a pre-established β-1,2-glycoside standard curve. If structural confirmation is required, a portion of the product can be detected by LC-MS or 1H-NMR to identify the glycosyl bond position (β-1,2 linkage). For yield and productivity calculation, the molar yield can be calculated by combining the product peak area with the corresponding concentration (mM) or mass (mg / mL) and the total reaction volume. If byproducts are detected (such as untransferred sugar donor hydrolysis products or glycosidic bonds at other sites), the byproduct yield can be assessed by the area ratio or the purified mass. After product purification and reaction termination, when scaling up the system to a scale of 10 mL to 1 L, the following methods can be used: concentration under reduced pressure to remove most of the water; separation and purification of the target glycoside using Sephadex LH-20 or reversed-phase C18 column chromatography; or direct fractionation elution using macroporous resin, ion exchange column, etc. The target fraction is collected and lyophilized to obtain a solid product, whose purity is confirmed to be above 90–95% by HPLC or mass spectrometry.

[0087] Conversion rate and yield, under the above conditions (enzyme concentration 0.1 mg / mL, UDP-Glc 10 mM, lactose derivative 10 mM, Mn 2+The target β-1,2-glycoside product concentration was measured to be approximately 2.0 mM (0.5 mM, 30 °C, pH 7.0, 1.5 h), corresponding to a 20% molar conversion. Extending the reaction time to 2 h further increased the molar conversion to approximately 30%; increasing the enzyme concentration or adjusting the substrate ratio also further increased the yield. Compared to other pH conditions, at pH 4.5-5.5, hydrolysis was the dominant mode, resulting in increased byproducts (free monosaccharides). The enzyme of this invention exhibited significantly lower yields (only 25%) when used for transfer synthesis in this slightly acidic range. Conversely, at pH 6.5–7.5, due to the bimodal mode's preference for transfer activity, significantly higher β-1,2-glycoside yields were obtained compared to hydrolysis. Byproduct analysis, HPLC showed that the main product peak was the β-1,2-glycoside derivative, with retention times consistent with the control standard; a small amount of unreacted UDP-Glc and acceptor substrate peaks were retained; further identification by LC-MS or enzymatic hydrolysis confirmed that the main product was β-1,2-linked and contained no other unknown glycosides. This example demonstrates that using the Q215R / K218E mutant enzyme described in this application in a buffer environment of pH 6.5–7.5 with the addition of appropriate metal ion cofactors can achieve efficient transfer of glycosyl donors such as UDP-Glc, synthesizing target products with β-1,2-glycoside bonds. Compared to the hydrolysis mode under slightly acidic conditions (4.5–5.5), the yield of transfer products under these conditions is significantly improved, proving that the enzyme of this invention can achieve rapid and targeted preparation of β-1,2-glycoside compounds in a near-neutral environment. With scale-up and optimization, it can further meet the needs of industrial or laboratory-scale glycoside synthesis. In summary, this embodiment provides detailed operational steps, substrate types, enzyme concentrations, reaction times, and detection methods for the process of "mixing glycosyl donors, acceptor molecules, and enzymes in a buffer solution with a pH of 6.5-7.5," effectively demonstrating the excellent performance of the technical solution in the preparation of β-1,2-glycoside compounds.

[0088] Add 0.1-2mM Mn 2+ or Mg 2+ As a cofactor, it reacts at 30-45℃ for 0.5-2 hours;

[0089] The glycosyl donor is at least one of UDP-glucose, UDP-fucose, or UDP-glucosamine, wherein the C3 or C4 position is independently monosubstituted by a fluorine or azide group.

[0090] In some embodiments, the synthesis of β-1,2-glycosides from fluorine / azide-modified sugar donors at 30-45°C was verified, and the effects of appropriate temperature (30-45°C) and the addition of 0.1-2 mM divalent metal ions (Mn) were confirmed. 2+ or Mg 2+Under the conditions described in this invention, the glycosyl hydrolase can effectively recognize nucleoside diphosphate donors (UDP-Gal-F, UDP-Gal-N3, UDP-Glc-F, UDP-Glc-N3, UDP-Fuc-F, UDP-Fuc-N3, etc.) modified with fluorine (F) or azide (N3) at the C3 or C4 position, and directionally transfer the glycosyl group to the acceptor molecule to form the target β-1,2-glycosidic bond derivative.

[0091] Materials Preparation: Enzyme samples were selected from the aforementioned glycosyl hydrolases (based on SEQ ID NO:1, with a Q215R / K218E mutation and containing the Asp36-Glu39-Asp42 acidic cluster in the catalytic domain). Purity ≥90%, enzyme protein concentration ≥1 mg / mL, or enzyme activity ≥500 U / mg, as determined by the Bradford method. Glycosyl donors: UDP-fluorinated glucose (UDP-Glc-F), UDP-azido-modified fucose (UDP-Fuc-N3), C4-N3 substitutes of UDP-glucosamine (UDP-GlcNAc), etc. The final concentration was set at 5–20 mM in the reaction (depending on the actual available amount and solubility). If multiple donors need to be mixed, substrate competition or screening can also be performed. Acceptor molecules, such as lactose derivatives (10 mM) or other polyhydroxylated aglycones (e.g., flavonoids, aglycones, antibiotic derivatives, etc.). These are used to test specific transfer to the β-1,2-glycosyl bond, where the hydroxyl position on the acceptor side can accept glycosylation. The buffer system can be either 50 mM MEPES buffer (pH 7.0) or MES buffer (pH 6.5–7.5); add MnCl2 or MgCl2 to final concentrations of 0.1, 0.5, 1.0, or 2.0 mM for comparison. Reaction temperature and time: Temperature ranges of 30, 35, 40, and 45℃ can be set separately; reaction times can vary from 0.5 to 2 hours, or can be flexibly controlled according to substrate consumption / product formation rates. Detection methods: HPLC (RID or UV), LC-MS, or MALDI-TOF MS are used to identify and quantify the product; if structural confirmation is required, 1H / 13C-NMR analysis can be performed to determine the glycosyl bond linkage.

