Modified glycosyl transferase and application thereof in improvement of heparin synthesis stability and catalytic efficiency

Abstract

The invention discloses modified glycosyl transferase and application thereof in improvement of heparin synthesis stability and catalytic efficiency, and belongs to the technical field of enzyme engineering. According to the present invention, on the basis of the heparin synthase 2 (PmHS2) derived from the Pasteurella multocida strain (Pasteurella multocida), the N end is subjected to truncation so as to construct the enzyme mutant PmHS2, such that the enzyme mutant PmHS2 can be constructed, the truncated mutant enzyme prepared by the invention has the characteristics of high expression level and high catalytic efficiency, and enzymatic property analysis on the truncated mutant enzyme finds that compared with a wild enzyme, the truncated mutant enzyme has better tolerance to high temperature and extreme acid pH and has faster conversion rate within the same reaction time. The invention lays a foundation for rational design and practical application of the enzyme and enzyme engineering production of heparin.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, and in particular to an N-terminal truncated mutant enzyme of a glycosyltransferase heparin skeleton synthase PmHS2 and an application thereof in improving the stability and catalytic activity of the enzyme in a sugar chain extension process. Background Art

[0002] Glycosaminoglycan (GAG) is a complex biomacromolecule, a linear anionic acidic polysaccharide composed of repeating disaccharide units composed of hexuronic acid or hexose and hexosamine arranged in sequence. Glycosaminoglycans are of various types and widely distributed. According to the composition, connection mode and sulfation pattern of sugar units, they can be divided into four categories: hyaluronic acid, dermatan sulfate / chondroitin sulfate, heparan sulfate / heparin and keratan sulfate. Glycosaminoglycans are widely present in animal tissue cells in the form of free or bound to core proteins, and play important physiological roles, such as controlling cell proliferation and differentiation, regulating cell-to-cell interactions, etc. Therefore, they are widely used in the fields of medicine, food, and beauty.

[0003] Heparin, as an important glycosaminoglycan, is a mucopolysaccharide sulfate formed by alternating repeating disaccharide units composed of D-β-glucuronic acid (or La-iduronic acid) and aminoglucose. Heparin has been discovered for more than a hundred years and is widely used as an anticoagulant and antithrombotic drug. It has the function of enhancing the activity of the natural serine protease inhibitor antithrombin III, and can catalyze and inhibit all serine proteases in the endogenous coagulation pathway. Therefore, it has anticoagulant and antithrombotic effects. It can also mediate important physiological processes such as vascular coagulation, angiogenesis, development and viral infection by binding to other proteins. It also has the functions of lowering blood lipids, anti-inflammatory, and inhibiting bacterial adhesion. It can also be used clinically to treat diseases such as angina pectoris, nephrotic syndrome, severe burns, rheumatoid arthritis, etc. Its demand ranks among the top in the field of international biotechnology drugs.

[0004] The sources of heparin are mainly divided into animal extraction, chemical synthesis and chemical enzymatic synthesis. Animal-extracted heparin is mainly extracted from the small intestinal mucosa of pigs and cattle, but due to the differences in extraction methods and the fact that the structure and chain length of the basic unit of heparin from animals are easily affected by factors such as animal species, age, health status and extraction site, this may lead to poor uniformity of the extracted heparin chain length and unclear structure, which may cause heparin contamination. In addition, the structure of heparin is relatively complex. Although the sugar chain structure can be precisely controlled when the sugar chain is extended by chemical synthesis, the synthesis conditions are harsh and there are many steps. It is necessary to consider protection, deprotection, activation and coupling steps, as well as regional selectivity and stereoselectivity. It is difficult, has low yield and is not environmentally friendly. For example, the chemical synthesis of fondaparinux sodium pentasaccharide requires more than 60 steps of reaction, and the yield is only 0.1%. In contrast, the chemical enzymatic synthesis of heparin is a combination of chemical and enzymatic methods. The enzyme reaction conditions required for the synthesis process are mild, specific, simple and efficient.

