Property-changed glycogen synthase mutant and application thereof

Abstract

The invention provides a property-changed glycogen synthase mutant and application thereof, and relates to the technical field of enzyme engineering. According to the invention, rational design and site-directed mutagenesis are carried out on wild glycogen synthase, so that an enzyme mutant with enhanced heat resistance is obtained. Compared with a wild-type enzyme, the mutant shows remarkably improved catalytic activity in a temperature range of 30-46 DEG C, effectively overcomes the application bottleneck that a natural enzyme is easy to inactivate under a mild hot condition, can efficiently catalyze an alpha-1, 4-glucosidic bond synthesis reaction taking ADP-glucose as a donor, and has a wide application prospect. The lactic acid can be used as a core biocatalyst for constructing a stable multi-enzyme catalysis system, and shows excellent industrial application prospects in green synthesis of bio-based chemicals represented by lactic acid.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a glycogen synthase mutant with altered properties and its applications. Background Technology

[0002] Glycogen synthase (glgA, EC 2.4.1.21) is a key enzyme in the glycogen biosynthesis pathway. Its core function is to catalyze the transfer of glucose from uridine diphosphate glucose (UDP-Glucose) to the non-reducing end of the growing glycogen primer, forming an α-1,4-glycosidic bond. This reaction is not only a core process for energy storage in organisms, but also provides an important foundation for the industrial enzymatic synthesis of linear or branched α-glucans.

[0003] With its highly efficient and specific catalytic ability to form α-1,4-glycosidic bonds, glgA has shown great application potential in the green synthesis of functional polysaccharides, pharmaceutical carriers, food additives, and biodegradable materials. However, natural glgA enzymes (especially those derived from model bacteria such as Escherichia coli) generally suffer from poor thermal stability, which severely restricts their industrial application: the reaction needs to be carried out at low temperatures (usually below 37°C), which not only leads to a slow reaction rate but also increases production costs due to increased system viscosity and decreased mass transfer efficiency; the enzyme is easily and rapidly inactivated in batch production or continuous flow reactors, with a short half-life, requiring frequent replenishment of enzyme preparations, further increasing the cost of use; at the same time, the medium-low temperature reaction environment also facilitates microbial growth, which may affect the purity and quality stability of the product.

[0004] Current methods for improving the stability of glgA have many limitations. For example, screening homologous enzymes from thermophilic bacteria often faces problems such as low enzyme activity and a narrow substrate spectrum. Enzyme immobilization technology may be complex or result in the masking of the enzyme's active site, thus affecting catalytic efficiency. Therefore, there is an urgent need in this field to develop a glgA mutant with significantly enhanced thermal stability and high catalytic activity to overcome the application bottlenecks of natural enzymes and promote their large-scale application in related industrial fields. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention has obtained a series of enzyme mutants with enhanced structural stability and heat resistance through rational design and site-directed mutagenesis of wild-type glycogen synthase, effectively overcoming the application bottleneck of natural enzymes being easily inactivated under mild heat conditions.

[0006] The first object of the present invention is to provide a mutant of glycogen synthase, wherein the thermostable mutant is modified by any of the following mutations in the amino acid sequence shown in SEQ ID NO. 1 as the parent sequence:

[0007] The tyrosine at position 170 is mutated to proline;

[0008] The glutamine at position 178 is mutated to proline;

[0009] The valine at position 248 is mutated to isoleucine;

[0010] The glutamic acid at position 250 is mutated to proline;

[0011] The lysine at position 251 is mutated to glutamic acid;

[0012] The alanine at position 274 is mutated to lysine;

[0013] The alanine at position 283 is mutated to glutamic acid;

[0014] The proline at position 293 is mutated to leucine;

[0015] The valine at position 310 is mutated to isoleucine;

[0016] The glutamine at position 452 is mutated to methionine;

[0017] The alanine at position 457 is mutated to glutamic acid.

[0018] Furthermore, the mutant undergoes any of the following mutations with the amino acid sequence shown in SEQ ID NO.1 as the parent sequence:

[0019] The lysine at position 251 is mutated to glutamic acid and the glutamine at position 452 is mutated to methionine;

[0020] The 251st lysine was mutated to glutamic acid, the 452nd glutamine was mutated to methionine, and the 170th tyrosine was mutated to proline.

[0021] The 251st lysine was mutated to glutamic acid, the 452nd glutamine was mutated to methionine, the 170th tyrosine was mutated to proline, and the 248th valine was mutated to isoleucine.

