A recombinant glycerokinase, its production method and use

Abstract

The application provides a recombinant glycerol kinase, a production method and application thereof, and the recombinant glycerol kinase has an amino acid sequence as shown in SEQ ID NO. 1. The production method comprises the following steps: culturing a recombinant host cell or a recombinant strain capable of secreting the recombinant glycerol kinase, and separating the recombinant glycerol kinase from the obtained culture product. The recombinant glycerol kinase has the characteristics of high expression amount, high enzyme activity and good thermal stability, the production method has the characteristics of good enzyme production stability, and the recombinant glycerol kinase has a good application prospect in preparation of a triglyceride detection reagent and / or kit.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a recombinant glycerol kinase and a production method and application thereof. BACKGROUND

[0002] Triglyceride, widely existing in nature, is usually stored in the form of non-hydrated in animals and plants, is one of main forms of energy acquisition and storage of animals and human bodies, and is also a main component of fat in food. The increase of triglyceride in serum is common in familial lipid metabolism disorder, nephrotic syndrome, diabetes, hypothyroidism, etc., and the determination of triglyceride is helpful for understanding blood lipid metabolism, liver function (liver is one of organs for synthesizing triglyceride) and diagnosis of diseases caused by atherosclerosis. Common detection methods of triglyceride include enzyme method / dry chemical method and electrochemical method. Compared with other detection methods, the glycerol kinase method for detecting triglyceride has the characteristics of simple operation, rapidness and accuracy, and can be automatically operated in batches on a biochemical analyzer, and is a routine way for clinical laboratories to determine triglyceride.

[0003] The basic principle of the triglyceride enzyme method for detection is that lipoprotein esterase (LPL) is used to hydrolyze triglyceride (TG) in serum into glycerol and fatty acid, and glycerol is phosphorylated by glycerol kinase (GK) and adenosine triphosphate (ATP). The 3-phosphoglycerol (G-3-P) is oxidized by phosphoglycerol oxidase (GPO), and then 4-aminoantipyrine (4-AAP) and 4-chlorophenol (the three are collectively referred to as PAP) are reacted with peroxidase (POD) to develop color. The absorbance value at a specific wavelength is monitored, and the content of triglyceride can be calculated. Glycerol kinase (Glycerol Kinase, EC 2.7.1.30) is a key enzyme for detecting TG by enzyme method, and is mainly derived from microorganisms such as Escherichia coli and Bacillus which use glycerol as a carbon source. However, these natural bacteria have problems such as unstable enzyme production, poor activity and poor solubility, resulting in poor comprehensive performance. SUMMARY

[0004] The present application provides a recombinant glycerol kinase and a production method and application thereof. The recombinant glycerol kinase has the characteristics of high expression amount, high enzyme activity and good thermal stability. The production method has the characteristic of good enzyme production stability. The recombinant glycerol kinase has a good application prospect in the preparation of a triglyceride detection reagent and / or kit.

[0005] The above object of the present application is achieved by the following technical solutions.

[0006] In a first aspect, the present application provides a recombinant glycerol kinase, which has an amino acid sequence as shown in SEQ ID NO. 1.

[0007] In some embodiments of the present invention, the recombinant glycerol kinase is obtained by mutation of wild-type glycerol kinase with an amino acid sequence as shown in SEQ ID NO.3. Preferably, it is obtained by designing the wild-type glycerol kinase amino acid sequence (as shown in SEQ ID NO.3) using the Wemol biomedical digital intelligent computing platform through the Protein Design (ProteinMPNN) module.

[0008] In some embodiments of the present invention, the nucleotide fragment encoding the amino acid sequence shown in SEQ ID NO.3 has the base sequence shown in SEQ ID NO.4.

[0009] In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleotide fragment encoding the recombinant glycerol kinase described in the first aspect.

[0010] In some embodiments of the present invention, the nucleotide fragment has a base sequence as shown in SEQ ID NO.2.

[0011] Thirdly, the present invention provides a recombinant expression vector comprising the nucleic acid molecule described in the second aspect.

[0012] In some embodiments of the present invention, the recombinant expression vector is formed by recombination of an expression vector and the nucleic acid molecules described in the second aspect.

[0013] In some embodiments of the present invention, the expression vector is selected from the pET series expression vectors, preferably pET-32a plasmid, pET-28a plasmid or pET-41a plasmid.

[0014] Fourthly, the present invention provides a recombinant host cell comprising the nucleic acid molecule described in the second aspect or the recombinant expression vector described in the third aspect.

[0015] In some embodiments of the present invention, the host cell includes a prokaryotic cell.

[0016] Fifthly, the present invention provides a recombinant strain comprising the recombinant host cell described in the fourth aspect.

[0017] In some embodiments of the present invention, the recombinant strain is prepared by transforming a host bacterial strain into the recombinant expression vector described in the third aspect.

[0018] In some embodiments of the present invention, the host bacterial strain is selected from Escherichia coli or Bacillus, preferably Escherichia coli.