[0005] PmHS2 is a bifunctional enzyme with both N-acetylglucosamine transferase activity and glucuronyl transferase activity, and has both the N-terminal GT2 family β1-4-glucuronyl aminotransferase domain (GlcA-T) and the C-terminal GT45 family α1-4-N-acetylglucosamine transferase domain (GlcNAc-T). Since PmHS2 is easy to express in Escherichia coli, it has become the preferred enzyme for the chemoenzymatic synthesis of heparin. Further improving the catalytic activity, stability and tolerance of the enzyme will help to more efficiently synthesize the heparin backbone sugar chain. Summary of the invention

[0006] Purpose of the invention: In view of the deficiencies of the prior art, the present invention provides a modified glycosyltransferase. Compared with the wild-type enzyme, the modified glycosyltransferase has better tolerance to high temperature and extremely acidic pH and a faster conversion rate within the same reaction time, laying a foundation for the rational design and practical application of carbohydrases and the production of heparin by enzyme engineering.

[0007] The present invention also provides application of the modified glycosyltransferase.

[0008] Technical solution: To achieve the above-mentioned purpose of the invention, the present invention discloses a modified glycosyltransferase, wherein the modified glycosyltransferase is based on heparin synthase PmHS2 and the N-terminal is truncated to obtain a mutant enzyme △PmHS2, and the amino acid sequence of the mutant enzyme △PmHS2 is shown in any one of SEQ ID NO.3-6.

[0009] The modified glycosyltransferase encoding gene of the present invention is characterized in that the encoding gene is represented by any nucleotide sequence of SEQ ID NO.7-10.

[0010] The recombinant vector of the present invention contains the coding gene of the modified glycosyltransferase.

[0011] The recombinant vector is based on the plasmid vector pET-15b, and the coding gene of the modified glycosyltransferase is inserted into the plasmid vector.

[0012] The recombinant bacteria of the present invention and the recombinant vector thereof.

[0013] The recombinant bacteria uses Escherichia coli as a host cell, and the recombinant vector is transformed into the host cell to obtain the recombinant bacteria.

[0014] The modified glycosyltransferase or the encoding gene of the modified glycosyltransferase or the recombinant vector or recombinant bacteria of the present invention are used in the process of sugar chain synthesis.

[0015] The modified glycosyltransferase or the modified glycosyltransferase encoding gene or the recombinant vector or recombinant bacteria of the present invention are used in the synthesis of heparin backbone sugar chains.

[0016] The modified glycosyltransferase of the present invention or the coding gene of the modified glycosyltransferase or the recombinant vector or recombinant bacteria are used in heparin synthesis.

[0017] The application uses p-nitrophenyl-β-D-glucuronic acid (GlcA-pNP), GlcNAc-GlcA-pNP or other glucuronic acid derivatives as acceptor substrates; and uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) or its derivatives as donor substrates.

[0018] The invention provides a modified glycosyltransferase: a highly active heparin synthase PmHS2 truncated mutant enzyme ΔPmHS2, wherein the N-terminal amino acid sequence is truncated.

[0019] Preferably, the heparin synthase mutant is an enzyme having an amino acid sequence as shown in SEQ ID NO.1 and a nucleotide sequence as shown in SEQ ID NO.2, subjected to at least one of the mutations (1) to (4):

[0020] (1) Selecting to truncate the N-terminal positions 1 to 84, and finally obtaining the truncated mutant enzyme Δ84PmHS2, the amino acid sequence of which is shown in SEQ ID NO.4;

[0021] (2) truncating the N-terminal positions 1 to 89 to obtain the truncated mutant enzyme Δ89PmHS2, whose amino acid sequence is shown in SEQ ID NO.5;

[0022] (3) Selecting to truncate the N-terminal positions 1 to 93, and finally obtaining the truncated mutant enzyme △93PmHS2, whose amino acid sequence is shown in SEQ ID NO.6;

[0023] (4) The N-terminal positions 1 to 96 were truncated to obtain the truncated mutant enzyme Δ96PmHS2, whose amino acid sequence is shown in SEQ ID NO.3.

[0024] Further preferably, the heparin synthase mutant is obtained by subjecting the enzyme having the amino acid sequence shown in SEQ ID NO.1 to the mutation (4), wherein the amino acid sequence of the mutant enzyme with 96 amino acids truncated at the N-terminus is as shown in SEQ ID NO.3, encoding 557 amino acids.