[0022] The 251st lysine is mutated to glutamic acid, the 452nd glutamine is mutated to methionine, the 170th tyrosine is mutated to proline, the 248th valine is mutated to isoleucine, and the 149th alanine is mutated to leucine.

[0023] The 251st lysine is mutated to glutamic acid, the 452nd glutamine is mutated to methionine, the 170th tyrosine is mutated to proline, the 248th valine is mutated to isoleucine, the 149th alanine is mutated to leucine, and the 350th glycine is mutated to alanine.

[0024] The 251st lysine is mutated to glutamic acid, the 452nd glutamine is mutated to methionine, the 170th tyrosine is mutated to proline, the 248th valine is mutated to isoleucine, the 149th alanine is mutated to leucine, the 350th glycine is mutated to alanine, and the 419th serine is mutated to threonine.

[0025] The following mutations occurred: lysine at position 251 was replaced by glutamic acid; glutamine at position 452 was replaced by methionine; tyrosine at position 170 was replaced by proline; valine at position 248 was replaced by isoleucine; alanine at position 149 was replaced by leucine; glycine at position 350 was replaced by alanine; serine at position 419 was replaced by threonine; and glutamine at position 178 was replaced by proline.

[0026] Furthermore, the amino acid sequence of the heat-resistant mutant is shown in any one of SEQ ID NO.2-19.

[0027] Furthermore, the mutant has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology with the above-mentioned amino acid sequence, and has glycogen synthase activity.

[0028] A second objective of this invention is to provide a gene encoding the aforementioned mutant.

[0029] A third objective of this invention is to provide an expression vector containing the aforementioned genes.

[0030] A fourth objective of the present invention is to provide a host cell comprising the above-described expression vector.

[0031] Furthermore, the host cell is a non-plant cell.

[0032] Furthermore, the host cell is Escherichia coli.

[0033] A fifth objective of this invention is to provide the use of the above-mentioned mutant, the above-mentioned gene, the above-mentioned expression vector, or the above-mentioned host cell in the catalytic generation of α-1,4-glycosidic bonds.

[0034] A sixth object of the present invention is to provide a method for producing lactic acid, comprising the step of adding the above-mentioned mutant to a reaction system to carry out a reaction.

[0035] Furthermore, the reaction system also contains ADP-glucose and dextrin.

[0036] Furthermore, the reaction temperature is 30-46℃.

[0037] Furthermore, the reaction system also contains phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase.

[0038] Furthermore, the reaction system also contains magnesium ions and zinc ions.

[0039] The beneficial effects of this invention are:

[0040] This invention utilizes protein engineering to obtain a heat-resistant mutant of glycogen synthase. Within a temperature range of 30-46℃, its catalytic activity is significantly superior to the wild type, solving the core problem of the natural enzyme's high heat sensitivity. This fundamental improvement in thermal properties allows the mutant to operate stably over a wider temperature range, effectively increasing reaction rates, reducing system viscosity, and inhibiting microbial contamination. This significantly shortens the production cycle, increases production intensity, and reduces overall operating costs, providing a key enzyme element for continuous industrial production. Using the obtained heat-resistant mutant as the catalytic core, it can efficiently and stably catalyze the formation of α-1,4-glycosidic bonds. When used in conjunction with a multi-enzyme coupling system consisting of pyruvate kinase, lactate dehydrogenase, etc., it can achieve efficient and targeted conversion from ADP-glucose to lactate, providing a stable and efficient catalytic solution for the green biomanufacturing of platform chemicals such as lactate. Detailed Implementation

[0041] The following specific embodiments further illustrate the present invention so that those skilled in the art can better understand and implement it, but the embodiments are not intended to limit the present invention.

[0042] The amino acid sequence of the wild-type glycogen synthase (WT) involved in this invention is shown in SEQ ID NO.1, specifically as follows:

[0043] MQVLHVCSEMFPPLKTGGLADVIGALPAAQIADGVDARVLLPAFPDIRRGVTDAQVVSRRDTFAGHITLLFGHYNGVGIYLIDAPHLYDRPGSPYHDTNLFAYTDNVLRFALLGWVGAE MASGLDPFWRPDVVHAHDWHAGLAPAYLAARGRPAKSVFTVHNLAYQGMFYAHMNDIQLPWSFFNIHGLEFNGQISFLKAGLYYADHITAVSPTYAREITEPQFAYGMEGLLQQRHRE GRLSGVLNGVDEKIWSPETDLLLASRYTRDTLEDKAENKRQLQIAMGLKVDDKVPLFAVVSRLTSQKGLDLVLEALPGLLEQGGQLALLGAGDPVLQEGFLAAAAEYPGQVGVQIGYHE AFSHRIMGGADVILVPSRFEPCGLTQLYGLKYGTLPLVRRTGGLADTVSDCSLENLADGVASGFVFEDSNAWSLLRAIRRAFVLWSRPSLWRFVQRQAMAMDFSWQVAAKSYRELYYRLK

