An N-acetylglucosamine isomerase mutant and its application

Abstract

This invention provides an N-acetylglucosamine isomerase mutant and its applications. The N-acetylglucosamine isomerase mutant is obtained by mutating any one or more of the following sites in the wild-type N-acetylglucosamine isomerase: glutamic acid at position 73, glutamine at position 110, leucine at position 171, alanine at position 172, or alanine at position 198. Compared with the wild-type N-acetylglucosamine isomerase, the N-acetylglucosamine isomerase mutant constructed in this invention exhibits higher catalytic activity, with an increase in enzyme activity of 27-55%. The modified N-acetylglucosamine isomerase mutant of this invention can be better applied to the preparation of N-acetylneuraminic acid and is more suitable for industrial applications.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to an N-acetylglucosamine isomerase mutant and its applications. Background Technology

[0002] N-acetylglucosamine isomerase and N-acetylneuraminic acid lyase can be coupled to produce N-acetylneuraminic acid. However, during the production of N-acetylneuraminic acid, the activity of N-acetylneuraminic acid isomerase decreases, which seriously affects the yield of N-acetylneuraminic acid. In order to increase the yield of N-acetylneuraminic acid, it is necessary to develop an N-acetylneuraminic acid isomerase with high stability. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide an N-acetylglucosamine isomerase mutant to address the shortcomings of the prior art.

[0004] The technical problem that this invention also aims to solve is the application of the above-mentioned N-acetylglucosamine isomerase mutant in the preparation of N-acetylmnosamine and in the preparation of N-acetylneuraminic acid.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] An N-acetylglucosamine isomerase mutant, wherein the amino acid sequence of the N-acetylglucosamine isomerase mutant is obtained by mutating any one or more of the following positions in the wild-type N-acetylglucosamine isomerase: glutamic acid at position 73, glutamine at position 110, leucine at position 171, alanine at position 172, or alanine at position 198.

[0007] Specifically, glutamic acid at position 73 (Glu) is mutated to glycine (Gly), glutamine at position 110 (Gln) is mutated to cysteine ​​(Cys), leucine at position 171 (Leu) is mutated to isoleucine (Ile), alanine at position 172 (Ala) is mutated to glycine (Gly), and alanine at position 198 (Ala) is mutated to aspartic acid (Asp).

[0008] Preferably, an N-acetylglucosamine isomerase mutant is obtained by double mutation of the wild-type N-acetylglucosamine isomerase at two sites: alanine at position 172 and alanine at position 198.

[0009] Among them, alanine at position 172 (Ala) is mutated to glycine (Gly), and alanine at position 198 (Ala) is mutated to aspartic acid (Asp).

[0010] The wild-type N-acetylglucosamine isomerase is derived from N-acetylglucosamine 2-epimerase in Anabaena sp.CH1, and its amino acid sequence is shown in SEQ ID No.:1. The corresponding nucleotide sequence encoding the wild-type N-acetylglucosamine isomerase is shown in SEQ ID No.:2.

[0011] The N-acetylglucosamine isomerase mutant has an amino acid sequence as shown in any one of SEQ ID No.:3-8.

[0012] Preferably, the N-acetylglucosamine isomerase mutant has the amino acid sequence shown in SEQ ID No.:8.

[0013] The N-acetylglucosamine isomerase mutant has a nucleotide sequence as shown in any one of SEQ ID No.:9-14.

[0014] Preferably, the N-acetylglucosamine isomerase mutant has the nucleotide sequence shown in SEQ ID No.:14.

[0015] In the effect test on N-acetylglucosamine isomerase mutants, it was found that compared with wild-type N-acetylglucosamine isomerase, all mutants had higher enzyme activities than WT, with enzyme activities increasing by 27-55%, exhibiting higher catalytic activity. The modification sites are located at sites including activity pockets and stability-related sites, making the enzyme more stable and enhancing its activity.