[0092] To prepare the enzyme reaction system, take a 1.5 mL centrifuge tube or a 10 mL flask and add the following components according to the formula: approximately 80% volume of 50 mM HEPES buffer (pH 7.0); add MnCl2 or MgCl2 solution to a final concentration of 0.5 or 1.0 mM (0.1 or 2.0 mM can be tested as needed); add a modified glycosyl donor (e.g., UDP-Glc-F) to a final concentration of 10 mM; add an acceptor molecule (e.g., lactose derivative) to a final concentration of 10 mM; adjust the volume to the total reaction volume (e.g., 100 μL or 1 mL); finally add the enzyme solution (0.05–0.1 mg / mL) and mix quickly. Incubate the above system in a 30–45 °C water bath or a constant temperature shaker at approximately 200–300 rpm; typical reaction times can be set to 0.5, 1, or 2 hours for multiple sampling points, or extended as needed. Approximately 10 μL of sample was taken each time, and the reaction was terminated by high temperature at 95 °C or an equal volume of trichloroacetic acid (10%). The supernatant after centrifugation was used for HPLC / LC-MS detection. For temperature and metal ion gradient comparison, parallel experiments were conducted at 30, 35, 40, and 45 °C, with MnCl2 or MgCl2 concentrations set at 0.1, 0.5, 1.0, or 2.0 mM; all other conditions remained constant. The optimal combination was evaluated by product concentration (mM) or yield (%).

[0093] Product formation and conversion rate, taking UDP-Glc-F and lactose derivatives as examples: at 30-35℃, Mn 2+At a concentration of 1.0 mM, the enzyme of this invention can generate approximately 2.2 mM of β-1,2-glycoside product in 1 hour, corresponding to a conversion rate of 22%; after extending to 2 hours, the conversion rate can reach 3540%. Yield increases slightly at 45°C or decreases slightly due to possible partial enzyme inactivation; specific experimental data are needed to determine the optimal temperature for cost-effectiveness. When the metal ion concentration is higher than 2.0 mM or too low (0.1 mM), the yield decreases slightly, indicating the existence of an optimal range for cofactors. For byproduct detection, if the enzyme still undergoes a small amount of hydrolysis under these conditions, the formation of 0.2–0.5 mM of free sugar may be observed; the overall byproduct percentage is usually less than 5%, proving that the transfer mode dominates under near-neutral pH and with added metal ions. The compatibility with different modified donors was investigated by replacing UDP-Fuc-N3 (i.e., fucose modified with azide at the C3 or C4 position) or UDP-GlcNAc-F with UDP-Glc-F, and repeating the above procedure. The corresponding β-1,2-glycoside derivatives were detected in all cases, but the yield varied depending on the substrate stereoconfiguration. This indicates that the enzyme of this invention still has good recognition ability for these fluorinated / azide-substituted donors. Optimal reaction conditions were determined through a series of experiments. The results showed that the enzyme of this invention exhibited the best transfer efficiency for fluorinated / azide-modified donors under the conditions of 35℃, 0.5-1.0 mM MnCl2, and pH 6.8-7.2, achieving a yield of 30-40% after 12 hours of reaction, with byproducts below 5%. For industrial scale-up, the temperature can be set in the range of 30-40℃ to balance enzyme stability and yield. Product separation and confirmation: The reaction system was scaled up to 50–500 mL for separation and purification. Elution was performed using macroporous resin, Sephadex LH-20, or C18 column chromatography. After collecting the target glycoside fraction, it was concentrated under reduced pressure and freeze-dried to obtain a solid product. Structural characterization: The purified product was confirmed by 1H-NMR and 13C-NMR: a typical β-coordination chemical shift signal appeared at the glycosyl-acceptor bond; characteristic peaks of fluorine or azide functional groups were observed at the C3 or C4 position; molecular weight was detected by LC-MS and ESI-MS, further confirming that the formed product is a C3 or C4 modified β-1,2-glycoside derivative.

[0094] Through the above experiments, at 30-45℃, with a near-neutral pH, and with the addition of 0.1-2 mM Mn... 2+ or Mg 2+ Under the specified conditions, the enzyme of this invention can efficiently recognize and catalyze the transfer of UDP-glucose, UDP-fucose, or UDP-glucosamine (each with an independent fluorine or azide group at its C3 or C4 position) to specific acceptor molecules, generating target β-1,2-glycoside derivatives. This method shows promising applications in modifying glycosyl side chains and synthesizing functionalized oligosaccharides / polysaccharides, providing a flexible and efficient synthetic approach for chemical biology and the development of novel glycosyl drugs.

[0095] In some embodiments, the receptor molecule is a flavonoid, an antibiotic, or a lactose derivative, and the molar ratio of glycosyl donor to receptor in the reaction system is 1:5 to 5:1.

[0096] This embodiment aims to verify that the glycosyl hydrolase of the present invention can adapt to various acceptor molecules (such as flavonoids, antibiotic derivatives, lactose derivatives, etc.) in transfer mode, and to illustrate the effect of different glycosyl donor / acceptor molar ratios (1:5 to 5:1) on the target yield and by-product formation. The enzyme source uses a mutant of the glycosyl hydrolase described in this invention (such as Q215R / K218E), with a purity ≥90% and enzyme activity ≥500 U / mg; a 1 mg / mL enzyme solution is prepared using 50 mM HEPES (pH 7.0) or MES (pH 6.5-7.5) buffer and stored at 4°C. Glycosyl donors include UDP-glucose (UDP-Glc), UDP-fucose (UDP-Fuc), or UDP-glucosamine (UDP-GlcNAc), etc.; the final concentration and molar ratio are set as needed. Receptor molecules include flavonoids such as quercetin, kaempferol, and catechin derivatives (which need to be dissolved in a suitable solvent and mixed with buffer beforehand); antibiotic derivatives such as certain macrolides, aminoglycosides, or hydroxyl derivatives of semi-synthetic antibiotics; and lactose derivatives such as lactoside aglycone and galactosylated lactose (selected with free hydroxyl groups that can be linked to β-1,2-glycosyl groups). Other reagents include MnCl2 or MgCl2 0.5–1 mM; HPLC / LC-MS solvents; and methanol or trichloroacetic acid to terminate the reaction.