[0044] Methods for determining glycogen synthase activity:

[0045] Using adenosine diphosphate glucose (ADPG) as a substrate, the reaction mixture consisted of 100 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM ZnSO4, 0.1 mM EDTA, 3 mM ADPG, 0.1 mg / mL dextrin, 3 mM phosphoenolpyruvate (PEP), 5 U / mL pyruvate kinase (PK), 15 U / mL lactate dehydrogenase (LDH), and 10 mM NADH. After adding an appropriate amount of GLGA to initiate the reaction, the consumption of NADH was monitored in real time at 340 nm at 30℃ using a microplate reader, and the kinetic curve of absorbance change over time was recorded. One unit (U) of glycogen synthase (glgA) activity is defined as the amount of enzyme required to consume 1 μmol of NADH per minute under the above experimental conditions.

[0046] The primer sequences (5'-3') involved in the following examples are shown below:

[0047] Y170P-F: CCTATCAAGGCATGTTTCCAGCACATCACATGAATGACATC;

[0048] Y170-R:AAACATGCCTTGATAGGCCAGGT;

[0049] Q178P-F:ATCACATGAATGACATCCCATTGCCATGGTCATTCTTTAA;

[0050] Q178-R:GATGTCATTCATGTGATG;

[0051] V248I-F:GGCGTACTGAACGGCATCGACGAGAAAATCTGGAGTC;

[0052] V248I-R:GCCGTTCAGTACGCCGGAAAG;

[0053] E250P-F:CTGAACGGCGTGGACCCAAAAATCTGGAGTCCAGAGACG;

[0054] E250P-R:GTCCACGCCGTTCAGTACG;

[0055] K251E-F:AACGGCGTGGACGAGGAGATCTGGAGTCCAGAGACG;

[0056] K251E-R:CTCGTCCACGCCGTTCAGTAC;

[0057] A274K-F:ATACGTTGGAAGATAAAAAAGAAAATAAGCGCCAGTTACA;

[0058] A274K-R:TTTATCTTCCAACGTATCGC;

[0059] A283E-F:AGCGCCAGTTACAAATCGAAATGGGGCTTAAGGTTGACGA;

[0060] A283E-R:GATTTGTAACTGGCGCTTAT;

[0061] P293L-F:AGGTTGACGATAAAGTGCTTCTTTTTGCAGTGGTGAGCCG;

[0062] P293L-R:CACTTTATCGTCAACCTTAA;

[0063] V310L-F: AGAAAGGTCTCGACCTGATCCTGGAAGCCTTACCGGGTCT;

[0064] V310L-R: CAGGTCGAGACCTTTCTGGC;

[0065] Q452M-F: CTGTGGCGGTTTGTGATGCGTCAGGCTATGGCAATGG;

[0066] Q452M-R: CACAAACCGCCACAGTGAAGG;

[0067] A457E-F: TGCAACGTCAGGCTATGGAGATGGATTTTAGCTGGCAGGT;

[0068] A457E-R: CATAGCCTGACGTTGCACAA;

[0069] A149L-F: CTGCGTATCTGGCGCTTCGCGGGCGTCCG;

[0070] A149L-R: CGCCAGATACGCAGGCGCAAG;

[0071] G350A-F: TACCCCGGTCAGGTGGCAGTTCAGATTGGCTATC;

[0072] G350A-R: CACCTGACCGGGGTATTCCGct;

[0073] S419T-F:GCAGATGGCGTCGCCACAGGGTTTGTCTTTGAAGATAG;

[0074] S419T-R:GGCGACGCCATCTGCAAGG.

[0075] Example 1: Design of Mutants

[0076] I. Structural Analysis

[0077] Structural weakness analysis: Based on high-resolution 3D structures obtained using GLGA (such as X-ray diffraction or cryo-electron microscopy structures), and combined with structural visualization tools, a systematic evaluation of their overall conformation and local features is conducted. The analysis focuses on secondary structure composition, domain boundaries, active center conformation, and surface properties to form a preliminary assessment of structural stability.

