CN110938607B

CN110938607B - Glycerol-3-phosphate oxidase with good thermal stability and application thereof in kit

Info

Publication number: CN110938607B
Application number: CN201911306224.4A
Authority: CN
Inventors: 邹炳德; 邹继华; 章玉胜; 汪屹; 张帅; 张永
Original assignee: Medical System Biotechnology Co ltd
Current assignee: Medical System Biotechnology Co ltd
Priority date: 2019-12-18
Filing date: 2019-12-18
Publication date: 2023-03-28
Anticipated expiration: 2039-12-18
Also published as: CN110938607A

Abstract

The invention discloses glycerol-3-phosphate oxidase with good thermal stability, the amino acid sequence of which is the sequence shown in SEQ ID No.2, and the polynucleotide sequence for coding the glycerol-3-phosphate oxidase with good thermal stability is the sequence shown in SEQ ID No. 1; the recombinant glycerol-3-phosphate oxidase obtained by the invention has the characteristic of good thermal stability. After 30min of treatment at 60 ℃, the enzyme activity retention rate is 52.3 percent; after the treatment for 3 days at 37 ℃, the enzyme activity retention rate is 92.7 percent, the recombinant glycerol-3-phosphate oxidase obtained by the invention has good thermal stability, can improve the thermal stability of the enzyme in a kit, and is beneficial to improving the stability of the kit. Meanwhile, the dosage of the enzyme in triglyceride can be reduced, and the production cost of the kit is reduced.

Description

Glycerol-3-phosphate oxidase with good thermal stability and application thereof in kit

Technical Field

The invention relates to the field of xx, in particular to glycerol-3-phosphate oxidase with good thermal stability and application thereof in a kit.

Background

The invention relates to the technical field of enzymes for diagnosis, in particular to a preparation method and application of glycerol-3-phosphate oxidase with good thermal stability.

Triglyceride (TG) is a constituent of lipid, and is a lipid formed of glycerol and 3 fatty acids. Triglycerides belong to neutral fats, and the human body stores a large amount of glycerides, the primary function of which is to provide energy for cell metabolism, and 90-95% of the glycerides in plasma are triglycerides.

The triglyceride has important clinical significance in the detection and monitoring of the blood fat of human bodies. According to the standard division of 'Chinese adult dyslipidemia prevention and treatment guidelines' in 2007, fasting (fasting for 12 hours) triglyceride less than or equal to 1.70Mmol is a proper level; 1.70-2.25 Mmol is edge rising; the molar mass of the compound is not less than 2.26 Mmol. The elevation of triglyceride can be seen in various diseases such as hyperlipoproteinemia, diabetes, gout, obstructive jaundice, hypothyroidism, pancreatitis and the like. It is now clinically accepted that elevated triglycerides are a risk factor for the development of coronary heart disease, and dietary management or drug therapy should be given when elevated.

The chemistry for clinical detection of TG has been eliminated due to poor specificity. Triglyceride detection is currently largely replaced by enzymatic methods. The enzyme method is rapid and accurate, the operation is simple, and the large-batch sample detection can be carried out on an automatic biochemical analyzer. The reaction principle of TG detection by the enzyme method is as follows: the TG to be detected is decomposed into fatty acids and glycerol under the action of Lipoprotein Lipase (LPL); glycerol is subjected to phosphorylation reaction with ATP in a kit under the action of Glycerol Kinase (GK) to produce Glycerol-3-phosphate, the Glycerol-3-phosphate is further subjected to oxidation by oxygen under the action of Glycerol-3-phosphate Oxidase (GPO) to generate hydrogen peroxide (H2O 2), and the hydrogen peroxide (H2O 2) is generated by the product of the oxidation reaction with a chromogen substance in the presence of Trinder reaction, namely Peroxidase (POD) and a color-developing agent 4-aminoantipyrine (4-AAP), and the content of TG is determined through the change of an absorption peak under visible light.

