N-acetylglucosamine transferase acetrs-k23g and use thereof

Abstract

The application discloses N-acetylglucosamine transferase AcetRS-K23G and an application thereof, and belongs to the technical field of functional enzymes. The amino acid sequence of the N-acetylglucosamine transferase AcetRS-K23G is shown as SEQ ID NO. 3. The application of the N-acetylglucosamine transferase AcetRS-K23G in the preparation of chitooligosaccharide. The N-acetylglucosamine transferase AcetRS-K23G is obtained by mutation and modification of N-acetylglucosamine transferase AcetRS, and can be used for producing chitooligosaccharide with a polymerization degree of 5-7, especially chitoseptasaccharide, and the yield of the chitoseptasaccharide can reach 42.01 mg / L, so that the N-acetylglucosamine transferase AcetRS-K23G is an ideal functional enzyme for industrialized production of chitoseptasaccharide. The application widens the product spectrum of N-acetylglucosamine transferase, and can be used for preparing chitooligosaccharide with a high polymerization degree, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to an N-acetylglucosamine transferase, AcetRS-K23G, 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). Conventional N-acetylglucosaminyltransferases can only produce chitosan oligosaccharides with a degree of polymerization (degree of polymerization) of 3–5, resulting in a very limited product degree of polymerization. It is difficult to achieve the production of chitosan oligosaccharides with a degree of polymerization greater than 5, and a high proportion of chitosan oligosaccharides with a degree of polymerization of 7 cannot be produced. Therefore, based on N-acetylglucosaminyltransferase, obtaining an N-acetylglucosaminyltransferase with high specificity for producing chitosan oligosaccharides with a degree of polymerization of 7 through rational design strategies has broad application prospects.

[0003] CN 119842658 A discloses an N-acetylglucosamine transferase, AcetRS-R346D, and its applications. The mutant N-acetylglucosamine transferase AcetRS was obtained by mutating N-acetylglucosamine transferase AcetRS, which can be used to produce chitosan oligosaccharides with a degree of polymerization of 4–7, especially (GlcNAc)7. However, the proportion of chitoheptaose in its enzymatic hydrolysis products is only 15%, which is still not ideal. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides an N-acetylglucosamine transferase AcetRS-K23G 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-K23G, has the amino acid sequence shown in SEQ ID NO.3.

[0007] The application of the N-acetylglucosamine transferase AcetRS-K23G in the preparation of chitosan oligosaccharides, wherein the chitosan oligosaccharides are any one or more of chitopentose, chitohexaose and chitoheptaose.

[0008] Preferably, the chitin oligosaccharide is chitinhexasaccharide.

[0009] Further, in specific applications, the encoding gene of N-acetylglucosamine transferase AcetRS-K23G is introduced into Bacillus subtilis to construct a recombinant engineered bacterium expressing N-acetylglucosamine transferase AcetRS-K23G; fermentation of this recombinant engineered bacterium yields a fermentation broth containing chitosan oligosaccharides; the nucleotide sequence of the encoding gene of N-acetylglucosamine transferase AcetRS-K23G is shown in SEQ ID NO.4. The principle of producing chitosan oligosaccharides is as follows: using GlcNAc and UDP-GlcNAc produced by Bacillus subtilis itself as substrates, chitosan oligosaccharides are prepared under the action of N-acetylglucosamine transferase AcetRS-K23G.

[0010] 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.

[0011] The N-acetylglucosamine transferase AcetRS-K23G of this invention is obtained by mutating N-acetylglucosamine transferase AcetRS. After mutation, the total yield of chitosan oligosaccharides is significantly increased, reaching 85.72 mg / L; it exhibits product specificity and can be used to produce chitosan oligosaccharides with a degree of polymerization of 5-7, especially chitoheptaose, with a yield of up to 42.01 mg / L and a very high proportion of chitoheptaose in the product, reaching 50.41%, making it an ideal functional enzyme for the industrial production of chitoheptaose. This invention broadens the product spectrum of N-acetylglucosamine transferase and can be used to prepare high-degree-of-polymerization chitosan oligosaccharides, showing broad application prospects.

