Property-changed phosphoglucomutase mutant and application thereof

Abstract

The invention relates to a property-changed phosphoglucomutase mutant and application thereof, and belongs to the technical field of enzyme engineering. According to the invention, the phosphoglucomutase is mutated at different sites, the property of the obtained phosphoglucomutase mutant is changed, especially the thermal stability of the phosphoglucomutase mutant is obviously improved, the residual enzyme activity of the phosphoglucomutase mutant at 30 DEG C is also greatly improved at 65 DEG C, and the residual enzyme activity of the phosphoglucomutase mutant at 65 DEG C is also greatly improved. Therefore, the phosphoglucomutase mutant disclosed by the invention can still keep good enzyme activity in a biological process needing to be carried out at high temperature, and has wide application value in industrial application.

Description

Technical Field

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

[0002] Phosphoglucosidase (PGM, EC 5.4.2.2) is a key enzyme in carbohydrate metabolism, reversibly catalyzing the interconversion between glucose-1-phosphate (G-1-P) and glucose-6-phosphate (G-6-P). This reaction is a core step in glycogen and starch synthesis and degradation, as well as galactose metabolism, and therefore has enormous industrial application potential in the in vitro synthesis of glyconucleotides, functional oligosaccharides, and structurally complex carbohydrate compounds.

[0003] However, naturally derived PGMs (such as those from E. coli) typically have low optimal reaction temperatures and poor thermal stability. This poses a significant challenge for their industrial applications: they are prone to inactivation in high-temperature bioprocesses (such as the glycosylation conversion after high-temperature starch liquefaction), resulting in low reaction efficiency, high enzyme usage costs, and susceptibility to microbial contamination. While enzyme immobilization technology can improve operational stability to some extent, it often leads to enzyme activity loss. Therefore, developing PGM mutants with inherently high thermal stability is the fundamental solution to these problems. However, current technologies still lack heat-resistant PGM mutants. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problem of poor thermostability of phosphogluconomutase in the prior art.

[0005] To address the aforementioned technical problems, this invention provides a phosphoglucomutase mutant with altered properties and its applications. This invention involves mutations and deletions at different sites in the phosphoglucomutase, resulting in mutant phosphoglucomutases with altered properties, particularly in their thermal stability. Not only is the residual enzyme activity significantly increased at 30°C, but it also shows a substantial increase at 65°C. Therefore, it can maintain good enzyme activity even in bioprocesses requiring high temperatures, making it widely applicable in industrial settings.

[0006] The first objective of this invention is to provide a phosphoglucose mutase mutant, which is a phosphoglucose mutase with an amino acid sequence as shown in SEQ ID NO.1, modified by any of the following methods:

[0007] (1) The asparagine at position 125 is mutated to isoleucine (N125I);

[0008] (2) The isoleucine at position 50 is mutated to glutamic acid (I50E);

[0009] (3) The isoleucine at position 50 is mutated to arginine (I50R);

[0010] (4) Threonine at position 177 is mutated to serine (T177S).

[0011] Furthermore, SEQ ID NO.1:

[0012] MKKILSFDIDNTLNEPKMPIFPEMAELLATLSQKYIIAPISGQKYDQFLIQIINNLPESANLDNFHLFVAQGTQYYAHKDGEWKQVFNYALTDEQANAIMGALEKAAKELGHWDESVLAPGDEINE NRESMIAYSAIGQKAGVEAKQAWDPDMTKRNEIAKLASQYAPEFEFEVAGTTTINGFVPGQNKEFGMNHLMEELNVTKEEILYFGDMTQPGGNDYPVVQMGIETITVRDWKETAAILKAIIAMEEA.

[0013] Furthermore, the amino acid sequence of the glucose-phosphodiester mutant also includes an amino acid sequence having the above-mentioned mutation site and having more than 80% homology with the amino acid sequence that has undergone the above-mentioned mutation, preferably having 85% homology, more preferably having 90% homology, even more preferably having 95% homology, and even more preferably having 99% homology.

[0014] A second objective of this invention is to provide a gene encoding the aforementioned glucose-phosphodiesterase mutant.

[0015] A third objective of this invention is to provide a recombinant plasmid carrying the aforementioned gene.

[0016] A fourth objective of this invention is to provide a recombinant cell expressing the above-described glucose-1,5-phosphate mutase mutant.

[0017] Furthermore, the recombinant cells are bacteria or fungi.

[0018] A fifth objective of this invention is to provide the use of the above-described glucose-1-phosphate phosphate mutant, the above-described gene, the above-described recombinant plasmid, or the above-described recombinant cell in the preparation of glucose-1-phosphate.

[0019] Furthermore, the application involves adding a phosphoglucomutase mutant or an expression system containing a phosphoglucomutase mutant to a system containing glucose-1-phosphate for reaction.

[0020] Furthermore, the system also includes metal ions.

[0021] The sixth object of the present invention is to provide a method for hydrolyzing glucose-6-phosphate, wherein the method involves adding the above-mentioned glucose phosphate mutase mutant to a system containing glucose-6-phosphate for reaction.

[0022] Furthermore, the temperature of the reaction is greater than or equal to 65°C.