[0016] The technical method and preparation process used in this invention involves constructing a vector plasmid containing the wild-type N-acetylglucosamine isomerase gene using techniques known in the art. Then, a site for targeted mutation and the type of mutated amino acid are selected, and corresponding primers are synthesized. Using the N-acetylglucosamine isomerase gene vector plasmid as a template, the mutated DNA fragment is amplified by PCR. The resulting fragment is then amplified into a full-length mutated gene by PCR. This full-length mutated gene is cloned into an appropriate vector and transformed into suitable host cells. Positive clones with high N-acetylglucosamine isomerase activity are selected through culture and screening. Finally, plasmid DNA is extracted from the positive clones and subjected to DNA sequencing analysis to determine the introduced mutation.

[0017] Specifically, the method for constructing the above-mentioned N-acetylglucosamine isomerase mutant includes the following steps:

[0018] (1) The N-acetylglucosamine isomerase gene anAGE was synthesized from the whole gene. The fragment after PCR amplification and gel recovery was ligated into the pET28a vector by enzyme digestion to obtain the plasmid pET28a-anAGE.

[0019] (2) Using the plasmid pET28a-anAGE obtained in step (1) as a template, segmented PCR was performed using wild-type primers and mutant site primers. After purifying and recovering the segmented PCR product, overlap PCR was performed using the segmented PCR product as a template to obtain the N-acetylglucosamine isomerase gene with the mutant site.

[0020] (3) The N-acetylglucosamine isomerase gene with mutation site obtained in step (2) is digested and ligated into the pET28a vector to construct recombinant plasmids. The recombinant plasmids are then transformed into competent Escherichia coli Rosetta (DE3) to obtain N-acetylglucosamine isomerase mutants.

[0021] In step (3), the recombinant plasmids include pET28a-anAGE-E73G, pET28a-anAGE-Q110C, pET28a-anAGE-L171I, pET28a-anAGE-A172G, and pET28a-anAGE-A198D. Using the recombinant plasmid pET28a-anAGE-A172G as a template, the alanine Ala at position 198 in the amino acid sequence is mutated to aspartic acid Asp to obtain the N-acetylglucosamine isomerase double mutant anAGE-A172G-A198D.

[0022] The N-acetylglucosamine isomerase mutant described in this invention can be used in the form of unpurified crude enzyme, or in a partially purified or purified form, or as a solid-phase enzyme / solid-phase cell.

[0023] The application of N-acetylglucosamine isomerase mutants in the preparation of N-acetymannosamine is also within the scope of this invention.

[0024] Specifically, using N-acetylglucosamine as a substrate, the described N-acetylglucosamine isomerase mutant is subjected to an enzymatic reaction with N-acetylglucosamine. Those skilled in the art will understand that the applications described in this invention may also include the use of other reagents and materials required for the preparation of N-acetymannosamine.

[0025] The application of N-acetylglucosamine isomerase mutants in the preparation of N-acetylneuraminic acid is also within the scope of this invention.

[0026] Specifically, using N-acetylglucosamine and sodium pyruvate as substrates, the described N-acetylglucosamine isomerase mutant is subjected to an enzymatic reaction with N-acetylglucosamine, sodium pyruvate, and N-acetylneuraminic acid lyase. Those skilled in the art will understand that the applications described in this invention may also include the use of other reagents and materials required for the preparation of N-acetylneuraminic acid.

[0027] Specifically, such as Figure 1 As shown, the modified enzyme was relatively stable during the 48-hour reaction, and the yield increased with time, reaching equilibrium after 18 hours, and was significantly improved compared to the wild-type fungus.

[0028] The preparation of N-acetylglucosamine and N-acetylneuraminic acid can be carried out using conventional methods known in the art, provided that the N-acetylglucosamine isomerase mutant described in this invention is used as the enzyme required for the corresponding step in each method.

