A phosphoglycerate oxidase mutant with improved thermal stability and its application

Abstract

The present invention discloses a phosphoglycerol oxidase mutant with improved thermal stability and its application, wherein an amino acid sequence such as SEQ ID NO.1 is used as a parent, and the 249th or 451st position of the parent is mutated. The phosphoglycerol oxidase mutant with improved thermal stability has improved stability and catalytic activity at the same time.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a glycerol phosphate oxidase mutant with improved thermal stability and application thereof. Background Art

[0002] The concentration of triglycerides in serum is one of the important indicators in the clinical diagnosis of heart disease and hyperlipidemia. The normal reference value of triglycerides in serum is 0.45-1.69mmol / L. The commonly used method for clinical analysis and detection of triglycerides is the glycerol phosphate oxidase method, which has the advantages of high specificity, simplicity, trace amount and easy to achieve rapid and automated detection. Glycerol phosphate oxidase (L-α-Glycerophosphate Oxidase, GPO, EC1.1.3.21) is one of the key enzymes required for the triglyceride determination kit, which can specifically catalyze the oxidation of α-glycerol phosphate to produce dihydroxyacetone phosphate and hydrogen peroxide. Glycerol phosphate oxidase (GPO) can be prepared from organisms of different sources. Currently, GPO used for in vitro diagnosis is mainly derived from microorganisms, such as yeast (Saccharomyces sp.), Enterococcus sp., Pediococcus sp., Streptococcus sp., Lactobacillus sp. and Escherichia coli.

[0003] The preparation method of glycerol phosphate oxidase (GPO) is mainly through wild bacteria fermentation and Escherichia coli recombinant expression. For example, Lu Xu et al. fermented and cultured the selected streptococci and purified them to obtain high-purity GPO (Journal of Sichuan University (Natural Science Edition), 2006, 43 (6), 1379-1383), Bao Lingsheng et al. fermented and cultured wild Enterococcus faecalis and purified them to obtain GPO with an apparent molecular weight of 67 kDa, and the enzyme activity reached 54.3 U / mg (2008, 11, 15-18), Zheng et al. recombinantly expressed the GPO gene from Enterococcus faecalis in Escherichia coli and purified it to obtain recombinant GPO (protein expression and purification, 2011, 75 (2): 133-137). However, these wild bacteria-derived GPOs have the disadvantages of complicated fermentation and purification process, low expression level, and poor thermal stability. Although heterologous expression of wild-type GPO genes increases the yield of recombinant protein and simplifies the purification process, the recombinant wild-type GPO still has the disadvantage of poor enzyme thermal stability.

[0004] At present, the glycerol phosphate oxidase (GPO) used by in vitro diagnostic reagent manufacturers in my country is basically imported from foreign companies such as Roche and Toyobo of Japan. Although the GPO produced by these companies has improved the stability of the enzyme to a certain extent through chemical modification and protection agent optimization, the problem of low enzyme thermal stability still exists, resulting in insufficient long-term storage stability of the triglyceride diagnostic reagents produced. Summary of the invention

[0005] The purpose of the present invention is to provide a glycerol phosphate oxidase mutant with improved thermal stability and application thereof, wherein the stability and catalytic activity are improved simultaneously.

[0006] The present invention adopts the following technical scheme: a phosphoglycerate oxidase mutant with improved thermal stability, with an amino acid sequence such as SEQ ID NO.1 as a parent, and a mutation is made at position 249 or position 451 of the parent.

[0007] In the above-mentioned glycerol phosphate oxidase mutant with improved thermal stability, the serine at position 249 of the parent is substituted with proline; or the glutamic acid at position 451 of the parent is substituted with methionine.

[0008] The present invention also discloses a gene encoding the above-mentioned phosphoglycerate oxidase mutant with improved thermal stability, and the nucleotide sequence of the gene is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0009] The invention also discloses a recombinant plasmid containing the gene.

[0010] The invention also discloses a host cell expressing the mutant or containing the recombinant plasmid.

[0011] The present invention also discloses the use of the above-mentioned glycerol phosphate oxidase mutant with improved thermal stability, or the above-mentioned gene, or the above-mentioned recombinant plasmid, or the above-mentioned host cell in the production of a triglyceride detection agent.

[0012] The beneficial effects of the present invention are as follows: the catalytic efficiency of the phosphate oxidase mutant provided by the present invention is increased by 1.4 times, and the residual enzyme activity of the mutant enzyme is 86% after incubation at 50°C for 30 minutes, which is 3.3 times higher than the residual enzyme activity of the wild-type enzyme; and 1.5 times higher than the residual enzyme activity of GPO produced by Roche. The thermal stability and catalytic activity of glycerol phosphate oxidase are significantly improved, which solves the problem of low thermal stability of glycerol phosphate oxidase and lays a foundation for its application in clinical diagnostic reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the SDS-PAGE electrophoresis diagram of the purification results of wild-type glycerol phosphate oxidase.

