N-acetylglucosamine transferase AcetRL-R349H and application thereof

Abstract

The invention discloses N-acetylglucosamine transferase AcetRL-R349H and an application thereof, and belongs to the technical field of functional enzymes. The amino acid sequence of the N-acetylglucosamine transferase AcetRL-R349H is as shown in SEQ ID NO. 5 (sequence identifier number 5). The invention further discloses the application of the N-acetylglucosamine transferase AcetRL-R349H in the preparation of the chitosan oligosaccharide. The N-acetylglucosamine transferase AcetRL-R349H disclosed by the invention is obtained by carrying out mutation modification on the N-acetylglucosamine transferase AcetRL, and can be used for producing the chitosan oligosaccharide with the polymerization degree of 4 to 6, especially (GlcNAc) 6. The product spectrum of the N-acetylglucosamine transferase is widened, and the N-acetylglucosamine transferase can be used for preparing high-polymerization-degree chitosan oligosaccharide and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to N-acetylglucosamine transferase AcetRL-R349H and application thereof, belonging to the technical field of functional enzymes. Background Art

[0002] Chitosan oligosaccharide, also known as chitosan oligosaccharide, is a functional oligosaccharide with a degree of polymerization of 2 to 10, which is formed by connecting N-acetylglucosamine (GlcNAc) through β-1,4 glycosidic bonds. Chitosan oligosaccharide has biological activities such as anti-oxidation, anti-tumor, prevention of atherosclerosis, and promotion of plant growth. The degree of polymerization of chitosan oligosaccharide has a great influence on its biological activity.

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

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

[0005] The present invention is achieved through the following technical solutions: An N-acetylglucosamine transferase AcetRL-R349H, whose amino acid sequence is shown in SEQ ID NO.5.

[0006] The application of the N-acetylglucosamine transferase AcetRL-R349H in the preparation of chito-oligosaccharides.

[0007] Furthermore, the chito-oligosaccharide is any one or more of chitotetraose, chitopentaose or chitohexaose.

[0008] Furthermore, in specific applications, the coding gene of N-acetylglucosamine transferase AcetRL-R349H is introduced into Bacillus subtilis to construct a recombinant engineered bacterium expressing N-acetylglucosamine transferase AcetRL-R349H; the recombinant engineered bacterium is fermented to obtain a fermentation liquid containing chitosan oligosaccharides. The principle of producing chitosan oligosaccharides is: using GlcNAc and UDP-GlcNAc produced by Bacillus subtilis itself as substrates, chitosan oligosaccharides are prepared under the action of N-acetylglucosamine transferase AcetRL-R349H.

[0009] Further, the Bacillus subtilis is Bacillus subtilis 168 strain. Bacillus subtilis 168 strain 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 AcetRL-R349H of the present invention is obtained by mutation modification of the N-acetylglucosamine transferase AcetRL. After mutation modification, the N-acetylglucosamine transferase AcetRL has product specificity and can be used to produce chito-oligosaccharides with a degree of polymerization of 4 to 6, especially (GlcNAc)6. The present invention broadens the product spectrum of the N-acetylglucosamine transferase, can be used to prepare chito-oligosaccharides with a high degree of polymerization, and has broad application prospects.

[0011] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : Schematic diagram of HPLC analysis results, wherein AcetRL represents N-acetylglucosamine transferase AcetRL, and AcetRL-R349H represents N-acetylglucosamine transferase AcetRL-R349H.

[0013] Figure 2 : Comparison of product composition and yield of fermentation broth, wherein AcetRL represents N-acetylglucosamine transferase AcetRL, and AcetRL-R349H represents N-acetylglucosamine transferase AcetRL-R349H.

[0014] Figure 3 : Schematic diagram of the CavityPlus pocket prediction of N-acetylglucosamine transferase AcetRL.

[0015] Figure 4 : 3D diagram of the docking analysis results of N-acetylglucosamine transferase AcetRL and chitotetraose molecules.

[0016] Figure 5 : 2D diagram of the docking analysis results of N-acetylglucosamine transferase AcetRL and chitotetraose molecules.

[0017] Figure 6 : Comparison of product composition and yield of fermentation broth containing each mutant.