[0025] Preferably, the application is to use p-nitrophenyl-β-D-glucuronic acid (GlcA-pNP) or a glucuronic acid derivative as an acceptor substrate when constructing a disaccharide; and to use uridine diphosphate-N-acetylglucosamine or its derivative (UDP-GlcNAc) as a donor substrate, specifically including uridine diphosphate-N-acetylglucosamine, including 4-azido-uridine diphosphate-N-acetylglucosamine, 4-amino-uridine diphosphate-N-acetylglucosamine, 4-fluoro-uridine diphosphate-N-acetylglucosamine, 4-thiol-uridine diphosphate-N-acetylglucosamine, 3-azido-uridine diphosphate-N-acetylglucosamine, 3-amino-uridine diphosphate-N-acetylglucosamine The invention relates to one or more of glucose, 3-fluoro-uridine diphosphate-N-acetylglucosamine, 3-thiol-uridine diphosphate-N-acetylglucosamine, 2-azido-uridine diphosphate-N-acetylglucosamine, 2-amino-uridine diphosphate-N-acetylglucosamine, 2-fluoro-uridine diphosphate-N-acetylglucosamine, 2-thiol-uridine diphosphate-N-acetylglucosamine, 6-azido-uridine diphosphate-N-acetylglucosamine, 6-amino-uridine diphosphate-N-acetylglucosamine, 6-fluoro-uridine diphosphate-N-acetylglucosamine, and 6-thiol-uridine diphosphate-N-acetylglucosamine; the molar ratio of the glycosyl acceptor to the glycosyl donor is 2:1 to 1:5.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0027] The present invention is based on heparin skeleton synthase PmHS2 for the first time. Through specific amino acid analysis and design, the amino acid at the N-terminus of PmHS2 is selected to be truncated to obtain a truncated mutant enzyme △PmHS2, wherein the truncated enzyme △PmHS2 (△96PmHS2, △PmHS2 in the following text refers to △96PmHS2 unless otherwise specified) is finally obtained by truncating from position 1 to position 96. Through induced expression and enzymatic property analysis, it is found that compared with the wild-type enzyme, the enzyme has better tolerance to high temperature and extremely acidic pH, and has a faster conversion rate within the same reaction time. The present invention lays a foundation for the rational design and practical application of carbohydrases and the production of heparin by enzyme engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the plasmid map of pET15b-PmHS2;

[0029] Figure 2 is the SDS-PAGE image of purified PmHS2 and Δ96PmHS2;

[0030] Figure 3 is the thin layer chromatogram of the supernatant after the disaccharide reaction catalyzed by △PmHS2;

[0031] Figure 4 This is the reaction formula for disaccharide synthesis catalyzed by heparin skeleton synthase;

[0032] Figure 5 It is a comparison chart of the conversion rate of disaccharide reaction catalyzed by PmHS2 and △PmHS2 within 6 hours;

[0033] Figure 6 It is a comparison chart of the conversion rate of disaccharide reaction catalyzed by △84PmHS2, △89PmHS2, △93PmHS2 and △96PmHS2 after 6 hours;

[0034] Figure 7 It is a comparison chart of pH stability of PmHS2 and △PmHS2;

[0035] Figure 8 It is a comparison chart of the temperature stability of PmHS2 and △PmHS2;

[0036] Fig. 9 It is the reaction formula of the trisaccharide reaction catalyzed by heparin backbone synthase;

[0037] Fig.10 It is a stacked line graph of the relative conversion rate of trisaccharide reaction catalyzed by Pm8S2 and △PmHS2 within 6 hours;

[0038] Fig.11 It is a trisaccharide synthesis reaction formula of a dual enzyme cascade catalyzed by glucose dehydrogenase and heparin skeleton synthase;

[0039] Fig.12 It is a comparison chart of the conversion rate of trisaccharide reaction in 24 hours of dual enzyme cascade catalyzed by glucose dehydrogenase and heparin skeleton synthase;

[0040] Fig.13 It is a disaccharide synthesis reaction formula of a three-enzyme cascade catalyzed by N-acetylhexosamine kinase, glucosamine-1-phosphate uridyl transferase and heparin skeleton synthase;