[0014] Figure 2 Analysis of candidate mutated amino acid residues and mutant screening results based on semi-rational design;

[0015] Among them: A is the minimum free energy heat map after virtual saturation mutation of candidate amino acid residues; B is the residual enzyme activity after incubation of wild-type enzyme and positive mutant at 50℃ for 30min.

[0016] Figure 3 is the SDS-PAGE electrophoresis of the purified phosphoglycerate oxidase mutant protein;

[0017] Wherein, M represents the protein molecular weight standard; 1 represents the purified phosphoglycerate oxidase mutant S249P; and 2 represents the purified phosphoglycerate oxidase mutant E451M.

[0018] Figure 4 The figure shows the thermal stability comparison between the glycerol phosphate oxidase mutant S249P, the wild-type enzyme and the GPO produced by Roche.

[0019] Figure 5 The figure compares the pH tolerance of the glycerol phosphate oxidase mutant S249P and the wild-type enzyme. DETAILED DESCRIPTION

[0020] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] The present invention discloses a phosphoglycerate oxidase mutant with improved thermal stability, wherein an amino acid sequence such as SEQ ID NO.1 is used as a parent, and the 249th or 451st position of the parent is mutated, specifically, the serine at the 249th position of the parent is replaced by proline, or the glutamic acid at the 451st position of the parent is replaced by methionine.

[0022] The nucleotide sequence of the parental coding gene is shown in SEQ ID NO.2.

[0023] The present invention also discloses a gene encoding the above-mentioned phosphoglycerate oxidase mutant with improved thermal stability, and the nucleotide sequence of the gene is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0024] The invention also discloses a recombinant plasmid containing the gene.

[0025] In one embodiment, the expression vector of the recombinant plasmid includes but is not limited to pET series, pACYC series or pGEX series, etc. In one embodiment, pET-28a(+) is used as the vector.

[0026] The invention also discloses the mutant or the host cell of the recombinant plasmid.

[0027] In one embodiment, Escherichia coli (E. coli) is used as a host to express the glycerol phosphate oxidase mutant.

[0028] The present invention also discloses the use of the above-mentioned glycerol phosphate oxidase mutant with improved thermal stability, or the above-mentioned gene, or the above-mentioned recombinant plasmid, or the above-mentioned host cell in the production of a triglyceride detection agent.

[0029] The preparation method of the phosphoglycerate oxidase mutant with improved thermal stability is as follows:

[0030] Using the crystal structure of phosphoglycerate oxidase from Streptococcus (PDB ID: 2RGO), a simulated mutant with improved thermal stability was obtained by computer-aided rational design.

[0031] The target mutants were obtained by plasmid amplification using site-directed mutagenesis primers.

[0032] Specifically, the host cell monoclone containing the recombinant plasmid is inoculated into 5 mL of liquid LB medium and cultured at 37° C. overnight to obtain a seed solution;

[0033] The seed liquid was inoculated into LB medium at a 1% inoculum, and cultured at 37°C until the biomass OD600 reached 0.6-0.8, then the inducer was added and cultured at 30°C for 12-16 hours, and the cells were collected by centrifugation;

[0034] The cells collected by centrifugation were ultrasonically disrupted, and the disrupted liquid was centrifuged at 4°C and 15000 rpm for 15 min to obtain a crude enzyme solution;

[0035] The crude enzyme solution was subjected to nickel column affinity chromatography and gel filtration column desalting to obtain pure glycerol phosphate oxidase enzyme.

[0036] The present invention performs rational molecular modification on streptococcal glycerol oxidase, performs site-directed mutagenesis and screening on multiple sites such as 109, 112, 150, 245, 248, 249, 451, 456, and finally obtains a glycerol oxidase mutant with improved thermal stability and catalytic activity.

[0037] The specific experimental and verification process is as follows:

[0038] Example 1

[0039] Expression and purification of recombinant wild-type glycerol phosphate oxidase GPO:

[0040] Glycerol phosphate oxidase from Streptococcus sp. was recombinantly expressed in E. coli. The required cells and reagents are as follows: the expression plasmid pET-28a(+) and E. coli DH5α and BL21(DE3) are all homemade and can also be purchased; nickel affinity chromatography filler (Ni Sepharose 6Fast Flow) and gel filtration filler (Sephadex G-25Medium) are purchased from GE Company, and the column is loaded as needed during protein purification; the remaining reagents are domestically produced or imported analytical grade.

[0041] The protein sequence of glycerol phosphate oxidase GPO from Streptococcus sp. was obtained from the online database (PDB ID: 2RGO), and the amino acid sequence is shown in SEQ ID NO.1. The GPO gene sequence (nucleotide sequence is shown in SEQ ID NO.2) was obtained according to the codon preference of Escherichia coli and synthesized artificially. The synthesized GPO gene was connected to the Escherichia coli expression vector pET-28a(+) through the NdeⅠ and NotⅠ restriction sites to construct the recombinant plasmid pET-28a-GPO. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) to obtain a recombinant bacterium expressing wild-type glycerol phosphate oxidase.

