Alpha-2,3-sialyltransferase mutants and uses thereof

Abstract

This invention discloses an α-2,3-sialyltransferase mutant and its applications, relating to the field of microbial genetic engineering technology. The α-2,3-sialyltransferase mutant possesses at least one of the following mutations: T188E, A210C, introduction of a stop codon at any position between positions 274 and 285, mutation at amino acid position 190, F65S, and F284L. This mutant can sialylate a wider range of substrates, exhibiting particularly good catalytic activity towards complex oligosaccharides on glycoproteins or glycolipids. Compared to the wild type, the α-2,3-sialyltransferase mutant exhibits enhanced catalytic activity and product conversion rate. It possesses the technical advantages of high catalytic efficiency (kcat) and fewer substrate hydrolysis side reactions.

Description

Technical Field

[0001] This invention relates to the field of microbial genetic engineering technology, and more specifically, to an α-2,3-sialic acid transferase mutant and its applications. Background Technology

[0002] α-2,3-Sialyltransferases are key glycosyltransferases that catalyze the transfer of sialic acid to the ends of oligosaccharides, glycolipids, and complex glycoproteins to form α-2,3-sialic acid glycosidic bonds. Terminal α-2,3-sialylation modification plays a crucial role in maintaining physiological homeostasis, regulating immune recognition, and determining the circulating half-life and stability of glycoproteins in vivo.

[0003] In industrial applications, mammalian-derived sialyltransferases often face challenges in expression and high costs. Microbial-derived α-2,3-sialyltransferases, on the other hand, may exhibit significant drawbacks in practical biocatalysis and industrial applications, including low catalytic efficiency (kcat), undesirable substrate hydrolytic activity, and weak catalytic ability towards complex glycoconjugates and therapeutic proteins with high steric hindrance. These limitations in enzymatic performance severely restrict the application of existing technologies in protein drug development and large-scale production, hindering the formation of efficient and universally applicable biocatalytic platforms.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide an α-2,3-sialic acid transferase mutant and its application to solve at least one of the above-mentioned problems.

[0006] This invention is implemented as follows: In a first aspect, the present invention provides an α-2,3-sialyltransferase mutant, which is based on the amino acid sequence shown in SEQ ID NO.1 and mutated by at least one of the following: T188E, A210C, introducing a stop codon at any position between positions 274 and 285, mutating amino acid position 190, F65S and F284L; Among them, the 190th amino acid has any of the following mutations: F190M, F190Q, F190A, and F190V.

[0007] Secondly, the present invention provides a nucleic acid molecule that encodes the aforementioned α-2,3-sialic acid transferase mutant.

[0008] Thirdly, the present invention provides a recombinant vector comprising the aforementioned nucleic acid molecules.

[0009] Fourthly, the present invention provides a recombinant bacterium comprising the above-mentioned nucleic acid molecule or the above-mentioned recombinant vector.

[0010] Fifthly, the present invention provides the use of α-2,3-sialyltransferase mutants, nucleic acid molecules, recombinant vectors, or recombinant bacteria in any of the following: (1) Sialylation of oligosaccharides or complex oligosaccharides; (2) Sialylation of protein substrates or lipids containing monosaccharides, oligosaccharides or glycans; (3) Azide sialic acid glycosylation of protein substrates or lipids or other biomacromolecules containing monosaccharides, oligosaccharides or glycans for labeling glycans on cell surfaces or for dynamic tracking and imaging in living cells.

[0011] In a sixth aspect, the present invention provides the use of α-2,3-sialyltransferase mutants, nucleic acid molecules, recombinant vectors or recombinant bacteria in the preparation of drugs or formulations for prolonging the half-life of drugs or formulations; Preferably, the drug is selected from blood-derived VWF concentrate, recombinant VWF, recombinant human erythropoietin and its derivatives, α-1-antitrypsin, TNFR-IgG immunoadhesin molecules or recombinant thrombomodulin, and the formulation is selected from a model protein, with the model protein selected from AsialoFetuin.

[0012] In a seventh aspect, the present invention provides a method for sialyl glycosylation of a substrate, comprising the following steps: mixing the above-mentioned α-2,3-sialyltransferase mutant or the above-mentioned recombinant bacteria with a recipient substrate and a donor substrate, wherein the recipient substrate is a monosaccharide, oligosaccharide, or polysaccharide containing galactose (Gal) or N-acetylgalactosamine (GalNAc), or a glycoprotein or glycolipid macromolecule containing the above-mentioned sugar, and the donor substrate is CMP-sialic acid or a derivative thereof.

[0013] The present invention has the following beneficial effects: This invention employs a directed evolution strategy to successfully develop a highly active α-2,3-sialyltransferase mutant. Compared to the wild type, the α-2,3-sialyltransferase with a specific amino acid mutation combination can more efficiently sialylate substrates, essentially "installing" sialic acid to the glycan terminus to complete glycosylation modification. It also exhibits excellent catalytic activity, particularly for complex oligosaccharides on glycoproteins or glycolipids. Compared to the wild type, the α-2,3-sialyltransferase mutant demonstrates enhanced catalytic activity and product conversion rate. It possesses the technical advantages of high catalytic efficiency (kcat) and fewer substrate hydrolysis side reactions. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 A technical roadmap for obtaining mutants with enhanced enzyme activity through high-throughput screening combined with dominant site analysis; Figure 2 The relative enzyme activity data are for some single-site (a) and combined mutants (b) compared with the wild-type CstI1-285. Figure 3 SDS-PAGE gel images of CstI wild-type 1-285 (WT) and M8 mutant; Figure 4 To use HPLC method to detect donor substrate lactose ( Figure 4 a) 3′-sialyllactose Figure 4 b) Standard, 6′-sialyllactose Figure 4 c) Standards and CstI M8 mutants ( Figure 4 d) Graph showing the detection results of glycosidic bond types in the reaction system for catalytic lactose sialylation; Figure 5 The figure shows the results of enzyme kinetic parameter determination for CstI wild-type 1-285 (WT) and M8 mutant; Figure 6 Statistical plot of the hydrolysis rate of substrate CMP-sia by CstI wild-type 1-285 (WT), M8 mutant and PmST1 (M144D); Figure 7 The HPLC fluorescence (FLR) analysis of sialylated G2 oligosaccharides yields the following chromatograms: (a) Chromatogram of the unmodified G2 glycan control (substrate only); (b) Chromatogram of the reaction mixture after sialylation of the M8 variant. Figure 8 LC-MS glycosylation analysis of wild-type CstI and M8 mutants of asialofetuin after enzymatic sialylation and non-natural modification. Figure 8 The figure above shows the initial LC-MS glycosylation of the substrate desialylated fetoglobulin, which is mainly composed of complex N-glycan chains with galactose-terminated biantennary (G2) and triantennary (G3). Figure 8The images in the middle are, in order, the glycosylation of fetal proteins after the reaction catalyzed by wild-type CstI (1-285) and the glycosylation of fetal proteins after the reaction catalyzed by the M8 mutant. After the reaction catalyzed by wild-type CstI (1-285), it is shown that the degree of sialylation is incomplete, and there are still a large number of incompletely capped intermediate glycoforms. After the reaction catalyzed by the M8 mutant of this invention, it is shown that the complex biantennary and triantennary glycan chains are almost completely sialylated, and there are no obvious unmodified intermediate residues. Figure 8 The figure below shows the glycosylation profile of fetal globulin after the reaction of the M8 mutant of this invention with a non-natural donor (CMP-9-azidosialic acid), indicating that the enzyme successfully achieved a high proportion of non-natural azidosialylation modification on the surface of the glycoprotein. Figure 9 To illustrate the results of an orthogonal fluorescent labeling experiment for incorporating azidosialic acid into desialofetuin using the CstI M8 variant (lanes from left to right: 1. Desialofetuin incubated with Cy5-DBCO, unlabelable; 2. Sialized fetuin incubated with Cy5-DBCO, unlabelable; 3. Azidosialic fetuin incubated with Cy5-DBCO, labelable, showing red fluorescence; 4. Desialofetuin, as control; 5. Sialized fetuin, as control; 6. Azidosialic fetuin, as control; 7. Protein marker; 8. Sialyltransferase CstI M8). Figure 10 Using CstI M8 to sialylate galactose-modified glycoproteins and perform azide-dependent fluorescent labeling; Figure 11 A schematic diagram of in vivo circulation assessment of AF647-labeled glycoproteins in mice, and in vivo serum clearance curves of non-sialylated desialylated fetal protein and M8 sialylated fetal protein. Figure 12 A statistical graph showing the stepwise conversion of antibody glycoform over time by sializing the monoclonal antibody Belimumab with the CstI M8 variant and analyzing the antibody glycoform over time using HPLC. Figure 13 A schematic diagram of sequential sugar engineering and sialylation of human IVIG catalyzed by the CstI M8 variant; Figure 14 SDS-PAGE analysis of the purified CstI truncated variant (a) and its relative enzyme activity (b). Detailed Implementation