[0041] Fig.14 It is a comparison chart of the conversion rate of disaccharide reaction in 6 hours in the three-enzyme cascade catalyzed by N-acetylhexosamine kinase, glucosamine-1-phosphate uridyltransferase and heparin skeleton synthase. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0043] The reagents used in the examples of the present invention, unless otherwise specified, can be obtained from commercial sources. Experimental methods without specific conditions in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0044] Experimental operations not described in detail in the present invention can be carried out according to conventional experimental operations in the art. UDP-GlcNAc: purchased from Sigma, catalog number: U4375, CAS: 91183-98-1,

[0045]

[0046] GlcA-pNP: purchased from Sigma, catalog number: N1627, CAS: 10344-94-2,

[0047]

[0048] GlcNAc-GlcA-pNP: A mixture containing 100 mM pH 7.5 Tris-HCl, 10 mM MnCl2, 5 mM UDP-GlcA, 5 mM GlcA-pNP and excess PmHS2 was incubated at 37°C for 12 hours to synthesize heparin disaccharide GlcNAc-GlcA-pNP. After centrifugation, the supernatant was purified by preparative chromatography, and the product was analyzed by mass spectrometry (MS) and high-performance liquid chromatography (HPLC). For details of the preparation, see the literature: Fabricating Bimetal Organic Material Capsules with aCommodious Microenvironment and Synergistic Effect for Glycosyltransferas, ACS Appl. Mater. Interfaces 2022, 14, 22, 26034–26043,

[0049]

[0050] UDP-GlcA: trisodium uridine 5'-diphosphoglucuronic acid, purchased from Sigma, product number: U6751, CAS: 63700-19-6,

[0051]

[0052] UDP-GlcNAc: purchased from Sigma, catalog number: U4375, CAS: 91183-98-1,

[0053]

[0054] N-acetylhexosamine kinase NahK (ANC68241.1) and N-acetylglucosamine uridyltransferase GlmU (ACO75977.1) were artificially synthesized according to the sequences on NCBI and obtained by fermentation according to the literature (Zheng, J.; Xu, H.; Li, BZ; Sohail, M.; Bi, JJ; Zhang, FM; Linhardt, RJ; Huang, H.; Zhang, X. Spatially Segregated MOF Bioreactor Enables Versatile Modular Glycoenzyme Assembly for Hierarchical Glycan Library Construction. ACS Appl. Mater. Interfaces 2023, 15(16): 19807-19816.).

[0055] Culture medium:

[0056] LB medium (liquid): 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0057] TB medium (liquid): 13.33 g / L peptone, 26.67 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 5.61 g / L glycerol.

[0058] In the following examples, the HPLC detection method used used a C18 column from Agilent, and the high performance liquid chromatograph was purchased from Agilent, the instrument model was Agilent 1260-LC, and the ultraviolet absorption of each component at 262nm and 310nm after the above-mentioned heparin skeleton synthase catalyzed product was separated by the chromatographic column was detected.

[0059] Example 1

[0060] Construction of recombinant plasmid pET15b-PmHS2 and truncated recombinant plasmid pET15b-△96PmHS2

[0061] (1) Construction of recombinant plasmid pET15b-PmHS2

[0062] The heparin synthase PmHS2 (GenBank: AAQ55110.1) from Pasteurella multocida, whose amino acid sequence is shown in SEQ ID NO.1 and nucleotide sequence is shown in SEQ ID NO.2, was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and primers Nde1-PmHS2-F and BamH1-PmHS2-R (Table 1) were designed for amplification. Subsequently, the amplified product and the pET-15b vector were double-digested with NdeI and BamHI. The digested products were separated by 1% agarose gel electrophoresis. After purification and recovery, the target gene was ligated to the vector backbone using T4 DNA ligase to finally obtain a recombinant plasmid. The target gene PmHS2 was constructed on the pET-15b plasmid and named pET15b-PmHS2 ( Figure 1 ).

[0063] (2) Construction of truncated recombinant plasmid pET15b-△96PmHS2

[0064] The recombinant plasmid pET15b-PmHS2 constructed in step (1) was used as a template and primers were designed. The primer sequences used are shown in Table 1. Nde1-△96PmHS2-F and BamH1-△96PmHS2-R were used as primers to amplify the target gene △96PmHS2. The PCR product of the target gene and the commercial pET15b plasmid backbone were double-digested with BamH1 and Nde1. The digested products were subjected to 1% agarose gel electrophoresis and purified and recovered. The target gene and the backbone were treated with T4 DNA ligase to obtain the recombinant plasmid pET15b-△PmHS2. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, and the recombinant strain was constructed. The recombinant strain was spread on an LB plate containing ampicillin antibiotics and cultured at 37°C overnight. The positive clone was picked and the plasmid was extracted. After sequencing was correct, the △96PmHS2 positive transformant was obtained by activation.