[0042] The recombinant E. coli was streaked onto a solid LB plate containing kanamycin (35 μg / mL) and cultured at 37°C to obtain a single clone. A single clone was then picked and cultured in 5 mL of liquid LB medium at 37°C with shaking overnight. The overnight culture seed solution was inoculated into LB medium at a 1% inoculum and cultured at 37°C until the biomass OD 600 After reaching 0.6-0.8, add the inducer isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.3mM at 30℃ for induction for 12-16h, and collect the bacteria by centrifugation at 4℃. Add the collected bacteria into the disruption solution (1g wet bacteria is added to 8mL disruption solution for suspension) for ultrasonic disruption (power 450W, ultrasonic 8s, interval 15s) for 30min, then centrifuge at 15000rpm 4℃ for 15min to collect the supernatant. After filtering the supernatant through a filter membrane (0.22μm), apply it to a nickel affinity chromatography column (Ni Sepharose 6Fast Flow), use an equilibrium buffer (PBS, containing 40mM imidazole) to elute the impurities, and then use an elution buffer (containing 400mM imidazole) to elute the target protein. The eluate is then desalted through a gel filtration column (Sephadex G-25Medium) and collected to obtain purified recombinant phosphoglycerol oxidase. The purity of the purified protein was over 95% as determined by SDS-PAGE. Figure 1As shown in the figure, M represents the protein molecular weight standard; 1 represents the total protein of the bacteria after induction; 2 represents the total protein of the supernatant after the bacteria are broken; 3 represents the precipitate after the bacteria are broken; and 4 represents the purified wild-type glycerol phosphate oxidase.

[0043] Example 2

[0044] Construction of phosphoglycerate oxidase mutant library:

[0045] Based on the three-dimensional structure of glycerol phosphate oxidase GPO from Streptococcus sp. (PDBID: 2RGO), the protein analysis tool FoldX was used for computer-aided design. The amino acids at the flexible sites in the tertiary structure of GPO were subjected to virtual saturation mutations, and the unfolding free energy change (ΔΔG) after mutation was calculated. The mutation sites with ΔΔG < -0.92 kcal / mol were selected. The minimum free energy heat map after virtual saturation mutation of candidate amino acid residues is shown in the attached figure. Figure 2 As shown in A. The virtual screening results of the mutation sites are as follows: Asp109Ile / Met, Ala112Gly, Glu150Arg, Thr245Gln / Leu, Val248Arg / Leu, Ser249Pro / Trp, Glu451Met, Thr456Leu / Phe.

[0046] The target mutant was obtained by PCR amplification using site-directed mutagenesis primers with the plasmid pET-28a-GPO containing the wild-type glycerol phosphate oxidase gene (see Example 1 for the plasmid construction process) as a template. The PCR amplification procedure is as follows: pre-denaturation at 98°C for 2min; denaturation at 98°C for 10s, annealing at 56°C for 30s, extension at 72°C for 90s, 25 cycles; extension at 72°C for 5min. The above product was digested with DpnI at 37°C for 3h, and the digested product was then transformed into Escherichia coli competent DH5α to obtain positive clones. After the single clone was correctly sequenced, different mutants were obtained, and the recombinant plasmids of 13 mutants were extracted for use. The specific primer sequences are shown in Table 1:

[0047] Table 1 Primers used for site-directed mutagenesis

[0048]

[0049]

[0050] Example 3

[0051] Expression and screening of phosphoglycerate oxidase mutants:

[0052] The glycerol phosphate oxidase (GPO) mutant plasmid obtained in Example 2 was transformed into Escherichia coli BL21 (DE3) for GPO mutant expression. The specific steps are as follows:

[0053] (1) Preparation of Escherichia coli strains expressing GPO mutants. The screened positive strains were streaked on LB solid medium (35 μg / mL kanamycin) until a single clone grew out, and a single clone was picked and inoculated into liquid LB medium, and cultured in a shaker at 37°C and 200 rpm for 12-16 h to obtain seed liquid.

[0054] (2) Induced expression and screening of mutant enzymes. The induced expression conditions of the 13 mutant enzymes were consistent with those of the wild-type GPO, as shown in Example 1. After the induction expression of the wild-type GPO and the mutants was completed, the bacteria were collected by centrifugation at 4°C. The collected bacteria were added to the disruption solution (1 g of wet bacteria was added to 8 mL of disruption solution for suspension) and ultrasonically disrupted (power 450 W, ultrasound 8 s, interval 15 s) for 30 min, and then centrifuged at 15000 rpm and 4°C for 15 min to collect the supernatant for use.