[0029] Beneficial Effects: This invention improves the catalytic activity of N-acetylglucosamine isomerase from Anabaena sp. CH1 through genetic engineering. Compared to wild-type N-acetylglucosamine isomerase, the mutant N-acetylglucosamine isomerase of this invention exhibits 27-55% increased enzyme activity. In the production of N-acetylneuraminic acid, the modified enzyme is more stable during the reaction, with increased yield, and a significant improvement over the wild-type strain. The N-acetylglucosamine isomerase mutant constructed in this invention is more suitable for industrial applications. Attached Figure Description

[0030] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0031] Figure 1 Example of 48-hour response against WT and mutant bacteria Detailed Implementation

[0032] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0033] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0034] Unless otherwise specified, all conditions described in the following examples are based on standard conditions or conditions recommended by the manufacturer. Unless otherwise stated, percentages are volume percentages.

[0035] Example 1: Cloning of N-acetylglucosamine isomerase gene and construction of plasmid pET28a-anAGE

[0036] Based on the N-acetylglucosamine isomerase DNA sequence from the DNA sequence database (EC 5.1.3.8), the DNA sequence was optimized using Gene Designer 2.0 software according to the codon preferences of the host cell. Primers anAGE-T1 and anAGE-B1 were designed based on this gene sequence (Table 1).

[0037] The above DNA sequence was synthesized to obtain plasmid Top10-anAGE. Using plasmid Top10-anAGE as a template, PCR was performed with primers anAGE-T1 and anAGE-B1 to amplify a 1167bp product.

[0038] The PCR reaction conditions were as follows: 300 ng plasmid Top10-anAGE, 60 ng primers (anAGE-T1+anAGE-B1), 5 μL 10x buffer, 4 μL 2.5 mM dNTP, 1 μL PrimerStar polymerase (TaKaRa), and the reaction volume was adjusted to 50 μL with sterile water.

[0039] The PCR amplification program was as follows: 95℃ for 5 minutes, 30 cycles: 94℃ for 30 seconds, 55℃ for 30 seconds and 72℃ for 1.5 minutes, and finally 72℃ for 10 minutes.

[0040] The wild-type N-acetylglucosamine isomerase gene was obtained by amplification reaction. The PCR product was purified using a DNA gel extraction kit (TaKaRa) and the purified 1167 bp fragment was isolated. After digestion with XhoI and NotI (TaKaRa), the fragment was ligated into the pET28a vector using T4 DNA ligase (TaKaRa) to obtain the plasmid pET28a-anAGE. This plasmid was transformed into competent *E. coli* Rosetta (DE3) and cultured at 37°C on LB agar plates containing 34 mg / L chloramphenicol and 20 mg / L kanamycin. Single colonies were picked, and the pET28a-anAGE plasmid was extracted using a plasmid DNA purification kit (TaKaRa). The sequence was confirmed by DNA sequencing.

[0041] Example 2: Site-directed mutagenesis at amino acid site 73 of N-acetylglucosamine isomerase

[0042] Site-directed mutagenesis techniques are described in the book “PCR Protocols (John MS Bartlett and David Stirling. Totowa, NJ: Humana Press, 2003)”.

[0043] Using the plasmid pET28a-anAGE constructed in Example 1 as a template, primer pairs 73T1 and 73B1 (Table 1) were designed to mutate glutamic acid (Glu) at position 73 of the original amino acid sequence to glycine (Gly), thus obtaining the mutant anAGE-E73G.

[0044] Specifically, using plasmid pET28a-anAGE as a template, template fragment 73-1 was amplified using primers anAGE-T1 and 73B1; template fragment 73-2 was amplified using primers anAGE-B1 and 73-T1. The PCR reaction conditions were as follows: 300 ng plasmid pET28a-anAGE, 60 ng primers (anAGE-T1+73B1) or 60 ng primers (anAGE-B1+73-T1), 5 μL 10x buffer, 4 μL 2.5 mM dNTPs, 1 μL PrimerStar polymerase (TaKaRa), and the reaction volume was adjusted to 50 μL with sterile water.