[0065] The above PCR reaction system: 25ul KOD Buffer, 5ul dNTP, 1ul upstream primer, 1ul downstream primer, 1ul template, 1ul KOD fx, 16ul sterile water.

[0066] PCR reaction conditions: (1) Pre-denaturation: 95°C, 3 min; (2) Denaturation: 95°C, 15 s; (3) Annealing: 55°C, 15 s; (4) Extension: 68°C, 1 min, set 28 to 33 cycles; (5) Final extension stage: 68°C, 5 min. Store at 4°C.

[0067] (3) Construction of truncated recombinant plasmid pET15b-△84PmHS2

[0068] The steps were similar to (2), except that the primers were different: Nde1-△84PmHS2-F and BamH1-△84PmHS2-R, see Table 1;

[0069] (4) Construction of truncated recombinant plasmid pET15b-△89PmHS2

[0070] The steps were similar to (2), except that the primers were different: Nde1-△89PmHS2-F and BamH1-△89PmHS2-R, see Table 1;

[0071] (5) Construction of truncated recombinant plasmid pET15b-△93PmHS2

[0072] The steps are similar to (2), except that the primers are different: Nde1-△93PmHS2-F and BamH1-△93PmHS2-R, see Table 1.

[0073] After sequencing, it was selected to truncate the N-terminal positions 1 to 84, and finally obtained the truncated mutant enzyme △84PmHS2, whose amino acid sequence is shown in SEQ ID NO.4; it was selected to truncate the N-terminal positions 1 to 89, and finally obtained the truncated mutant enzyme △89PmHS2, whose amino acid sequence is shown in SEQ ID NO.5; it was selected to truncate the N-terminal positions 1 to 93, and finally obtained the truncated mutant enzyme △93PmHS2, whose amino acid sequence is shown in SEQ ID NO.6; it was selected to truncate the N-terminal positions 1 to 96, and finally obtained the truncated mutant enzyme △96PmHS2, whose amino acid sequence is shown in SEQ ID NO.3.

[0074] Table 1 Primer sequence list

[0075]

[0076]

[0077] Example 2

[0078] Protein induction expression and purification

[0079] (1) Inducible expression of proteins

[0080] Taking mutant △PmHS2 as an example, a single colony of the positive transformant of mutant △PmHS2 (△96PmHS2) obtained in Example 1 was inoculated into a sterile LB liquid culture medium containing ampicillin, and cultured at a temperature of 37°C and a rotation speed of 220 rpm for 12 h to obtain a seed solution; the seed solution was inoculated into a TB liquid culture medium at an inoculum of 1% (v / v), and cultured in a shaking incubator at 37°C and 220 rpm. When the culture solution OD 600 When the pH reached 0.6-0.8, IPTG (final concentration 0.2 mM) was added, and the culture was carried out overnight in a shaker at 18°C ​​and 200 rpm to obtain a fermentation broth;

[0081] (2) Protein purification

[0082] Take 20 mL of fermentation broth and centrifuge at 12000 rpm for 3 min to collect the cells, and resuspend the cells in 20 mL of pH 8 phosphate buffer, ultrasonically disrupt on ice, and collect the supernatant after centrifugation to obtain △PmHS2 crude enzyme solution;

[0083] Use nickel column to purify through the histidine tag on the protein. After filtering the crude enzyme solution and adding it to the nickel column, wash away the impurities with 5-10 column volumes of washing buffer (20mM imidazole, 0.5M NaCl, 50mM NaH2PO4, pH8), and elute with elution buffer (250mM imidazole, 0.5M NaCl, 50mM NaH2PO4, pH8) to obtain the target protein. The collected target protein was subjected to SDS-PAGE protein electrophoresis. The results are shown in Figure 2 The purified protein was added to glycerol at a final concentration of 20% and stored in a -80°C refrigerator.