[0055] 1 mL of supernatant from the cell crushing of wild-type GPO and 13 mutants was added to a 1.5 mL centrifuge tube, and the GPO enzyme activity of each supernatant was measured (see Example 5 for the enzyme activity measurement method), and the activity of each enzyme that was not heat-treated was set to 100%. At the same time, the supernatant of the wild-type and 13 mutants was treated at 50°C for 30 minutes, and the residual activity of each enzyme after heat treatment was measured. The ratio of the residual activity of each enzyme after heat treatment to the enzyme activity of the enzyme before treatment was calculated, which was the residual enzyme activity rate of the enzyme after heat treatment. The results are shown in FIG. Figure 2 As shown in B. The residual enzyme activity of the wild-type GPO after treatment at 50°C for 30 minutes was only 26%. Compared with the wild-type enzyme, the thermal stability of mutants D109I, E115R, S249P, S249W and E451M was significantly improved. GPO mutants S249P and E451M with residual enzyme activities greater than 50% after treatment at 50°C for 30 minutes were selected. The residual enzyme activities of S249P and E451M mutants after heat treatment were 86% and 67%, respectively. Next, according to the wild-type GPO expression and purification method of Example 1, S249P and E451M mutant proteins were purified, and the purity of the purified proteins detected by SDS-PAGE exceeded 95%. The results are shown in Figure 3 Wherein M represents the protein molecular weight standard; 1 represents the purified phosphoglycerate oxidase mutant S249P; 2 represents the purified phosphoglycerate oxidase mutant E451M.

[0056] According to the GPO enzyme activity determination method of Example 5, the specific activities of wild-type GPO and S249P and E451M mutants were determined. The results showed that the specific activity of wild-type GPO was 47.2U / mg, and the Km was 2.17mM (pH 6.5); the specific activity of mutant S249P was 66.5U / mg, and the Km was 2.24mM (pH 6.5); the specific activity of mutant E451M was 41.5U / mg, and the Km was 2.53mM (pH 6.5). The results showed that compared with the wild-type GPO, the specific activity of mutant S249P increased by 1.4 times, and the specific activity of mutant E451M decreased slightly. Compared with the wild-type GPO, the Km values ​​of the two mutants did not change significantly. Since mutant S249P has higher specific activity and thermal stability than E451M, the enzymatic properties of mutant S249P were further analyzed.

[0057] Example 4

[0058] Analysis of enzymatic properties of mutant S249P:

[0059] (1) Thermal stability analysis of mutant S249P

[0060] The purified wild-type GPO and mutant S249P protein solutions were subjected to buffer replacement using a gel filtration column (Sephadex G-25 Medium), and the desalting buffer was 100 mM phosphate buffer (pH 6.5). The protein concentration after desalting was determined using the BCA method and the protein concentration was adjusted to 1 mg / mL. The protein solution was then incubated at different temperatures for 30 min, and the absorbance change of the reaction solution at 500 nm was determined using a microplate reader on a 96-well plate to calculate the residual enzyme activity after treatment at different temperatures. The enzyme activity determination method is described in Example 5. The activities of the untreated wild-type and mutant S249P were taken as 100% respectively, and the residual enzyme activity after treatment at different temperatures was calculated. The results are shown in FIG. Figure 4 As shown. Compared with the wild-type GPO, the thermal stability of mutant S249P is significantly improved. Especially under high temperature conditions exceeding 45°C, the thermal stability of mutant S249P is greatly improved. After treatment at 45°C for 30 minutes, the activity of mutant S249P is not affected, while the activity of wild-type GPO only remains 65%; after treatment at 50°C for 30 minutes, the activity of mutant S249P remains 86%, while the activity of wild-type GPO only remains 26%; after treatment at 55°C for 30 minutes, the activity of mutant S249P remains 57%, while the wild-type GPO is completely inactivated. The above research results show that compared with the wild-type GPO, the thermal stability of mutant S249P has been greatly improved.

[0061] In addition, the thermal stability of mutant S249P and Roche GPO was compared. The protein solution (1 mg / mL) was incubated at different temperatures for 30 min, and the residual enzyme activity after treatment at different temperatures was measured. The residual enzyme activity after treatment at different temperatures was calculated with the untreated mutant S249P and Roche GPO activities as 100%, respectively. The results are shown in Figure 2. Figure 4 As shown. Compared with Roche GPO, the thermal stability of mutant S249P is significantly improved. After treatment at 50°C for 30 minutes, the activity of mutant S249P remains 86%, while the activity of Roche GPO remains 56%; after treatment at 55°C for 30 minutes, the activity of mutant S249P remains 57%, while the activity of Roche GPO remains only 23%; the above results show that compared with Roche GPO, the thermal stability of mutant S249P has also been greatly improved. In addition, the specific activity of Roche GPO was determined to be 58U / mg protein, and the specific activity of mutant S249P (66.5U / mg protein) was slightly higher than that of Roche GPO.