The specific reaction steps are divided into the following four parts:

GPO plays an important role in the enzymatic TG determination method. Which catalyzes the oxidation of glycerol-3-phosphate to H ₂ O ₂ The method is often the rate-limiting step of the kit reaction, the dosage and the enzyme activity of the enzyme determine the sensitivity and precision of the enzyme method determination to a great extent, and the thermal stability of the enzyme also determines the storage stability of the kit to a great extent. However, the existing GPO in the market has poor thermal stability, and is difficult to meet the temperature influence of the detection kit in the transportation and clinical application processes, so that the sensitivity and precision of the kit in clinical detection are reduced, and the application of the kit is limited.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the prior art, provide a preparation method of glycerol-3-phosphate oxidase (GPO) with good thermal stability, and provide application of the GPO in a triglyceride detection kit.

The technical solution of the invention is as follows: a GPO with good thermal stability is based on that GPO gene sequences extracted from Pediococcus acidilacteici strains at present are taken as templates, error-prone PCR is adopted for random mutation to obtain a series of mutant genes, a GPO mutation library is constructed and is introduced into E.Coli BL21 (DE 32), the mutant strains are subjected to plate heat treatment and then subjected to high-throughput active screening to obtain 8-10 mutant strains with good thermal stability, and the mutant strains are determined by shake flask fermentation and rescreening to determine 1 GPO with good thermal stability and a recombinant expression strain thereof. The enzyme still has 52.3% retention rate after heat treatment at 60 ℃ for 30min, and 92.7% retention rate after heat treatment for 3d at 37 ℃. The GPO with good thermal stability has an amino acid sequence shown in SEQ ID No.2, and a nucleotide sequence for coding the GPO has a sequence shown in SEQ ID No. 1.

The glycerol-3-phosphate oxidase with good thermal stability is obtained by the following ways:

(1) Carrying out error-prone PCR amplification mutation by taking the GPO gene sequence of the Pediococcus acidilactici strain as a template to obtain a series of mutant genes and establishing a GPO random mutation library.

(2) The obtained GPO mutant library was ligated under PET22b T7 promoter, introduced into e.coli expression strain BL21 (DE 32), and recombinant-expressed.

(3) After heat treatment is carried out on the strain plate of the recombinant expression, 8-10 GPO with better thermal stability are obtained through color reaction and high-throughput screening.

(4) And (3) performing shake flask rescreening on 8-10 GPO recombinant expression strains with good thermal stability to determine 1 GPO strain with the best thermal stability.

The strain of Pediococcus acitilacticii in nature contains the gene sequence of glycerol-3-phosphate oxidase. The invention designs primer amplification by taking the gene sequence as a template, namely 5 'TACAGTAAAGTAATAAT-3' and 5 'CTAGATTATCTGAGATAG-3', the fragment is subjected to error-prone PCR amplification mutation to obtain a GPO gene sequence containing random mutation, the mutation fragment is recovered, enzyme digestion is carried out by amH I and Bam I, the fragment is connected to PET22b containing a T7 promoter by T4 ligase, the fragment is transformed into an E.coli expression strain BL21 (DE 32), the E.coli expression strain is coated on an LB plate containing 10-200 mg/L Amp resistance and cultured overnight at 37 ℃ to form a monoclonal colony, the monoclonal colony is copied and transferred to another LB plate containing 0.05-1 mM IPTG by a method, and the culture is induced at 25 ℃ for 10-12 h to induce the GPO to be fully recombined and express the GPO, and the expressed GPO has a series of random mutation. And then adding cell lysate, treating for 30min at 60 ℃, uniformly spraying enzyme activity reaction liquid on a flat plate after the treatment is finished, carrying out warm bath treatment at 37 ℃, observing the color change of the monoclonal colony after 10min, and selecting the monoclonal with obvious color change to obtain 8-10 purple monoclonal, namely GPO recombinant expression strains with better thermal stability. It is composed ofThe medium cell lysate consists of 1-100 mM Tris-HCl, 0.2-1 g/L CTAB, 1-20 mM KCl, 30-100 mM MgCl ₂ 0.1-1 g/L sodium deoxycholate, pH7.5; the enzyme activity reaction liquid consists of 10-100 mM Tris-HCl, 1-20 mM glycerol-3-phosphoric acid, 2-100U/ml POD, 2-50 mM TOOS, 1-10 mM4-AAP, and pH7.0-8.0.

