N-acetylglucosamine transferase AcetRS-R346D and application thereof

Abstract

The invention discloses an N-acetylglucosamine transferase AcetRS-R346D and an application thereof, and belongs to the technical field of functional enzymes. And the amino acid sequence of the N-acetylglucosamine transferase AcetRS-R346D is as shown in SEQ ID NO. 3. The invention further discloses the application of the N-acetylglucosamine transferase AcetRS-R346D in the preparation of the chitosan oligosaccharide. The N-acetylglucosamine transferase AcetRS-R346D disclosed by the invention is obtained by carrying out mutation modification on the N-acetylglucosamine transferase AcetRS, and can be used for producing the chitosan oligosaccharide with the polymerization degree of 4 to 7, especially (GlcNAc) 7. 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 AcetRS-R346D 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 AcetRS-R346D 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 AcetRS-R346D, whose amino acid sequence is shown in SEQ ID NO.3.

[0006] The application of the N-acetylglucosamine transferase AcetRS-R346D in the preparation of chito-oligosaccharides.

[0007] Furthermore, the degree of polymerization of the chito-oligosaccharide is 4-7.

[0008] Furthermore, in specific applications, the coding gene of N-acetylglucosamine transferase AcetRS-R346D is introduced into Bacillus subtilis to construct a recombinant engineered bacterium expressing N-acetylglucosamine transferase AcetRS-R346D; 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 AcetRS-R346D.

[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 AcetRS-R346D of the present invention is obtained by mutation modification of the N-acetylglucosamine transferase AcetRS. After mutation modification, the N-acetylglucosamine transferase AcetRS has product specificity and can be used to produce chito-oligosaccharides with a degree of polymerization of 4 to 7, especially (GlcNAc)7. 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 AcetRS represents N-acetylglucosamine transferase AcetRS, and AcetRS-R346D represents N-acetylglucosamine transferase AcetRS-R346D.

[0013] Figure 2 : Comparison of product composition and yield of fermentation broth, wherein AcetRS represents N-acetylglucosamine transferase AcetRS, and AcetRS-R346D represents N-acetylglucosamine transferase AcetRS-R346D.

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

[0015] Figure 4 : 2D diagram of the docking analysis results of N-acetylglucosamine transferase AcetRS and chitopentaose molecules.

[0016] Figure 5 : Comparison of product composition and yield of fermentation broth containing each mutant.

[0017] Figure 6 :2D diagram of the docking analysis results of N-acetylglucosamine transferase AcetRS-R346D and chitosan. DETAILED DESCRIPTION

[0018] 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.

[0019] 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.

[0020] The Escherichia coli DH5α strain used in the present invention was purchased from Beijing Qingke Biotechnology Co., Ltd.

[0021] The Bacillus subtilis 168 strain used in the present invention was purchased from China General Microbiological Culture Collection Center (CGMCC).

[0022] Example 1 Mining of N-acetylglucosamine transferase AcetRS In order to explore N-acetylglucosamine transferases with product specificity, the present invention screened a gene from Rhizobium GRH2 ( Rhizobium sp. The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO.1, and the gene is named as N-acetylglucosamine transferase AcetRS. After the preferred codon optimization of Bacillus subtilis, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0023] The amino acid sequence of N-acetylglucosamine transferase AcetRS is shown in SEQ ID NO.1, as shown below: MDLLNTIGIGAVSCYALLSTAHKSMQTLYAQPKDQSSASEDFAFLPSVDIIVPCYNENPHTFSECLASIANQDYAGKLRVYVVDDGSANREKLERVHHTYAGDPRFDFILL RENVGKRKAQIAAIRGSSGDLVLNVDSDSTLASDVVTKLALKMQNPEIGAAMGQLTASNRNDTWLTRLIDMEYWLACNEERAAQARFGAVMCCCGPCAMYRRSALLSLLDQ YESQFFRGKPSDFGEDRHLTILMLKAGFRTDYVPDAIAATVVPDRMGPYLRQQLRWARSTFRDTLLALRLLPGLDHYITLDVIGQNLGPLLLALAVLTGVLQVALTATVPL WTVMMIASMTMIRCAVAAVRARQLRFLVFSLHTPINLFFLLPMKAYALCTLSNSDWLSRSSPANKTSAGGEHPTTEASAGGTSGNATPLRRLNLARDSSTVTPAGVYSDD.

[0024] The nucleotide sequence of the gene encoding N-acetylglucosamine transferase AcetRS 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 AcetRS 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 connection product was transformed into Escherichia coli DH5α competent cells. Positive transformants were screened using LB plates containing sodium ampicillin. After the clones were verified by colony PCR, positive clones were selected for sequencing to obtain recombinant plasmids.

[0027] (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.

[0028] (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.

[0029] Example 3 Determination of the product of N-acetylglucosamine transferase AcetRS 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.

[0030] HPLC analysis results are as follows Figure 1 As shown, the product composition and yield are compared. Figure 2 As shown, the products of N-acetylglucosamine transferase AcetRS are chitotetraose, chitopentose, and chitohexaose.

[0031] Example 4 Modification of N-acetylglucosamine transferase AcetRS From the results of Example 3, N-acetylglucosamine transferase AcetRS can produce chitotetraose, chitopentose and chitohexaose, and 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.

[0032] To determine 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 pocket of N-acetylglucosamine transferase AcetRS. The results are as follows: Figure 3 As shown; Autodock software was used to dock the substrate (chitopentose) with the enzyme molecule, and the molecular docking results were analyzed using Discovery Studio Client software. The results are shown in Figure 4 shown.