[0016] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0017] In a first aspect, the present invention provides an α-2,3-sialyltransferase mutant, which is based on the amino acid sequence shown in SEQ ID NO.1 and mutated by at least one of the following: (1) T188E; (2) A210C; (3) Introduce a stop codon at any position from the 274th to the 285th position; (4) Mutate the 190th amino acid; (5) F65S; and (6) F284L; Among them, the 190th amino acid has any of the following mutations: F190M, F190Q, F190A, and F190V.

[0018] Mutating the above-mentioned sites of wild-type α-2,3-sialyltransferase can significantly enhance the enzyme activity of α-2,3-sialyltransferase.

[0019] For example, α-2,3-sialyltransferase mutants have any of the following mutations: T188E and A210C; A210C and the introduction of a stop codon at any position between positions 274 and 285; T188E and a mutation at amino acid position 190; T188E, A210C and a mutation at amino acid position 190; T188E and the introduction of a stop codon at any position between positions 274 and 285; A210C and a mutation at amino acid position 190; and the introduction of a stop codon at any position between positions 274 and 285. Mutation of amino acid 190; T188E, A210C and the introduction of a stop codon at any position between positions 274 and 285; A210C, the introduction of a stop codon at any position between positions 274 and 285 and the mutation of amino acid 190; T188E and A210C, and the introduction of a stop codon at any position between positions 274 and 285 and the mutation of amino acid 190; F65S and F284L; F284L and T188E; F65S and T188E, but not limited to the above combinations.

[0020] The amino acid sequence shown in SEQ ID NO.1 is the original sequence of α-2,3-sialyl transferase, derived from CstI (1-285 amino acids) of Campylobacter jejuni. In this invention, to overcome the activity bottleneck of the wild-type enzyme, the inventors adopted a directed evolution strategy and successfully developed a highly active mutant, M8. The specific technical route is as follows: First, using the wild-type CstI amino acid sequence as a template, a deep mutation scanning (DMS) saturated mutant library covering the entire sequence was designed and constructed using molecular biology techniques, and transformed into the screening strain (pACKC18-NmCSS+JM107 NanA-, which was obtained from Stephen Withers' laboratory); second, the screening strain was incubated with fluorescent substrate and washed using the fluorescence activated cell sorting (FACS) ultra-high-throughput screening method based on intracellular dual fluorescent substrate accumulation. This method converts the catalytic activity of the target enzyme into a single-cell level fluorescence signal, thereby enriching a mutant cell population with high sialyltransferase activity using flow cytometry. Subsequently, combined with next-generation sequencing (NGS) technology, deep sequencing and data mining were performed on the mutant libraries before and after screening. By comparing changes in mutation frequency, a series of dominant single-point mutations that can significantly enhance enzyme activity were identified. Furthermore, the candidate mutants were re-screened using an enzyme coupling method based on CMP release to eliminate false positive interference. Finally, based on the verification of single-point mutations, the dominant mutation sites and C-terminal truncation modifications were systematically combined to successfully obtain a combined mutant with optimal catalytic performance, named M8. Compared to the wild-type sequence, this mutant contains the following mutations: the codon at the C-terminus of α-2,3-sialyltransferase, amino acid 277, is mutated to a stop codon, resulting in a truncation mutation at positions 277-285, and three key amino acid substitution mutations, T188E, F190M, and A210C (i.e., the combined mutation is: CstI 1-276 + T188E + F190M + A210C).

[0021] Compared to the wild type, α-2,3-sialyltransferases with specific amino acid mutation combinations can sialylate a wider range of substrates, essentially "installing" sialic acid to the glycan termini to complete glycosylation modification. They exhibit particularly good catalytic activity on complex oligosaccharides in glycoproteins or glycolipids. Compared to the wild type, α-2,3-sialyltransferase mutants demonstrate enhanced catalytic activity and product conversion rates. They offer technical advantages such as high catalytic efficiency (kcat) and fewer hydrolytic side reactions of both substrates and products.

[0022] The specific amino acid sequence of SEQ ID NO.1 is as follows: MTRTRMENELIVSKNMQNIIIAGNGPSLKNINYKRLPREYDVFRCNQFYFEDKYYLGKKIKAVFFNPGVFLQQYHTAKQLILKNEYEIKNIFCSTFNLPFIESNDFLHQFYNFFPDAKLGYEVIENLKEFYAYIKYNEIYFNK RITSGVYMCAIAIALGYKTIYLCGIDFYEGDVIYPFEAMSTNIKTIFPGIKDFKPSNCHSKEYDIEALKLLKSIYKVNIYALCDDSILANHFPLSININNNFTLENKHNNSINDILLTDNTPGVSFYKNQLKADNKIMLNFY.