Selecting 8-10 monoclonal colonies with obvious color change, inoculating the colonies into 10-50ml 2YT liquid culture medium, culturing at 37 ℃ until OD is 0.6-1.0, inoculating the colonies into 100-500ml 2YT liquid culture medium, culturing at 37 ℃ until OD is 0.6-1.0, adding IPTG with final concentration of 0.05-1 mM, cooling to 25 ℃, inducing and culturing for 5-15 h, and centrifuging to collect thalli. Taking centrifugally collected thalli, adding 2-10 volumes of cell lysate, adding a proper amount of Ni filler substrate, vertically mixing overnight, centrifuging for 5-20 min at 5000-12000 revolutions, discarding supernatant, adding 5-12 volumes of washing buffer1 into the precipitate, vertically mixing overnight, centrifuging for 5-20 min at 5000-12000 revolutions, discarding supernatant, adding 2-10 volumes of washing buffer2 into the precipitate, vertically mixing overnight, centrifuging for 5-20 min at 5000-12000 revolutions, and taking supernatant, thus obtaining GPO enzyme solution with higher purity. Wherein the cell lysate consists of 10-100 mM Tris-HCl, 0.02-2% SDS, 100-2000U/ml lysozyme, 0.1-2% TritonX-100; washing buffer1 consists of 10-100 mM Tris-HCl, 0.2-2M NaCl, 20-70 mM imidazole and pH7.0-8.5; washing buffer2 consists of 10-100 mM Tris-HCl, 0.2-2M NaCl, 200-1000 mM imidazole, and pH 6.0-8.0.

Each of the GPO enzyme solutions obtained above was subjected to a thermostability experiment separately. Treating the enzyme solution by 60 ℃ water bath for 30min, taking 0.1-1 ml of untreated and heat-treated enzyme solution, adding 10 times of enzyme activity reaction solution, keeping the temperature for 10min by 37 ℃ warm bath, adding 0.1 times of 100 TCA solution to terminate the reaction, measuring the absorbance change at 546nm, and comparing the heat treatment with the untreated enzyme solution to obtain 1 GPO recombinant expression strain with good thermal stability. The enzyme activity reaction solution is 10-100 mM Tris-HCl, 1-20 mM glycerol-3-phosphoric acid, 2-100U/ml POD, 2-50 mM TOOS, 1-10 mM4-AAP, and pH7.0-8.0.

1 strain of glycerol-3-phosphate oxidase recombinant expression strain with good thermal stability obtained by the shake flask re-screening is measured for GPO thermal stability of the expression.

After the enzyme solution is treated by warm bath at 60 ℃ for 30min, 0.1-1 ml of the enzyme solution is added into 10 times of the enzyme activity reaction solution, the reaction is carried out for 10min at 37 ℃, and 0.1 time of 100 percent TCA is immediately added to terminate the reaction. Taking 1ml of reaction solution, measuring the absorbance change value by using the 546nm wavelength under a spectrophotometer, and comparing the absorbance change value with the untreated enzyme solution to obtain the enzyme, wherein the enzyme activity retention rate is 52.3% after the enzyme is treated for 30min at 60 ℃. Similarly, the enzyme solution was treated with 37 ℃ bath for 3 days, and the enzyme activity retention was determined to be 92.7%. The enzyme activity reaction solution is 10-100 mM Tris-HCl, 1-20 mM glycerol-3-phosphoric acid, 2-100U/ml POD, 2-50 mM TOOS, 1-10 mM4-AAP, and pH7.0-8.0.