[0084] Example 3

[0085] Heparin synthase PmHS2 and △96PmHS2 enzyme activity verification reaction system

[0086] Table 2 Heparin synthase enzyme activity verification reaction system

[0087]

[0088] by Figure 4 The disaccharide reaction shown was used as a model, with commercial GlcA-pNP as the acceptor substrate, UDP-GlcNAc as the donor substrate, and heparin backbone synthase PmHS2 and △96PmHS2 as proteases to react. The conversion rate of the glycosylation reaction was detected within 6 hours of reaction at the optimal reaction temperature of 37°C under the same enzyme concentration (conversion rate = 1-remaining glycosyl donor after reaction / glycosyl donor before reaction). The reaction system is shown in Table 2. The reaction systems in Table 2 were mixed, reacted at 37°C for 6 hours, and then the reaction solution was heated at 95°C for 5 minutes to inactivate the protease to terminate the reaction. The reaction solution was filtered with a 0.22μm filter membrane and 80% methanol: 20% phosphoric acid aqueous solution (0.1% phosphoric acid) was used as the mobile phase. HPLC detection was performed at a flow rate of 1ml / min. The results are shown in Table 2. Figure 5 In the same time, the conversion of △96PmHS2 was faster. At 2h, the conversion rate of △96PmHS2 reached more than 95%. At 6h, the glycosylation conversion rate of △96PmHS2 was 1.3 times that of PmHS2. Figure 3 This is the thin layer chromatogram of the supernatant after the disaccharide reaction catalyzed by △PmHS2.

[0089] Example 4

[0090] Heparin synthase △84PmHS2, △89PmHS2, △93PmHS2 and △96PmHS2 enzyme activity verification reaction system

[0091] Example 4 is the same as Example 3, except that the proteases used are △84PmHS2, △89PmHS2, △93PmHS2 and △96PmHS2, respectively. Figure 6 As shown, with the conversion rate of △96PmHS2 being 100%, the conversion rate of △84PmHS2 is 82.2%, that of △89PmHS2 is 85.6%, and that of △93PmHS2 is 94.5%. The results show that the enzyme activity of △96PmHS2 is the best. In the following examples, △96PmHS2 is the most preferred enzyme, abbreviated as △PmHS2, and is compared with PmHS2.

[0092] In addition, further increasing the number of N-terminal amino acids of the truncated heparin synthase PmHS2 (110 amino acids) will significantly reduce the protease activity of the resulting △PmHS2.

[0093] Example 5

[0094] Study on pH Stability of Heparin Skeleton Synthase PmHS2 and △PmHS2

[0095] Table 3 Reaction system for stability study of heparin skeleton synthase

[0096] UDP-GlcNAc GlcA-pNP <![CDATA[Mn 2+ ]]> Tris-HCl Protein Final concentration 3mM 2.5mM 1mM 50mM 0.14mg / mL

[0097] The commercial GlcA-pNP was used as the acceptor substrate, UDP-GlcNAc as the donor substrate, and the heparin backbone synthase PmHS2 and △PmHS2 as the protease to react. The glycosylation reaction conversion rate was detected at the same enzyme concentration, the optimal reaction temperature of 37°C, and the pH of 2-10 after 15 hours of reaction. The reaction system is shown in Table 3. The reaction system in Table 3 was mixed, and after reacting at 37°C for 15 hours, the reaction solution was heated at 95°C for 5 minutes to inactivate the protease to terminate the reaction. The reaction solution was filtered with a 0.22μm filter membrane and the ratio of 80% methanol: 20% phosphoric acid aqueous solution (0.1% phosphoric acid) was used as the mobile phase. HPLC detection was performed at a flow rate of 1ml / min. The results are shown in Table 3. Figure 7 Compared with PmHS2, △PmHS2 has a wider tolerance to pH, exhibits better tolerance to acidic environments, and also has excellent conversion rate at lower pH (pH2-5).