[0023] The α-2,3-sialyltransferase mutant has any one of the following mutations: (1) T188E and A210C, and the α-2,3-sialyltransferase mutant also has any of the following mutations: F190M, F190Q, F190A and F190V; (2) T188E and A210C, and deletion mutations of amino acids at positions 277-285; (3) For the deletion mutation of amino acids at positions 277-285, the α-2,3-sialyltransferase mutant also has any of the following mutations: F190M, F190Q, F190A and F190V; (4) Introduce a stop codon at the 279th position; (5) Introduce a stop codon at the 275th position; (6) Introduce a stop codon at the 277th position; (7) Introduce a stop codon at the 278th position; (8) Introduce a stop codon at the 280th position.

[0024] In a preferred embodiment of the present invention, the α-2,3-sialyltransferase mutant also has at least one of the following mutations: E9R, Y33H, R35M, D41A, Y54A, T76I, A77R, I88F, D105M, E129F, F141S, R144E, G148M, K161M, Y164T, D195G, N200A, E209R, L213E, N211E, N233G, S238V, N240P, and amino acid deletion mutations at positions 266-275.

[0025] All of the above mutation combinations exhibit good catalytic activity and product conversion rate.

[0026] Secondly, the present invention provides a nucleic acid molecule that encodes the aforementioned α-2,3-sialic acid transferase mutant.

[0027] As used herein, the term "nucleic acid molecule" refers to a sequence of nucleoside or nucleotide monomers composed of natural bases, sugars, and interglycosylation (backbone) bonds. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid molecules of this invention can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences and may contain natural bases, including adenine, guanine, cytosine, thymine, and uracil. Modified bases may also be present. Examples of these modified bases include nitrogenous and denitrogenated adenine, guanine, cytosine, thymine, and uracil; and xanthine and hypoxanthine.

[0028] Given the known amino acid sequence of the α-2,3-sialyltransferase mutant, those skilled in the art can easily obtain the nucleic acid sequence encoding the α-2,3-sialyltransferase mutant based on the degeneracy of the codons.

[0029] Thirdly, the present invention provides a recombinant vector comprising the aforementioned nucleic acid molecules.

[0030] The term "vector" is used herein in its most common sense and includes any intermediate medium for nucleic acids that enables the nucleic acids to be introduced, for example, into prokaryotic and / or eukaryotic cells, and, where appropriate, integrated into the genome. Vectors of this type preferably replicate and / or are expressed in cells. The term "vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, or other vectors well known in the art. The term "plasmid," as used herein, generally refers to a construct of extrachromosomal genetic material, typically a circular double-stranded DNA that can replicate independently of chromosomal DNA. Any plasmid and vector can be used as long as it can replicate and be stable within a host.

[0031] For example, the expression vector is the pET28a vector.

[0032] Fourthly, the present invention provides a recombinant bacterium comprising the above-mentioned nucleic acid molecule or the above-mentioned recombinant vector.

[0033] The recombinant bacteria can be prokaryotes or eukaryotes. Prokaryotes can be selected from genera such as Enterobactergenus, Escherichia genus, Erwinia genus, Serratia genus, Providencia genus, Corynebacterium genus, Brevibacterium genus, or Lactobacillus. For example, Escherichia coli can be selected.

[0034] Eukaryotic organisms, such as those selected from yeast, fungi, or insect cells.

[0035] Fifthly, the present invention provides the use of α-2,3-sialyltransferase mutants, nucleic acid molecules, recombinant vectors, or recombinant bacteria in any of the following: (1) Sialylation of oligosaccharides or complex oligosaccharides; (2) Sialylation of protein substrates or lipids containing monosaccharides, oligosaccharides or glycans; (3) Azide sialic acid glycosylation of protein substrates or lipids or other biomacromolecules containing monosaccharides, oligosaccharides or glycans for labeling glycans on cell surfaces or for dynamic tracking and imaging in living cells.

[0036] Uses (1) For: using monosaccharide, oligosaccharide or polysaccharide chains with non-reducing terminal galactose (Gal) or N-acetylgalactosamine (GalNAc) as acceptor substrates and CMP-Neu5Ac or other CMP-sialic acid derivatives as donor substrates for enzymatic sialylation modification.

[0037] The α-2,3-sialyltransferase mutant provided by this invention can efficiently transfer sialic acid to the ends of oligosaccharides, glycolipids, and complex glycoproteins to form α-2,3-sialyl glycosidic bonds. This α-2,3-sialyltransferase mutant has the technical advantages of high efficiency and universal applicability.

[0038] In a preferred embodiment of the present invention, oligosaccharides or complex oligosaccharides in glycoprotein substrates or glycolipids are sialylated.

[0039] In a sixth aspect, the present invention provides the use of α-2,3-sialyltransferase mutants, nucleic acid molecules, recombinant vectors or recombinant bacteria in the preparation of drugs or formulations for prolonging the half-life of drugs or formulations.

[0040] The drug is selected from blood-derived VWF concentrate, recombinant VWF, recombinant human erythropoietin and its derivatives, α-1-antitrypsin, TNFR-IgG immunoadhesin molecules, recombinant thrombomodulin, or human intravenous immunoglobulin IVIG. The formulation is selected from a model protein, and the model protein is selected from AsialoFetuin.

[0041] Protein drugs, for example, are selected from protein drugs used to treat autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, multiple sclerosis, inflammatory bowel disease, and psoriasis.

[0042] The inventors have discovered that the α-2,3-sialyltransferase mutant provided by this invention can prolong the retention time of protein drugs in the body and improve the efficacy of the drugs.

[0043] In a seventh aspect, the present invention provides a method for sialyl glycosylation of a substrate, comprising the following steps: mixing the above-mentioned α-2,3-sialyltransferase mutant or the above-mentioned recombinant bacteria with a recipient substrate and a donor substrate, wherein the recipient substrate is a monosaccharide, oligosaccharide, or polysaccharide containing galactose (Gal) or N-acetylgalactosamine (GalNAc), or a glycoprotein or glycolipid macromolecule containing the above-mentioned sugar, and the donor substrate is CMP-sialic acid or a derivative thereof.

[0044] In a preferred embodiment of the present invention, the receptor substrate is selected from natural proteins, natural lipids, or non-natural substrates; the non-natural substrate is a sialic acid derivative. The natural protein is selected from glycoproteins, proteins whose glycan chains are covalently linked. This includes, but is not limited to, N-linked glycoproteins, O-linked glycoproteins, GPI-anchored proteins, C-linked proteins, S-linked proteins, and O-GlcNAc glycosylated proteins.

[0045] Donor substrates, for example, are selected from CMP-9 azidosialic acid.