[0062] (2) pH stability analysis of mutant S249P protein:

[0063] The purified wild-type GPO and mutant S249P protein solutions were buffer replaced using a gel filtration column (Sephadex G-25 Medium). The desalting buffer was 20 mM phosphate buffer (pH 7.4). The protein concentration after desalting was determined using the BCA method and the protein concentration was adjusted to 5 mg / mL. Then 100 mM buffers of different pH values ​​were prepared. The buffers selected for different pH ranges are as follows: acetic acid-sodium acetate buffer (pH 4-6), phosphate buffer (pH 6.5-7.4), and Tris-HCl buffer (pH 8-10). 5 mg / mL of wild-type GPO and mutant S249P proteins were diluted to 0.5 mg / mL using buffers of different pH values, and then incubated at 4°C for 12 hours to determine the activity of wild-type GPO and mutant S249P after incubation under different pH conditions. The highest enzyme activity of wild-type GPO and mutant S249P was taken as 100%, and the residual enzyme activity after incubation under different pH conditions was calculated. The results are shown in the figure. Figure 5 As shown. The results showed that the stability of mutant S249P in acidic and neutral solutions was not significantly different from that of wild-type GPO. However, in alkaline solution (pH>7.4), the stability of mutant S249P was significantly improved. After incubation at 4°C for 12 hours in a pH 9 solution, the enzyme activity of mutant S249P remained 69%, while that of wild-type GPO remained only 49%.

[0064] Example 5

[0065] Glycerol phosphate oxidase activity assay:

[0066] The principle of the assay of glycerol phosphate oxidase (GPO) activity is as follows: the reaction substrate sodium α-glycerophosphate generates hydrogen peroxide (H 2 O 2 ), then H 2 O 2 The quinone imine is generated by 4-aminoantipyrine under the catalysis of horseradish peroxidase. The absorbance change of the colored quinone imine is measured by an ELISA instrument at 500nm. The concentration change of the colored quinone imine is calculated according to the molar extinction coefficient, thereby calculating the GPO enzyme activity. Under this detection condition, the extinction coefficient of quinone imine is 13.3mM -1 cm -1 The enzyme activity was defined as the amount of enzyme required to catalyze the production of 1 μmol of quinoneimine in 1 min at 37 °C.

[0067] The specific steps are as follows:

[0068] (1) Reagent preparation:

[0069] α-Sodium glycerol phosphate stock solution (0.2 M): weigh 5.1 g of sodium α-glycerol phosphate (TCI, product code: G0096) and dissolve it in 100 mL of phosphate buffer (100 mM, pH 6.5) containing 0.13% Triton X-100;

[0070] 4-aminoantipyrine solution (0.1%): 20 mg 4-aminoantipyrine was dissolved in 20 mL pure water and stored in a brown bottle at 4°C;

[0071] Phenol solution (0.1%): 20 mg of phenol was dissolved in 20 mL of water and stored in a brown bottle at 4°C;

[0072] Horseradish peroxidase solution (25 U / mL): Dissolve 10 mg horseradish peroxidase in 1 mL water and store at -80°C after aliquoting;

[0073] Bovine serum albumin (0.2%): 40 mg of bovine serum albumin was dissolved in 20 mL of pure water and prepared before use.

[0074] Enzyme activity assay method and steps:

[0075] (1) Preparation of reaction solution. Prepare 10 mL of reaction solution according to the following proportions: 5 mL of α-phosphoglycerate sodium solution; 1 mL of 4-aminoantipyrine solution; 2 mL of phenol solution; 2 mL of horseradish peroxidase solution; use bovine serum albumin (0.2%) to dilute the enzyme to 20 ug / mL.

[0076] (2) Add the above reaction solution to a 96-well plate, 200 μL per well, and incubate at 37°C for 5 min.

[0077] (3) Add 10 μL of GPO diluted enzyme solution (0.2 μg / well) to each well of a 96-well plate. After shaking for 15 s in a microplate reader, the reaction was immediately started. The reaction was monitored at a wavelength of 500 nm for 10 min. The enzyme activity was calculated based on the molar extinction coefficient of the reaction product quinone imine.