[0018] Figure 7 : 3D diagram of the docking analysis results of N-acetylglucosamine transferase AcetRL-R349H and chitopentaose molecules.

[0019] Figure 8 : 2D diagram of the docking analysis results of N-acetylglucosamine transferase AcetRL-R349H and chitopentaose molecules. DETAILED DESCRIPTION

[0020] 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. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0021] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods available in the prior art.

[0022] Example 1 Mining of N-acetylglucosamine transferase AcetRL In order to explore N-acetylglucosamine transferase with product specificity, the present invention screened a gene from Rhizobium rapae ( Rhizobium loti ) and may have N-acetylglucosamine transferase activity (ID: Q8KJI5_RHILI), the amino acid sequence of the protein expressed by it is shown in SEQ ID NO.1, and it is named N-acetylglucosamine transferase AcetRL. After Bacillus subtilis preferred codon optimization, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0023] The amino acid sequence of N-acetylglucosamine transferase AcetRL is shown in SEQ ID NO.1, as shown below: MTLFATASTVAICSYALLSTVYKTAQVFYTLPTNVPPTSGDPPGGEPWPSVDVIIPCYNEAPRTLSDCLASIASQEYAGKLQVYVVDDGSANRDALVGVHEEYAGD PRFNFIALPKNVGKRKAQIAAIRRSCGDLVLNVDSDTILAPDVITRLALKMQDQAVGAAMGQLAASNRSETWLTRLIDMEYWLACNEERAAQARFGAVMCCCGPCA MYRRSALVSLLDQYETQRFRGKPSDFGEDRHLTILKAGFRTEYVPEAVAATVVPNSMGPYLRQQLRWARSTFRDTLLAFQLLRGLNIYLTLDVIGQNIGPLLLSLSILAGLAQLVTTGTAPWTACLMIAMTIVRCSVAAFRARQLRFLGFSLHTLINIFLLLPLKAYALCTLSNSDWLSRSSAANVTDTGGTSSKPNLVGSDAAYSEQQ.

[0024] The nucleotide sequence of the gene encoding N-acetylglucosamine transferase AcetRL is shown in SEQ ID NO.2, as shown below (direction 5'-3'):

[0025] Example 2 Construction of recombinant engineering bacteria expressing N-acetylglucosamine transferase AcetRL Here are the steps: (1) The gene shown in SEQ ID NO. 2 was synthesized and PCR amplified to obtain a large number of gene fragments.

[0026] (2) Construction of recombinant expression vector The gene fragment obtained by the above amplification was connected with the pP43NMK cloning vector using seamless cloning technology, and the ligation product was transformed into Escherichia coli ( E. coli )DH5α competent cells. Use LB plates containing ampicillin sodium to screen positive transformants. After the clones are verified by colony PCR using primers NMK-F and NMK-R, positive clones are picked and sequenced to obtain recombinant plasmids.

[0027] The nucleotide sequence of the primer NMK-F is shown in SEQ ID NO.3, as shown below (direction 5'-3'): cgtgtcaaagaaattattgaatcacaaaaaacagg.

[0028] The nucleotide sequence of the primer NMK-R is shown in SEQ ID NO.4, as shown below (direction 5'-3'): gcatgtgggtgatgtagatctcg.

[0029] (3) Construction of recombinant engineered bacteria The correctly sequenced Escherichia coli DH5α strain was cultured, the recombinant plasmid was extracted and transformed into the Bacillus subtilis 168 strain, and the constructed engineered strain was grown on a kanamycin sulfate resistance plate.

[0030] (4) Fermentation of recombinant engineered strains A single colony of the recombinant engineered strain was picked up and placed in an LB medium tube for culture for 16 h, then transferred to 50 mL of M9 medium and fermented at 37°C and 150 rpm for 48 h, and the fermentation broth was collected.

[0031] Example 3 Determination of the product of N-acetylglucosamine transferase AcetRL The fermentation broth collected in Example 2 was analyzed and the product was identified using high performance liquid chromatography (HPLC). HPLC is a high performance size exclusion chromatography method equipped with a photodiode array detector (PDA, SPD-M40, Shimadzu). The system used a Shodex Asahipak NH2P-50 4E column (Shodex) 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) and filtered with a 0.22 μm filter to obtain a supernatant; 10 μL of the supernatant was injected into the HPLC system. The product components were qualitatively identified based on the corresponding standard curve.