[0045] The PCR amplification program was as follows: 95℃ for 5 minutes, 30 cycles: 94℃ for 30 seconds, 55℃ for 30 seconds and 72℃ for 1.5 minutes, and finally 72℃ for 10 minutes.

[0046] Template fragments 73-1 and 73-2 were amplified and subjected to 1% agarose gel electrophoresis (1 g agarose was placed in 100 mL of 1×TBE, heated to boiling in a microwave oven, 10 μL of GelRed dye was added, and the gel was solidified to obtain a gel). The PCR products were purified using a DNA gel extraction kit (TaKaRa). The full-length gene was then amplified using primers anAGE-T1 and anAGE-B1. The PCR reaction conditions were as follows: 200 ng template fragment 73-1, 200 ng template fragment 73-2, 60 ng primers (anAGE-T1 + anAGE-B1), 5 μL 10x buffer, 4 μL 2.5 mM dNTPs, 1 μL PrimerStar polymerase (TaKaRa), and the reaction volume was adjusted to 50 μL with sterile water.

[0047] The PCR amplification program was as follows: 95℃ for 5 minutes, 30 cycles: 94℃ for 30 seconds, 55℃ for 30 seconds and 72℃ for 1.5 minutes, and finally 72℃ for 10 minutes.

[0048] The full-length mutant gene was obtained by amplification reaction. It was then subjected to 1% agarose electrophoresis (1g agarose was placed in 100mL 1×TBE, heated to boiling in a microwave oven, 10μL of GelRed dye was added, and the gel was solidified to obtain a gel). The PCR product was purified using a DNA gel extraction kit (TaKaRa) to isolate and purify the full-length mutant gene anAGE-E73G with a size of 1167pb.

[0049] The mutants anAGE-Q110C (glutamine at position 110 is mutated to cysteine), anAGE-L171I (leucine at position 171 is mutated to isoleucine), anAGE-A172G (alanine at position 172 is mutated to glycine), and anAGE-A198D (alanine at position 198 is mutated to aspartic acid) were constructed using a similar method as described above. The primers used are shown in Table 1.

[0050] Example 3: Construction of plasmid pET28a-anAGE-E73G

[0051] The mutant gene anAGE-E73G and the vector pET28a were digested with XhoI+NotI (TaKaRa). The product was then subjected to 1% agarose gel electrophoresis (1 g agarose was placed in 100 mL of 1×TBE, heated to boiling in a microwave oven, 10 μL of GelRed dye was added, and the gel was solidified). The product was purified using a DNA fragment purification kit (TaKaRa) for separation and purification. The digested pET28a vector was ligated with T4 DNA ligase (TaKaRa) and transformed into competent E. coli Rosetta (DE3). The cells were cultured at 37°C on LB agar plates containing 34 mg / L chloramphenicol and 20 mg / L kanamycin. Single colonies were picked, and the plasmid pET28a-anAGE-E73G was extracted using a plasmid DNA purification kit (TaKaRa). The sequence was determined by DNA sequencing.

[0052] Plasmids pET28a-anAGE-Q110C, pET28a-anAGE-L171I, pET28a-anAGE-A172G, and pET28a-anAGE-A198D were constructed using a similar method described above.

[0053] Example 4: Construction of the double mutant combination anAGE-A172G-A198D of N-acetylglucosamine isomerase

[0054] Site-directed mutagenesis techniques are described in the book “PCR Protocols (John MS Bartlett and David Stirling. Totowa, NJ: Humana Press, 2003)”.

[0055] Using the plasmid pET28a-anAGE-A172G constructed in Example 3 as a template, the alanine (Ala) at position 198 in the amino acid sequence was mutated to aspartic acid (Asp) to obtain the mutant anAGE-A172G-A198D.

