N-Acetylglucosaminyltransferase AcetRL-R349H and Its Application

Abstract

The present invention discloses an N - acetylglucosaminyltransferase AcetRL - R349H and its application, belonging to the technical field of functional enzymes. The amino acid sequence of the N - acetylglucosaminyltransferase AcetRL - R349H is as shown in SEQ ID NO.5. The application of the N - acetylglucosaminyltransferase AcetRL - R349H in the preparation of chito - oligosaccharides. The N - acetylglucosaminyltransferase AcetRL - R349H of the present invention is obtained by mutating and modifying the N - acetylglucosaminyltransferase AcetRL, and can be used for producing chito - oligosaccharides with a degree of polymerization of 4 - 6, especially (GlcNAc)6. The present invention broadens the product spectrum of N - acetylglucosaminyltransferase and can be used for preparing chito - oligosaccharides with a high degree of polymerization, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to an N-acetylglucosaminyltransferase AcetRL-R349H and its application, belonging to the technical field of functional enzymes. Background Art

[0002] Chito-oligosaccharides, also known as chitosan oligosaccharides, are functional oligosaccharides with a degree of polymerization of 2-10 formed by N-acetylglucosamine (GlcNAc) linked by β-1,4 glycosidic bonds. Chito-oligosaccharides have biological activities such as antioxidant, anti-tumor, prevention of atherosclerosis, and promotion of plant growth. The degree of polymerization of chito-oligosaccharides has a great influence on their biological activities.

[0003] N-acetylglucosaminyltransferase is a glycosyltransferase that can produce chito-oligosaccharides using the substrates GlcNAc and uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc). However, the N-acetylglucosaminyltransferases in the prior art can only produce chito-oligosaccharides with a degree of polymerization of 3-5, and the degree of polymerization of the products is very limited, making it difficult to produce chito-oligosaccharides with a degree of polymerization greater than 5. Therefore, based on N-acetylglucosaminyltransferase, by means of rational design strategies, obtaining N-acetylglucosaminyltransferases with the ability to produce chito-oligosaccharides with a higher degree of polymerization has broad application prospects. Summary of the Invention

[0004] In view of the above prior art, the present invention provides an N-acetylglucosaminyltransferase AcetRL-R349H and its application, belonging to the technical field of functional enzymes.

[0005] The present invention is achieved by the following technical solutions:

[0006] An N-acetylglucosaminyltransferase AcetRL-R349H, whose amino acid sequence is as shown in SEQ ID NO.5.

[0007] The application of the N-acetylglucosaminyltransferase AcetRL-R349H in the preparation of chito-oligosaccharides.

[0008] Furthermore, the chito-oligosaccharides are any one or more of chitotetraose, chitopentaose or chitohexaose.

[0009] Furthermore, in specific applications, the coding gene of N-acetylglucosaminyltransferase AcetRL-R349H is introduced into Bacillus subtilis to construct a recombinant engineering bacterium expressing N-acetylglucosaminyltransferase AcetRL-R349H; the recombinant engineering bacterium is fermented to obtain a fermentation broth containing chitooligosaccharides. The principle of producing chitooligosaccharides is as follows: Using GlcNAc and UDP-GlcNAc produced by Bacillus subtilis itself as substrates, chitooligosaccharides are prepared under the action of N-acetylglucosaminyltransferase AcetRL-R349H.

[0010] Furthermore, the Bacillus subtilis is Bacillus subtilis strain 168. Bacillus subtilis strain 168 is a Gram-positive, rod-shaped bacterium and is 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.

[0011] The N-acetylglucosaminyltransferase AcetRL-R349H of the present invention is obtained by mutating and modifying N-acetylglucosaminyltransferase AcetRL. After the mutation and modification, it has product specificity and can be used to produce chitooligosaccharides with a degree of polymerization of 4-6, especially (GlcNAc)6. The present invention broadens the product spectrum of N-acetylglucosaminyltransferase and can be used to prepare chitooligosaccharides with a high degree of polymerization, and has broad application prospects.

[0012] All the terms and phrases used in the present invention have the general meanings well-known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Schematic diagram of the HPLC analysis results, where AcetRL represents N-acetylglucosaminyltransferase AcetRL, and AcetRL-R349H represents N-acetylglucosaminyltransferase AcetRL-R349H.

[0014] Figure 2 : Comparison chart of the product composition and yield of the fermentation broth, where AcetRL represents N-acetylglucosaminyltransferase AcetRL, and AcetRL-R349H represents N-acetylglucosaminyltransferase AcetRL-R349H.

[0015] Figure 3 : Schematic diagram of the CavityPlus pocket prediction of N-acetylglucosaminyltransferase AcetRL.