[0012] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0013] Figure 1 : A schematic diagram comparing the product composition and yield of N-acetylglucosamine transferase AcetRS and its mutants in fermentation broth. In this diagram, CTOS represents chitosan oligosaccharide, CTOS4 represents chitosan tetrasaccharide, CTOS5 represents chitosan pentasaccharide, CTOS6 represents chitosan hexasaccharide, and CTOS7 represents chitosan heptasaccharide.

[0014] Figure 2 3D diagram showing the docking results of N-acetylglucosamine transferase AcetRS-K23G with chitosan molecules.

[0015] Figure 3Analysis of the docking results between N-acetylglucosamine transferase AcetRS-K23G and chitosan molecules (2D diagram). Detailed Implementation

[0016] 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.

[0017] 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.

[0018] Example 1: Modification of N-acetylglucosamine transferase AcetRS

[0019] Previous studies have shown that N-acetylglucosamine transferase AcetRS can produce chitotetrasaccharides, chitopentoses, and chitohexasaccharides, but cannot produce chitooligosaccharides with higher degrees of polymerization. Therefore, this invention attempts to mutate and modify it to obtain an N-acetylglucosamine transferase with a higher degree of polymerization capacity for producing chitooligosaccharides.

[0020] The amino acid sequence of N-acetylglucosamine transferase AcetRS is shown in SEQ ID NO.1, as follows:

[0021] MDLLNTIGIGAVSCYALLSTAHKSMQTLYAQPKDQSSASEDFAFLPSVDIIVPCYNENPHTFSECLASIANQDYAGKLRVYVVDDGSANREKLERVHHTYAGDPRFDFILL RENVGKRKAQIAAIRGSSGDLVLNVDSDSTLASDVVTKLALKMQNPEIGAAMGQLTASNRNDTWLTRLIDMEYWLACNEERAAQARFGAVMCCCGPCAMYRRSALLSLLDQ YESQFFRGKPSDFGEDRHLTILMLKAGFRTDYVPDAIAATVVPDRMGPYLRQQLRWARSTFRDTLLALRLLPGLDHYITLDVIGQNLGPLLLALAVLTGVLQVALTATVPL WTVMMIASMTMIRCAVAAVRARQLRFLVFSLHTPINLFFLLPMKAYALCTLSNSDWLSRSSPANKTSAGGEHPTTEASAGGTSGNATPLRRLNLARDSSTVTPAGVYSDD.

[0022] The nucleotide sequence of the gene encoding N-acetylglucosamine transferase AcetRS is shown in SEQ ID NO.2, as shown below (direction 5'-3'):

[0023]

[0024] Based on sequence alignment and the position of the active amino acid residues, Lys23, a key pocket amino acid in the compressed substrate channel, was selected as the key mutation site. The amino acid at position 23 was mutated from lysine to a hydrophobic and nonpolar side chain amino acid, resulting in nine mutants, tentatively named: AcetRS-K23G, AcetRS-K23A, AcetRS-K23V, AcetRS-K23L, AcetRS-K23I, AcetRS-K23M, AcetRS-K23F, AcetRS-K23W, and AcetRS-K23P.

[0025] The original enzyme and recombinant plasmids of the nine mutants 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 identified according to the corresponding standard curve.

[0026] A comparative diagram of the product composition and yield of N-acetylglucosamine transferase AcetRS and its mutants is shown below. Figure 1 As shown. By Figure 1 It is evident that AcetRS-K23G, AcetRS-K23A, and AcetRS-K23W all possess the ability to produce chitoheptaose. Among them, AcetRS-K23G exhibits the highest chitoheptaose yield at 42.01 mg / L, and also the highest chitoheptaose percentage at 50.41%, significantly superior to AcetRS-K23A and AcetRS-K23W, and also significantly superior to AcetRS-R346D disclosed in CN 119842658 A (whose chitoheptaose yield is 9.85 mg / L). Its total chitooligosaccharide yield is also significantly increased to 85.72 mg / L, only slightly lower than AcetRS-K23A. AcetRS-K23A has the highest chitohexaose percentage and the highest total chitooligosaccharide yield at 87.36 mg / L.

[0027] This invention selected AcetRS-K23G for further research and named the mutant N-acetylglucosamine transferase AcetRS-K23G.

[0028] The construction process of N-acetylglucosamine transferase AcetRS-K23G is as follows:

[0029] The 23rd amino acid of N-acetylglucosamine transferase AcetRS was changed from lysine to glycine. Specifically, a site-directed mutagenesis was performed at residue Lys23 of N-acetylglucosamine transferase AcetRS, changing the codon for the 23rd amino acid in the gene encoding N-acetylglucosamine transferase AcetRS from the lysine codon "aaa" to the glycine codon "ggc".