[0056] Specifically, using plasmid pET28a-anAGE-A172G as a template, the template fragment 73G198D-1 was amplified using primers anAGE-T1 and 198-B1 (Table 1); the template fragment 73G198D-2 was amplified using primers anAGE-B1 and 198-T1. The PCR reaction conditions were as follows: 300 ng plasmid pET28a-anAGE-A172G, 60 ng primers (anAGE-T1+198-B1) or 60 ng primers (anAGE-B1+198-T1), 5 μL 10x buffer, 4 μL 2.5 mM dNTPs, 1 μL PrimerStar polymerase (TaKaRa), and the reaction volume was adjusted to 50 μL with sterile water.

[0057] The PCR amplification program was as follows: 95℃ for 5 minutes, 30 cycles: 94℃ for 30 seconds, 55℃ for 30 seconds and 72℃ for 1.5 minutes, and finally 72℃ for 10 minutes.

[0058] Template fragments 73G198D-1 and 73G198D-2 were amplified and subjected to 1% agarose gel electrophoresis (1g agarose was placed in 100mL 1×TBE, heated to boiling in a microwave oven, 10μL of GelRed dye was added, and the gel was solidified). The PCR products were purified using a DNA gel extraction kit (TaKaRa). The full-length gene was then amplified using primers anAGE-T1 and anAGE-B1. The PCR reaction conditions were as follows: 200ng template fragments 73G198D-1 and 73G198D-2, 60ng primers (anAGE-T1 + anAGE-B1), 5μL 10x buffer, 4μL 2.5mM dNTPs, 1μL PrimerStar polymerase (TaKaRa), and the reaction volume was adjusted to 50μL with sterile water.

[0059] The PCR amplification program was as follows: 95℃ for 5 minutes, 30 cycles: 94℃ for 30 seconds, 55℃ for 30 seconds and 72℃ for 1.5 minutes, and finally 72℃ for 10 minutes.

[0060] The full-length mutant gene was obtained from the amplification reaction and subjected to 1% agarose gel electrophoresis (1 g agarose was placed in 100 mL of 1×TBE, heated to boiling in a microwave oven, 10 μL of GelRed dye was added, and the gel was solidified to obtain a gel). The PCR product was purified using a DNA gel extraction kit (TaKaRa) to isolate and purify the full-length mutant gene anAGE-A172G-A198D, which has a size of 1167 pb.

[0061] The mutant gene anAGE-A172G-A198D and the vector pET28a were digested with XhoI+NotI (TaKaRa). The resulting gel was obtained by 1% agarose gel electrophoresis (1 g agarose was placed in 100 mL of 1×TBE, heated to boiling in a microwave oven, 10 μL of ElRed dye was added, and the gel was solidified). The product of anAGE-A172G-A198D digestion was purified using a DNA fragment purification kit (TaKaRa) for further separation and purification. The digested pET28a vector was ligated with T4 DNA ligase (TaKaRa) and transformed into competent E. coli Rosetta (DE3). The cells were cultured at 37°C on LB agar plates containing 34 mg / L chloramphenicol and 20 mg / L kanamycin. Single colonies were picked, and the plasmid pET28a-anAGE-A172G-A198D was extracted using a plasmid DNA purification kit (TaKaRa). The sequence was confirmed by DNA sequencing.

[0062] Table 1. Primers used in the construction of the above mutants.

[0063]

[0064] Example 5: Effect test of N-acetylglucosamine isomerase mutant

[0065] E. coli containing the mutant expression vector and the wild-type expression vector were cultured in TB medium until OD100. 600 When the concentration of the bacterial cell reached 0.6-0.8, IPTG was added to a final concentration of 0.2 mM to induce protein expression. After culturing for 24 hours, the bacterial cells were collected for use in the catalytic reaction.