The invention takes Pediococcus acidilactaci glycerol-3-phosphate oxidase gene sequence as a template, constructs a library by random mutation through an error-prone PCR technology, is connected to PET22b after enzyme digestion, then is transformed into E.coli BL21 (DE 32), and obtains 1 glycerol-3-phosphate oxidase recombinant expression strain with good thermal stability through flat heat treatment high-throughput screening and shake flask fermentation rescreening, thereby being capable of inducing and expressing GPO.

Through DNA sequencing reaction, the nucleotide coding sequence of the enzyme is shown in SEQ ID No1, and compared with the original gene sequence, 8 nucleotide sequences are changed, namely, 245A → T, 262G → C, 534T → C, 628G → C, 748A → T, 785T → G, 825C → G and 1001T → A. The amino acid sequence of glycerol-3-phosphate oxidase having good thermal stability obtained by this mutation is shown in SEQ ID No.2, and it was found that 6 amino acid sequences were changed from the original amino acid sequence, i.e., asp → Val at position 82, ala → Pro at position 88, gly → Arg at position 210, thr → Ser at position 250, val → Gly at position 262 and Ile → Asn at position 334.

The enzyme can be applied to a triglyceride detection kit and is used for detecting the coupling reaction of triglyceride. The triglyceride detection kit consists of two parts, namely R1 and R2. Wherein R1 is: 1 to 500mM Tris-HCl buffer, 0.5 to 10mM TOOS, 0.001 to 0.2mM MgCl _2、 1-100U/mL lipoprotein lipase, 0.1 &2 percent of stabilizer, pH7.2-8.2. R2 is: 1 to 500mM Tris-HCl buffer solution, 0.2 to 5mM 4-AAP, 0.5 to 20mM ATP, 5 to 50U/mL glycerol kinase, 1 to 50U/mL peroxidase, 5 to 100U/mL glycerol-3-phosphate oxidase, 0.1 to 2 percent stabilizer and pH7.5 to 9.5. The kit is used for detecting triglyceride, has good accuracy, precision and linear range, and good stability, and has good performance indexes when being placed for 1 year at 37 ℃ and 4 ℃ with accelerated heat storage.

The invention has the advantages that:

(1) The recombinant glycerol-3-phosphate oxidase obtained by the invention has the characteristic of good thermal stability. After 30min of treatment at 60 ℃, the enzyme activity retention rate is 52.3 percent; after the treatment for 3 days at 37 ℃, the enzyme activity retention rate is 92.7 percent.

(2) The recombinant glycerol-3-phosphate oxidase obtained by the invention has good thermal stability, can improve the thermal stability of the enzyme in the kit, and is beneficial to improving the stability of the kit. Meanwhile, the dosage of the enzyme in triglyceride can be reduced, and the production cost of the kit is reduced.

(4) The recombinant glycerol-3-phosphate oxidase obtained by the invention can be efficiently expressed in escherichia coli genetic engineering bacteria, and the escherichia coli genetic engineering bacteria have the advantages of simple culture mode, high expression level, and quick and simple purification. The enzyme has low production cost, can be produced in large scale, and effectively reduces the production cost of the triglyceride detection kit.

Drawings

FIG. 1 is a linear regression plot of the application of GPO to the triglyceride detection kit in the examples.

FIG. 2 is a regression graph showing the linear relationship of GPO after accelerated treatment at 37 ℃ in the triglyceride detection kit applied with the GPO in the example.

Detailed Description

The present invention will be described in further detail below by way of examples, but the present invention is not limited to only the following examples:

example 1

Designing a primer sequence according to a glycerol-3-phosphate oxidase gene sequence in a Pediococcus acidilactici strain:

a forward primer: 5 'TACAGTAAAGTAATAAT-3',

reverse primer: 5' CTAGATTATCTGAGAGATAG-3

The primers are applied, error-prone PCR is used for amplification, and the error-prone PCR amplification system is as follows:

10. Mu.l amplification buffer 20. Mu.l

dNTP mix 30. Mu. Mol

Primer 60pmol

Template 0.5ug

Error-prone TaqDNA polymerase 2.5U/L

Mg ²⁺ 0.15Mmol/L

Mn ²⁺ 6Mmol

Double distilled water was added to 200. Mu.l.