[0078] SEQ ID NO.1

[0079] MFSNKTRQDSIQKMQQEELDLLIIGGGITGAGVAVQAAASGIKTGLIEMQDFAE

[0080] GTSSRSTKLVHGGIRYLKTFDVEVVADTVGERAVVQGIAPHIPKPDPMLLPIYE

[0081] DEGATTFNMFSVKVAMDLYDKLANVTGTKYENYTTLTPEEVLEREPFLKKEGL

[0082] KGAGVYLDFRNNDARLVIDNIKKAAEDGAYLVSKMKAVGFLYEGDQIVGVKA

[0083] RDLLTDEVIEIKAKLVINTSGPWVDKVRNLNFTRPVSPKMRPTKGIHLVVDAK

[0084] KLPVPQPTYFDTGKQDGRMVFAIPRENKTYFGTTDTDYQGDFTDPKVTQEDV

[0085] DYLLDVINHRYPEANITLADIEASWAGLRPLLIGNSGSDYNGGDNGSISDKSFN

[0086] KVVDTVSEYKENKVSRAEVEDVLNHLENSRDEKAPSTISRGSSLEREPDGLLT

[0087] LSGGKITDYRKMAEGALRLIRQLLKEEYGIETKEIDSKKYQISGGNFDPTKLEE

[0088] TVTELAKEGVAAGLEEEDATYIADFYGTNARRIFELAKEMAPYPGLSLAESAR

[0089] LRYGLEEEMVLAPGDYLIRRTNHLLFERDQLDEIKQPVIDAIAEYFGWTEEEKAQQTKRLEALIAESDLRELKGEK。

[0090] SEQ ID NO.2

[0091] ATGTTCTCCAACAAGACTCGCCAGGACTCTATTCAGAAGATGCAGCAGGAG

[0092] GAACTGGATCTGCTGATTATCGGTGGTGGTATCACTGGTGCAGGTGTAGCAG

[0093] TGCAGGCAGCTGCTAGCGGTATCAAAACCGGCCTGATTGAGATGCAGGATT

[0094] TCGCTGAAGGTACCTCCTCCCGTTCCACCAAACTGGTTCATGGTGGTATCCG

[0095] TTATCTGAAAACCTTCGACGTTGAGGTGGTTGCGGACACCGTTGGCGAACG

[0096] TGCTGTTGTACAGGGTATCGCTCCGCACATCCCGAAACCGGACCCGATGCT

[0097] GCTGCCGATCTACGAGGACGAAGGTGCGACGACCTTCAACATGTTCAGCGT

[0098] GAAGGTAGCTATGGATCTGTACGACAAACTGGCCAACGTAACTGGTACCAA

[0099] ATACGAAAACTACACCCTGACCCCTGAAGAAGTTCTGGAACGTGAACCGTT

[0100] TCTGAAGAAGGAAGGCCTGAAAGGTGCAGGTGTGTATCTGGATTTCCGCAA

[0101] CAACGATGCGCGTCTGGTGATTGACAACATCAAAAAAGCAGCGGAAGACG

[0102] GCGCTTACCTGGTCTCTAAAATGAAAGCAGTTGGTTTCCTGTACGAAGGTG

[0103] ACCAGATCGTGGGTGTGAAGGCACGTGACCTGCTGACGGATGAAGTGATC

[0104] GAGATCAAAGCCAAACTGGTCATCAACACTTCTGGCCCGTGGGTGGACAA

[0105] AGTTCGCAACCTGAATTTCACCCGTCCGGTGTCTCCGAAAATGCGTCCGAC

[0106] CAAAGGCATCCACCTGGTTGTTGACGCGAAAAAACTGCCGGTACCGCAGC

[0107] CGACCTACTTCGACACGGGCAAACAGGACGGCCGCATGGTGTTTGCAATCC

[0108] CGCGTGAAAACAAAACCTACTTCGGTACCACTGACACCGACTACCAGGGT

[0109] GATTTCACGGACCCGAAAGTTACTCAAGAGGACGTAGACTACCTGCTGGAT

[0110] GTTATCAATCATCGCTATCCGGAAGCTAACATCACTCTGGCTGACATCGAAG

[0111] CTTCTTGGGCTGGCCTGCGTCCGCTGCTGATTGGTAACTCCGGCTCTGACTA

[0112] CAACGGTGGTGATAATGGTTCTATTTCCGACAAATCTTTCAACAAAGTTGTG

[0113] GACACTGTGTCTGAATACAAAGAAAACAAAGTTTCCCGCGCGGAAGTGGA

[0114] AGATGTACTGAACCACCTGGAAAACAGCCGTGATGAAAAAGCCCCTAGCA

[0115] CGATCAGCCGCGGTAGCTCTCTGGGAACGTGAACCGGATGGTCTGCTGACCC

[0116] TGTCCGGTGGTAAAATCACTGATTATCGTAAAATGGCGGAAGGTGCTCTGC

[0117] GCCTGATCCGTCAGCTGCTGAAAAGGAATATGGTATCGAAACTAAAAGAAA

[0118] TCGACTCCAAAAAATCCAGATCAGCGGTGGCAACTTTGACCCGACTAAAC

[0119] TGGAAGAAACTGTAACCGAGCTGGCGAAAAGAGGCGTTGCTGCGGGTCTG

[0120] GAAGAAGAAGACGCGACGTACATCGCTGATTTCTACGGTACTAACGCGCGC

[0121] CGCATCTTTGAACTGGCGAAGGAGATGGCTCCATATCCAGGCCTGAGCCTG

[0122] GCAGAAAGCGCTCGTCTGCGCTACGGCCTGGAGGAAGAAATGGTTCTGGC

[0123] TCCGGGCGACTACCTGATCCGTCGTACTAACCACCTGCTGTTGAGCGTGA