[0098] Example 6

[0099] Study on the Temperature Stability of Heparin Skeleton Synthase PmHS2 and △PmHS2

[0100] The commercial GlcA-pNP was used as the acceptor substrate, UDP-GlcNAc was used as the donor substrate, and the heparin backbone synthase PmHS2 and △PmHS2 were used as the protease to react. The relative glycosylation reaction conversion rate was detected after 15 hours of reaction at 47°C, which was higher than the optimal temperature of 37°C, at the same enzyme concentration and pH 7. The reaction system is shown in Table 3. The reaction system in Table 3 was mixed, and after reacting at 47°C for 15 hours, the reaction solution was heated at 95°C for 5 minutes to inactivate the protease to terminate the reaction. The reaction solution was filtered with a 0.22μm filter membrane and 80% methanol: 20% phosphoric acid aqueous solution (0.1% phosphoric acid) was used as the mobile phase. HPLC detection was performed at a flow rate of 1ml / min. The results are shown in Table 3. Figure 8 As shown, if the glycosylation reaction conversion rate of △PmHS2 is set to 100%, the glycosylation reaction conversion rate of PmHS2 is only 65.31%. Compared with PmHS2, △PmHS2 has a wider temperature tolerance and shows better tolerance to high temperature environment.

[0101] Example 7

[0102] Activity verification reaction system of heparin skeleton synthase PmHS2 and △PmHS2 catalyzing trisaccharide synthesis

[0103] Table 4 Trisaccharide synthesis reaction system of heparin skeleton synthase

[0104] UDP-GlcA GlcNAc-GlcA-pNP <![CDATA[Mn 2+ ]]> Tris-HCl Protein Final concentration 3mM 2.5mM 1mM 72mM 0.05mg / mL

[0105] by Fig. 9 The trisaccharide reaction shown in the figure was used as a model, with the synthesized GlcNAc-GlcA-pNP as the acceptor substrate, UDP-GlcA as the donor substrate, and the heparin skeleton synthase PmHS2 and △PmHS2 as the protease to react, and the relative conversion rate of the glycosylation reaction within 6 hours of reaction at the optimal reaction temperature of 37°C under the same enzyme concentration was detected. The reaction system is shown in Table 4. The reaction systems in Table 4 were mixed, reacted at 37°C for 6 hours, and then the reaction solution was heated at 95°C for 5 minutes to inactivate the protease to terminate the reaction. The reaction solution was filtered with a 0.22μm filter membrane and the ratio of 85% methanol: 15% phosphoric acid aqueous solution (0.1% phosphoric acid) was used as the mobile phase. HPLC detection was performed at a flow rate of 1ml / min. The results are shown in Table 4. Fig.10 As shown, the conversion rate of △PmHS2 is always much higher than that of PmHS2 within 6 hours, and its conversion rate can reach 100% in 0.5 hours, while that of PmHS2 is less than 40%.

[0106] Example 8

[0107] Dual enzyme cascade: trisaccharide synthesis reaction system catalyzed by glucose dehydrogenase (UDH) and heparin skeleton synthase

[0108] Table 5 Trisaccharide synthesis reaction system catalyzed by glucose dehydrogenase and heparin skeleton synthase

[0109]

[0110] by Fig.11 The trisaccharide reaction catalyzed by the dual enzyme cascade shown is used as a model, with the synthesized GlcNAc-GlcA-pNP as the acceptor substrate, the UDP-GlcA synthesized by UDH-catalyzed UDP-Glc as the donor substrate, and the heparin skeleton synthase PmHS2 and △PmHS2 as the second-step protease to react, and the conversion rate of the disaccharide to trisaccharide reaction within 24 hours of reaction at the optimal reaction temperature of 37°C under the same enzyme concentration is detected. The reaction system is shown in Table 5. The reaction systems in Table 5 are mixed, reacted at 37°C for 24 hours, and then the reaction solution is heated at 95°C for 5 minutes to inactivate the protease to terminate the reaction. The reaction solution is filtered with a 0.22μm filter membrane and 85% methanol: 15% phosphoric acid aqueous solution (0.1% phosphoric acid) is used as the mobile phase. HPLC detection is performed at a flow rate of 1ml / min. The results are shown in Table 5. Fig.12 As shown, the conversion rate of △PmHS2 was much higher than that of PmHS2 within 24 hours, and reached the plateau phase faster than PmHS2, proving that the modified enzyme has higher activity in actual industrial production.