The PCR reaction conditions are as follows: 5min at 92 ℃; 30s at 92 ℃, 30s at 55 ℃, 2min at 72 ℃ and 35 cycles; 10min at 72 ℃ and 4 DEG C

And (3) carrying out agarose gel electrophoresis on the 5 microliter of PCR amplification product, observing a target product band at a position of 58-60kb, and determining to obtain a PCR amplification band. Purifying and recovering the PCR amplification product by a DNA recovery kit to obtain a gene fragment, carrying out enzyme digestion by amH I and Bam I, connecting the enzyme fragment to PET22b containing a T7 promoter by T4 ligase, transforming the enzyme fragment into an E.coli expression strain BL21 (DE 32), coating the E.coli expression strain onto an LB plate containing 100mg/L AMP, and culturing overnight at 37 ℃ to obtain a series of GPO random mutation expression single colonies for subsequent screening.

Example 2

The single clones on LB plates of the series of mutant expression strains obtained in example 1 were transferred by replica transfer by the stamp method to LB plates containing 100mg/L AMP at a final concentration of 0.2mM IPTG, and cultured at 25 ℃ for 8 hours to induce the expression of glycerol-3-phosphate oxidase.

Cell lysate is evenly sprayed on a single colony plate expressing GPO, and then the plate is placed in a water bath kettle at 60 ℃ for heat treatment for 30min. The composition of the cell lysate was 20mM Tris-HCl, 0.5g/L CTAB, 10mM KCl, 50mM MgCl ₂ 0.2g/L sodium deoxycholate, pH7.5.

And uniformly spraying enzyme activity reaction liquid on the LB flat plate subjected to heat treatment, and fully reacting for 10min. The enzyme activity reaction solution comprises the following components: 100mM Tris-HCl, 20mM glycerol-3-phosphate, 20U/ml POD, 10mM TOOS, 5mM 4-AAP, pH7.5.

After the reaction is finished, directly observing the color reaction around the single colony by naked eyes, selecting 9 single colonies which are obvious in reaction and dark purple in total, and rescreening the single colonies by using a subsequent shake flask.

Example 3

9 single clones obtained by the primary screening of the plate of example 2 were inoculated into 20ml of 2YT liquid medium, cultured at 37 ℃ until OD is 0.8, transferred into 100ml of 2YT liquid medium, added with IPTG at final concentration of 0.2mM when OD is 0.6, and cultured at 25 ℃ for 8 hours.

Centrifuging fermentation liquor at 8200rpm for 15min, collecting thalli, adding 8 times of cell lysate, adding 10ml of Ni filler substrate, vertically mixing overnight, performing 8200 rotation centrifugation for 10min, discarding supernatant, adding 8 times of washing buffer1 into the precipitate, vertically mixing overnight, performing 9000 rotation centrifugation for 10min, discarding supernatant, adding 8 times of washing buffer2 into the precipitate, vertically mixing overnight, performing 10000 rotation centrifugation for 5min, taking supernatant, and obtaining supernatant which is pure glycerol-3-phosphate oxidase dilute enzyme solution for subsequent stability rescreening experiments. Wherein the cell lysate is 100mM Tris-HCl, 1% SDS, 500U/ml lysozyme, 1% TritonX-100; washing buffer1 consists of 20mM Tris-HCl, 0.8M NaCl and 40mM imidazole, and the pH value is 7.0; washing buffer2 was composed of 50mM Tris-HCl, 0.5M NaCl, 500mM imidazole, pH7.0.

Example 4

The group 9 glycerol-3-phosphate oxidase enzyme solutions obtained in example 3 were subjected to a thermostability experiment.