[0124] CCAGCTGGACGAAATCAAACAGCCGGTTATCGATGCGATCGAATACTT

[0125] CGGCTGGACTGAGGAGGAAAAGCTCAGCAGACTAAACGTCTGGAGGCTCTGATCGCGGAGAGCGATCTGCGCGAACTGAAAGGTGAAAAA。

[0126] SEQ ID NO.3

[0127] ATGTTCTCCAACAAAGACTCGCCAGACTCTATTCAGAAGATGCAGCAGGAG

[0128] GAACTGGATCTGCTGATTATCGGTGGTGGTATCACTGGTGCAGGTGTAGCAG

[0129] TGCAGGCAGCTGCTAGCGGTATCAAAACCGGCCTGATTGAGATGCAGGATT

[0130] TCGCTGAAGGTACCTCCTCCCGTTCCACCAAACTGGTTCATGGTGGTATCCG

[0131] TTATCTGAAAACCTTCGACGTTGAGGTGGTTGCGGACACCGTTGGCGAACG

[0132] TGCTGTTGTACAGGGTATCGCTCCGCACATCCCGAAACCGGACCCGATGCT

[0133] GCTGCCGATCTACGAGGACGAAGGTGCGACGACCTTCAACATGTTCAGCGT

[0134] GAAGGTAGCTATGGATCTGTACGACAAACTGGCCAACGTAACTGGTACCAA

[0135] ATACGAAAACTACACCCTGACCCCTGAAGAAGTTCTGGAACGTGAACCGTT

[0136] TCTGAAGAAGGAAGGCCTGAAAGGTGCAGGTGTGTATCTGGATTTCCGCAA

[0137] CAACGATGCGCGTCTGGTGATTGACAACATCAAAAAAGCAGCGGAAGACG

[0138] GCGCTTACCTGGTCTCTAAAATGAAAGCAGTTGGTTTCCTGTACGAAGGTG

[0139] ACCAGATCGTGGGTGTGAAGGCACGTGACCTGCTGACGGATGAAGTGATC

[0140] GAGATCAAAGCCAAACTGGTCATCAACACTTCTGGCCCGTGGGTGGACAA

[0141] AGTTCGCAACCTGAATTTCACCCGTCCGGTGCCGCCGAAAATGCGTCCGAC

[0142] CAAAGGCATCCACCTGGTTGTTGACGCGAAAAAACTGCCGGTACCGCAGC

[0143] CGACCTACTTCGACACGGGCAAACAGGACGGCCGCATGGTGTTTGCAATCC

[0144] CGCGTGAAAACAAAACCTACTTCGGTACCACTGACACCGACTACCAGGGT

[0145] GATTTCACGGACCCGAAAGTTACTCAAGAGGACGTAGACTACCTGCTGGAT

[0146] GTTATCAATCATCGCTATCCGGAAGCTAACATCACTCTGGCTGACATCGAAG

[0147] CTTCTTGGGCTGGCCTGCGTCCGCTGCTGATTGGTAACTCCGGCTCTGACTA

[0148] CAACGGTGGTGATAATGGTTCTATTTCCGACAAATCTTTCAACAAAGTTGTG

[0149] GACACTGTGTCTGAATACAAAGAAAACAAAGTTTCCCGCGCGGAAGTGGA

[0150] AGATGTACTGAACCACCTGGAAAACAGCCGTGATGAAAAAGCCCCTAGCA

[0151] CGATCAGCCGCGGTAGCTCTCTGGAACGTGAACCGGATGGTCTGCTGACCC

[0152] TGTCCGGTGGTAAAATCACTGATTATCGTAAAATGGCGGAAGGTGCTCTGC

[0153] GCCTGATCCGTCAGCTGCTGAAAGAGGAATATGGTATCGAAACTAAAGAAA

[0154] TCGACTCCAAAAAATCCAGATCAGCGGTGGCAACTTTGACCCGACTAAAC

[0155] TGGAAGAAACTGTAACCGAGCTGGCGAAAAGAGGCGTTGCTGCGGGTCTG

[0156] GAAGAAGAAGACGCGACGTACATCGCTGATTTCTACGGTACTAACGCGCGC

[0157] CGCATCTTTGAACTGGCGAAGGAGATGGCTCCATATCCAGGCCTGAGCCTG

[0158] GCAGAAAGCGCTCGTCTGCGCTACGGCCTGGAGGAAGAAATGGTTCTGGC

[0159] TCCGGGCGACTACCTGATCCGTCGTACTAACCACCTGCTGTTGAGCGTGA

[0160] CCAGCTGGACGAAATCAAACAGCCGGTTATCGATGCGATCGAATACTT

[0161] CGGCTGGACTGAGGAGGAAAAGCTCAGCAGACTAAACGTCTGGAGGCTCTGATCGCGGAGAGCGATCTGCGCGAACTGAAAGGTGAAAAA。

[0162] SEQ ID NO.4

[0163] ATGTTCTCCAACAAAGACTCGCCAGACTCTATTCAGAAGATGCAGCAGGAG

[0164] GAACTGGATCTGCTGATTATCGGTGGTGGTTATCACTGGTGCAGGTGTAGCAG

[0165] TGCAGGCAGCTGCTAGCGGTATCAAAACCGGCCTGATTGAGATGCAGGATT

[0166] TCGCTGAAGGTACCTCCTCCGTTCCACCAAACTGGTTCATGGTGGTATCCG

[0167] TTATCTGAAAACCTTCGACGTTGAGGTGGTTGCGGACACCGTTGGCGAACG

[0168] TGCTGTTGTACAGGGTATCGCTCCGCACATCCCGAAACCGGACCCGATGCT

[0169] GCTGCCGATCTACGAGGACGAAGGTGCGACGACCTTCAACATGTTCAGCGT