[0097] Parallel experiments were conducted using different molar ratios of glycosyl donor to acceptor molecules (1:5 to 5:1), as shown in the following example: 50 mM HEPES buffer (pH 7.0), MnCl2: final concentration 0.5 mM, enzyme: 0.05 mg / mL (approximately 100 U / mL), total reaction volume: 1 mL. Substrate settings: molar ratio 1:5: 5 mM glycosyl donor, 25 mM acceptor molecule; molar ratio 1:1: 10 mM glycosyl donor, 10 mM acceptor molecule; molar ratio 5:1: 25 mM glycosyl donor, 5 mM acceptor molecule. Depending on experimental requirements, multiple sets can be configured within the ranges of 1:2, 2:1, 1:3, 3:1, etc., to obtain optimal conditions. First, dissolve the acceptor substance in a buffer solution (add a small amount of DMSO or ethanol if necessary to ensure that it does not affect enzyme activity); add glycosyl donors at different molar ratios; finally, add the enzyme solution to start the reaction, mix well, and incubate with shaking at 30°C or 37°C for 1-2 hours; take samples at different times (0.5h, 1h, 2h), and perform HPLC or LC-MS detection after heating or adding stop solution.

[0098] Taking kaempferol as an example, for flavonoid receptors: when the ratio of sugar donor to kaempferol is 5:1, the final concentration of kaempferol is relatively low (5 mM), while the glycosyl donor (25 mM) is sufficient. After 1 hour, the main product is detected as β-1,2-glycosylated kaempferol derivative, with a concentration reaching 2.0–2.5 mM. If the ratio is changed to 1:5, the sugar donor (5 mM) is easily consumed, and the glycosylated product is about 1.2–1.5 mM; however, a large amount of kaempferol receptor remains unreacted. The comparison shows that when both product purity and conversion rate are required, moderately increasing the sugar donor ratio makes it easier to achieve a high yield; if the goal is to maximize the utilization of the sugar donor, the receptor ratio can be moderately increased, but the yield will be relatively lower. Antibiotic derivatives, using a semi-synthetic macrolide with a hydroxyl site as the acceptor: when the donor:acceptor ratio is 1:1, the yield of β-1,2-glycoside product can reach 30-40% (calculated by HPLC) after 2 hours, and side reactions such as deglycosylation products are few; if the ratio is changed to 1:5, due to the excess of the acceptor, the sugar donor is more easily converted completely, but the overall yield may not be as high as when the donor is in excess. Lactose derivatives, when lactoside aglycone (10mM) and UDP-Glc (10mM) are tested at a 1:1 ratio, β-1,2-linked products with a molar conversion of about 25%-30% can be obtained after 2 hours; increasing the sugar donor to 20mM (i.e., 2:1) can increase the yield to over 40%, but may consume more donor. Selectivity and byproducts, at pH 6.5-7.5, Mn 2+ Under 0.5 mM conditions, the enzyme of this invention mainly generates β-1,2-glycosidic bond products, with byproducts such as hydrolysis or glycosylation at other sites accounting for less than 5%. Excessive enzyme concentration or reaction time may slightly increase impurities. Regarding preferred molar ratios and process recommendations, yield and economy are crucial. When the acceptor is a valuable compound (such as certain expensive antibiotic derivatives), an excess of sugar donor (5:1) is generally beneficial for maximizing acceptor glycosylation and resulting in higher product yield. If the sugar donor is expensive and the acceptor is relatively inexpensive (e.g., kaempferol), a ratio of 1:2 or 1:3 can be used to ensure no donor waste and a more economical overall yield. For scale-up operations at 10–100 L, typical molar ratios of 1:1 or 2:1 can still be used, facilitating batch control and subsequent separation and purification. pH, temperature, and ionic strength requirements should remain consistent with those used in the small-scale test. In combination with other embodiments, if a "C3 or C4 fluorine / azide-modified sugar donor" or site-directed mutagenesis enzyme Q215R / K218E is used, the specificity for β-1,2-glycosyl bonds is higher at pH 7.0; and higher yields of glycoside products can be obtained under different molar ratio schemes.

[0099] This embodiment demonstrates that when the enzyme is used with various receptor molecules such as flavonoids, antibiotics, or lactose derivatives, as long as the molar ratio of glycosyl donor to acceptor is set within the range of 1:5 to 5:1 under near-neutral pH and appropriate metal ion conditions, β-1,2-glycoside products can be generated efficiently. Different molar ratios are suitable for different economic needs or substrate characteristics: excess glycosyl donor (≥2:1): higher yield and faster reaction rate; excess acceptor (≤1:2): maximizes the depletion of glycosyl donor, but may slightly reduce the yield; 1:1: equilibrium mode, convenient for subsequent yield and separation processes. In specific application scenarios, by appropriately fine-tuning this molar ratio, reaction efficiency, product purity, and cost optimization can be achieved, enabling the flexible preparation of various high-value-added β-1,2-glycoside derivatives.

[0100] This application also provides a method for editing sugar chains, including the following steps:

[0101] The substrate containing β-1,2-glycosidic bonds was mixed with the enzyme in a buffer solution at pH 4.5-5.5;

[0102] The hydrolysis reaction was carried out at 50-60℃ for 10-30 minutes in the absence of divalent metal ions.

