HEPARIN SKELETON SYNTHASE AND ITS MUTANTS AND APPLICATION

Abstract

A heparin skeleton synthase originates from Neisseria animaloris, with an amino acid sequence as shown in SEQ ID NO.2 and a nucleotide sequence of the coding gene as shown in SEQ ID NO.1. Its recombinant expression level is 6.8 times that of the existing heparin skeleton synthase KfiA from Escherichia coli K5, and total enzyme activity per fermentation liquor is 5.22 times that of the heparin skeleton synthase KfiA. The heparin skeleton synthase mutants obtained through site-directed mutagenesis of the sites No. 16, No. 25, No. 30, No. 111, No. 165, and No. 172 in the amino acid sequence of the said heparin skeleton synthase all have high expression levels.

Claims

1. A mutant heparin skeleton synthase (NaGlcNAc-T) is derived from a wild-type NaGlcNAc-T, wherein the mutant NaGlcNAc-T is selected from the group consisting of NaGlcNAc-T (C16L), NaGlcNAc-T (N25P), NaGlcNAc-T (I30L), NaGlcNAc-T (I111S), NaGlcNAc-T (S165K) and NaGlcNAc-T (S172A), wherein the wild-type NaGlcNAc-T has the amino acid sequence as shown in SEQ ID NO.2 encoded by a cDNA having the nucleotide sequence as shown in SEQ ID NO.1; the NaGlcNAc-T (C16L) has the amino acid sequence as shown in SEQ ID NO.4 encoded by a cDNA having the nucleotide sequence as shown in SEQ ID NO.3, the NaGlcNAc-T (N25P) has the amino acid sequence as shown in SEQ ID NO.6 encoded by a cDNA having the nucleotide sequence as shown in SEQ ID NO.5, the NaGlcNAc-T (I30L) has the amino acid sequence as shown in SEQ ID NO.8 encoded by a cDNA having the nucleotide sequence as shown in SEQ ID NO.7, the NaGlcNAc-T (I111S) has the amino acid sequence as shown in SEQ ID NO.10 encoded by a cDNA having the nucleotide sequence as shown in SEQ ID NO. 9, the NaGlcNAc-T (S165K) has the amino acid sequence as shown in SEQ ID NO.12 encoded by a cDNA having the nucleotide sequence as shown in SEQ ID NO.11 and the NaGlcNAc-T (S172A) has the amino acid sequence as shown in SEQ ID NO.14 encoded by a cDNA having the nucleotide sequence as shown in SEQ ID NO.13.

2. The mutant heparin skeleton synthase NaGlcNAc-T according to claim 1, wherein the NaGlcNAc-T (C16L) is obtained by mutating cysteine-16 of the wild-type NaGlcNAc-T into leucine through a site-directed mutagenesis.

3. The mutant heparin skeleton synthase NaGlcNAc-T according to claim 1, wherein wherein the NaGlcNAc-T (N25P) is obtained by mutating asparagine-25 of the wild-type NaGlcNAc-T into proline through a site-directed mutagenesis.

4. The mutant heparin skeleton synthase NaGlcNAc-T according to claim 1, wherein the NaGlcNAc-T (I30L) is obtained by mutating isoleucine-30 of the wild-type NaGlcNAc-T into leucine through a site-directed mutagenesis.

5. The mutant heparin skeleton synthase NaGlcNAc-T according to claim 1, wherein the NaGlcNAc-T (I111S) is obtained by mutating isoleucine-111 of the wild-type NaGlcNAc-T into serine through a site-directed mutagenesis.

6. The mutant heparin skeleton synthase NaGlcNAc-T according to claim 1, wherein the NaGlcNAc-T (S165K) is obtained by mutating serine-165 of the wild-type NaGlcNAc-T into lysine through a site-directed mutagenesis.

7. The mutant heparin skeleton synthase NaGlcNAc-T according to claim 1, wherein the NaGlcNAc-T (S172A) is obtained by mutating serine-172 of the wild-type NaGlcNAc-T into alanine through a site-directed mutagenesis.

8. A recombinant vector which is obtained by inserting a cDNA into a plasmid vector; wherein the cDNA is selected from the group consisting of nuclei acids having the nucleotide sequences as shown as SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11 or SEQ ID NO. 13; the plasmid vector is pET30a(+).

