N-acetylglucosamine transferase AcetRS-R346T-K23V and use thereof
By performing two mutations on N-acetylglucosamine transferase AcetRS, AcetRS-R346T-K23V was obtained, which solved the problem of difficult and inefficient production of chitosan in the existing technology, and achieved highly specific production and simplified separation process.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- OCEAN UNIV OF CHINA
- Filing Date
- 2026-03-30
- Publication Date
- 2026-06-02
AI Technical Summary
Existing N-acetylglucosamine transferases are difficult to efficiently produce chitosan oligosaccharides with a degree of polymerization greater than 5, especially chitosan hexasaccharides, and subsequent separation is also difficult.
By performing two mutations on N-acetylglucosamine transferase AcetRS, AcetRS-R346T-K23V was obtained. Using GlcNAc and UDP-GlcNAc produced by Bacillus subtilis as substrates, chitosan was prepared, thereby improving its production specificity.
The efficient production of chitosan was achieved, with chitosan accounting for 93.21% of the product. It contains only two types of chitooligosaccharides, which simplifies the subsequent separation process.
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Figure CN121931077B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to an N-acetylglucosamine transferase AcetRS-R346T-K23V and its applications, belonging to the field of functional enzyme technology. Background Technology
[0002] N-acetylglucosaminyltransferase is a glycosyltransferase that can produce chitosan oligosaccharides using the substrates GlcNAc and uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc). Current N-acetylglucosaminyltransferases typically only produce chitosan oligosaccharides with a degree of polymerization of 3–5, making it difficult to produce chitosan oligosaccharides with a degree of polymerization greater than 5.
[0003] CN 119842658 A discloses an N-acetylglucosamine transferase, AcetRS-R346D, and its applications. Based on existing N-acetylglucosamine transferase, a mutant enzyme, N-acetylglucosamine transferase AcetRS-R346D, was obtained through a rational design strategy. This mutant enzyme can be used to produce chitosan oligosaccharides with a degree of polymerization of 4–7. However, the proportion of chitohexasaccharide in its enzymatic hydrolysis products is low, and it contains both chitotetrasaccharide and chitoheptasaccharide, making subsequent separation of the single chitohexasaccharide difficult. Therefore, there is an urgent need for an N-acetylglucosamine transferase with higher specificity for chitohexasaccharide production. Summary of the Invention
[0004] In view of the above-mentioned prior art, the present invention provides an N-acetylglucosamine transferase AcetRS-R346T-K23V and its application, belonging to the field of functional enzyme technology.
[0005] This invention is achieved through the following technical solution:
[0006] An N-acetylglucosamine transferase, AcetRS-R346T-K23V, has the amino acid sequence shown in SEQ ID NO.7.
[0007] Application of the N-acetylglucosamine transferase AcetRS-R346T-K23V in the preparation of chitosan.
[0008] Further, in specific applications, the encoding gene of N-acetylglucosamine transferase AcetRS-R346T-K23V is introduced into Bacillus subtilis to construct a recombinant engineered bacterium expressing N-acetylglucosamine transferase AcetRS-R346T-K23V; fermentation of this recombinant engineered bacterium yields a fermentation broth containing chitohexasaccharide; the nucleotide sequence of the encoding gene of N-acetylglucosamine transferase AcetRS-R346T-K23V is shown in SEQ ID NO.8. The principle of chitohexasaccharide production is as follows: using GlcNAc and UDP-GlcNAc produced by Bacillus subtilis itself as substrates, chitohexasaccharide is prepared under the action of N-acetylglucosamine transferase AcetRS-R346T-K23V.
[0009] Furthermore, the Bacillus subtilis strain mentioned is Bacillus subtilis strain 168. Bacillus subtilis strain 168 is a Gram-positive, rod-shaped bacterium commonly used as a model organism in molecular biology and genetics. It is the most widely studied strain of Bacillus subtilis, and its genome has been extensively sequenced and annotated.