[0046] In a preferred embodiment of the present invention, the conditions for sialic acid glycosylation include pH 4.0 to 11.0, temperature 0 to 50 degrees Celsius, donor substrate concentration 0.01 mM to 200 mM, acceptor substrate concentration 0.01 mM to 1000 mM, and incubation time 0.1 hours to 24 hours.

[0047] For example, the donor substrate concentration can be 0.01mM~1mM, 5-10mM, 15-50mM, or 60-200mM, the acceptor substrate concentration can be 0.01mM~1000mM, and the incubation time can be 0.1 hours~1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11-15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours.

[0048] When the acceptor substrate is an oligosaccharide containing galactose (Gal) or N-acetylgalactosamine (GalNAc), and the donor substrate is CMP-sialic acid or its derivative, incubate at 37°C for 10 minutes.

[0049] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0050] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0051] Example 1 This embodiment screens for α-2,3-sialic acid transferase mutants.

[0052] 1. The original sequence of the α-2,3-sialic acid transferase selected in this study is CstI (amino acids 1-285) from Campylobacter jejuni, and the specific amino acid sequence is shown in SEQ ID NO.1. The wild-type sequence can also be obtained from https: / / www.uniprot.org / uniprotkb / Q9RGF1 / entry#sequences.

[0053] In this invention, to overcome the activity bottleneck of wild-type enzymes, a directed evolution strategy was adopted, and a highly active mutant M8 was successfully developed. The specific technical route is described in [reference needed]. Figure 1 As shown, the specific technologies are as follows: First, using the wild-type CstI amino acid sequence as a template, a deep mutation scanning (DMS) saturated mutant library covering the entire sequence was designed and constructed using molecular biology techniques, and transformed into the screening strain (pACKC18-NmCSS+JM107 NanA - this strain originated from Stephen Withers' laboratory). Second, a high-throughput screening method based on fluorescence activated cell sorting (FACS) using intracellular dual-fluorescent substrate accumulation was employed, incubating the screening strain with fluorescent substrates and then washing them. This method can convert the catalytic activity of the target enzyme into a single-cell level fluorescence signal, thereby enriching a mutant cell population with high sialyltransferase activity using flow cytometry. Subsequently, combined with next-generation sequencing (NGS) technology, deep sequencing and data mining were performed on the mutant libraries before and after screening. By comparing changes in mutation frequency, a series of advantageous single-point mutations that could significantly enhance enzyme activity were identified. Furthermore, a CMP-based enzyme coupling method was used to re-screen the initial candidate mutants to eliminate false positive interference.

[0054] The specific methods used in the mutant screening process are as follows: 1. Construction of CstI saturated mutant library Using the wild-type cstI gene as a template, a deep mutation scan (DMS) library covering residues 2 to 285 of CstI was constructed. Saturation mutations were introduced at each of the 284 target sites using NNK degenerate codons (where N = A, C, G, or T, K = G or T). A pool of NNK-substituted DNA fragments was obtained from a commercial supplier, followed by PCR amplification and purification.

[0055] For library assembly, the pUC18 cloning vector was double-digested using BamHI and KpnI restriction enzymes. The amplified fragments were seamlessly assembled into the linearized target vector using homologous recombination cloning. The assembled plasmid library was transformed into *E. coli* TOP10 cells. After 45 minutes of incubation, the transformed cells were plated onto solid LB agar plates containing ampicillin. All resulting colonies were scraped and collected for mass plasmid extraction.

[0056] 2. Fluorescence-activated cell sorting (FACS) screening The CstI mutant library was screened for activity using fluorescence activated cell sorting (FACS) using the previously described cell-based assay method. *E. coli* JM107 NanA- cells, which lack the Neu5Ac aldehyde enzyme (nanA) and β-galactosidase (LacZ) genes to prevent intracellular degradation of sialic acid and lactose, were used as chassis cells. To synthesize the donor substrate CMP-sialic acid intracellularly, the pACKC18-NmCSS plasmid containing CMP-sialic acid synthase was transformed into JM107NanA- cells to prepare competent cells. The CstI mutant library prepared in the previous step was then transformed into these competent cells. The transformants were cultured and transferred to LB medium containing ampicillin (100 μg / ml) and chloramphenicol (25 μg / ml) for amplification (37°C, 220 rpm). The amplification was achieved when the OD600 of the culture reached 0.5–0.7. Protein expression was induced with 1 mM MIPTG, and the culture was incubated overnight at 20 °C in a shaker. Cells were collected and resuspended in M9 basal medium containing 0.5 mM fluorescent acceptor substrates (BODIPY-lactose and coumarin-lactose) and incubated for 30 min. Cells were then washed with LB medium and PBS (pH 7.4) to remove excess substrate and diluted in PBS for flow cytometry. Cell sorting was performed using a BD FACSAria II flow cytometer (BD Biosciences) using sterile PBS as the sheath solution. Cells were activated using 508 nm and 405 nm lasers, and fluorescence emission was detected at 515 nm (BODIPY channel) and 450 nm (coumarin channel). Sorted cells were collected in LB medium, plated on selective agar plates containing ampicillin and chloramphenicol, and incubated overnight. Colonies were collected, and plasmid DNA was extracted for the next round of FACS screening.

[0057] Relative enzyme activity data of some single-site and combined mutants compared with CstI1-285 wild type are referenced. Figure 2As shown, with the enzyme activity of wild-type CstI1-285 as 1, single-point mutations and combined mutations were tested using the enzyme coupling method. The combination with the highest enzyme activity was CstI1-276 + T188E + F190M + A210C. Single-point mutations of F65S, T188E, F190Q, K279stop, F284L, A210C, and CstI1-276 all significantly increased enzyme activity. Among the combined mutations, CstI1-276 + T188E+A210C, CstI1-276 + F190M, CstI1-276 + F190V, and CstI1-276 + F190Q significantly increased enzyme activity.

[0058] Following the above method, based on the verification of single-point mutation, the advantageous mutation site was systematically combined with C-terminal truncation modification, and the combined mutant with the best catalytic performance—M8—was successfully obtained.

[0059] Compared to the wild-type sequence, this mutant contains the following mutations: the C-terminal codon at amino acid 277 is mutated to a stop codon, resulting in a truncation mutation of amino acids 277-285 in the transferase, as well as three key amino acid substitution mutations at three sites: T188E, F190M, and A210C (i.e., the combined mutation is: CstI 1-276 + T188E + F190M + A210C).

[0060] Example 2 In this embodiment, a recombinant vector and recombinant bacteria expressing the α-2,3-sialic acid transferase mutant from Example 1 were constructed, and the α-2,3-sialic acid transferase mutant was prepared.