[0078] Thermal flexibility analysis: Temperature factor (B factor) data are extracted from the crystal structure, and the distribution map of B factor of main chain atoms is drawn to identify residue regions with significant structural fluctuations; Molecular dynamics simulations are performed at three temperatures (300K, 340K, and 380K), and by comparing the differences in structural fluctuations (RMSF) of various parts of the protein after simulation, hot spots with relatively large structural changes due to heat are identified.

[0079] Interaction identification: Examine non-covalent interactions such as hydrogen bonds, salt bridges, and hydrophobic stacking to identify regions where the interaction strength is weaker than the average value and determine the unstable structural point.

[0080] II. Mutation Strategy

[0081] Electrostatic and hydrophobic optimization: Mutations are designed to optimize salt bridges (ion pairs). Surface residues at appropriate distances are mutated into salt bridge pairs to form stronger and more stable electrostatic pairings. Simultaneously, hydrophobic stacking in the core region is optimized to enhance structural strength.

[0082] Enhancing loop stability: To address the weakness of highly flexible loop regions, proline mutations are introduced. The unique rigid structure of proline restricts the conformational freedom of the main chain, fixing the peptide chain conformation, reducing local thermal motion, and thus increasing the protein's melting temperature. Mutations are also made in daunoglycine, which causes structural instability, to moderately increase structural rigidity.

[0083] Based on the above strategy, GLGA single-site mutants were obtained, and GLGA multi-site combined mutants were obtained by combining the predicted key amino acid sites.

[0084] Example 2: Construction of a strain containing a gene expressing a glycogen synthase mutant

[0085] Primers were designed based on different mutation sequences of GLGA. The PCR product and linearized vector pET21b were mixed in a specific ratio using a DNA Assembly Cloning Kit, and transformation was performed at 50°C for 15 min under the catalysis of recombinase. The assembled plasmid was transformed into *E. coli* DH5a competent cells, plated on plates containing AMP resistance, and incubated overnight at 37°C. After incubation, single clones were picked for PCR verification. Positive clones were cultured overnight in test tubes, and the plasmid was extracted and transformed into *E. coli* BL21(DE3) competent cells. The cells were then plated on plates containing AMP resistance and incubated overnight at 37°C to obtain the protein-expressing recombinant strain.

[0086] Example 3: Expression of glycogen synthase mutant

[0087] Host bacterial activation culture: E. coli BL21(DE3) containing the GLGA wild-type / mutant expression vector plasmid was streaked onto amp-resistant LB solid medium and incubated overnight at 37°C. Single colonies were picked and inoculated into test tubes containing 5 mL of LB liquid medium. The test tubes were then placed in a rotary shaker at 200 rpm and incubated at 37°C for 16 h.

[0088] Fermentation culture: Inoculate 5 mL of the activated bacterial culture into a 2 L Erlenmeyer flask containing 1 L LB liquid medium with AMP resistance, and place it in a rotary shaker at 200 r / min and incubate at 37°C until OD. 600 The concentration should be between 0.8 and 1.0. After lowering the culture temperature to 16°C, add IPTG to a final concentration of 0.5 mM and continue induction under these conditions for 12-18 hours.

[0089] Separation and purification: After overnight culture, centrifuge to remove the supernatant. Collect the precipitate in a 50 mL centrifuge tube for protein purification. Resuspend the precipitate in 30 mL of 25 mM Tris-HCl and 500 mM NaCl buffer, and vortex to mix. Add 3-5 mL of TieChui lysis buffer to the bacterial suspension, mix, and incubate at room temperature for 30-60 min until the bacterial suspension becomes clear. Centrifuge at 4°C, remove the precipitate, and pour the supernatant into a prepared nickel column for binding. Wash with 100 mL of 25 mM Tris-HCl, 500 mM NaCl, and 25 mM imidazole buffer, and then elute the protein with 15 mL of 25 mM Tris-HCl (500 mM NaCl, 250 mM imidazole) buffer. Dialyze the 15 mL protein eluent overnight in 25 mM Tris-HCl and 500 mM NaCl buffer. The 15 mL protein eluent after dialysis was passed through an ultrafiltration tube and concentrated to 0.5–1 mL at 3500 tpm and 4°C. Protein concentration was determined using the Bradford method, ensuring all mutant proteins were homogenized before detection.