9. The recombinant vector according to claim 8 wherein the recombinant vector is transformed into a host cell; the host cell is Escherichia coli.

10. A method for producing a heparin disaccharide by adding the mutant heparin skeleton synthase NaGlcNAc-T of claim 1 in substrates, wherein the substrates comprise GlcA-pNP and UDP-GlcNAc, the heparin disaccharide has a structure of GlcNAc-GlcA-pNP.

11. The method according to claim 10, wherein the mutant heparin skeleton synthase NaGlcNAc-T is purified from a recombinant Escherichia coli that is transformed with an expression vector; the expression vector containing a mutant heparin skeleton synthase NaGlcNAc-T cDNA.

12. The method according to claim 11, wherein the mutant heparin skeleton synthase NaGlcNAc-T cDNA is selected from the group consisting of nuclei acids having the nucleotide sequences as shown as SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11 or SEQ ID NO. 13.

13. The method according to claim 11, wherein the expression vector is pET30a(+).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 shows the SDS-PAGE testing results of the soluble expression and purification of the heparin skeleton synthase NaGlcNAc-T and its mutants in recombinant Escherichia coli;

[0036] Where: M is the marker; the rest of the strips are the purified recombinant proteins expressed by the NaGlcNAc-T and its mutants, with NaKfiA corresponding to NaGlcNAc-T, C16L corresponding to NaGlcNAc-T (C16L), N25P corresponding to NaGlcNAc-T (N25P), I30L corresponding to NaGlcNAc-T (I30L), I111S corresponding to NaGlcNAc-T (I111S), S165K corresponding to NaGlcNAc-T (S165K), and S172A corresponding to NaGlcNAc-T (S172A);

[0037] FIG. 2 is the protein quantitative standard curve of the heparin skeleton synthase NaGlcNAc-T;

[0038] FIG. 3 is the bar graph of the expression levels and receptor substrate reaction conversion rates of the heparin skeleton synthase NaGlcNAc-T and the heparin skeleton synthase KfiA;

[0039] FIG. 4 is the HPLC chromatogram of the reaction products of the heparin skeleton synthase NaGlcNAc-T;

[0040] FIG. 5 is the bar graph of the total enzyme activity of the heparin skeleton synthase NaGlcNAc-T and the heparin skeleton synthase KfiA;

[0041] FIG. 6 is the bar graph of the receptor substrate reaction conversion rates of the heparin skeleton synthase NaGlcNAc-T and its mutants;

[0042] FIG. 7 is the mass spectrogram of the reaction product GlcNAc-GlcA-pNP of the heparin skeleton synthase NaGlcNAc-T;

[0043] FIG. 8 is the .sup.1H-H COSY of the reaction product GlcNAc-GlcA-pNP of the heparin skeleton synthase NaGlcNAc-T;

[0044] FIG. 9 shows the reaction conversion rate curve of the receptor substrate when the heparin skeleton synthase NaGlcNAc-T is catalyzing oligosaccharide synthesis at different pH values in vitro.

[0045] FIG. 10 shows the reaction conversion rate bar graph of the receptor substrate when the heparin skeleton synthase NaGlcNAc-T is catalyzing oligosaccharide synthesis under the action of different metal ions in vitro.

[0046] FIG. 11 shows the reaction conversion rate curve of the receptor substrate when the heparin skeleton synthase NaGlcNAc-T is catalyzing oligosaccharide synthesis at different temperatures in vitro.

[0047] FIG. 12 shows the reaction conversion rate bar graph of the receptor substrate when the heparin skeleton synthase NaGlcNAc-T is catalyzing oligosaccharide synthesis based on different donor substrates in vitro.

[0048] Where: N.D. denotes that the substrate cannot be catalyzed by the NaGlcNAc-T.

EMBODIMENTS

[0049] The technical solution disclosed in the present invention is further described as follows with reference to the embodiments and drawings. However, the present invention is not limited thereto. Unless otherwise specified, the technical means used in the invention are all known to those skilled in the field.