[0111] Example 9

[0112] Three-enzyme cascade: a trisaccharide synthesis reaction system catalyzed by N-acetylhexosamine kinase (NahK), glucosamine-1-phosphate uridylyltransferase (GlmU) and heparin skeleton synthase

[0113] Table 6 Disaccharide synthesis reaction system of N-acetylhexosamine kinase, glucosamine-1-phosphate uridyl transferase and heparin skeleton synthase

[0114]

[0115] by Fig.13 The disaccharide reaction catalyzed by the three-enzyme cascade shown is used as a model, with commercial GlcA-pNP as the acceptor substrate, UDP-GlcNAc synthesized by NahK and GlmU as the donor substrate, and heparin backbone synthase PmHS2 and △PmHS2 as the third-step protease to react, and the conversion rate of monosaccharide to disaccharide within 6 hours of reaction at the optimal reaction temperature of 37°C under the same enzyme concentration is detected. The reaction system is shown in Table 6. The reaction system in Table 6 is mixed, and after reacting at 37°C for 6 hours, the reaction solution is heated at 95°C for 5 minutes to inactivate the protease to terminate the reaction. The reaction solution is filtered with a 0.22μm filter membrane and 80% methanol: 20% phosphoric acid aqueous solution (0.1% phosphoric acid) is used as the mobile phase. HPLC detection is performed at a flow rate of 1ml / min. The results are shown in Table 6. Fig.14As shown, the conversion rate of △PmHS2 was higher than that of PmHS2 within 6 h, and reached the plateau phase faster than PmHS2.

[0116] Example 10

[0117] Example 10 is the same as Example 3, except that the molar ratio of the glycosyl acceptor to the glycosyl donor is 2:1, and after reacting for 6 hours under the system of Example 3, the reaction is terminated by treating at 95° C. for 5 minutes. The reaction solution is centrifuged, and the supernatant is separated by TLC.

[0118] Embodiment 11

[0119] Example 11 is the same as Example 10, except that the molar ratio of the glycosyl acceptor to the glycosyl donor is 1:5, and after reacting for 6 hours under the system of Example 9, the reaction is terminated by treating at 95° C. for 5 minutes. The reaction solution is centrifuged, and the supernatant is separated by TLC.

Claims

1. A modified glycosyltransferase, characterized in that: The modified glycosyltransferase is based on heparin synthase PmHS2 and the N-terminal is truncated to obtain a mutant enzyme △PmHS2, and the amino acid sequence of the mutant enzyme △PmHS2 is shown in any one of SEQ ID NO.3-6.

2. A gene encoding the modified glycosyltransferase according to claim 1, characterized in that: The coding gene is shown by any nucleotide sequence of SEQ ID NO.7-10.

3. A recombinant vector, characterized in that: It contains the gene encoding the modified glycosyltransferase according to claim 2.

4. The recombinant vector according to claim 3, characterized in that The recombinant vector is based on the plasmid vector pET-15b, and the coding gene of the modified glycosyltransferase is inserted into the plasmid vector.

5. A recombinant bacterium, characterized in that: It contains the recombinant vector according to claim 3.

6. The recombinant bacterium according to claim 5, characterized in that The recombinant bacteria uses Escherichia coli as a host cell, and the recombinant vector is transformed into the host cell to obtain the recombinant bacteria.

7. Use of the modified glycosyltransferase according to claim 1 or the coding gene of the modified glycosyltransferase according to claim 2 or the recombinant vector according to claim 3 or the recombinant bacteria according to claim 4 in the process of sugar chain synthesis.

8. Use of the modified glycosyltransferase according to claim 1 or the coding gene of the modified glycosyltransferase according to claim 2 or the recombinant vector according to claim 3 or the recombinant bacteria according to claim 4 in the synthesis of heparin backbone sugar chains.

9. Use of the modified glycosyltransferase according to claim 1 or the coding gene of the modified glycosyltransferase according to claim 2 or the recombinant vector according to claim 3 or the recombinant bacteria according to claim 4 in heparin synthesis.

10. The use according to claim 9, characterized in that: The application preferably uses p-nitrophenyl-β-D-glucuronic acid (GlcA-pNP), GlcNAc-GlcA-pNP or other glucuronic acid derivatives as the acceptor substrate; uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) or its derivatives as the donor substrate, and the molar ratio of the glycosyl acceptor to the glycosyl donor is 2:1 to 1:5.

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