Treating the enzyme solution with 60 deg.C water bath for 30min, adding 1ml enzyme activity reaction solution into 0.1ml enzyme solution after untreated and heat treatment, keeping temperature at 37 deg.C for 10min, and measuring absorbance change at 546 nm. The absorbance change is shown in Table 1. Comparing the thermal stability data, it can be seen that the thermal stability of strain No. 8 is higher, i.e. GPO expression strain with good thermal stability is obtained for expressing GPO enzyme with good thermal stability. The enzyme activity reaction solution was 20mM Tris-HCl, 5mM glycerol-3-phosphate, 20U/ml POD, 5mM TOOS, 2mM 4-AAP, pH8.0.

TABLE 1 thermal stability data for different GPO mutants

Example 5

The strain No. 8 obtained in example 4 was rescreened in a shake flask, and purified to prepare a glycerol-3-phosphate oxidase enzyme solution. Strain No. 8 was inoculated into 10ml of 2YT liquid medium, cultured at 37 ℃ until OD was about 1.0, transferred to 200mL of 2YT liquid medium, cultured at 37 ℃ until OD was about 0.6, added with IPTG at a final concentration of 0.3mM, and induced at 25 ℃ for 6 hours.

Centrifuging fermentation liquor at 6000rpm for 20min, collecting thalli, adding cell lysate with 2 times of volume, adding 10ml of Ni filler substrate, vertically mixing overnight, rotating and centrifuging for 12min at 8000, discarding supernatant, adding washing buffer1 with 2 times of volume into the sediment, vertically mixing overnight, rotating and centrifuging for 12min at 8000, discarding supernatant, adding washing buffer2 with 2 times of volume into the sediment, vertically mixing overnight, rotating and centrifuging for 10min at 8000, taking supernatant, wherein the obtained supernatant is high-purity glycerol-3-phosphate oxidase enzyme solution, and recovering 5ml of GPO enzyme solution in total. Wherein the cell lysate consists of 20mM Tris-HCl, 0.5% SDS, 500U/ml lysozyme, 0.2% Triton X-100; washing buffer1 consists of 50mM Tris-HCl, 0.3M NaCl and 50mM imidazole, and the pH value is 7.0; washing buffer2 was composed of 50mM Tris-HCl, 0.3M NaCl, 400mM imidazole, pH7.0.

Example 6

The glycerol-3-phosphate oxidase prepared in example 5 was measured for stability data after heat treatment.

GPO enzyme solution is treated by water bath at 60 ℃ for 30min, 0.1ml is taken, 1ml of color-changing reaction solution is added, the temperature bath is carried out at 37 ℃ for 10min, 0.1ml of 100% TCA is added to terminate the reaction, the absorbance change value under 546nm is measured, the color-changing reaction solution added with pure water is used as a blank control, and the experiment is repeated three times. The GPO enzyme solution is taken out, and is subjected to water bath at 37 ℃ for 3d, and the absorbance change value is measured. The absorbance change values of the GPO enzyme solution after heat treatment and untreated are shown in Table 2, the average value is calculated, the enzyme activity retention rate is 52.3 percent after the enzyme is treated for 30min at 60 ℃, and the enzyme activity retention rate is 92.7 percent after the enzyme is treated for 3d at 37 ℃. The enzyme-activated reaction solution had a composition of 100mM Tris-HCl, 20mM glycerol-3-phosphate, 100U/ml POD, 20mM TOOS, 10mM4-AAP, pH7.0.