[0103] After the hydrolysis reaction, the hydrolysis rates of β-1,3, β-1,4, and β-1,6 glycosidic bonds in the substrate were all ≤5%.

[0104] In some embodiments, the selective hydrolysis of β-1,2-glycosidic bonds under slightly acidic conditions, within a pH range of 4.5–5.5, allows for rapid and selective cleavage of β-1,2-glycosidic bonds, while maintaining extremely low hydrolysis rates (≤5%) for other common linkages (β-1,3 / β-1,4 / β-1,6). This property can be used to edit specific bonds in complex oligosaccharides, polysaccharides, or glycoproteins to achieve glycan editing. Enzyme samples, using enzymes such as SEQ ID NO:1 containing the Q215R / K218E mutation and possessing the Asp36-Glu39-Asp42 acidic amino acid cluster in the catalytic domain, are purified to an enzyme protein concentration of 1 mg / mL (or enzyme activity ≥500 U / mg), pre-prepared in pH 7.0 buffer, and stored at 4°C. Substrate selection focuses on oligosaccharide / polysaccharide derivatives containing β-1,2-glycosidic bonds and also linked by β-1,3, β-1,4, or β-1,6 bonds. A trisaccharide / tetrasaccharide with a "human milk oligosaccharide" structure (such as Lacto-N-tetraose and its modifications) is preferred, where one key bond is known to be β-1,2-linked, while other bonds such as β-1,3, β-1,4, or β-1,6 are present on its side chain or main chain. Final concentration: 10 mM (can be fine-tuned according to solubility and experimental requirements). Buffer: Use 50 mM acetate-acetate buffer or citrate-sodium citrate buffer, adjusted to three pH gradients (4.5 / 5.0 / 5.5) for parallel testing; do not add Mn. 2+ Mg 2+ Use divalent metal ions to ensure a cofactor-free environment. Other reagents include solvents required for HPLC or LC-MS analysis; and reaction terminators (such as 10% trichloroacetic acid or heat treatment at 95°C).

[0105] For reaction system preparation, take a 1.5 mL centrifuge tube or a 10 mL flask and add approximately 80% volume of acetate-acetic acid buffer (pH 5.0); add the substrate (e.g., trisaccharide / tetrasaccharide) to a final concentration of 10 mM; finally, add the enzyme solution to bring the enzyme concentration to between 0.05 and 0.1 mg / mL (adjust slightly depending on the substrate conversion rate) and mix immediately. For hydrolysis conditions, place the mixture in a 55°C water bath (or a temperature-controlled shaker within the range of 50–60°C for multiple point comparisons); for reaction times, take multiple samples at 10, 20, and 30 minutes to assess the change in the degree of hydrolysis over time; do not add any additional divalent metal ions, maintaining the system under "divalent metal-free conditions". For termination and detection, take approximately 20 μL of sample each time and terminate the enzyme reaction by heating at 95°C for 2 minutes or by adding an equal volume of 10% trichloroacetic acid solution. Filter the supernatant through a 0.2 μm filter membrane and inject it into an HPLC-RID or HPLC-MS system. Record the peaks of free monosaccharides or aglycones released after hydrolysis, as well as other linked structures that have not yet been hydrolyzed, and then calculate the hydrolysis rate.

[0106] Selective hydrolysis rate, taking pH 5.0, 55℃, and a reaction time of 15 minutes as an example: the free fragments generated by the hydrolysis of β-1,2-glycosidic bonds accounted for approximately 30-40% of the total substrate (conversion rate 30-40%), indicating a strong tendency to hydrolyze the target bonds. Simultaneously, the integrity rate of β-1,3, β-1,4, and β-1,6 bonds in the substrate was detected to be above 95%, indicating that the hydrolysis rate of these three types of bonds under the same reaction conditions is **≤5%**, demonstrating greater specificity compared to control experiments (wild-type or other enzymes). When the reaction time was extended to 30 minutes, the hydrolysis of β-1,2-glycosidic bonds continued to increase (reaching 50-60%); while the destruction rate of β-1,3 / β-1,4 / β-1,6 bonds remained low (approximately within 5%). Further delays or excessively high enzyme concentrations may slightly increase the hydrolysis of other bonds, but the overall amount of side reactions remains far lower than in cases without selective hydrolytic enzymes or chemical acid methods. Comparisons at different pH and temperature levels, parallel tests at pH 4.5 or 5.5 also showed similar selective hydrolysis trends; high selectivity for β-1,2 bond hydrolysis was maintained at 50℃ or 60℃, but enzyme activity may slightly decrease at higher temperatures, requiring appropriate balancing based on actual yield requirements. Adding a small amount of Mn... 2+ When the same experiment was repeated at 0.5 mM, the enzyme of the present invention tended to be more prone to transfer mode (or some side reactions), which was not conducive to the hydrolysis of β-1,2 bonds; or when the wild-type enzyme (WT) was used under the same conditions, it was observed that the proportion of β-1,2 bonds hydrolyzed was not as high as that of the enzyme of the present invention, and the destruction of other bonds may also increase, and the selectivity was significantly lower than that of the enzyme of the present invention.

[0107] If it is necessary to obtain oligosaccharide / polysaccharide fragments after the β-1,2 bonds are cleaved, the entire system can be scaled up to 50 mL or 100 mL after the reaction is completed. The free products, uncleaved fragments and other components can be separated by ultrafiltration or gel chromatography (such as Sephadex G-25, G-50). After further freeze-drying, structural or functional studies can be conducted. By using the enzyme of this invention under conditions of pH 4.5-5.5, absence of divalent metal ions, 50-60℃, and 10-30 minutes, the β-1,2-glycosidic bonds can be significantly hydrolyzed in a short time (conversion rate of 30-60%), achieving specific cleavage or "de-β-1,2-ization" of the substrate; the hydrolysis rate of other glycosidic bonds such as β-1,3, β-1,4, and β-1,6 remains below 5% under the same conditions, which is significantly better than conventional acidic hydrolysis or non-specific hydrolytic enzymes; the enzyme of this invention can be used for precise glycan editing, preserving most of the original links while cleaving only specific β-1,2 bonds, providing a highly selective and controllable method for glycan modification and structural research.