[0170] GAAGGTAGCTATGGATCTGTACGACAAACTGGCCAACGTAACTGGTACCAA

[0171] ATACGAAAACTACACCCTGACCCCTGAAGAAGTTCTGGAACGTGAACCGTT

[0172] TCTGAAGAAGGAAGGCCTGAAAGGTGCAGGTGTGTATCTGGATTTCCGCAA

[0173] CAACGATGCGCGTCTGGTGATTGACAACATCAAAAAAGCAGCGGAAGACG

[0174] GCGCTTACCTGGTCTCTAAAATGAAAGCAGTTGGTTTCCTGTACGAAGGTG

[0175] ACCAGATCGTGGGTGTGAAGGCACGTGACCTGCTGACGGATGAAGTGATC

[0176] GAGATCAAAGCCAAACTGGTCATCAACACTTCTGGCCCGTGGGTGGACAA

[0177] AGTTCGCAACCTGAATTTCACCCGTCCGGTGTCTCCGAAAATGCGTCCGAC

[0178] CAAAGGCATCCACCTGGTTGTTGACGCGAAAAAACTGCCGGTACCGCAGC

[0179] CGACCTACTTCGACACGGGCAAACAGGACGGCCGCATGGTGTTTGCAATCC

[0180] CGCGTGAAAAAAAACCTACTTCGGTACCACTGACACCGACTACCAGGGT

[0181] GATTTCACGGACCCGAAGTTACTCAAGAGGACGTAGACTACCTGCTGGAT

[0182] GTTATCAATCATCGCTATCCGGAAGCTAACATCACTCTGGCTGACATCGAAG

[0183] CTTCTTGGGCTGGCCTGCGTCCGCTGCTGATTGGTAACTCCGGCTCTGACTA

[0184] CAACGGTGGTGATAATGGTTCTATTTCGACAAATCTTTCAACAAAGTTGTG

[0185] GACACTGTGTCTGAATACAAAAGAAAACAAAGTTTCCCGCGCGGGAAGTGGA

[0186] AGATGTACTGAACCACCTGGAAAACAGCCGTGAATGAAAAGCCCCTAGCA

[0187] CGATCAGCCGCGGTAGCTCTCTGGGAACGTGAACCGGATGGTCTGCTGACCC

[0188] TGTCCGGTGGTAAAATCACTGATTATCGTAAAATGGCGGAAGGTGCTCTGC

[0189] GCCTGATCCGTCAGCTGCTGAAAGAGATGTATGGTATCGAAACTAAAAGAAA

[0190] TCGACTCCAAAAAATCCAGATCAGCGGTGGCAACTTTGACCCGACTAAAC

[0191] TGGAAGAAACTGTAACCGAGCTGGCGAAAAGAGGCGTTGCTGCGGGTCTG

[0192] GAAGAAGAAGACGCGACGTACATCGCTGATTTCTACGGTACTAACGCGCGC

[0193] CGCATCTTTGAACTGGCGAAGGAGATGGCTCCATATCCAGGCCTGAGCCTG

[0194] GCAGAAAGCGCTCGTCTGCGCTACGGCCTGGAGGAAGAAATGGTTCTGGC

[0195] TCCGGGCGACTACCTGATCCGTCGTACTAACCACCTGCTGTTTGAGCGTGA

[0196] CCAGCTGGACGAAATCAAACAGCCGGTTATCGATGCGATCGCAGAATACTT

[0197] CGGCTGGACTGAGGAGGAAAAAGCTCAGCAGACTAAACGTCTGGAGGCTC

[0198] TGATCGCGGAGAGCGATCTGCGCGAACTGAAAGGTGAAAAA。

Claims

1. A phosphoglycerate oxidase mutant with improved thermal stability, characterized in that: Taking the amino acid sequence SEQ ID NO.1 as the parent, mutating the 249th or 451st position of the parent; The serine at position 249 of the parent is replaced by proline; or the glutamic acid at position 451 of the parent is replaced by methionine.

2. A gene encoding a phosphoglycerate oxidase mutant with improved thermal stability as claimed in claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID NO.3 or SEQ ID NO.

4.

3. A recombinant plasmid containing the gene according to claim 2.

4. A host cell expressing the mutant according to claim 1, or containing the recombinant plasmid according to claim 3.

5. Use of the glycerol phosphate oxidase mutant with improved thermal stability as claimed in claim 1, or the gene as claimed in claim 2, or the recombinant plasmid as claimed in claim 3, or the host cell as claimed in claim 4 in the production of a triglyceride detection agent.

Images