TABLE 2 thermal stability data of GPO treated at 60 deg.C for 30min and at 37 deg.C for 3 days

Example 7

The glycerol-3-phosphate oxidase solution prepared in example 5 was added to a triglyceride assay kit, and the accuracy, precision, linear relationship, and other indicators of the kit were analyzed. The kit R1 comprises: 50mM Tris-HCl buffer, 20mM TOOS, 0.2mM MgCl _2、 Lipoprotein lipase 40U/mL, 0.4% stabilizer, pH7.2. The kit R2 comprises: 100mM Tris-HCl buffer, 5mM 4-AAP, 10mM ATP, 20U/mL glycerol kinase, 20U/mL peroxidase, 50U/mL glycerol-3-phosphate oxidase, 1% stabilizer, pH7.5. The accuracy of the kit is determined by determining the assigned quality control under the condition of normal calibration of the kit, comparing the determined value with the quality control, and determining that the accuracy is qualified within the deviation of +/-5%. And the precision measurement is to continuously measure the high-value sample and the low-value sample for 20 times respectively under the condition of normal calibration of the kit, calculate CV after statistical analysis, and judge that the precision is qualified when the CV is less than or equal to 5 percent. The linear assay is a method in which, under normal calibration of the kit, a 12mM linear high value is mixed with physiological saline in a ratio of 10 ² More than or equal to 0.995 is regarded as qualified. The results of accuracy and precision measurements are shown in Table 3, and the results of linear relationship measurements are shown in FIG. 1. As can be seen from the table, the deviation of the quality control low value and the quality control high value is 3.94 percent and 1.54 percent respectively, and the accuracy is qualified; CV of the low value sample and CV of the high value sample are respectively 2.49 percent and 2.00 percent, and the precision is qualified. As can be seen from FIG. 1, linear regression R ² =0.9993, the linear relationship is good.

Table 3: accuracy and precision determination result of GPO applied to triglyceride detection kit

Example 8

The glycerol-3-phosphate oxidase enzyme solution prepared in example 5 was added to a triglyceride test kit, and the accuracy, precision, linear relationship and other indicators of the kit after heat storage were analyzed to examine the stability of the kit. The kit R1 comprises: 20mM Tris-HCl buffer, 10mM TOOS, 0.1mM MgCl _2、 Lipoprotein lipase 20U/mL, stabilizer 0.2%, pH7.0; the kit R2 comprises: 20mM Tris-HCl buffer, 5mM 4-AAP, 5mM ATP, 30U/mL glycerol kinase, 10U/mL peroxidase, 20U/mL glycerol-3-phosphate oxidase, 0.5% stabilizer, pH7.2. Reagents R1, R2 were placed at 37 ℃ for accelerated storage for 7d, thermal storage was complete, and under normal calibration conditions, accuracy, precision and linearity were determined. The specific measurement method was the same as in example 7. As can be seen from the table, the deviation of the quality control low value and the quality control high value is 2.59 percent and-1.16 percent respectively, and the accuracy is qualified; the CV of the low value sample and the high value sample are respectively 2.44 percent and 2.08 percent, and the precision is qualified. As can be seen from FIG. 2, linear regression R ² =0.9992, the linear relationship is good.

Table 3: accuracy and precision determination results of GPO applied to triglyceride detection kit after accelerated treatment at 37 DEG C

The above are merely characteristic embodiments of the present invention, and do not limit the scope of the present invention in any way. All technical solutions formed by adopting the equivalent exchange or the equivalent substitution fall within the protection scope of the present invention.

Sequence listing

<110> Meikang Biotechnology Ltd

<120> preparation of glycerol-3-phosphate oxidase with good thermal stability and application of glycerol-3-phosphate oxidase in kit

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Glu Met Gln Asp Phe Ala Glu Gly Thr Ser Ser Arg Ser Thr Lys Leu

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Val His Gly Gly Ile Arg Tyr Leu Lys Asn Phe Asp Val Glu Val Val

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Ser Thr Phe Asn Met Phe Ser Val Lys Val Ala Met Asn Leu Tyr Asp

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Gln Leu Ala Gly Val Thr Asp Pro Lys Tyr Thr Asn Tyr Thr Ile Ser

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Ser His Val Lys Ala Gln His Ile Leu His Asp Glu Asn Gly Asn Val

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Pro Thr Tyr Phe Asp Ser Gly Gln Gln Asp Gly Arg Met Val Phe Ala