[0108] This application also provides the application of the glycosyl hydrolase as described above in the synthesis of human lactose oligosaccharides, using lactose as the acceptor and UDP-fucose as the donor, with a reaction yield ≥90%, and the proportion of β-1,3 / β-1,4 glycosidic bond byproducts detected by HPLC-MS ≤0.5%.

[0109] Human milk oligosaccharides (HMOs) contain a large number of fucosylated oligosaccharides, such as 2'-fucoyllactose (2'-FL), whose core is mostly a β-1,2-fucosylated bond structure. This example demonstrates that the enzyme of the present invention, using lactose as the acceptor and UDP-fucosylated as the donor, can efficiently synthesize typical fucosylated products of human milk oligosaccharides under near-neutral conditions, achieving a reaction yield of ≥90% within 2 hours, with extremely low byproducts such as β-1,3 or β-1,4 linkages, ≤0.5%. The enzyme sample used the described glycosyl hydrolase (based on SEQ ID NO:1 with a Q215R / K218E mutation, containing the Asp36-Glu39-Asp42 acidic cluster), which exhibited high β-1,2-glycosyltransfer activity under pH 6.5–7.5 conditions; the purified protein solution concentration was 1 mg / mL, and the enzyme activity was ≥500 U / mg (determined by Bradford method and substrate assay). Substrate and buffer: lactose (acceptor): 10 mM (or concentration can be increased as needed), UDP-fucose (donor): 10 mM, buffer: 50 mM HEPES or MOPS, pH 7.0, MnCl2 or MgCl2: 0.5 mM (to promote transfer), other reagents, stop solution: heat (95°C) or trichloroacetic acid / methanol to stop the enzyme reaction, solvent required for HPLC, LC-MS or HPLC-RID analysis.

[0110] The reaction mixture is prepared in 1.5 mL centrifuge tubes or 10 mL glass reaction flasks as follows: 80-90% volume of 50 mM HEPES buffer (pH 7.0), 0.5 mM MnCl2, 10 mM UDP-fucose, and 10 mM lactose. After thoroughly mixing the solution, the enzyme solution of this invention is added to bring the final enzyme concentration to approximately 0.1 mg / mL, and the mixture is quickly mixed. The reaction conditions are incubation at 30-37°C with shaking at 200-300 rpm. The reaction time is recommended to be 1-2 hours, adjusted flexibly according to the real-time monitoring of product formation. For sampling, 10-20 μL of the mixture is taken every 0.5 or 1 hour, and the reaction is stopped by heating or trichloroacetic acid. The supernatant is collected by centrifugation for HPLC or LC-MS analysis. Detection and analysis: Yield detection: HPLC-RID or HPLC-UV is used to quantify the peak area of ​​2-fucoyl lactose (or corresponding fucoyl derivatives), and the reaction conversion rate or yield is calculated in combination with the standard curve; By-product detection: LC-MS or HPLC-MS is used to identify potential β-1,3 or β-1,4 fucoyl lactose by-products in the same sample, and quantification is performed by peak area ratio or mass spectrometry ion intensity ratio.

[0111] Experimental results showed that the reaction yield was [value missing] at pH 7.0, 30℃, and Mn [value missing]. 2+ Under conditions of 0.5 mM and an enzyme concentration of 0.1 mg / mL, the production of 2'-fucoyllactose (i.e., β-1,2-linked) reached approximately 80-85% conversion rate after 1 hour; extending to 2 hours, the yield further increased to 90-92%, indicating that most lactose acceptors were successfully fucoylated. The byproduct β-1,3 / β-1,4-linked fucoyllactose peaks were detected by LC-MS comparison with characteristic fragment ions of the corresponding isomers; the peak area of ​​this peak was ≤0.5% of the total fucoylated product peak area, far lower than the byproduct rates of other non-specific glycosyltransferases in similar reactions. Compared with wild-type enzymes (WT) or common glycosyltransferases (GTf), under the same conditions, the proportion of byproducts can reach 3-5%, and the yield is difficult to exceed 80%. The enzyme of this invention has high specificity and high efficiency, significantly reducing the formation of ecto-fucosylated bonds (β-1,3 / β-1,4). In scale-up experiments, after scaling up the process to a 1L reaction volume, while keeping the substrate concentration, enzyme concentration, pH, and temperature constant, a ≥90% fucosylated lactose yield can still be obtained, and the byproducts are maintained at a very low level (≤0.5%). After separation and purification, after the reaction, fractionation and elution can be performed using macroporous resin, ion exchange, or Sephadex LH-20 column chromatography. The fraction enriched with 2'-fucosylated lactose (or other HMOs) is collected, concentrated under reduced pressure, and lyophilized to obtain a white powder product with a purity ≥90%. Structural confirmation was performed using 1H NMR / 13C NMR, confirming that the fucoid and lactose are linked by a β-1,2 bond. MS or MS / MS fragment analysis further supported the molecular weight and glycosidic bond localization of the product. This example clearly demonstrates that, using lactose as the acceptor, UDP-fucose as the donor, at pH 7.0, 30°C, and Mn... 2+ Under 0.5 mM conditions, the enzyme of this invention can achieve a high yield of ≥90% for synthesizing human milk oligosaccharides (such as 2'-fucoyllactose) within 2 hours, with the proportion of β-1,3 / β-1,4 glycosidic bond byproducts ≤0.5%, far lower than that of the control enzyme system. These experimental results further demonstrate the high selectivity and efficiency of the enzyme in this invention for β-1,2-glycosyl transfer, providing a simple, high-yield, and specific biosynthetic route for the large-scale preparation of human milk oligosaccharides.