[0016] Figure 4 : 3D analysis diagram of the docking results of N-acetylglucosaminyltransferase AcetRL and chitotetraose molecules.

[0017] Figure 5 : 2D diagram of the docking result analysis of N-acetylglucosaminyltransferase AcetRL with chitotetraose molecule.

[0018] Figure 6 : Diagram for comparing the product composition and yield of the fermentation broth containing each mutant.

[0019] Figure 7 : 3D diagram of the docking result analysis of N-acetylglucosaminyltransferase AcetRL-R349H with chitopentaose molecule.

[0020] Figure 8 : 2D diagram of the docking result analysis of N-acetylglucosaminyltransferase AcetRL-R349H with chitopentaose molecule. Detailed implementation mode

[0021] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0022] The instruments, reagents, and materials involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, and materials existing in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods and detection methods existing in the prior art.

[0023] Example 1 Mining of N-acetylglucosaminyltransferase AcetRL

[0024] In order to mine N-acetylglucosaminyltransferase with product specificity, the present invention screened a gene (ID: Q8KJI5_RHILI) from Rhizobium leguminosarum ( Rhizobium loti ) that may have N-acetylglucosaminyltransferase activity through Uniprot search and comparison. The amino acid sequence of the protein expressed by it is shown in SEQ ID NO.1 and is named N-acetylglucosaminyltransferase AcetRL. After optimization with Bacillus subtilis-preferred codons, the nucleotide sequence of this gene is shown in SEQ ID NO.2.

[0025] The amino acid sequence of N-acetylglucosaminyltransferase AcetRL is shown in SEQ ID NO.1 as follows:

[0026] MTLFATASTVAICSYALLSTVYKTAQVFYTLPTNVPPTSGDPPGGEPWPSVDVIIPCYNEAPRTLSDCLASIASQEYAGKLQVYVVDDGSANRDALVGVHEEYAGDPRFNFIALPKNVGKRKAQIAAIRRSCGDLVLNVDSDTILAPDVITRLALKMQDQAVGAAMGQLAASNRSETWLTRLIDMEYWLACNEERAAQARFGAVMCCCGPCAMYRRSALVSLLDQYETQRFRGKPSDFGEDRHLTILMLKAGFRTEYVPEAVAATVVPNSMGPYLRQQLRWARSTFRDTLLAFQLLRGLNIYLTLDVIGQNIGPLLLSLSILAGLAQLVTTGTAPWTACLMIAAMTIVRCSVAAFRARQLRFLGFSLHTLINIFLLLPLKAYALCTLSNSDWLSRSSAANVTDTGGTSSKPNLVGSDAAYSEQQ。

[0027] The nucleotide sequence of the coding gene of N-acetylglucosaminyltransferase AcetRL is shown in SEQ ID NO.2 as follows (direction 5'-3'):

[0028]

[0029] Example 2 Construction of Recombinant Engineered Bacteria Expressing N-Acetylglucosaminyltransferase AcetRL

[0030] The steps are as follows:

[0031] (1) Synthesize the gene shown in SEQ ID NO.2 entirely by gene synthesis, and perform PCR amplification to obtain a large amount of gene fragments.

[0032] (2) Construction of the recombinant expression vector

[0033] The amplified gene fragments are ligated to the pP43NMK cloning vector using seamless cloning technology, and the ligation product is transferred into Escherichia coli ( E.coli ) DH5α competent cells. Positive transformants are screened using LB plates containing sodium ampicillin. After verifying the clones by colony PCR using primers NMK-F and NMK-R, positive clones are picked for sequencing to obtain the recombinant plasmid.

[0034] The nucleotide sequence of the primer NMK-F is as shown in SEQ ID NO.3, as follows (direction 5'-3'):

[0035] cgtgtcaaagaaattattgaatcacaaaaaacagg.

[0036] The nucleotide sequence of the primer NMK-R is as shown in SEQ ID NO.4, as follows (direction 5'-3'):

[0037] gcatgtgggtgatgtagatctcg.

[0038] (3) Construction of the recombinant engineered bacteria

[0039] Culture the correctly sequenced Escherichia coli DH5α strain, extract the recombinant plasmid and transform it into Bacillus subtilis 168 strain. The constructed engineered strain grows on the kanamycin sulfate resistance plate.

[0040] (4) Fermentation of the recombinant engineered strain

[0041] Pick a single colony of the recombinant engineered strain from the plate into a test tube of LB medium and culture for 16 h, then transfer it to 50 mL of M9 medium and ferment at 37°C and 150 rpm for 48 h, and collect the fermentation broth.