[0050] Upon search in the bioinformatics database through Blast sequence alignment, the author of the invention has found that the amino acid sequence formed by a gene of the Neisseria animaloris (ATCC29858) is 55.7% homologous to that of the previously reported heparin skeleton synthase KfiA from Escherichia coli K5. Therefore, it is speculated that the protein product expressed by this gene may have heparin skeleton synthase activity. Upon protein expression in the Escherichia coli expression system, the gene is named NaGlcNAc-T, with a nucleotide sequence as shown in SEQ ID NO.1, and the protein product expressed by it is named NaGlcNAc-T, with an amino acid sequence as shown in SEQ ID NO.2.

[0051] Additionally, the author of the invention has conducted sequence homology analysis for the searched KfiA homologous sequence by performing multiple sequence alignment with EMBL Clustal Omega and analyzing the highly conserved region of the amino acid sequence with the Jalview software. Also, the author has performed protein simulation modeling for NaGlcNAc-T with Swiss-Model and predicted the active center of the enzyme with HotSpot Wizard 2.0. The findings show that sites No. 16, No. 25, No. 30, No. 111, No. 165, and No. 172 in the amino acid sequence of NaGlcNAc-T are located near the active center and the highly conserved region, and site-directed mutagenesis of these sites is very likely to improve the catalytic activity of the NaGlcNAc-T. Hence, with reference to the dominant amino acids at these six sites according to the homology analysis, the invention has designed six mutants, respectively NaGlcNAc-T (C16L), NaGlcNAc-T (N25P), NaGlcNAc-T (I30L), NaGlcNAc-T (I111S), NaGlcNAc-T (S165K), and NaGlcNAc-T (S172A). Among them, the mutant NaGlcNAc-T (C16L) is with a nucleotide sequence as shown in SEQ ID NO.3 and an amino acid sequence as shown in SEQ ID NO.4; the mutant NaGlcNAc-T (N25P) is with a nucleotide sequence as shown in SEQ ID NO.5 and an amino acid sequence as shown in SEQ ID NO.6; the mutant NaGlcNAc-T (I30L) is with a nucleotide sequence as shown in SEQ ID NO.7 and an amino acid sequence as shown in SEQ ID NO.8; the mutant NaGlcNAc-T (I111S) is with a nucleotide sequence as shown in SEQ ID NO.9 and an amino acid sequence as shown in SEQ ID NO.10; the mutant NaGlcNAc-T (S165K) is with a nucleotide sequence as shown in SEQ ID NO.11 and an amino acid sequence as shown in SEQ ID NO.12; the mutant NaGlcNAc-T (S172A) is with a nucleotide sequence as shown in SEQ ID NO.13 and an amino acid sequence as shown in SEQ ID NO.14.

[0052] The substrate saccharide reagents used in the present invention are all purchased from the Sigma Company. All the plasmids of NaGlcNAc-T and its mutants are synthesized by Nanjing Genscript Company. The BL21 (DE3) competent cells containing pGro7 molecular chaperones are purchased from Takara. The HPLC testing method used is YMC's amino column method, the liquid phase system is from Shimadzu JAPAN, and the UV testing system is SPD-20A. The ultraviolet absorption of the components from the catalysate of the NaGlcNAc-T and its mutants upon separation by chromatographic column is tested at 310 nm and 254 nm respectively under the HPLC mobile phase conditions as shown in Table 1:

TABLE-US-00001 TABLE 1 The HPLC analysis method used to test the heparin oligosaccharide Time/min 1M dipotassium phosphate Ultrapure water 0  0% 100% 30  60%  40% 31 100%  0% 33 100%  0% 43  0% 100%

Embodiment 1. Recombinant Protein Expression and Purification of the Heparin Skeleton Synthase NaGlcNAc-T and its Mutants

[0053] Construction of Expressing Strains

[0054] The recombinant plasmids pET30a(+)-NaGlcNAc-T synthesized by Nanjing Genscript Company are transformed into the Escherichia coli BL21 (DE3) competent cells containing pGro7 molecular chaperones and then cultured on LB plates containing kanamycin (100 μg/mL) and chloramphenicol (37 μg/mL) for 12 h. After that, transformants are screened (negative control experiments are carried out at the same time) to obtain the positive ones.

[0055] Following the above method, positive transformants are obtained for the six mutants, NaGlcNAc-T (C16L), NaGlcNAc-T (N25P), NaGlcNAc-T (I30L), NaGlcNAc-T (I111S), NaGlcNAc-T (S165K), and NaGlcNAc-T (S172A) respectively.