[0066] A standard reaction mixture (10 ml) was prepared: 20 g / L N-acetylglucosamine, 20 g / L MgCl2·6H2O, 1 g / L ATP, 1‰ Triton X-100, and 10 g / L N-acetylglucosamine isomerase. The above reaction mixture was permeabilized and stirred at 200 rpm for 6 h at 37 °C. The pH was maintained at 7.5 by adding 5 mol / L alkaline solution. The enzyme activity of the mutant and wild-type bacteria was evaluated (catalytic activity was defined as the amount of SA produced per 1 g of enzyme-containing wet bacterial cells within 1 minute). Table 2 lists the enzyme activities of wild-type N-acetylglucosamine isomerase and various N-acetylglucosamine isomerase mutants.

[0067] The experimental results are shown in Table 2. During the catalytic reaction, all mutants exhibited higher activity than WT, demonstrating superior catalytic activity. The modified sites included both activity pocket sites and stability-related sites, which made the enzyme more stable and enhanced its activity.

[0068] Table 2 Enzyme activities of wild-type N-acetylglucosamine isomerase and various N-acetylglucosamine isomerase mutants

[0069] Sequence List Sequence Number N-acetylglucosamine isomerase Enzyme activity (%) SEQ ID No.:2 WT 100 SEQ ID No.:3 pET28a-anAGE-E73G 130 SEQ ID No.:4 pET28a-anAGE-Q110C 137 SEQ ID No.:5 pET28a-anAGE-L171I 127 SEQ ID No.:6 pET28a-anAGE-A172G 145 SEQ ID No.:7 pET28a-anAGE-A198D 150 SEQ ID No.:8 pET28a-anAGE-A172G-A198D 155

[0070] Example 6: Production of N-acetylneuraminic acid from a 50 mL N-acetylglucosamine system

[0071] N-acetylneuraminic acid was prepared according to the following reaction system: 50 g / L N-acetylglucosamine isomerase, 50 g / L N-acetylneuraminic acid lyase, 20 g / L MgCl2·6H2O, 1‰ Triton X-100, 120 g / L N-acetylglucosamine, and 70 g / L pyruvate. The above reaction permeate was stirred at 200 rpm for 48 h at 37 °C, and the pH was maintained at 7.5 by adding 5 mol / L alkaline solution. The yield and enzyme stability of the mutant and wild-type strains were evaluated.

[0072] The results are as follows Figure 1 As shown in the figure, the modified enzyme is relatively stable during the 48-hour reaction, and the yield increases with time, reaching equilibrium after 18 hours, and is significantly higher than that of the wild-type fungus.

[0073] This invention provides an idea and method for N-acetylglucosamine isomerase mutants and their applications. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A mutant of N-acetylglucosamine isomerase, characterized in that, The amino acid sequence of the N-acetylglucosamine isomerase mutant is obtained by mutating the wild-type N-acetylglucosamine isomerase at the 172nd alanine and / or the 198th alanine, wherein the 172nd alanine (Ala) is mutated into glycine (Gly), and the 198th alanine (Ala) is mutated into aspartic acid (Asp); The wild-type N-acetylglucosamine 2-epimerase is derived from Anabaena N-acetylglucosamine 2-epimerase in sp. CH1, the amino acid sequence of which is shown as SEQ ID No. 1, and the nucleotide sequence encoding the wild-type N-acetylglucosamine 2-epimerase is shown as SEQ ID No.

2.

2. The mutant N-acetylglucosamine isomerase according to claim 1, wherein The nucleotide sequence of the N-acetylglucosamine isomerase mutant is shown in any one of SEQ ID No. 12-14.

3. Use of the N-acetylglucosamine isomerase mutant of claim 1 or 2 in the preparation of N-acetylneuraminic acid.

4. Use according to claim 3, characterized in that, The N-acetylglucosamine isomerase mutant of claim 1 or 2 is subjected to an enzymatic reaction with N-acetylglucosamine and pyruvic acid as substrates, together with N-acetylglucosamine, pyruvic acid, and N-acetylneuraminic acid lyase.

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