[0061] CstI mutants and wild-types were expressed in *E. coli* BL21(DE3) using the pET-28a(+) expression vector. Cells were cultured in LB medium containing 50 μg / mL kanamycin at 37°C until OD600 reached 0.6–0.8, induced with 0.1 mM IPTG, and expressed at 18°C ​​for 16 h. Cells were harvested (4000×g, 20 min, 4°C), resuspended in buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 20 mM imidazole), and lysed by high-pressure homogenization. After centrifugation (8000×g, 60 min, 10°C), the supernatant was loaded onto a Ni-NTA affinity column, washed with buffer, and eluted with buffer containing 250 mM imidazole. The eluted proteins were concentrated by ultrafiltration to replace the buffer and analyzed by SDS-PAGE. Protein concentration was determined by absorbance at 280 nm using a NanoDrop spectrometer. The purified protein was stored in 50% (v / v) glycerol at -20 °C.

[0062] Compared to the wild type, this mutant also increases the yield of protein from the α-2,3-sialyltransferase mutant.

[0063] Reference Figure 3 As shown, the left lanes of the gel image, from left to right, contain: protein marker, precipitate after disruption of the wild-type CstI1-285 expression strain, supernatant after disruption of the wild-type CstI1-285 expression strain (repeat 1), supernatant after disruption of the wild-type CstI1-285 expression strain (repeat 2), and CstI1-285 wild-type protein eluted with imidazole. The right lanes of the gel image, from left to right, contain: protein marker, precipitate after disruption of the CstI M8 expression strain, supernatant after disruption of the CstI M8 expression strain, and CstI M8 protein eluted with imidazole.

[0064] The donor substrate lactose was detected using HPLC. Figure 4 a) 3′-sialyllactose Figure 4 b) Standard, 6′-sialyllactose Figure 4 c) Standards and CstI M8 mutants ( Figure 4 d) The reaction system for catalytic lactose sialylation was analyzed to detect the type of glycosidic bonds in the substances in the system after the reaction.

[0065] The specific HPLC detection method was as follows: 5 mM aqueous solutions of lactose, 3′-sialylated lactose, and 6′-sialylated lactose standards were prepared separately. A separate reaction system was prepared for the sialylation of lactose substrates catalyzed by the CstI M8 mutant (5 mM lactose, 5 mM CMP-sialic acid, 20 mM Hepes 7.0). Purified CstI M8 enzyme was added to initiate the reaction, and the mixture was immediately and thoroughly mixed and incubated at 37°C for 10 minutes. The reaction was then terminated by adding 2-aminobenzamide (2-AB) solution (dissolved in 72% acetonitrile and 28% acetic acid) and 2-pyridineborane (2-PB) solution (dissolved in methanol) in a 1:1:1 (v / v / v) ratio to the reaction mixture. Simultaneously, fluorescent derivatization was performed via reductive amination. The mixture was incubated at 40°C for 2 hours for derivatization.

[0066] Subsequently, 10 μL of the derivatization mixture was diluted with 20 μL of ultrapure water and 120 μL of pure acetonitrile. The supernatant was collected after centrifugation for HPLC analysis. Chromatographic separation was performed using a HALO Glycan column (2.7 μm, 4.6 mm × 150 mm). Detection was performed at an excitation wavelength of 330 nm and an emission wavelength of 420 nm. The peak positions of lactose, 3′-sialyllactose, and 6′-sialyllactose were different. The product of CstI M8 mutant-catalyzed sialylation of the lactose substrate had the same peak position as 3′-sialyllactose, while no product was visible at the peak position of 6′-sialyllactose.

[0067] Figure 4 The results showed that the CstI M8 mutant only added α-2,3-sialic acid glycosidic bonds, instead of 2,6-sialic acid glycosidic bonds.

[0068] Example 3 This embodiment describes the enzyme kinetics determination of the sialyl transferase mutant from Example 2.

[0069] Enzyme kinetics were determined using high-performance liquid chromatography (HPLC) at 37°C in 20 mM HEPES buffer (pH 7.0). Initial rates were measured by maintaining one substrate at saturation (30 mM) while varying the concentration of another substrate (CMP-Neu5Ac donor or Lactose acceptor) (0–30 mM). Acceptor substrate, donor substrate, and pH buffer were pre-prepared in PCR tubes and equilibrated in a PCR instrument at 37°C. The purified enzyme was added to initiate the reaction, which was immediately and thoroughly mixed and incubated at 37°C for 10 min. The reaction was then terminated by adding 2-aminobenzamide (2-AB) solution (dissolved in 72% acetonitrile and 28% acetic acid) and 2-pyridineborane (2-PB) solution (dissolved in methanol) in a 1:1:1 (v / v / v) ratio to the reaction mixture. Fluorescence derivatization was performed simultaneously by reductive amination. Incubate at 40°C for 2 hours to carry out derivatization.

[0070] Subsequently, 10 μL of the derivatization mixture was diluted with 20 μL of ultrapure water and 120 μL of pure acetonitrile. The supernatant was collected after centrifugation for HPLC analysis. Chromatographic separation was performed using a HALO Glycan column (2.7 μm, 4.6 mm × 150 mm). Fluorescently labeled glycans were detected at an excitation wavelength of 330 nm and an emission wavelength of 420 nm.

[0071] The results are shown in Table 1 and... Figure 5 As shown, the results indicate that, compared to the wild-type (WT), the M8 mutant has a higher conversion number and can catalyze the production of more products from the substrate. It significantly enhances catalytic activity and enzyme activity for different substrates (CMP-Neu5Ac donor or Lactose acceptor).

[0072] Table 1: Determination of enzyme kinetic parameters of CstI wild-type 1-285 (WT) and M8 mutant.

[0073]

[0074] Example 4 This embodiment tests the substrate hydrolase activity of CstI1-285 wild type and its mutant M8.

[0075] Many microbial sialyl transferases, such as PmST1, possess inherent donor hydrolysis activity for the donor substrate CMP-Sia, meaning they catalyze the hydrolysis of CMP-Sia to produce free sialic acid and CMP without relying on the acceptor. This side reaction consumes valuable donor substrate, reducing the yield of the target product. For certain difficult-to-catalyze acceptor substrates, the donor hydrolysis problem of sialyl transferases is particularly prominent, becoming one of the key bottlenecks limiting their application.

[0076] PmST1, an α2-3 sialyl transferase derived from Pasteurella multocida, is a bacterial sialyl transferase with broad substrate heterogeneity, capable of catalyzing a variety of CMP-sialic acid donors and different types of acceptor substrates. However, wild-type PmST1 exhibits significant donor hydrolytic activity and sialidase activity, limiting its application in the synthesis of complex sugar substrates. To address this issue, researchers (ACS Chem. Biol. 2012, 7, 1232) A single-point mutant of PmST1, PmST1 (M144D), was designed (1240), which significantly reduced the hydrolytic activity of the donor substrate and the sialidase activity of the product, while basically retaining the α2-3 sialic acid transfer activity, thus becoming a highly efficient catalyst.