275 280 285

Ile Pro Arg Glu Gly Lys Thr Tyr Phe Gly Thr Thr Asp Thr Asp Tyr

290 295 300

Gln Gly Asp Phe Leu His Pro Arg Val Glu Gln Ala Asp Val Asp Tyr

305 310 315 320

Leu Leu Lys Thr Ile Asn Asp Arg Tyr Pro Glu Ala Glu Asn Thr Ile

325 330 335

Asp Asp Val Glu Ala Ser Trp Ala Gly Leu Arg Pro Leu Ile Ala Ser

340 345 350

Asn Gly Ser Ser Asp Tyr Asn Gly Gly Asn Ser Gly Lys Ile Thr Ala

355 360 365

Gln Ser Phe Asp Glu Val Ile Ala Ile Val Asp Lys Tyr Glu Lys Gln

370 375 380

Leu Val Ser Arg Glu Asp Val Glu Asp Val Leu Asn His Leu Glu Ser

385 390 395 400

Thr Leu Ser Glu Asn Lys Pro Asn Pro Ser Ala Val Ser Arg Gly Ser

405 410 415

Ser Leu Asp Val Ala Gln Asp Gly Leu Ile Thr Leu Ala Gly Gly Lys

420 425 430

Ile Thr Asp Tyr Arg Lys Met Ala Ala Gly Ala Ile Ala Leu Ile Ala

435 440 445

Lys Leu Leu Lys Gln Arg Phe Asn Arg Asn Phe Asn Ser Ile Asp Ser

450 455 460

Lys His Leu Gln Val Ser Gly Gly Asp Ile Asp Pro Gln Asn Val Asp

465 470 475 480

Ala Thr Leu Ser Phe Phe Ala Lys Gln Gly Ile Glu Ser Gly Leu Ser

485 490 495

Lys Pro Glu Ala Glu Lys Ile Ala Asn Leu Phe Gly Ser Asn Ala Ala

500 505 510

Arg Val Phe Ser Gln Ile Gly Arg Leu Pro Ala Ala Pro Gly Leu Ser

515 520 525

Leu Ala Glu Thr Ile Ser Leu His Tyr Ser Met Glu Glu Glu Met Thr

530 535 540

Leu Thr Pro Val Asp Tyr Leu Leu Arg Arg Thr Asn His Leu Leu Phe

545 550 555 560

His His Asp Ala Ile Ala Asp Leu Gln Gln Gly Val Val Asp Glu Met

565 570 575

Ala Arg Tyr Phe Asn Trp Ser Ala Glu Gln Lys Gln Gln His Ser Gln

580 585 590

Glu Leu Gln Asp Val Ile Ala Glu Ala His Leu Asp Tyr Leu Arg

595 600 605

Claims

1. Glycerol-3-phosphate oxidase with good thermal stability is characterized in that the amino acid sequence of the glycerol-3-phosphate oxidase is shown in SEQ ID No. 2.

2. A polynucleotide encoding glycerol-3-phosphate oxidase having good thermal stability according to claim 1, wherein the nucleotide sequence is represented by SEQ ID No. 1.

3. The use of glycerol-3-phosphate oxidase with good thermal stability as defined in claim 1 in the preparation of a triglyceride detection kit, wherein said triglyceride detection kit comprises two parts, R1 and R2:

wherein R1 is:

1-500 mM Tris-HCl buffer solution;

0.5～10mM TOOS；

0.001～0 .2mM MgCl2；

1-100U/mL lipoprotein lipase;

0.1-2% of stabilizer;

pH7.2～8.2；

r2 is:

1-500 mM Tris-HCl buffer solution;

0.2～5mM 4-AAP；

0.5～20mM ATP；

5-50U/mL glycerol kinase;

1-50U/mL peroxidase;

5-100U/mL glycerol-3-phosphate oxidase;

0.1-2% of stabilizer;

pH7.5～9.5。