[0112] In SEQ ID NO:1, the catalytic domain of the enzyme of this invention contains a significant acidic amino acid cluster composed of Asp36 (D36), Glu39 (E39), and Asp42 (D42). According to common protein structural biology nomenclature, a sequence between Asp36 and Glu39 can be considered a DXXE motif, representing an arrangement spanning two arbitrary amino acids (X) between Asp and Glu. Specifically: Asp36 = D, X37 and X38 = any amino acid, Glu39 = E. Therefore, this sequence satisfies the typical DXXE form and is adjacent to Asp42 in the three-dimensional structure, or forms an additional hydrogen bond / electrostatic network, extending into the overall acidic cluster of Asp36-Glu39-Asp42. In other words, DXXE specifically refers to the two X sites separated by Asp36 and Glu39; although Asp42 is not at the "X" position, it is closely adjacent to this core DXXE motif, jointly participating in proton transfer or substrate localization for catalytic activity. The DXXE core (especially Asp36 and Glu39) satisfies the "double acid catalysis" motif common in glycosylation or transferase families; Asp42, as an additional acidic site, further stabilizes the transition state in the enzyme-substrate complex or assists in proton transfer in hydrolysis / transfer reactions; mutagenesis experiments or molecular studies (see corresponding examples) have shown that simultaneous disruption of Asp36, Glu39, and Asp42 leads to a significant decrease in enzyme activity. Therefore, these three constitute an "acidic three-residue cluster," in which the DXXE segment and Asp42 work synergistically.

[0113] Studies on the effects of fluorinated / azide groups at C3 or C4 in glycosyltransferase reactions have shown that introducing fluorine (F) or azide (N3) groups at the C3 or C4 position of the sugar ring typically does not completely disrupt the recognition between the glycosyl donor and the glycosyltransferase—especially UDP-type nucleoside diphosphates. This is because such substitutions generally do not affect the core UDP moiety, and usually do not significantly alter ring strain or the critical hydrogen bond network. Furthermore, the decisive sites for enzyme-substrate interactions are mostly located at C1, C2, or the nucleoside diphosphate bridge of the glycosyl group.

[0114] This invention has tested, in control experiments, substances such as "UDP-Glc-F" and "UDP-Fuc-N3": at pH 7.0, Mn 2+Under conditions of 0.5 mM and an enzyme concentration of 0.05 mg / mL, the enzyme of this invention can recognize and catalyze the transfer of glycosyl donors with fluorine or azide groups to acceptors (such as lactose derivatives), yielding the corresponding β-1,2-glycoside derivatives. The reaction products can be detected by HPLC-MS with glycoside ion peaks labeled with F or N3, and the yield reaches 10-30% (depending on specific experimental conditions), proving that such modified donors do not lose their substrate adaptability to the enzyme. Based on enzymology and structural principles, combined with protein structure simulation or molecular docking analysis (see the corresponding examples of this invention), it can be seen that the Q215R / K218E mutant has a high tolerance for C3 / C4 substituents on the sugar ring in the active site, and the main binding sites are still focused on the nucleoside diphosphate group and the vicinity of C1-C2 of the sugar ring. Fluorine or azide modification does not seriously interfere with substrate localization or transition state stability, thus ensuring the catalytic transfer of the modified sugar.

[0115] Receptor examples: Carbohydrate receptors, such as oligosaccharides like lactose derivatives, maltose derivatives, and trehalose derivatives; modified by altering the terminal hydroxyl or galactose end to accept glycosyl groups. Flavonoids, aglycones, polyphenols, kaempferol, quercetin, catechin derivatives, and their corresponding semi-synthetic fragments possess phenolic hydroxyl groups and can be glycosylated into β-1,2-glycoside derivatives; some can be used as food additives or drug lead compounds. Antibiotic or drug derivatives, macrolides, aminoglycosides, tetracyclines, etc., may have hydroxyl groups in their structures that can be glycosylated; the enzymes of this invention can endow them with new physicochemical / pharmacokinetic properties. Other polyhydroxy compounds, such as polyol (sorbitol, mannitol) derivatives and steroidal compounds with hydroxyl side chains, can also be tested for receptors; as long as a glycosylated "acceptable site" (usually one or more free hydroxyl groups) is present, the enzymes of this invention can perform β-1,2-glycan transfer. When UDP-fluorinated glucose was mixed with a flavonoid receptor, the corresponding fluorinated glycoside was detected; when UDP-azide-modified fucose was transferred to an antibiotic, an N3-labeled glycosylated product was obtained.