[0077] To evaluate the hydrolytic activity of the CstI M8 mutant of this invention on CMP-sialic acid donor substrates, the following experiments were conducted using wild-type CstI 1-285 (WT) and PmST1 (M144D) as controls: Three groups of 4.5 mM CMP-Sia solutions were prepared, and CstI 1-285 wild-type, CstI M8 mutant, and PmST1 (M144D) were added respectively, with a final concentration of 0.04 mg / mL for each enzyme. During the reaction, samples were taken every 30 minutes, and the remaining concentration of CMP-Sia was detected by HPLC at a wavelength of 272 nm. The experimental results showed that ( Figure 6The hydrolytic activity of both wild-type CstI1-285 and the CstI M8 mutant on CMP-Sia donor substrates was significantly lower than that of PmST1(M144D). Under the same reaction conditions, PmST1(M144D) hydrolyzed approximately 80% of 4.5 mM CMP-Sia within 60 minutes, while wild-type CstI1-285 and the CstI M8 mutant hydrolyzed only approximately 30% of CMP-Sia within the same timeframe. These results indicate that the CstI M8 mutant possesses lower donor hydrolytic activity and has a significant advantage in the enzymatic synthesis of sialylated products.

[0078] Example 5 This embodiment tests the ability of the sialyltransferase mutant sialylated complex oligosaccharide G2 (G2-Glycan, glycogene.com, catalog number: GN-3007).

[0079] The reaction was carried out at 37°C in 20 mM Tris buffer (pH 9.0) for 30 minutes. The final concentrations of each component in the reaction system were: 1 mM G2-glycan, 0.1 g / L CstI M8 purified enzyme, 5 mM CMP-Sia, and 20 mM Tris pH 9.0. The acceptor substrate, donor substrate, and pH buffer were pre-prepared in PCR tubes and equilibrated in a PCR instrument at 37°C. The purified enzyme was added to initiate the reaction, and the mixture was immediately and thoroughly mixed and incubated at 37°C for 30 minutes. The reaction was then terminated by adding 2-aminobenzamide (2-AB) solution (dissolved in 72% acetonitrile and 28% acetic acid) and 2-pyridineborane (2-PB) solution (dissolved in methanol) in a 1:1:1 (v / v / v) ratio to the reaction mixture. Simultaneously, fluorescent derivatization was performed by reductive amination. The mixture was incubated at 40°C for 2 hours for derivatization. The sialylation of G2 oligosaccharides was then analyzed using HPLC fluorescence (FLR). Figure 7 As shown, Figure 7 In the figure, 'a' represents the chromatogram of the unmodified G2 polysaccharide control (substrate only). Figure 7 In the figure, b is a chromatogram of the reaction mixture after sialylation of the M8 mutant, indicating complete conversion to the sialylated product. The results show that the M8 mutant provided by this invention has the ability to sialylate complex oligosaccharides.

[0080] Example 6 This embodiment tests the conversion effect of sialyltransferase mutant M8 on sialyl glycosylation of asialofetuin.

[0081] Specifically, in 20 mM Tris-HCl (pH 9.0) and 100 mM NaCl, 10 mg / mL -1 A solution of asialofetuin was prepared at a concentration of [specific concentration not specified]. Sialization (150 μL) consisted of 1 mg asialofetuin, 60 mM CMP-sialic acid, and 50 μg CstI M8 (same as in Example 2), and was incubated at 37 °C for 4 hours. After ultrafiltration to remove small molecules, His-labeled CstI M8 was removed using Ni-NTA resin to obtain sialofetuin.

[0082] Method for detecting glycan chains in glycoprotein substrates and glycoprotein products: An N-glycan kit (manufacturer: HanHai New Enzymes (www.hzymes.com), catalog number: HBP003106L) is used to release and label N-glycans in glycoproteins or antibodies. Refer to the instruction manual for specific procedures. In short, adjust the sample to a concentration of 2 mg / mL. - ¹, the sample was incubated with PNGase F at 50°C for 10 min, and then labeled for 15 min at 65°C with a mixture of 2-AB (2-aminobenzamide) / labeling reducing agent solution / formic acid (1:1:1) to obtain 2-aminobenzamide-labeled glycans, followed by elution in 75% acetonitrile. The sample was diluted for HPLC analysis. Chromatographic separation was performed using an ACQUITY Glycan BEH Amide column. The HPLC buffer conditions were: ammonium formate buffer (50 mM, pH 4.4) (aqueous phase) and acetonitrile (organic phase) as the mobile phase, with a flow rate of 0.4 mL / min. The concentration gradient was achieved by increasing the aqueous phase from 25% to 46% over a 35-minute period, followed by washing the column with high aqueous phase and reequilibrating to 25% aqueous phase. The fluorescently labeled glycans were detected at an excitation wavelength of 330 nm and an emission wavelength of 420 nm. Based on the LC-MS data, the types and proportions of glycans represented by each absorption peak were distinguished.

[0083] Results reference Figure 8 As shown, Figure 8 The LC-MS analysis results show that the highly active mutant M8 of this invention exhibits excellent catalytic ability towards complex glycoprotein substrates. In the fields of modern biopharmaceuticals and protein engineering, the glycosylation state of proteins (such as recombinant antibodies, fusion proteins, and various cytokines), especially the integrity of terminal sialylation, directly determines the in vivo pharmacokinetic properties and clinical efficacy of the drug molecule.

[0084] When the galactose residues at the ends of glycoproteins are exposed (e.g.) Figure 8Asialofetuin, a macromolecule rich in G2 and G3 glycans, is readily recognized by the asialofetuin receptor (ASGPR) on the surface of liver cells, leading to rapid endocytosis and clearance of the drug in the bloodstream. Completely covering the glycan ends with sialic acid molecules via in vitro enzymatic reactions effectively masks galactose clearance signals, significantly prolongs the serum half-life of therapeutic proteins, enhances protein conformational stability, and reduces their potential immunogenicity in vivo.

[0085] The complex N-glycan chains with multiple tentacles on the surface of natural glycoproteins present certain steric hindrances, making it difficult for existing wild-type sialyltransferases to achieve complete end-capping modification during in vitro catalysis (e.g., Figure 8 (The incompletely modified state is shown). The M8 mutant of this invention can achieve nearly 100% complete sialylation of the biantennary and triantennary N-glycan chains (e.g., Figure 8 (As shown).

[0086] More importantly, such as Figure 8 As shown, the M8 mutant also exhibits extremely high catalytic conversion rates for non-natural sialic acid donors carrying orthogonal functional groups such as azides. This characteristic enables the installation of "click-handles" on the surface of intact protein macromolecules, thus providing an enzymatic tool for applications such as peptide targeted delivery and medical molecular imaging probes. Example 7 This embodiment tests the activity of the sialyltransferase mutant M8 on the non-natural substrate CMP-9 azidesialyl.