[0056] (2) Recombinant Protein Expression and Purification of the NaGlcNAc-T and its Mutants

[0057] Single colonies of the NaGlcNAc-T positive transformants are picked and inoculated on a 30 mL sterile LB culture medium (containing 100 μg/mL kanamycin and 37 μg/mL chloramphenicol) for activated culture (37° C., 225 r/min); after activated culture overnight, the bacteria solution is then transferred into 1 L LB medium (containing 100 μg/mL kanamycin and 37 μg/mL chloramphenicol) at an inoculum size of 1% for propagation; the medium is shaken under the conditions of 37° C. and 225 r/min for about 4 hours until the OD.sub.600 reaches about 0.8; then, IPTG with a final concentration of 0.5 mM and 1 mg/mL L-arabinose are added for induced expression under the conditions of 22° C. and 225 r/min for 16-18 hours; then, bacterial cells are collected, re-suspended with equilibration buffer (20 mM Tris-HCl, pH=8.00; 0.5M NaCl; 10 mM imidazole), and crushed by ultrasonic wave on ice (operating for 3 s and pausing for 5 s alternately; amplitude: 33%; energy: 1500KJ; 4° C.) for 30 minutes; the crushed bacterial cells are then centrifuged at 12000 rpm for 20 min (4° C.); the resulting supernatant is filtered by 0.22 μm filter membrane and purified by nickel column; after loading, the samples are rinsed by the equilibration buffer and then the impurity washing buffer (20 mM Tris-HCl, pH=8.00; 0.5M NaCl; 40 mM imidazole) to remove the undesired miscellaneous proteins and finally eluted by the elution buffer (20 mM Tris-HCl, pH=8.00; 0.5M NaCl; 250 mM imidazole) to obtain the target protein. The purified protein is stored in 20% glycerin and sub-packed in tubes before being put into a −80° C. freezer.

[0058] Following the above method, purified recombinant proteins are obtained for the six mutants, NaGlcNAc-T (C16L), NaGlcNAc-T (N25P), NaGlcNAc-T (I30L), NaGlcNAc-T (I111S), NaGlcNAc-T (S165K), and NaGlcNAc-T (S172A) respectively.

[0059] The purified recombinant proteins of the NaGlcNAc-T and its mutants are identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as shown in FIG. 1. The results show that the NaGlcNAc-T and its mutants (NaGlcNAc-T (C16L), NaGlcNAc-T (N25P), NaGlcNAc-T (I30L), NaGlcNAc-T (I111S), NaGlcNAc-T (S165K), and NaGlcNAc-T (S172A)) are all successfully purified following the above method, with a relative purity of above 90%.

[0060] A BCA protein assay kit (Beyotime P0011) is used to quantify the NaGlcNAc-T: first, an appropriate amount of BCA working solution is prepared by mixing 50 parts of BCA reagent A and 1 part of BCA reagent B (50:1 by volume) evenly; then, the standard substance is added in the standard wells of a 96-well plate at the volume of 0, 4, 8, 12, 16, and 20 μL respectively and supplemented by standard dilution buffer to 20 μL each to prepare 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL standard substance solutions; then 20 μL samples are added in each sample well of the 96-well plate, and 200 μL BCA working solution is added in each well; after keeping them still for 20-30 minutes under 37° C., the A562 values are measured with ELIASA, based on which a standard curve is plotted; finally, the protein concentration of the samples is calculated according to the standard curve and the sample volume used.

[0061] The protein quantitative standard curve of the heparin skeleton synthase NaGlcNAc-T is shown in FIG. 2. According to the determination, the recombinant expression of NaGlcNAc-T is as high as 102 mg/L per liter of LB medium, which is 6.8 times that (about 15 mg/L) of the heparin skeleton synthase KfiA from Escherichia coli K5 (the two follow the same way of expression), as shown in FIG. 3. Therefore, the NaGlcNAc-T has a higher expression level and a great prospect of industrial application.

[0062] Upon expression level determination of the six mutants (NaGlcNAc-T (C16L), NaGlcNAc-T (N25P), NaGlcNAc-T (I30L), NaGlcNAc-T (I111S), NaGlcNAc-T (S165K), and NaGlcNAc-T (S172A) respectively following the above method, it is found that the six mutants also have higher expression levels than the heparin skeleton synthase KfiA from Escherichia coli K5.