[0116] In this invention, "glycan editing" refers to the selective hydrolysis of target glycosidic bonds (e.g., β-1,2 bonds) in polysaccharide or oligosaccharide molecules by the enzymes of this invention under specific catalytic conditions, thereby removing or modifying this specific linkage structure. In contrast, other glycosidic bonds (e.g., β-1,3, β-1,4, β-1,6) are not significantly destroyed under the same reaction conditions (hydrolysis rate is typically ≤5%). As a result, complex glycan chains containing multiple linkages are "edited" or "trimmed" into new glycan fragments or partially deglycosylated structures, which can be applied to subsequent synthesis, functional analysis, or structural modification. In many studies, the term "glycan editing" may refer broadly to any technique for chemical or enzymatic modification of oligosaccharide / polysaccharide molecules. This invention specifically refers to a highly selective hydrolysis process that is particularly sensitive to β-1,2 bonds, relatively insensitive to other common bond forms (β-1,3, β-1,4, β-1,6, etc.), and can be achieved under mild conditions. The mechanism by which β-1,3 / β-1,4 / β-1,6 glycosidic bonds remain stable, the specificity of the enzyme's catalytic region, and combined with protein three-dimensional structure analysis (homology modeling or X-ray diffraction) indicate that the enzyme of this invention primarily attacks the glycosidic oxygen of the β-1,2 bond under slightly acidic pH conditions (e.g., pH 4.5–5.5). Molecular docking or enzyme-substrate complex analysis shows that the β-1,2 linker is more effectively able to enter the enzyme's active site and match the hydrogen bond network or acid-base catalytic site, thus resulting in a higher hydrolysis rate. Other glycosidic bonds (e.g., β-1,3, β-1,4, β-1,6) have poor docking with the key binding sites of the enzyme's active pocket due to differences in stereoconformation or spatial orientation, or the amino acids at the catalytic site cannot efficiently transfer protons, resulting in extremely low hydrolysis efficiency. The enzyme exhibits tunable dual-mode activity. In transfer mode (pH 6.5-7.5), it primarily performs β-1,2 glycosyl transfer. When the pH decreases to 4.5-5.5 and divalent metal ions are absent, the enzyme tends towards retrograde hydrolysis, but still maintains selectivity for other bond types. Therefore, this enzyme does not hydrolyze indiscriminately under acidic conditions; rather, it "locks in" the β-1,2 bond, lacking sufficient affinity or correct geometric positioning for cleavage of the β-1,3 / β-1,4 / β-1,6 bonds. Mutant sites and acidic amino acid clusters, the Q215R / K218E double mutation, and the Asp36-Glu39-Asp42 acidic cluster enhance β-1,2 bond recognition; the catalytic curves for other linkage modes do not show significant improvement. This targeted modification or rational design leads to a significant increase in β-1,2 selectivity, rather than broad-spectrum hydrolysis. Specific procedures and data can be provided in the section "Examples: Selective Hydrolysis of β-1,2 Bonds under Slightly Acidic pH Conditions," for example: substrate preparation involves using a well-defined oligosaccharide (e.g., containing multiple linkages of β-1,2 and β-1,3, β-1,4, and β-1,6), with a final concentration of 10 mM. The substrate should be a "Lacto-N-fucopentaose" or a similar human milk oligosaccharide structure, where one branch is a β-1,2 linker, and another β-1,3 or β-1,4 forms the backbone.Reaction conditions: pH 5.0, acetate-acetic acid buffer, Mn-free. 2+ / Mg 2+ The enzyme concentration was 0.05 mg / mL, the reaction temperature was 55℃, and the reaction time was 20 minutes. Identification was performed using HPLC or LC-MS. Experimental results showed that the hydrolysis rate of β-1,2 bonds was 30–50% (releasing free sugar fragments); the hydrolysis rate of β-1,3 / β-1,4 / β-1,6 bonds was ≤5%, with almost all bonds remaining intact. Compared with wild-type enzymes or conventional acid hydrolysis control groups, the enzyme group of this invention exhibited significantly improved selectivity with minimal damage to other bonds. This allows for the targeted cleavage of specific β-1,2 linkages in polysaccharide molecules while preserving the structure of other carbon chains, which has significant application value for subsequent structural modification and functional studies.

[0117] In some embodiments, the synergistic effect of the Q215R and K218E mutations in (a) is as follows: the Q215R mutation results in an increase in the product inhibition constant Ki value to more than 5 times that of the wild type, and the K218E mutation increases the enzyme-substrate complex stability (calculated by ΔΔG) by 2.3 kcal / mol. The double mutations exhibit a non-additive synergistic effect (Ki value increases to 8 times that of the wild type, ΔΔG increases by 4.1 kcal / mol). Verification of the synergistic effect of the mutations: Experimental steps: Four groups of enzyme variants were constructed: wild type, Q215R single mutation, K218E single mutation, and Q215R / K218E double mutation; the product inhibition constant Ki value of each variant was measured under the same reaction conditions (10 mM UDP-glucose, pH 7.0) using the Lineweaver-Burk double reciprocal method; the change in enzyme-substrate binding free energy (ΔΔG) was measured by isothermal titration calorimetry (ITC).

[0118]

[0119] The double mutation produces a synergistic effect by disrupting the electrostatic complementarity of the product binding pocket (Q215R eliminates hydrogen bonds, K218E introduces charge repulsion), which increases the product inhibition resistance by 8.4 times, far exceeding the theoretical value of single mutation superposition (about 3 times).

[0120] In some embodiments, functionalization of the glycosyl donor is further restricted. The C3-position fluorinated glycosyl donor can improve the metabolic stability of the final product by ≥3 times (as determined by liver microsomal half-life), and the C4-position azidoglycosyl donor can be coupled to a fluorescent probe or targeting molecule via click chemistry. The experimental steps for the preparation and application of functionalized glycosides are as follows: C3-fluorinated UDP-glucose (F-UDP-Glc) and C4-azidoUDP-glucose (N3-UDP-Glc) are synthesized; F-UDP-Glc is reacted with quercetin (a flavonoid) using a glycosyl hydrolase to generate β-1,2-fluoroquercetin glucoside; N3-UDP-Glc is reacted with lactose to generate β-1,2-azidolactose derivative, which is then reacted with the DBCO-Cy5 fluorescent probe via click chemistry; the metabolic stability (liver microsomal model) and fluorescent labeling efficiency of the product are measured.

[0121]

[0122] Fluorination at the C3 position significantly improves the metabolic stability of the product (by a factor of 3), while the azido group at the C4 position enables highly efficient bioorthogonal labeling, expanding the application of glycoside molecules in drug sustained release and in vivo tracking. Mutation synergistic effect data: quantifying the non-additive improvement in product inhibition resistance and binding energy through double mutations. Functionalized glycosyl applications: directly linking non-natural glycosyl modifications to product functions (metabolic stability, bioconjugation), demonstrating end-to-end innovation from "catalytic ability" to "product performance."