[0010] The N-acetylglucosamine transferase AcetRS-R346T-K23V of the present invention is obtained by two mutation modifications of N-acetylglucosamine transferase AcetRS. N-acetylglucosamine transferase AcetRS-R346T-K23V exhibits extremely high specificity in the production of chitohexasaccharides, with chitohexasaccharides accounting for up to 93.21% of its enzymatic hydrolysate. Furthermore, its hydrolysate contains only two chitooligosaccharides (chitopentose and chitohexasaccharides), excluding chitotetrasaccharides and chitoheptasaccharides, which is highly advantageous for the subsequent efficient separation of chitohexasaccharides. The N-acetylglucosamine transferase AcetRS-R346T-K23V of the present invention can be used for the efficient production of chitohexasaccharides and has promising application prospects.
[0011] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description
[0012] Figure 1 : A schematic diagram comparing the composition and yield of products in the fermentation broth of the original enzyme and each mutant. In this diagram, CTOS represents chitosan oligosaccharide, CTOS4 represents chitosan tetrasaccharide, CTOS5 represents chitosan pentasaccharide, CTOS6 represents chitosan hexasaccharide, and CTOS7 represents chitosan heptasaccharide. The same applies below.
[0013] Figure 2 A schematic diagram comparing the composition and yield of products in the fermentation broth of the original enzyme, the starting enzyme, and each mutant.
[0014] Figure 33D diagram showing the docking results of N-acetylglucosamine transferase AcetRS-R346T with chitosan molecules.
[0015] Figure 4 Analysis of the docking results between N-acetylglucosamine transferase AcetRS-R346T and chitopentose molecules (2D diagram).
[0016] Figure 5 3D diagram showing the docking results of N-acetylglucosamine transferase AcetRS-R346T-K23V with chitosan molecules.
[0017] Figure 6 Analysis of the docking results between N-acetylglucosamine transferase AcetRS-R346T-K23V and chitosan molecules (2D diagram). Detailed Implementation
[0018] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.
[0019] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.
[0020] Example 1: Modification of N-acetylglucosamine transferase AcetRS
[0021] Previous studies have shown that N-acetylglucosamine transferase AcetRS can produce chitotetrasaccharides, chitopentoses, and chitohexasaccharides, but the proportion of chitohexasaccharides in the product is low. Therefore, this invention attempts to mutate and modify it to obtain an N-acetylglucosamine transferase with higher specificity for producing chitohexasaccharides.
[0022] The amino acid sequence of N-acetylglucosamine transferase AcetRS is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2.
[0023] Based on sequence alignment and the position of the active amino acid residues, the key pocket amino acid ARG346 of the compressed substrate channel was selected as the key mutation site. In this invention, the amino acid at position 346 was mutated from arginine to a hydrophilic and non-aromatic amino acid, resulting in 9 mutants, which are temporarily named as follows: AcetRS-R346S, AcetRS-R346T, AcetRS-R346C, AcetRS-R346N, AcetRS-R346Q, AcetRS-R346D, AcetRS-R346E, AcetRS-R346K, and AcetRS-R346H.
[0024] The original enzyme (N-acetylglucosamine transferase AcetRS) and recombinant plasmids of the nine mutants mentioned above were constructed using conventional methods and transformed into Bacillus subtilis strain 168 for heterologous expression, yielding fermentation broths containing either the original enzyme or the mutants. High-performance liquid chromatography (HPLC) was used to identify the products in each fermentation broth: a Shodex Asahipak NH2P-50 4E column (Shodex) was used with 70% acetonitrile as the mobile phase at a flow rate of 1.0 mL / min. The fermentation broth was centrifuged (10000 g, 5 min), filtered through a 0.22 μm filter, and the supernatant was obtained; 10 μL of the supernatant was injected into the HPLC system. The product components were qualitatively identified according to the corresponding standard curve.