Embodiment 2. Activity Verification of the Heparin Skeleton Synthase NaGlcNAc-T and its Mutants

[0063] GlcNAc Transferase Activity Verification of the Heparin Skeleton Synthase NaGlcNAc-T

[0064] A reaction system with the commercially available GlcA-pNP (final concentration: 0.2 mM) as the receptor substrate and the UDP-GlcNAc (final concentration: 0.3 mM) as the donor substrate is constructed as shown in Table 2; the reaction system is placed in a 37° C. water bath kettle to react for 4 hours and then heated by boiling water for 5 min to stop the reaction by inactivating the enzyme; then, the reaction solution is filtered by 0.22 μm filter membrane and detected by HPLC according to the method described in Table 1. The pNP group of the monosaccharide receptor shows specific absorption at the 310 nm UV test wavelength, and the flow rate of the mobile phase is 0.5 mL/min.

[0065] Following the same method, the GlcNAc transferase activity can be tested for the six mutants, NaGlcNAc-T (C16L), NaGlcNAc-T (N25P), NaGlcNAc-T (I30L), NaGlcNAc-T (I111S), NaGlcNAc-T (S165K), and NaGlcNAc-T (S172A) respectively.

TABLE-US-00002 TABLE 2 The reaction system for the GlcNAc transferase activity verification of NaGlcNAc-T and its mutants Tris- GlcA-pNP UDP-GalNAc Mn.sup.2+ HCl(pH 7.0) Protein Total 0.2 mM 0.3 mM 20 mM 50 mM 20 μg 400 μL

[0066] According to the testing results in FIG. 4, the NaGlcNAc-T and its mutants all have GlcNAc transferase activity and can transfer the GlcNAc group to the non-reducing end of the GlcA-pNP to produce the heparin disaccharide GlcNAc-GlcA-pNP. The reaction conversion rate of the receptor donor is as shown in FIG. 3. Based on the reaction conversion rate and the expression level of the protease, the total activity of protease can be calculated, as shown in FIG. 5. The total enzyme activity of the NaGlcNAc-T per fermentation liquor is 5.22 times that of the heparin skeleton synthase KfiA from Escherichia coli K5.

[0067] The activity of the six mutants is determined based on the reaction system in Table 2 and the above treatment method. Upon reaction for 1 h in a 37° C. water bath kettle, the reaction conversion rates of the donor substrate GlcA-pNP for the mutants are shown in FIG. 6. As can be seen from the results, the mutants have remained stable or improved to different extents compared to the NaGlcNAc-T in terms of activity, with the mutants NaGlcNAc-T (C16L) and NaGlcNAc-T (S165K) performing best. These mutants can serve as tool enzymes for heparin disaccharide chain synthesis with high efficiency

[0068] (2) Mass Spectrum Verification of the Heparin Disaccharide GlcNAc-GlcA-pNP

[0069] To verify whether the above active reaction products are of the GlcNAc-GlcA-pNP structure, an electrospray ionization mass spectrometry (ESI-MS) analysis is conducted. The active reaction is carried out on a large scale to obtain sufficient disaccharide products. Upon purification through P2 column, an MS analysis is conducted for the resulting products on Thermo LCQ-Deca. All samples of MS analysis are prepared by dissolving the products in 50% methanol. The MS experiments are conducted in a negative ion mode, the electrospray voltage is 5 kV, and the capillary temperature is 275° C.

[0070] As can be seen from the mass spectrometry analysis results in FIG. 7, the mass spectrometry shows peaks (517.02) with a molecular weight consistent with that of GlcNAc-GlcA-pNP (M.sub.w=518.14) after it removes a proton, as well as diploid peaks (1034.72), proving that the resulting product is GlcNAc-GlcA-pNP.

[0071] (3) NMR Verification of the Heparin Disaccharide GlcNAc-GlcA-pNP

[0072] About 1 mg of the above active reaction product GlcNAc-GlcA-pNP is dissolved in 500 μL heavy water, and .sup.1H-NMR is collected by a 600 M NMR spectrometer. The .sup.1H-H COSY spectrum is shown in FIG. 8, wherein the signal with a chemical shift value of 5.35 (d, J=4.15 Hz, 1H) indicates that the bond type between GlcNAc and GlcA is a bond, thus confirming that the synthesized disaccharide is a heparin skeleton.