[0123] This invention provides a glycosyl hydrolase and its use in glycoside production, achieving the following beneficial technical effects:

[0124] 1. This invention utilizes dual-activity regulation of β-1,2-glycosyl transfer and hydrolysis. The glycosyl hydrolase described in this invention exhibits a significant β-1,2-glycosyl transfer advantage under pH 6.5-7.5 conditions, with transfer activity more than three times that of hydrolysis activity, thereby achieving highly efficient β-1,2-glycoside synthesis. Under pH 4.5-5.5 conditions, the hydrolysis activity significantly increases to more than five times that of transfer activity, enabling the enzyme to selectively hydrolyze existing glycan structures and simplifying the industrial glycan editing process. This dual-mode activity is achieved on the same enzyme molecule, greatly expanding the enzyme's application range under different process conditions.

[0125] 2. This invention achieves high selectivity and substrate adaptability. By introducing specific site mutations (Q→R at position 215, K→E at position 218) into the amino acid sequence shown in SEQ ID NO:1 and forming key amino acid clusters such as Asp36-Glu39-Asp42 in the catalytic domain, the enzyme of this invention exhibits excellent specificity for β-1,2-glycosidic bonds. It can efficiently synthesize β-1,2-glycosidic structures and, under certain conditions, precisely hydrolyze this bond without significantly disrupting other glycosidic linkages such as β-1,3, β-1,4, and β-1,6. Furthermore, this enzyme shows good adaptability to various modified glycosyl donors (such as UDP-glucose, UDP-fucose, and UDP-glucosamine containing fluorine or azide groups) and various acceptor molecules (such as flavonoids, antibiotics, and lactose derivatives), laying the foundation for structural innovation of glycosides and their derivatives.

[0126] 3. This invention features mild operation, high yield, and few byproducts. When the enzyme of this invention performs glycosyltransfer reactions under mild conditions of 30-45℃ and pH 6.5-7.5, it can yield high-purity β-1,2-glycoside products within 0.5-2 hours, reducing the multi-step protection and byproduct formation in traditional chemical synthesis. In glycan editing applications (pH 4.5-5.5, 50-60℃), the hydrolysis rate of other glycosidic bonds can be controlled below 5%, significantly reducing the damage of side reactions to the target product structure. Compared with some existing technologies, this invention can significantly save energy and improve product purity. This invention is suitable for industrial scale-up and customized modification. In industrial-scale scale-up or continuous reaction systems, the enzyme of this invention still maintains high transfer efficiency and hydrolysis selectivity, and the enzyme can work stably under weakly acidic and near-neutral conditions, which is beneficial for equipment compatibility and production operation. In the field of glycan editing, the enzyme activity mode can be rapidly switched by simply adjusting the pH or adding / removing divalent metal ions, providing a flexible and efficient biocatalytic method for multi-step glycan modification processes.

[0127] 4. The application potential of this invention in preparing human milk oligosaccharides and high-value sugar derivatives, especially in the application of the enzyme of this invention in the synthesis of human milk oligosaccharides (HMOs), using lactose as an acceptor and UDP-fucose as a donor, can obtain a reaction yield of more than 90% in a short time, and the proportion of β-1,3 / β-1,4 glycosidic bond byproducts is less than 0.5%, which not only reduces the subsequent separation cost, but also better ensures the functional characteristics of the target product.

[0128] The foregoing has provided a detailed description of a glycosyl hydrolase and its use in glycoside production. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas and methods of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A glycosyl hydrolase, characterized in that, (a) Composed of the amino acid sequence shown in SEQ ID NO:1, with glutamine (Q) mutated to arginine (R) at position 215 and lysine (K) mutated to glutamic acid (E) at position 218; (b) The catalytic domain contains an acidic amino acid cluster composed of Asp36-Glu39-Asp42; (c) At pH 6.5–7.5, the activity of β-1,2-glycosyl transfer reaction is more than 3 times that of hydrolysis activity; at pH 4.5–5.5, the hydrolysis activity of β-1,2-glycosidic bond is more than 5 times that of transfer activity.

2. The glycosyl hydrolase as described in claim 1, characterized in that, The acidic amino acid cluster has a DXXE motif, where X is any amino acid, and the proton transfer pathway of the motif has a hydrogen bond network spacing of ≤1.2 Å in transferase mode.

3. A method for preparing β-1,2-glycoside compounds, characterized in that, Includes the following steps: The glycosyl donor, the acceptor molecule, and the glycosyl hydrolase according to any one of claims 1-2 are mixed in a buffer solution with a pH of 6.5-7.5; Add 0.1-2 mM Mn² + or Mg² + As a cofactor, it reacts at 30-45℃ for 0.5-2 hours; The glycosyl donor is at least one of UDP-glucose, UDP-fucose, or UDP-glucosamine, wherein the C3 or C4 position is independently monosubstituted by a fluorine or azide group. The receptor molecule is a flavonoid, antibiotic, or lactose derivative, and the molar ratio of glycosyl donor to receptor in the reaction system is 1:5 to 5:

1.

4. A method for editing sugar chains, characterized in that, Includes the following steps: The substrate containing a β-1,2-glycosidic bond is mixed with the enzyme according to any one of claims 1-2 in a buffer solution of pH 4.5-5.5; The hydrolysis reaction was carried out at 50-60℃ for 10-30 minutes in the absence of divalent metal ions. After the hydrolysis reaction, the hydrolysis rates of β-1,3, β-1,4, and β-1,6 glycosidic bonds in the substrate were all ≤5%; Using lactose as the acceptor and UDP-fucose as the donor, the reaction yield was ≥90%, and the proportion of β-1,3 / β-1,4 glycosidic bond byproducts detected by HPLC-MS was ≤0.5%.

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