[0090] Example 4: Determination of the activity of glycogen synthase and its mutant enzymes

[0091] In a reaction system containing 100 mM HEPES (pH=7.5), 5 mM MgCl2, 1 mM ZnSO4, 0.1 mM EDTA, and excess pyruvate kinase (PK) and lactate dehydrogenase (LDH), the substrate ADPG, dextrin, PEP, NADH, and the target GLGA were added to initiate the reaction. The ADP generated by the glgA-catalyzed reaction was consumed by the subsequent PK / LDH coupled enzyme system, accompanied by the oxidation of NADH. The enzyme activity (U) of glgA was determined by calculating the initial reaction rate by real-time monitoring of the decrease in NADH absorbance at 340 nm, defined as the amount of enzyme required to consume 1 μmol of NADH per minute.

[0092] The PCR instrument temperature was set to the highest incubation temperature (e.g., 46℃ for glgA mutant, 30℃ for control group), and incubated for 30 minutes. Protein samples after incubation were then analyzed. The activity of GLGA was determined using a PK / LDH coupling assay: ADP generated by GLGA catalyzes the consumption of NADH under the coupling reaction of PK and LDH. The rate of NADH consumption was detected spectrophotometrically at 340 nm to generate kinetic curves. Reaction rates were calculated for comparison, with the enzyme activity of wild-type WT at 30℃ as 100%. Single or combined GLGA mutants with improved thermostability compared to wild-type were screened, as shown in Table 1.

[0093] Table 1 Enzyme activity and thermostability of GLGA and its mutants

[0094]

[0095] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A mutant of glycogen synthase, characterized in that, The mutant undergoes any of the following mutations based on the amino acid sequence shown in SEQ ID NO.1, as in the parent sequence: The tyrosine at position 170 is mutated to proline; The glutamine at position 178 is mutated to proline; The valine at position 248 is mutated to isoleucine; The glutamic acid at position 250 is mutated to proline; The lysine at position 251 is mutated to glutamic acid; The alanine at position 274 is mutated to lysine; The alanine at position 283 is mutated to glutamic acid; The proline at position 293 is mutated to leucine; The valine at position 310 is mutated to isoleucine; The glutamine at position 452 is mutated to methionine; The alanine at position 457 is mutated to glutamic acid.

2. The mutant according to claim 1, characterized in that, The mutant undergoes any of the following mutations based on the amino acid sequence shown in SEQ ID NO.1, as in the parent sequence: The lysine at position 251 is mutated to glutamic acid and the glutamine at position 452 is mutated to methionine; The 251st lysine was mutated to glutamic acid, the 452nd glutamine was mutated to methionine, and the 170th tyrosine was mutated to proline. The 251st lysine was mutated to glutamic acid, the 452nd glutamine was mutated to methionine, the 170th tyrosine was mutated to proline, and the 248th valine was mutated to isoleucine. The 251st lysine is mutated to glutamic acid, the 452nd glutamine is mutated to methionine, the 170th tyrosine is mutated to proline, the 248th valine is mutated to isoleucine, and the 149th alanine is mutated to leucine. The 251st lysine is mutated to glutamic acid, the 452nd glutamine is mutated to methionine, the 170th tyrosine is mutated to proline, the 248th valine is mutated to isoleucine, the 149th alanine is mutated to leucine, and the 350th glycine is mutated to alanine. The 251st lysine is mutated to glutamic acid, the 452nd glutamine is mutated to methionine, the 170th tyrosine is mutated to proline, the 248th valine is mutated to isoleucine, the 149th alanine is mutated to leucine, the 350th glycine is mutated to alanine, and the 419th serine is mutated to threonine. The following mutations occurred: lysine at position 251 was replaced by glutamic acid; glutamine at position 452 was replaced by methionine; tyrosine at position 170 was replaced by proline; valine at position 248 was replaced by isoleucine; alanine at position 149 was replaced by leucine; glycine at position 350 was replaced by alanine; serine at position 419 was replaced by threonine; and glutamine at position 178 was replaced by proline.

3. The mutant according to claim 1 or 2, characterized in that: The amino acid sequence of the heat-resistant mutant is shown in any one of SEQ ID NO.2-19.

4. The gene encoding any of the mutants described in claims 1-3.

5. An expression vector comprising the gene of claim 4.

6. A host cell comprising the expression vector of claim 5.

7. The use of any mutant of claims 1-3, the gene of claim 4, the expression vector of claim 5, or the host cell of claim 6 in catalyzing the formation of α-1,4-glycosidic bonds.

8. A method for producing lactic acid, characterized in that: The step includes adding the mutant of any one of claims 1-3 to the reaction system to carry out the reaction.

9. The method according to claim 8, characterized in that: The reaction system also contains ADP-glucose and dextrin.

10. The method according to claim 8, characterized in that: The reaction system also contains phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase.

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