[0032] HPLC analysis results are as follows Figure 1 As shown, the product composition and yield are compared. Figure 2 As shown, it can be seen that the products of N-acetylglucosamine transferase AcetRL are chitotetraose and chitopentaose.

[0033] Example 4 Modification of N-acetylglucosamine transferase AcetRL From the results of Example 3, it can be seen that N-acetylglucosamine transferase AcetRL can only produce chitotetraose and chitopentaose. Although it has certain product specificity, the product spectrum is still not wide enough to meet market demand. Therefore, the present invention attempts to perform mutation modification on it in order to obtain N-acetylglucosamine transferase with higher polymerization degree chito-oligosaccharide production capacity.

[0034] To identify the key mutation sites, protein sequences from different sources were compared and analyzed, AlphaFold 2.0 was used to model the protein three-dimensional structure, and the CavityPlus database was used to predict the possible pockets of N-acetylglucosamine transferase AcetRL. The results are as follows: Figure 3 As shown; Autodock software was used to dock the substrate (chitotetraose) with the enzyme molecule, and the molecular docking results were analyzed using Discovery Studio Client software. The results are shown in Figure 4 , Figure 5 shown.

[0035] The amino acids at the intersection of the bottom of the pocket and the docking amino acids were selected, and based on the protein sequence comparison analysis of N-acetylglucosamine transferases from different sources, it was considered that ARG349 was the key site for controlling the polymerization degree of the product of N-acetylglucosamine transferase AcetRL. Therefore, the present invention selected the ARG349 site for saturation mutation, and the obtained 19 mutants were temporarily named: AcetRL-R349G, AcetRL-R349A, AcetRL-R349V, AcetRL-R349L, AcetRL-R L-R349I, AcetRL-R349M, AcetRL-R349F, AcetRL-R349W, AcetRL-R349P, AcetRL-R349S, AcetRL-R349T, AcetRL-R3 49C, AcetRL-R349Y, AcetRL-R349N, AcetRL-R349Q, AcetRL-R349D, AcetRL-R349E, AcetRL-R349K, AcetRL-R349H.

[0036] 19 mutant plasmids were constructed by conventional methods and transformed into Bacillus subtilis 168 strains for heterologous expression (the method is the same as in Example 2), and fermentation broths containing various mutants were obtained. The products of each fermentation broth were measured according to the method of Example 3 (with the fermentation broth of Example 2 as a control), and the results are as follows: Figure 6 As shown. Figure 6 It can be seen that multiple mutants have the ability to produce chitohexaose, but the ability varies greatly. Among them, the total chito-oligosaccharide production of AcetRL-R349Q is the highest, which is 113.78 mg / L, but the production of chitohexaose is only 11.35 mg / L; the production of chitohexaose of AcetRL-R349S and AcetRL-R349H is the highest, reaching 23.32 mg / L and 24.07 mg / L respectively, but the production of chitopentaose of AcetRL-R349H is 58.55 mg / L, which is higher than the chitopentaose production of AcetRL-R349S of 49.47 mg / L. Therefore, the ability of AcetRL-R349H to produce high-polymerization chito-oligosaccharides is better than that of AcetRL-R349S. The present invention selects AcetRL-R349H for subsequent research and names the mutant as N-acetylglucosamine transferase AcetRL-R349H.

[0037] The construction process of N-acetylglucosamine transferase AcetRL-R349H is as follows: The amino acid at position 349 of N-acetylglucosamine transferase AcetRL is changed from arginine to histidine, specifically by performing site-directed mutation on N-acetylglucosamine transferase AcetRL at residue ARG349, and changing the codon corresponding to the amino acid at position 349 on the coding gene of N-acetylglucosamine transferase AcetRL from the codon "CGC" for arginine to the codon "CAT" for histidine. The amino acid sequence of N-acetylglucosamine transferase AcetRL-R349H is shown in SEQ ID NO.5, and the nucleotide sequence of its coding gene is shown in SEQ ID NO.6.