[0023] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0024] This invention involves mutations and deletions at different sites in phosphoglucomutase, resulting in mutant phosphoglucomutases with altered properties, particularly in thermostability. The residual enzyme activity is significantly increased not only at 30°C but also at 65°C. Therefore, these mutants can maintain good enzyme activity even in bioprocesses requiring high temperatures, making them widely applicable in industrial settings. Attached Figure Description

[0025] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0026] Figure 1 This is a bar chart showing the residual enzyme activity of the mutant of the present invention at 30°C and 65°C. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0028] Methods for determining PGM activity:

[0029] An assay coupled with glucose-6-phosphate dehydrogenase (G6PDH) was used. The reaction system consisted of: 100 mM HEPES (pH 7.5), 100 mM NaCl, and 5 mM Mg. 2+ 10 μM Zn 2+ The reaction mixture consisted of 2 mM NAD+, 1 U / mL G6PDH, 1 mM glucose-1-phosphate (G-1-P), and an appropriate amount of pgm. The reaction was initiated at 30 °C, and the NADH production rate was monitored in real-time at 340 nm using spectrophotometry. One unit of enzyme activity (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of NADH per minute under the above conditions.

[0030] Example 1: Construction of a strain containing the gene expressing the PGM mutant of the present invention

[0031] This embodiment uses molecular dynamics simulations of PGM at three temperatures: 300K, 340K, and 380K. By comparing the differences in structural fluctuations (RMSF) at various sites of the protein after simulation, hotspot regions with relatively large structural changes due to heat were identified. Subsequently, consistency analysis was performed on thermostable homologous sequences, and high-probability amino acid types at various unstable sites were screened and used to replace existing residue types. Furthermore, partial mutations were designed to enhance internal hydrophobic interactions, increase internal hydrogen bonding interactions, deglycine-reacting of the helix structure, and increase surface salt bridge interactions. The mutants are shown in Table 2.

[0032] Primers were designed based on different mutation sites of PGM (as shown in Table 1). The PCR product and linearized vector 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 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 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 strain.

[0033] Table 1 Primers and their sequences

[0034]

[0035] Example 2: Expression of the PGM mutant of the present invention

[0036] Host bacterial activation culture: The culture containing the expression vector plasmid... E. coli BL21(DE 3) 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.

[0037] 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. 600The 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.

[0038] 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 TiChui lysis buffer to the bacterial suspension, mix, and incubate at room temperature for 30 min-1 h until the bacterial suspension becomes clear. Centrifuge at 4 °C, remove the precipitate, and pour the supernatant into a prepared nickel column for binding. After washing with 100 mL of 25 mM Tris-HCl, 500 mM NaCl, and 25 mM imidazole buffer, 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 concentrated to 0.5–1 mL using an ultrafiltration tube at 3500 rpm and 4°C. Protein concentration was determined using the Bradford method, ensuring all mutant proteins were homogenized before detection.

[0039] Example 3: Determination of the enzyme activity of PGM and its mutants according to the present invention

[0040] In a 100 μL reaction system, add 100 mM HEPES (pH 7.5), 100 mM NaCl, and 5 mM Mg. 2+ 10μM Zn 2+ 2 mM NAD+, 1 U / mL G6PDH, and 1 mM G-1-P were added. After preheating at 30℃, an appropriate amount of diluted PGM enzyme solution was added to start the reaction. The increase in NADH absorbance was immediately monitored at a wavelength of 340 nm, and the rate of the initial linear phase was recorded. By real-time monitoring of the increase in NADH absorbance at 340 nm and calculating the initial reaction rate, the enzyme activity (U) of PGM can be determined, defined as the amount of enzyme required to generate 1 μmol of NADH per minute.

[0041] Example 4: Determination of the thermal stability of the PGM mutant of the present invention

[0042] The purified wild-type and mutant PGM proteins were diluted to the same concentration with buffer. Equal volumes of protein solution were aliquoted and incubated in a PCR instrument at 65°C for 30 minutes. A control group (incubated at 30°C) was also included. After incubation, the high-temperature treated group was immediately cooled on ice. Subsequently, the enzyme activity of the heat-treated and control samples was measured according to the PGM enzyme activity assay method described in Example 3 (i.e., coupled with G6PDH to monitor the NADH generation rate). By comparing the activities of the two, the residual enzyme activity of the mutant at different temperatures was calculated to evaluate its effect on improving thermal stability.

[0043] Table 2 Thermal stability of different mutants at 30℃ and 65℃

[0044]

[0045] 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 phosphoglucomutase mutant, characterized in that, The phosphoglucomutase mutant is any one of the following modifications to the amino acid sequence shown in SEQ ID NO. 1: (1) Asparagine at position 125 is mutated to isoleucine; (2) Isoleucine at position 50 is mutated to glutamic acid; (3) Isoleucine at position 50 is mutated to arginine; (4) Threonine at position 177 is mutated to serine.

2. A gene encoding the phosphoglucomutase mutant of claim 1.

3. A recombinant plasmid carrying the gene of claim 2.

4. A recombinant cell expressing the phosphoglucomutase mutant of claim 1.

5. The recombinant cell of claim 4, wherein, The recombinant cell is a bacterium or a fungus.

6. Use of the phosphoglucomutase mutant of claim 1, the gene of claim 2, the recombinant plasmid of claim 3, or the recombinant cell of claim 4 or 5 in the preparation of glucose-1-phosphate.

7. Use according to claim 6, characterized in that, The use is adding the phosphoglucomutase mutant or an expression system containing the phosphoglucomutase mutant to a system containing glucose-1-phosphate to perform a reaction.

8. Use according to claim 7, characterized in that, The system further comprises metal ions.

9. A method of hydrolyzing glucose-6-phosphate, characterized in that, The method is adding the phosphoglucomutase mutant of claim 1 to a system containing glucose-6-phosphate to perform a reaction.

10. The method of claim 9, wherein, The system further comprises metal ions.

Images