[0087] To evaluate the biocatalytic performance on complex substrates, LC-MS was used to detect the ability of the glycoprotein substrate asialofetuin to undergo α-2,3-azidosialylation catalyzed by the CstI M8 enzyme. A schematic diagram of the CstI M8-based azidosialylation of galactose-modified glycoproteins and azido-dependent fluorescent labeling is shown below. Figure 10 As shown.

[0088] Specifically, in 20 mM Tris-HCl (pH 9.0) and 100 mM NaCl, 10 mg / mL -1A solution of asialofetuin was prepared at a concentration of [specific concentration not specified]. Sialization reaction (150 μL) contained 1 mg asialofetuin, 60 mM CMP-sialic acid or 30 mM CMP-9 azide-sialic acid, and 50 μg CstI M8 (same as in Example 6), and was incubated at 37 °C for 4 hours. After ultrafiltration to remove small molecules, His-labeled CstI M8 was removed using Ni-NTA resin. The resulting protein (approximately 6 mg mL⁻¹) was reacted with Cy5-DBCO (1 mM) at 37 °C for 10 hours. The product was analyzed by SDS-PAGE and in-gel fluorescence imaging, followed by Coomassie staining.

[0089] like Figure 9 As can be seen, the M8 mutant also exhibited highly efficient catalytic ability using the non-natural donor CMP-9-azidosialic acid. Subsequent Cy5-DBCO labeling and in-gel fluorescence imaging confirmed that these biological orthogonal handles were indeed mounted on the glycoprotein.

[0090] These results fully demonstrate the broad substrate tolerance, enhanced catalytic activity, and improved conversion rate of the CstI M8 mutant enzyme.

[0091] In other embodiments: Based on the above-mentioned highly efficient catalytic ability for non-natural donor substrates (such as CMP-9-azidosialic acid), the CstI M8 mutant of the present invention can be further widely applied to glycan editing and in-situ labeling on the surface of living cells or biological tissues. Specifically, terminal galactose (Gal) glycans are widely distributed on the surface of animal cells or pathological tissues. Utilizing the extremely high catalytic efficiency and steric hindrance tolerance of the CstI M8 mutant of the present invention, non-natural sialic acid with bioorthogonal groups such as azides can be specifically transferred to galactose residues on the surface of living cells or tissues under mild physiological conditions, thereby artificially introducing a click chemistry handle on the cell surface. Subsequently, using copper-free click chemistry (SPAAC), fluorescent labels, magnetic resonance contrast agents, affinity tags (such as biotin), or therapeutic drug molecules can be precisely coupled to the cell surface.

[0092] The potential application value of this typical application method in basic life sciences and translational medicine: (1) Molecular microscopy and medical diagnosis: It can achieve efficient in situ fluorescent labeling of galactose sites on the surface of cells or tissue sections, which can be used for screening of disease-specific glycotype markers, pathological diagnosis and precise tracing of tumor boundaries; (2) Live cell tracing and dynamic monitoring: Non-destructive live cell detection and dynamic tracking can be achieved by attaching fluorescent or other probes to the cell surface; (3) Engineered cell therapy and targeted delivery: This technology can be used as a tool to construct cell-drug conjugates, and to modify small molecule drugs on the surface of living target cells, providing technical support for novel targeted therapy or immune cell therapy.

[0093] Example 8 This example is a specific embodiment of M8 in prolonging the half-life of glycoproteins in mice.

[0094] To verify the effect of CstI M8 on the serum half-life of the glycoprotein fetuin after sialylation, we conducted the following experiments: To quantify the glycoprotein, both sialylated fetuin obtained using CstI M8 catalysis and unsialylated fetuin were covalently labeled with Alexa Fluor 647 (AF647). After injecting the fluorescently labeled proteins into mice via tail vein, peripheral blood samples were collected at specified time points, and the concentration of the glycoproteins was determined by measuring fluorescence intensity.

[0095] The results showed that unsialylated fetuin was rapidly cleared in vivo, with plasma concentrations decreasing sharply within the first 10 minutes after injection. In contrast, fetuin modified with CstI M8 enzymatic α-2,3-sialylation exhibited a significantly prolonged serum retention time, with significantly higher concentrations detected at all corresponding time points (see [link to study]. Figure 11 Quantitative fitting of the concentration-time curves revealed that the half-life of unsialylated fetuin was approximately 3.2 minutes. In contrast, fetuin modified with CstI M8 sialylation exhibited a prolonged half-life of 13.9 minutes and a 4.3-fold improvement in cycling stability. These findings demonstrate that enzymatic α-2,3-sialylation alone is sufficient to significantly delay the in vivo clearance of glycoproteins without the need for additional chemical modifications. This method is simple to perform and highly specific, highlighting the significant potential of the CstI α-2,3-sialyltransferase mutant M8 in regulating the in vivo half-life of glycoproteins.

[0096] Example 9 This embodiment tests the conversion effect of sialyltransferase mutant M8 on protein substrate sialyl glycosylation.

[0097] The sialyltransferase mutant M8 sialylated G2F-glycoform monoclonal antibody Belimumab (manufacturer: SparkJade, catalog number: SJ-BA1021-1mg*5) was used.

[0098] To evaluate the potential of the M8 variant in therapeutic antibody glycoengineering, the widely used monoclonal antibody Belimumab was selected as the model substrate. Belimumab, a fully human monoclonal antibody specifically targeting BLyS, was the first antibody to receive biological approval for the treatment of systemic lupus erythematosus (SLE) and lupus nephritis, and has been widely used to treat various rheumatic diseases.

[0099] This invention uses bovine β-1,4-galactosyltransferase 1 (β4GalT1) to completely convert Belimumab to the galactosylated G2F glycoform. Subsequently, it is sialylated using the CstI M8 variant. After a 12-hour reaction (with CMP-sialic acid feeding every 4 hours), approximately 50% monosialylated and 27% disialsialylated Belimumab were obtained. Figure 12 Under the same conditions, CstI1-285 wild-type (i.e., WT) could only achieve about 8% monosialylation and nearly 0% disialylation. The method for testing the product glycoform was the same as in Example 6.

[0100] This indicates that the CstI variant M8 can be effectively applied to the modification of the glycan chain in the Fc region of IgG antibodies, laying the foundation for its use in modifying antibody performance.