[0019] In some embodiments of the present application, the host strain is Escherichia coli BL21 (DE3) strain.

[0020] In a sixth aspect, the present application provides a method for producing the recombinant glycerol kinase of the first aspect, the method comprising the following steps:

[0021] culturing the recombinant host cell of the fourth aspect or the recombinant strain of the fifth aspect, and isolating the recombinant glycerol kinase from the resulting culture product.

[0022] In some embodiments of the present application, the culture conditions include that the culture medium is selected from LB medium, preferably liquid LB medium; the culture temperature is 36-40°C; and the inoculation amount is 0.5%-1.5%.

[0023] In some embodiments of the present application, the isolation includes subjecting the cells containing the recombinant glycerol kinase to a crushing treatment to release the recombinant glycerol kinase; preferably, the crushing treatment is performed in a high-pressure homogenizer.

[0024] In some embodiments of the present application, the isolation includes removing the precipitate after centrifugation, the centrifugation being performed at 10,000-20,000 rpm for 30-50 min.

[0025] In some embodiments of the present application, the production method further includes purifying the crude enzyme solution containing the recombinant glycerol kinase isolated from the resulting culture product; preferably, the purification is performed by column purification; more preferably, the purification process uses 20 mM PB buffer containing 250 mM imidazole and having a pH of 7.5 for elution.

[0026] In some embodiments of the present application, the purification further includes washing the sample loaded into the purification column with 20 mM PB buffer having a pH of 7.5 before elution.

[0027] In some embodiments of the present application, the purification further includes dialysis of the protein component collected after elution to remove excess imidazole.

[0028] In some embodiments of the present application, the purification is performed in a HisCap 6FF pre-packed column produced by Changzhou Tiandihuan Biotechnology Co., Ltd.

[0029] In a seventh aspect, the present application provides use of the recombinant glycerol kinase of the first aspect or produced by the production method of the sixth aspect in the preparation of a triglyceride detection reagent and / or kit.

[0030] In the eighth aspect, the present application provides a triglyceride detection kit, wherein the glycerol kinase used in the kit is the recombinant glycerol kinase of the first aspect and / or the recombinant glycerol kinase produced by the production method of the sixth aspect.

[0031] In some embodiments of the present application, the working concentration of the recombinant glycerol kinase in the kit is ≥0.4 KU / L, preferably 1.0-2.0 kU / L.

[0032] In some embodiments of the present application, the kit comprises the following reagent composition:

[0033] Good's buffer (pH = 7.2) with a working concentration of 50 mmol / L; 4-chloro-phenol with a working concentration of 4 mmol / L; Mg 2+ with a working concentration of 15 mmol / L; ATP with a working concentration of 2 mmol / L; recombinant triglyceride kinase with a working concentration of ≥0.4 KU / L; peroxidase with a working concentration of ≥2 KU / L; lipoprotein lipase with a working concentration of ≥2 KU / L; 4-amino-antipyrine with a working concentration of 0.5 mmol / L; glycerol-3-phosphate oxidase with a working concentration of ≥0.5 KU / L.

[0034] The present application has the following advantages:

[0035] The recombinant glycerol kinase of the present application has the characteristics of high expression amount, high enzyme activity and good thermal stability, and when used in the preparation of detection reagents or kits for triglyceride enzyme detection, it is beneficial to improve the stability of the detection reagents or kits, and at the same time, it can also reduce the amount of glycerol kinase used in the triglyceride detection reagents or kits, thereby reducing the detection cost or the production cost of the kits.

[0036] The recombinant glycerol kinase of the present application can be efficiently expressed in recombinant strains such as Escherichia coli, and the recombinant strain has the advantages of simple cultivation method, high enzyme expression amount, good enzyme stability and high enzyme activity. The production method of the present application is simple to operate and low in cost, and can produce high-purity glycerol kinase on a large scale to meet the demand for glycerol kinase in the production of triglyceride detection reagents and kits. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is the detection result of the expression amount of the recombinant glycerol kinase and the wild glycerol kinase.

[0038] Figure 2 is the purity identification result of the recombinant glycerol kinase, and the sample amount of GK in the figure is 4 μL, 6 μL, 8 μL and 12 μL from left to right.

[0039] Figure 3is the result of the determination of the loss of enzyme activity in the thermal stability analysis, and the sample amount of the GK sample in the figure is 4 μL, 6 μL, 8 μL and 12 μL, respectively.

[0040] Figure 4 is a linear relationship diagram of the determination of triglyceride by enzyme method using Roche glycerol kinase.

[0041] Figure 5 is a linear relationship diagram of the determination of triglyceride by enzyme method using the recombinant glycerol kinase of the application. DETAILED DESCRIPTION

[0042] The technology of the application is further illustrated by the following examples. These examples are an illustration and an example of the application, and do not limit the scope of the application in any form.