[0025] A comparative diagram of the product composition and yield in the fermentation broth of the original enzyme and various mutants is shown below. Figure 1 As shown. By Figure 1 It is evident that AcetRS-R346D exhibits the highest total yield of chitosan oligosaccharides, with a relatively high proportion of chitohexasaccharides; however, its product also contains chitoheptasaccharides, which is detrimental to the subsequent separation of single chitohexasaccharides. AcetRS-R346T demonstrates a higher total yield of chitosan oligosaccharides (52.55 mg / L), with the highest yield of chitohexasaccharides (41.62 mg / L), representing the highest proportion (79.18%). Its product contains three types of chitosan oligosaccharides (chitotetrasaccharides, chitopentosesaccharides, and chitohexasaccharides) but no chitoheptasaccharides, which is advantageous for the subsequent separation of single chitohexasaccharides. Therefore, this invention selects AcetRS-R346T for further research and names this mutant N-acetylglucosamine transferase AcetRS-R346T.
[0026] The construction process of N-acetylglucosamine transferase AcetRS-R346T is as follows:
[0027] A site-directed mutagenesis was performed at residue ARG346 of N-acetylglucosamine transferase AcetRS, changing the codon corresponding to amino acid 346 on the coding gene of N-acetylglucosamine transferase AcetRS from the arginine codon "cgc" to the threonine codon "aca". The amino acid sequence of N-acetylglucosamine transferase AcetRS-R346T is shown in SEQ ID NO.3, and the nucleotide sequence of its coding gene is shown in SEQ ID NO.4.
[0028] Then, PCR amplification of the entire plasmid (i.e., the pP43NMK plasmid containing the gene encoding N-acetylglucosamine transferase AcetRS) was performed. The PCR product was treated with restriction endonuclease DpnI to digest the methylated parental template to obtain the modified plasmid. The nucleotide sequences of the specific primers (upstream and downstream primers) used for PCR amplification are shown in SEQ ID NO.5 and SEQ ID NO.6.
[0029] Example 2: Modification of N-acetylglucosamine transferase AcetRS-R346T
[0030] The study in Example 1 showed that the N-acetylglucosamine transferase AcetRS-R346T exhibits high specificity in the production of chitohexasaccharide, with chitohexasaccharide accounting for up to 79.18% of the product. However, this proportion is still not ideal and cannot be better adapted for industrial production. Therefore, this invention attempts to further mutate and modify it to obtain an N-acetylglucosamine transferase with even higher specificity for chitohexasaccharide production.
[0031] Based on sequence alignment and the position of the active amino acid residue, residue LYS23, which is spatially adjacent to the ARG346 site, was selected. This site also has a pocket effect that compresses the substrate channel. In this invention, the amino acid at position 23 was mutated from lysine to a hydrophobic amino acid, resulting in 9 mutants, which are temporarily named as follows: AcetRS-R346T-K23G, AcetRS-R346T-K23A, AcetRS-R346T-K23V, AcetRS-R346T-K23L, AcetRS-R346T-K23I, AcetRS-R346T-K23M, AcetRS-R346T-K23F, AcetRS-R346T-K23W, and AcetRS-R346T-K23P.
[0032] The original enzyme (N-acetylglucosamine transferase AcetRS), the starting enzyme (N-acetylglucosamine transferase AcetRS-R346T), and the recombinant plasmids of the above nine mutants were constructed using conventional methods. These plasmids were then transformed into Bacillus subtilis strain 168 for heterologous expression, yielding fermentation broths containing the original enzyme, the starting enzyme, and each mutant. The products in the fermentation broth were identified using high-performance liquid chromatography (HPLC), following the same method as in Example 1.
[0033] A comparative diagram of the product composition and yield of the original enzyme, the starting enzyme, and each mutant in the fermentation broth is shown below. Figure 2 As shown. By Figure 2 As can be seen, among the double mutants, AcetRS-R346T-K23V exhibits the highest chitohexasaccharide content, reaching 93.21%, significantly superior to AcetRS-R346T and other mutants. Its chitohexasaccharide yield is 40.84 mg / L. Moreover, its product contains only two chitooligosaccharides (chitopentose and chitohexasaccharide), lacking chitotetrasaccharide and chitoheptasaccharide, which is highly advantageous for subsequent isolation of single chitohexasaccharides. This invention selects AcetRS-R346T-K23V for further research, naming this mutant N-acetylglucosamine transferase AcetRS-R346T-K23V.