[0042] Example 3 Determination of the Product of N-Acetylglucosaminyltransferase AcetRL

[0043] The fermentation broth collected in Example 2 was analyzed, and high performance liquid chromatography (HPLC) was used for product identification. HPLC is high performance size exclusion chromatography, equipped with a photodiode array detector (PDA, SPD-M40, Shimadzu). The system used a Shodex Asahipak NH2P-50 4E chromatographic column (Shodex), with 70% acetonitrile as the mobile phase and a flow rate of 1.0 mL / min. The fermentation broth was centrifuged (10000 g, 5 min) and filtered through a 0.22 μm filter to obtain the supernatant; 10 μL of the supernatant was injected into the HPLC system. The product components were qualitatively analyzed according to the corresponding standard curve.

[0044] The HPLC analysis results are as Figure 1 shown, and the product composition and yield are compared as Figure 2 shown. It can be seen that the products of N-acetylglucosaminyltransferase AcetRL are chitotetraose and chitopentaose.

[0045] Example 4 Modification of N-acetylglucosaminyltransferase AcetRL

[0046] From the results of Example 3, N-acetylglucosaminyltransferase AcetRL can only produce chitotetraose and chitopentaose. Although it has a certain product specificity, the product spectrum is still not wide enough to meet the market demand. Therefore, the present invention attempts to mutate and modify it in order to obtain N-acetylglucosaminyltransferase with the ability to produce chito-oligosaccharides with a higher degree of polymerization.

[0047] To determine the key mutation sites, the protein sequences from different sources were compared and analyzed. AlphaFold 2.0 was used for protein three-dimensional structure modeling, and the CavityPlus database was used to predict the potential pockets of N-acetylglucosaminyltransferase AcetRL. The results are as Figure 3 shown; the Autodock software was used for docking the substrate (chitotetraose) with the enzyme molecule, and the Discovery Studio Client software was used to analyze the molecular docking results. The results are as Figure 4 , Figure 5 shown.

[0048] Select the intersection amino acids of the amino acids at the bottom of the pocket and the docking amino acids, and conduct a protein sequence comparison analysis on N-acetylglucosaminyltransferases from different sources. It is considered that ARG349 is the key site controlling the degree of polymerization of the product of N-acetylglucosaminyltransferase AcetRL. Therefore, in the present invention, site-saturation mutagenesis is performed on the ARG349 site, and the 19 mutants obtained are temporarily named: AcetRL-R349G, AcetRL-R349A, AcetRL-R349V, AcetRL-R349L, AcetRL-R349I, AcetRL-R349M, AcetRL-R349F, AcetRL-R349W, AcetRL-R349P, AcetRL-R349S, AcetRL-R349T, AcetRL-R349C, AcetRL-R349Y, AcetRL-R349N, AcetRL-R349Q, AcetRL-R349D, AcetRL-R349E, AcetRL-R349K, AcetRL-R349H.

[0049] Construct 19 mutant plasmids by conventional methods and transform them into Bacillus subtilis strain 168 for heterologous expression respectively (the method is the same as in Example 2) to obtain fermentation broths containing each mutant. Measure the products of each fermentation broth according to the method of Example 3 (using the fermentation broth of Example 2 as a control), and the results are as Figure 6 shown. It can be Figure 6 seen that multiple mutants have the ability to produce chitohexaose, but there are significant differences in the level of ability. Among them, the total yield of chitooligosaccharides of AcetRL-R349Q is the highest, reaching 113.78 mg / L, but the yield of chitohexaose is only 11.35 mg / L; the yields of chitohexaose of AcetRL-R349S and AcetRL-R349H are the highest, reaching 23.32 mg / L and 24.07 mg / L respectively. However, the yield of chitopentaose of AcetRL-R349H is 58.55 mg / L, which is higher than the yield of chitopentaose of AcetRL-R349S, which is 49.47 mg / L. Therefore, the ability of AcetRL-R349H to produce high-polymerization-degree chitooligosaccharides is better than that of AcetRL-R349S. In the present invention, AcetRL-R349H is selected for subsequent research, and this mutant is named N-acetylglucosaminyltransferase AcetRL-R349H.

[0050] The construction process of N-acetylglucosaminyltransferase AcetRL-R349H is as follows:

[0051] The 349th amino acid of N-acetylglucosaminyltransferase AcetRL was changed from arginine to histidine. The specific method is as follows: site-directed mutagenesis of N-acetylglucosaminyltransferase AcetRL was carried out at residue ARG349, and the codon corresponding to the 349th amino acid on the coding gene of N-acetylglucosaminyltransferase AcetRL was changed from the arginine codon "CGC" to the histidine codon "CAT". The amino acid sequence of N-acetylglucosaminyltransferase AcetRL-R349H is shown in SEQ ID NO.5, and the nucleotide sequence of its coding gene is shown in SEQ ID NO.6.