[0101] Example 10 Intravenous immunoglobulin (IVIG) is a polyclonal IgG preparation extracted from the plasma of thousands of healthy donors and is widely used to treat immunodeficiency and various serious autoimmune diseases (such as Kawasaki disease and idiopathic thrombocytopenic purpura). Modern immunological research shows that the key anti-inflammatory activity of IVIG is highly dependent on the sialylation modification of the N-terminus of its Fc segment. Naturally extracted IVIG glycoforms exhibit heterogeneity (mainly galactose-deficient G0F and G1F), with a low proportion of sialylated glycoforms possessing anti-inflammatory activity. Furthermore, the Fc segment of the antibody is deeply embedded within the lumen of two heavy chains, resulting in significant steric hindrance. This invention utilizes the CstI M8 variant, which possesses high catalytic activity and a broad substrate spectrum, to successfully achieve sequential glycoengineering remodeling of human IVIG. The two-step enzymatic remodeling process and its glycoform distribution changes are as follows: Figure 13 As shown: Experimental Methods: The initial human intravenous immunoglobulin (IVIG) consisted of a heterogeneous mixture of G0F, G1F, and G2F glycoforms. First, complete galactosylation was catalyzed by β4GalT1 in the presence of UDP-Gal, yielding a homogeneous G2F intermediate. In subsequent steps, the CstI M8 variant was further sialylated using CMP-Neu5Ac to generate G2FS1 and G2FS2 substrates. Procedure Figure 13 The relative abundance of the final glycoform is noted in the annotation. The detection methods for the initial glycoform of IVIG, the glycoform of the G2F intermediate, and the glycoforms of the G2FS1 and G2FS2 products are the same as in Example 6.

[0102] This application example demonstrates that the CstI M8 mutant of this invention possesses outstanding practicality in the modification of extremely complex clinical antibody mixtures. Its application value lies in: through highly efficient in vitro enzymatic glycan reconstruction, it can increase the sialic acid abundance in natural IVIG products, providing a potential tool for IVIG performance optimization; and it proves that the CstI M8 mutant of this invention can achieve a certain degree of sialylation in challenging antibody substrates, such as human IVIG.

[0103] Experimental Example 11 This experimental example tests the enzyme activity of introducing a stop codon at any position between positions 274 and 285 of α-2,3-sialyltransferase.

[0104] Reference data on the relative enzyme activity of purified CstI truncated variants and CstI1-285 wild type Figure 14 As shown, the test method is the same as in Example 1.

[0105] SDS-PAGE analysis of purified CstI truncated variants and their relative enzyme activities Figure 14 Figure a shows SDS-PAGE of purified CstI variants with different C-terminal truncations. The purity of all variants was observed to be comparable.

[0106] The relative enzyme activities of different C-terminally truncated CstI variants were measured under the same reaction conditions. Figure 14 Figure b in the table shows the activity relative to the wild-type enzyme, representing the average of independently measured values. All activity comparisons have been normalized to the same protein concentration. The results show that introducing a stop codon at any position between positions 274 and 285 of α-2,3-sialyltransferase increases its activity.

[0107] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An α-2,3-sialyltransferase mutant, characterized in that, It is based on the amino acid sequence shown in SEQ ID NO.1 and involves at least one of the following mutations: (1) T188E; (2) A210C; (3) Introduce a stop codon at any position from the 274th to the 285th position; (4) Mutate the 190th amino acid; (5) F65S; and (6) F284L; Among them, the 190th amino acid has any of the following mutations: F190M, F190Q, F190A, and F190V.

2. The α-2,3-sialyltransferase mutant according to claim 1, characterized in that, The α-2,3-sialyltransferase mutant has any one of the following mutations: (1) T188E and A210C, and the α-2,3-sialyltransferase mutant has any of the following mutations: F190M, F190Q, F190A and F190V; (2) T188E and A210C, and deletion mutations of amino acids at positions 277-285; (3) For the deletion mutation of amino acids at positions 277-285, the α-2,3-sialyltransferase mutant also has any of the following mutations: F190M, F190Q, F190A and F190V; (4) Introduce a stop codon at the 279th position; (5) Introduce a stop codon at the 275th position; (6) Introduce a stop codon at the 277th position; (7) Introduce a stop codon at the 278th position; (8) Introduce a stop codon at the 280th bit; Preferably, the α-2,3-sialyltransferase mutant further has at least one of the following mutations: E9R, Y33H, R35M, D41A, Y54A, T76I, A77R, I88F, D105M, E129F, F141S, R144E, G148M, K161M, Y164T, D195G, N200A, E209R, L213E, N211E, N233G, S238V, N240P, and amino acid deletion mutations at positions 266-275.

3. A nucleic acid molecule, characterized in that, It encodes the α-2,3-sialic acid transferase mutant according to any one of claims 1-2.

4. A recombinant vector, characterized in that, It includes the nucleic acid molecule as described in claim 3.

5. A recombinant bacterium, characterized in that, It includes the nucleic acid molecule of claim 3 or the recombinant vector of claim 4.

6. Use of the α-2,3-sialyltransferase mutant according to any one of claims 1-2, the nucleic acid molecule according to claim 3, the recombinant vector according to claim 4, or the recombinant bacteria according to claim 5 in any one of the following: (1) Sialylation of monosaccharides, oligosaccharides or sugar chains; (2) Sialylation of protein substrates or lipids containing monosaccharides, oligosaccharides or glycans; (3) Azide sialic acid glycosylation of protein substrates or lipids or other biomacromolecules containing monosaccharides, oligosaccharides or glycans for labeling glycans on cell surfaces or for dynamic tracking and imaging in living cells.

7. The use of the α-2,3-sialyltransferase mutant as described in any one of claims 1-2, the nucleic acid molecule as described in claim 3, the recombinant vector as described in claim 4, or the recombinant bacteria as described in claim 5 in the preparation of a drug or formulation for prolonging the half-life of a drug or formulation; Preferably, the drug is selected from blood-derived VWF concentrate, recombinant VWF, recombinant human erythropoietin and its derivatives, α-1-antitrypsin, TNFR-IgG immunoadhesion molecules, recombinant thrombomodulin, or human intravenous immunoglobulin IVIG, and the formulation is selected from a model protein, wherein the model protein is selected from AsialoFetuin.

8. A method for sialyl glycosylation of a substrate, characterized in that, It includes the following steps: mixing the α-2,3-sialyltransferase mutant of any one of claims 1-2 or the recombinant bacteria of claim 5 with a recipient substrate and a donor substrate, wherein the recipient substrate is a monosaccharide, oligosaccharide, or polysaccharide containing galactose (Gal) or N-acetylgalactosamine (GalNAc), or a glycoprotein or glycolipid macromolecule containing the above sugars, and the donor substrate is CMP-sialic acid or a derivative thereof.

9. The method according to claim 8, characterized in that, The donor substrate is selected from CMP-9 azidosialic acid.

10. The method according to claim 8, characterized in that, The conditions for sialic acid glycosylation include a pH of 4.0 to 11.0, a temperature of 0 to 50 degrees Celsius, a donor substrate concentration of 0.01 mM to 200 mM, an acceptor substrate concentration of 0.01 mM to 1000 mM, and an incubation time of 0.1 hours to 24 hours.

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