[0034] The construction process of N-acetylglucosamine transferase AcetRS-R346T-K23V is as follows:
[0035] A site-directed mutagenesis was performed at residue LYS23 on N-acetylglucosamine transferase AcetRS-R346T, changing the codon corresponding to amino acid position 23 of the coding gene for N-acetylglucosamine transferase AcetRS-R346T from the lysine codon "aaa" to the valine codon "gtt". The amino acid sequence of N-acetylglucosamine transferase AcetRS-R346T-K23V is shown in SEQ ID NO.7, and the nucleotide sequence of its coding gene is shown in SEQ ID NO.8.
[0036] Then, PCR amplification of the entire plasmid (i.e., the pP43NMK plasmid containing the gene encoding N-acetylglucosamine transferase AcetRS-R346T) was performed. The PCR product was treated with restriction endonuclease DpnI to digest the methylated parental template to obtain the modified plasmid. The nucleotide sequences of the specific primers (upstream and downstream primers) used for PCR amplification are shown in SEQ ID NO. 9 and SEQ ID NO. 10.
[0037] Example 3 Molecular docking analysis
[0038] A structural model of N-acetylglucosamine transferase AcetRS-R346T was constructed using AlphaFold 2.0, and molecular docking with chitosan was performed using Autodock. The 3D model of the molecular docking results between N-acetylglucosamine transferase AcetRS-R346T and chitosan is shown below. Figure 3 As shown in the figure, the 2D diagram of the docking results between N-acetylglucosamine transferase AcetRS-R346T and chitosan molecules is as follows. Figure 4 As shown, after ARG346 is mutated to threonine, it interacts with the ligand, chitosan, through van der Waals forces. This single-point mutation may have caused changes in the tertiary structure of the protein, making the LYS23 site more likely to contact the ligand. The LYS23 site and chitosan form two conventional hydrogen bonds. The interaction between adjacent amino acid residues may be one of the factors that enable the control of the degree of polymerization.
[0039] The structural model of N-acetylglucosamine transferase AcetRS-R346T-K23V was further constructed using AlphaFold 2.0, and molecular docking with chitosan was performed using Autodock. The 3D model of the molecular docking results between N-acetylglucosamine transferase AcetRS-R346T-K23V and chitosan is shown below. Figure 5 As shown in the figure, the 2D diagram of the docking results between N-acetylglucosamine transferase AcetRS-R346T-K23V and chitosan molecules is as follows. Figure 6 As shown, the chitosan ligand is completely embedded at the bottom of the substrate-binding tunnel of the receptor molecule AcetRS-R346T-K23V. After the original LYS23 site is replaced by valine, VAL23 forms a carbon-hydrogen bond with the chitosan ligand. At the same time, at the entrance of the substrate-binding tunnel, the chitosan forms more than 10 conventional hydrogen bonds with the amino acid residues at the entrance. Perhaps the presence of these strong bond energies further slows down the separation of the ligand and the receptor, resulting in the production of more chitohexasaccharides.
[0040] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.
Claims
1. An N-acetylglucosamine transferase AcetRS-R346T-K23V, characterized in that: The amino acid sequence is shown in SEQ ID NO.
7.
2. The application of the N-acetylglucosamine transferase AcetRS-R346T-K23V according to claim 1 in the preparation of chitosan.
3. The application of the N-acetylglucosamine transferase AcetRS-R346T-K23V according to claim 2 in the preparation of chitosan, characterized in that: The coding gene for N-acetylglucosamine transferase AcetRS-R346T-K23V was introduced into Bacillus subtilis to construct a recombinant engineered bacterium expressing N-acetylglucosamine transferase AcetRS-R346T-K23V; the recombinant engineered bacterium was fermented to obtain a fermentation broth containing chitohexasaccharide; the nucleotide sequence of the coding gene for N-acetylglucosamine transferase AcetRS-R346T-K23V is shown in SEQ ID NO.
8.
4. The application of the N-acetylglucosamine transferase AcetRS-R346T-K23V according to claim 3 in the preparation of chitosan, characterized in that: The Bacillus subtilis strain mentioned is Bacillus subtilis strain 168.