[0052] The amino acid sequence of N-acetylglucosaminyltransferase AcetRL-R349H is shown in SEQ ID NO.5 and is as follows:

[0053] MTLFATASTVAICSYALLSTVYKTAQVFYTLPTNVPPTSGDPPGGEPWPSVDVIIPCYNEAPRTLSDCLASIASQEYAGKLQVYVVDDGSANRDALVGVHEEYAGDPRFNFIALPKNVGKRKAQIAAIRRSCGDLVLNVDSDTILAPDVITRLALKMQDQAVGAAMGQLAASNRSETWLTRLIDMEYWLACNEERAAQARFGAVMCCCGPCAMYRRSALVSLLDQYETQRFRGKPSDFGEDRHLTILMLKAGFRTEYVPEAVAATVVPNSMGPYLRQQLRWARSTFRDTLLAFQLLRGLNIYLTLDVIGQNIGPLLLSLSILAGLAQLVTTGTAPWTACLMIAAMTIVHCSVAAFRARQLRFLGFSLHTLINIFLLLPLKAYALCTLSNSDWLSRSSAANVTDTGGTSSKPNLVGSDAAYSEQQ.

[0054] The nucleotide sequence of the coding gene of N-acetylglucosaminyltransferase AcetRLR349H is shown in SEQ ID NO.6 and is as follows (orientation 5'-3'):

[0055]

[0056] Then, perform PCR amplification of the entire plasmid (i.e., the pP43NMK plasmid encoding the N-acetylglucosaminyltransferase AcetRL). Treat the PCR product with the restriction endonuclease DpnI to digest the methylated parental template to obtain the modified plasmid. The nucleotide sequences of the specific primers used for PCR amplification are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0057] The nucleotide sequence of the upstream primer is as shown in SEQ ID NO.7, as follows (direction 5'-3'):

[0058] cattgtgcattgtagcgttgcagcatttcg.

[0059] The nucleotide sequence of the downstream primer is as shown in SEQ ID NO.8, as follows (direction 5'-3'):

[0060] caacgctacaatgcacaatggtcattgctgc.

[0061] Example 5 Determination of the product of N-acetylglucosaminyltransferase AcetRL-R349H

[0062] Construct a recombinant engineering bacterium expressing N-acetylglucosaminyltransferase AcetRL-R349H and perform fermentation. Analyze the collected fermentation broth using the same method as in Example 3. The results of HPLC analysis are as Figure 1 shown, and the comparison of the product composition and yield is as Figure 2 shown. It can be seen that chitohexaose appears in the product of N-acetylglucosaminyltransferase AcetRL-R349H, and the yield is as high as 24.07 mg / L, which is not possessed by N-acetylglucosaminyltransferase AcetRL.

[0063] Continue to construct the structural model of N-acetylglucosaminyltransferase AcetRL-R349H using AlphaFold 2.0 and perform molecular docking with chitopentaose using Autodock. The results are as Figure 7 、 Figure 8 shown. The maximum product docked by N-acetylglucosaminyltransferase AcetRL is chitotetraose, but N-acetylglucosaminyltransferase AcetRL-R349H can achieve the docking of chitopentaose. The tolerance of N-acetylglucosaminyltransferase AcetRL-R349H to the embedding of chitopentaose may be the basis for the production of chitohexaose, and the extremely strong hydrogen bond interaction between the amino acid residues at the entrance of the active pocket and the ligand may be another key factor for the production of chitohexaose.

[0064] The above embodiments are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, and not to limit the scope disclosed herein. Modifications that are obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. An N-acetylglucosaminyltransferase AcetRL-R349H, characterized in that: Its amino acid sequence is as shown in SEQ ID NO.

5.

2. Use of N-acetylglucosaminyltransferase AcetRL-R349H according to claim 1 in the preparation of chito-oligosaccharide, characterized in that: The chito-oligosaccharide is any one or more than two of chitotetraose, chitopentaose or chitohexaose.

3. Use of N-acetylglucosaminyltransferase AcetRL-R349H according to claim 2 in the preparation of chitooligosaccharides, characterized in that: In specific applications, the coding gene of N-acetylglucosaminyltransferase AcetRL-R349H is introduced into Bacillus subtilis to construct a recombinant engineering bacterium expressing N-acetylglucosaminyltransferase AcetRL-R349H; the recombinant engineering bacterium is fermented to obtain a fermentation broth containing chito-oligosaccharide.

4. Use of N-acetylglucosaminyltransferase AcetRL-R349H according to claim 3 in the preparation of chitooligosaccharides, characterized in that: The Bacillus subtilis is Bacillus subtilis strain 168.

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