[0033] The key amino acid ARG346 with strong double traditional hydrogen bonding with chitopentaose was selected as the mutation site by selecting the amino acid at the intersection of the bottom of the pocket and the docking amino acid. Then, based on the comparative analysis of the protein sequences of N-acetylglucosamine transferases from different sources, it was further determined that ARG346 may be the key site for controlling the polymerization degree of the product of N-acetylglucosamine transferase AcetRS. Therefore, the present invention selects this site for semi-rational mutation, and changes the 346th amino acid from arginine to a hydrophilic amino acid with a polar charged side chain (aspartic acid, glutamic acid, histidine). The three mutants obtained are temporarily named: AcetRS-R346D, AcetRS-R346E, and AcetRS-R346H.

[0034] Three mutant plasmids were constructed by conventional methods and transformed into Bacillus subtilis 168 strain for heterologous expression (the method is the same as in Example 2), and fermentation broth containing each mutant was 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 5 As shown. Figure 5It can be seen that these three mutants all have the ability to produce chitoheptaose, among which AcetRS-R346D has the highest chitoheptaose yield of 9.85 mg / L, which is significantly better than AcetRS-R346E and AcetRS-R346H, and AcetRS-R346D also has the highest chitohexaose yield of 41.83 mg / L, which is also significantly better than AcetRS-R346E and AcetRS-R346H; therefore, the present invention selects AcetRS-R346D for subsequent research, and the mutant is named N-acetylglucosamine transferase AcetRS-R346D.

[0035] The construction process of N-acetylglucosamine transferase AcetRS-R346D is as follows: The amino acid at position 346 of N-acetylglucosamine transferase AcetRS is changed from arginine to aspartic acid, specifically by performing site-directed mutation on N-acetylglucosamine transferase AcetRS at residue ARG346, and changing the codon corresponding to the amino acid at position 346 on the coding gene of N-acetylglucosamine transferase AcetRS from the codon "aga" for arginine to the codon "gat" for aspartic acid. The amino acid sequence of N-acetylglucosamine transferase AcetRS-R346D is shown in SEQ ID NO.3, and the nucleotide sequence of its coding gene is shown in SEQ ID NO.4.

[0036] The amino acid sequence of N-acetylglucosamine transferase AcetRS-R346D is shown in SEQ ID NO.3, as shown below: MDLLNTIGIGAVSCYALLSTAHKSMQTLYAQPKDQSSASEDFAFLPSVDIIVPCYNENPHTFSECLASIANQDYAGKLRVYVVDDGSANREKLERVHHTYAGDPRFDFILL RENVGKRKAQIAAIRGSSGDLVLNVDSDSTLASDVVTKLALKMQNPEIGAAMGQLTASNRNDTWLTRLIDMEYWLACNEERAAQARFGAVMCCCGPCAMYRRSALLSLLDQ YESQFFRGKPSDFGEDRHLTILMLKAGFRTDYVPDAIAATVVPDRMGPYLRQQLRWARSTFRDTLLALRLLPGLDHYITLDVIGQNLGPLLLALAVLTGVLQVALTATVPL WTVMMIASMTMIDCAVAAVRARQLRFLVFSLHTPINLFFLLPMKAYALCTLSNSDWLSRSSPANKTSAGGEHPTTEASAGGTSGNATPLRRLNLARDSSTVTPAGVYSDD.

[0037] The nucleotide sequence of the gene encoding N-acetylglucosamine transferase AcetRSR346D is shown in SEQ ID NO.4, as shown below (direction 5'-3'):

[0038] Then, PCR amplification of the entire plasmid (i.e., the pP43NMK plasmid encoding the gene of N-acetylglucosamine transferase AcetRS) 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.5 and SEQ ID NO.6.

[0039] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.5, as shown below (direction 5'-3'): acaatgatcgattgcgcggttgcagc.

[0040] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.6, as shown below (direction 5'-3'): accgcgcaatcgatcattgtcattgatgc.

[0041] Example 5 Product determination of N-acetylglucosamine transferase AcetRS-R346D A recombinant engineered bacterium expressing N-acetylglucosamine transferase AcetRS-R346D 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, it can be seen that the product of N-acetylglucosamine transferase AcetRS-R346D contains chitoheptaose, which has never been reported before for this type of enzyme. The proportion of chitoheptaose in the total amount of chito-oligosaccharides is about 20%, which is not possessed by this type of N-acetylglucosamine transferase.

[0042] AlphaFold 2.0 was used to build the structural model of N-acetylglucosamine transferase AcetRS-R346D and Autodock was used to perform molecular docking with chitosan. The results are as follows: Figure 6 As shown. The largest product of N-acetylglucosamine transferase AcetRS docking is chitopentose, but N-acetylglucosamine transferase AcetRS-R346D can achieve the docking of chitohexaose. The number of traditional hydrogen bonds between the amino acid at the entrance of the active pocket of N-acetylglucosamine transferase AcetRS-R346D and chitohexaose increases, which makes the enzyme more capable of grabbing chitohexaose, which may be the key factor for the mutant to produce chitoheptaose.

[0043] 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 AcetRS-R346D, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

3.

2. Use of the N-acetylglucosamine transferase AcetRS-R346D according to claim 1 in the preparation of chito-oligosaccharides.

3. The use of N-acetylglucosamine transferase AcetRS-R346D according to claim 2 in the preparation of chito-oligosaccharides, characterized in that: The polymerization degree of the chito-oligosaccharide is 4-7.

4. The use of N-acetylglucosamine transferase AcetRS-R346D 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 AcetRS-R346D is introduced into Bacillus subtilis to construct a recombinant engineering bacterium expressing N-acetylglucosamine transferase AcetRS-R346D; the recombinant engineering bacterium is fermented to obtain a fermentation liquid containing chito-oligosaccharide.

5. The use of N-acetylglucosamine transferase AcetRS-R346D 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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