Embodiment 3 Characterization of the Heparin Skeleton Synthase NaGlcNAc-T and its Mutants

[0073] Determination of the Optimal Reaction pH Values of the Enzymes In Vitro

[0074] A reaction system as shown in Table 2 is used, and all conditions are maintained unchanged except for the pH value of the buffer solution. The Tris-HCl buffer is changed to citric acid/phosphate/Tris-HCl buffer of different pH values to set up 12 pH gradient points (4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.5) with each gradient consisting of three parallel groups, while the other conditions are maintained the same.

[0075] As can be seen from the results in FIG. 9, the enzymes are active in a wide range of pH (6.5-9.5), with the optimal pH value falling at 8.5.

[0076] (2) Determination of the Optimal Metal Ion for the Reaction of the Enzymes In Vitro

[0077] Except for the metal ion, all conditions of the reaction system are maintained unchanged. The Mn.sup.2+ is then changed to Mg.sup.2+, Mn.sup.2+, Ni.sup.2+, NH.sub.4.sup.+, Cu.sup.2+, Ca.sup.2+, K.sup.+, Ba.sup.2+, or Zn.sup.2+ of the same concentration for reaction. Three parallel experimental groups and a blank group are set up for each kind of metal ion. The other conditions are maintained the same.

[0078] As can be seen from the results in FIG. 10, the enzymes all have optimal catalytic activity in the presence of Mn.sup.2+, Mg.sup.2+, and Ni.sup.2+, while their activity will be reduced when the metal ions are absent.

[0079] (3) Study on the Influence of Reaction Temperature on Enzyme Activity

[0080] A reaction system as shown in Table 2 is used, and five temperature gradient points (4° C., 10° C., 20° C., 37° C., and 50° C.) are set up for enzyme reaction with each gradient consisting of three parallel groups. The influence of the reaction temperature on the enzyme activity is then measured. The other conditions are maintained the same.

[0081] As can be seen from the results in FIG. 11, the optimum reaction temperature of the enzymes is around 37° C.

[0082] Upon characterization of the six mutants NaGlcNAc-T (C16L), NaGlcNAc-T (N25P), NaGlcNAc-T (I30L), NaGlcNAc-T (I111S), NaGlcNAc-T (S165K), and NaGlcNAc-T (S172A) respectively following the above method, it is found that the six mutants have similar properties as the heparin skeleton synthase NaGlcNAc-T. They also have activity in a wide pH range (6.5-9.5), present the optimal catalytic activity in the presence of Mn.sup.2+, Mg.sup.2+, and Ni.sup.2+ and a reduced activity when the metal ions are absent, and have the optimum reaction temperature at 37° C.

Embodiment 4 Donor Specificity Study of the Heparin Skeleton Synthase NaGlcNAc-T

[0083] To determine the substrate specificity of the NaGlcNAc-T, a reaction system as shown in Table 2 with the commercially available GlcA-pNP as the receptor and the UDP-GlcNAc or another UDP-glucose of a similar structure (UDP-GalNAc, UDP-Glc, UDP-Gal, UDP-GlcNTFA, UDP-GalNAz, UDP-GlcNAz, and UDP-GlcNH.sub.2) is used for reactions. All the reactions are performed in a 37° C. water bath kettle for 4 hours. Finally, the reaction products are analyzed by HPLC according to the method described in Table 1.

[0084] As can be seen from the results in FIG. 12, when the receptor is the monosaccharide GlcA-pNP, the heparin skeleton synthase NaGlcNAc-T can also use UDP-GlcNTFA and UDP-GlcNAz as substrates in addition to the natural substrate UDP-GlcNAc in the experimental groups, but the extent of reaction is not that intensive.

[0085] Upon donor specificity study of the six mutants NaGlcNAc-T (C16L), NaGlcNAc-T (N25P), NaGlcNAc-T (I30L), NaGlcNAc-T (I111S), NaGlcNAc-T (S165K), and NaGlcNAc-T (S172A) respectively following the above method, it is found that the six mutants have similar donor specificity as the heparin skeleton synthase NaGlcNAc-T. In addition to the natural substrate UDP-GlcNAc, they can also use UDP-GlcNTFA and UDP-GlcNAz as substrates, provided however that the extent of reaction is not that intensive.