[0030] The amino acid sequence of N-acetylglucosamine transferase AcetRS-K23G is shown in SEQ ID NO.3, as follows:

[0031] MDLLNTIGIGAVSCYALLSTAHGSMQTLYAQPKDQSSASEDFAFLPSVDIIVPCYNENPHTFSECLASIANQDYAGKLRVYVVDDGSANREKLERVHHTYAGDPRFDFILL RENVGKRKAQIAAIRGSSGDLVLNVDSDSTLASDVVTKLALKMQNPEIGAAMGQLTASNRNDTWLTRLIDMEYWLACNEERAAQARFGAVMCCCGPCAMYRRSALLSLLDQ YESQFFRGKPSDFGEDRHLTILMLKAGFRTDYVPDAIAATVVPDRMGPYLRQQLRWARSTFRDTLLALRLLPGLDHYITLDVIGQNLGPLLLALAVLTGVLQVALTATVPL WTVMMIASMTMIRCAVAAVRARQLRFLVFSLHTPINLFFLLPMKAYALCTLSNSDWLSRSSPANKTSAGGEHPTTEASAGGTSGNATPLRRLNLARDSSTVTPAGVYSDD.

[0032] The nucleotide sequence of the gene encoding N-acetylglucosamine transferase AcetRS-K23G is shown in SEQ ID NO.4, as shown below (direction 5'-3'):

[0033]

[0034] Then, PCR amplification of the entire plasmid (i.e., the pP43NMK plasmid encoding the N-acetylglucosamine transferase AcetRS gene) was performed. The PCR product was treated 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.5 and SEQ ID NO.6.

[0035] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.5, as shown below (direction 5'-3'):

[0036] aacagcacatggctcaatgcaaacactgtacgcgc.

[0037] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.6, as shown below (direction 5'-3'):

[0038] gcattgagccatgtgctgttgacagcagtgcatagc.

[0039] Example 2 Molecular docking analysis of N-acetylglucosamine transferase AcetRS-K23G

[0040] A structural model of N-acetylglucosamine transferase AcetRS-K23G was constructed using AlphaFold 2.0, and molecular docking with chitosan was performed using Autodock. The 3D model of the N-acetylglucosamine transferase AcetRS-K23G molecular docking with chitosan is shown below. Figure 2 As shown in the figure, the 2D diagram of the docking results between N-acetylglucosamine transferase AcetRS-K23G and chitosan molecules is as follows. Figure 3 As shown in the figure, mutating Lys23 to glycine allows the non-reducing end of chitosan to connect to the bottom of the substrate catalytic channel. Simultaneously, the amino acid substitution significantly increases the substrate tunnel interface area, creating space for high-polymerization-degree ligands to enter the pocket and for further extension of the product sugar chain.

[0041] 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-K23G, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

2. The application of the N-acetylglucosamine transferase AcetRS-K23G according to claim 1 in the preparation of chitosan oligosaccharides, characterized in that: The chitin oligosaccharide is any one or more of chitinpentaose, chitinhexaose, and chitinheptaose.

3. The application of the N-acetylglucosamine transferase AcetRS-K23G according to claim 2 in the preparation of chitosan oligosaccharides, characterized in that: The chitin oligosaccharide is chitin heptaose.

4. The application of N-acetylglucosamine transferase AcetRS-K23G according to claim 2 or 3 in the preparation of chitosan oligosaccharides, characterized in that: In a specific application, the encoding gene of N-acetylglucosamine transferase AcetRS-K23G is introduced into Bacillus subtilis to construct a recombinant engineered bacterium expressing N-acetylglucosamine transferase AcetRS-K23G; the recombinant engineered bacterium is fermented to obtain a fermentation broth containing chitosan oligosaccharides; the nucleotide sequence of the encoding gene of N-acetylglucosamine transferase AcetRS-K23G is shown in SEQ ID NO.

4.

5. The application of the N-acetylglucosamine transferase AcetRS-K23G according to claim 4 in the preparation of chitosan oligosaccharides, characterized in that: The Bacillus subtilis strain mentioned is Bacillus subtilis strain 168.

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