[0038] The amino acid sequence of N-acetylglucosamine transferase AcetRL-R349H is shown in SEQ ID NO.5, as shown below: MTLFATASTVAICSYALLSTVYKTAQVFYTLPTNVPPTSGDPPGGEPWPSVDVIIPCYNEAPRTLSDCLASIASQEYAGKLQVYVVDDGSANRDALVGVHEEYAGD PRFNFIALPKNVGKRKAQIAAIRRSCGDLVLNVDSDTILAPDVITRLALKMQDQAVGAAMGQLAASNRSETWLTRLIDMEYWLACNEERAAQARFGAVMCCCGPCA MYRRSALVSLLDQYETQRFRGKPSDFGEDRHLTILKAGFRTEYVPEAVAATVVPNSMGPYLRQQLRWARSTFRDTLLAFQLLRGLNIYLTLDVIGQNIGPLLLSLSILAGLAQLVTTGTAPWTACLMIAMTIVHCSVAAFRARQLRFLGFSLHTLINIFLLLPLKAYALCTLSNSDWLSRSSAANVTDTGGTSSKPNLVGSDAAYSEQQ.

[0039] The nucleotide sequence of the gene encoding N-acetylglucosamine transferase AcetRLR349H is shown in SEQ ID NO.6, as shown below (direction 5'-3'):

[0040] Then, PCR amplification of the entire plasmid (i.e., the pP43NMK plasmid encoding the gene of N-acetylglucosamine transferase AcetRL) was performed. The PCR product was treated with restriction endonuclease DpnI to digest the methylated parent 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.

[0041] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.7, as shown below (direction 5'-3'): cattgtgcattgtagcgttgcagcatttcg.

[0042] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.8, as shown below (direction 5'-3'): caacgctacaatgcacaatggtcattgctgc.

[0043] Example 5 Product determination of N-acetylglucosamine transferase AcetRL-R349H A recombinant engineered bacterium expressing N-acetylglucosamine transferase AcetRL-R349H was constructed and fermented. The collected fermentation broth was analyzed in the same manner as in Example 3. The HPLC analysis results are shown in FIG. Figure 1 As shown, the product composition and yield are compared. Figure 2 As shown in the figure, chitosan appeared in the product of N-acetylglucosamine transferase AcetRL-R349H, and the yield was as high as 24.07 mg / L, which is not available in N-acetylglucosamine transferase AcetRL.

[0044] AlphaFold 2.0 was used to build the structural model of N-acetylglucosamine transferase AcetRL-R349H and Autodock was used to perform molecular docking with chitosan. The results are as follows: Figure 7 , Figure 8 As shown. The largest product of N-acetylglucosamine transferase AcetRL docking is chitotetraose, but N-acetylglucosamine transferase AcetRL-R349H can achieve the docking of chitopentaose. The fact that N-acetylglucosamine transferase AcetRL-R349H allows the embedding of chitopentaose may be the basis for the production of chitohexaose. At the same time, the amino acid residues at the entrance of the active pocket show extremely strong hydrogen bond interactions with the ligand, which may be another key factor in the production of chitohexaose.

[0045] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

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

5.

2. Use of the N-acetylglucosamine transferase AcetRL-R349H according to claim 1 in the preparation of chito-oligosaccharides.

3. The use of N-acetylglucosamine transferase AcetRL-R349H according to claim 2 in the preparation of chito-oligosaccharides, characterized in that: The chito-oligosaccharide is any one or more of chitotetraose, chitopentaose or chitohexaose.

4. The use of N-acetylglucosamine transferase AcetRL-R349H according to claim 2 or 3 in the preparation of chito-oligosaccharides, characterized in that: In specific application, the coding gene of N-acetylglucosamine transferase AcetRL-R349H is introduced into Bacillus subtilis to construct a recombinant engineering bacterium expressing N-acetylglucosamine transferase AcetRL-R349H; the recombinant engineering bacterium is fermented to obtain a fermentation liquid containing chito-oligosaccharide.

5. The use of N-acetylglucosamine transferase AcetRL-R349H according to claim 4 in the preparation of chito-oligosaccharides, characterized in that: The Bacillus subtilis is Bacillus subtilis 168 strain.

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