[0043] In the following examples, the enzyme activity of glycerol kinase is determined by using the coupled enzyme method, and the specific steps are as follows:

[0044] (1) Preparation of raw materials for reaction solution:

[0045] A solution: 0.3 M glycerol;

[0046] B solution: 20 U / mL glycerol-3-phosphate oxidase;

[0047] C solution: 1 KU / mL peroxidase;

[0048] D solution: 50 mM TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methyl aniline sodium salt);

[0049] E solution: 50 mM 4-AAP (4-aminoantipyrine);

[0050] F solution: 200 mM HEPES, pH = 7.9, containing 20 mM MgCl2 and 40 mM ATP.

[0051] (2) Preparation of reaction mixture:

[0052] A solution: 1.67 mL; B solution: 40 mL; C solution: 400 μL; D solution: 3 mL; E solution: 3 mL; F solution: 10 mL; and deionized water to 100 mL.

[0053] (3) Sample preparation: dilute the sample to 0.15-0.35 U / mL with enzyme diluent; the enzyme diluent is 20 mM potassium phosphate buffer, pH = 7.5.

[0054] (4) Glycerol kinase enzyme activity detection steps:

[0055] (4-1) Heat the reaction mixture in a 37°C metal bath for 2 min;

[0056] (4-2) Add 1 mL of the heated reaction mixture into a 1 mL cuvette;

[0057] (4-3) Add 0.02 mL of the diluted enzyme solution into the cuvette and mix well;

[0058] (4-4) Place the cuvette into a spectrophotometer and monitor the absorbance change (△As) at 555 nm within 1 min.

[0059] (5) Take the enzyme diluent as a blank control, i.e., follow the detection steps of step (4), and replace the diluted enzyme solution in step (4-3) with the enzyme diluent to measure the blank absorbance change (△Ab), and then calculate △A = △As-△Ab.

[0060] The formula for calculating the enzyme activity of glycerol kinase is:

[0061] GK (pmol / min / mg prot) = [(△A + 0.0041) ÷ 0.1326 × V1] ÷ (V1 × Cpr) ÷ T = 1.51 × (△A + 0.0041) ÷ Cpr

[0062] In the formula, Cpr represents the protein concentration in the sample, mg / mL.

[0063] The enzyme activity of glycerol kinase is quantified according to the absorbance change per minute, and the unit enzyme activity is defined as the amount of enzyme required to consume 1 pmol of glycerol per minute.

[0064] Example 1

[0065] I. Construction of a strain expressing recombinant glycerol kinase

[0066] 1. Obtain the wild-type glycerol kinase sequence SEQ ID NO. 3 derived from Cellulomonas sp. strain from databases such as NCBI, design a recombinant glycerol kinase through the Protein Design (Protein MPNN) module of the Wemol Biomedicine Digital Intelligent Computing Platform, and the amino acid sequence of the recombinant glycerol kinase is shown in SEQ ID NO. 1; the nucleotide fragment sequence encoding the recombinant glycerol kinase is shown in SEQ ID NO. 2.

[0067] 2. Synthesize the plasmid containing the nucleotide fragment with the sequence shown in SEQ ID NO. 2 by Nanjing Qikexin Biotechnology Co., Ltd., and the synthesized gene contains NdeI and XhoI enzyme digestion sites, and the amino terminal of the recombinant glycerol kinase is fused with a His tag; obtain the recombinant plasmid pET-28a-GK.

[0068] 3. Transform the recombinant plasmid pET-28a-GK into E. coli BL21(DE3) to obtain recombinant E. coli expressing recombinant glycerol kinase.

[0069] 4. Streak the recombinant E. coli containing the recombinant plasmid pET-28a-GK on a solid LB plate containing 50 μg / mL kanamycin, and incubate at 37°C for 14 h to obtain a recombinant E. coli monoclonal. Then pick the recombinant E. coli monoclonal from the solid LB plate into 5 mL liquid LB medium and incubate at 37°C overnight. The amino acid sequence of SEQ ID NO. 1 is:

[0070] AKYVLALDQGTTSSKAVIFDKNGNIVAEGEREHTLIHPAPGVLERNPREILEKT

[0071] REVIREALERGGLTAADIAAVGITNQRETAVVFDKNTGEPVTNAIVWQDTRTA

[0072] AIVKELGGPGGAMKYYDITGRPLSTYFSGPKIKLILDTVPGAKERAEKGELLFG

[0073] TTDTLVLWELTGGTDGGVHATDYTNASRTMFMDLETLKWNSEILKDFNIPEK

[0074] MMPEIKPNSIVFGTGRADGALPGVPIAGMAGDQQAATVGAAAFEEGEAFNDY

[0075] GTGNFLLLNTGTTIVRSKHGLLTTVLYKLGDEPPVYALEGSIAVGGGLPKWLR

[0076] ERLGMFEDVENIEELAAKVSSTGGLVVVAPYANLFEPYGKPNPIGALVGITAA

[0077] VNRNHLARAALRSIAFETKAVVDAMNADSGIKAKRLIVGGGLSNNELLMQDL

[0078] ADTLGIPVVVPANPELAALGAAYLAGIAVGFYGGKEDVKKKFKIKREFLPSRD

[0079] PALVERELRNWRTAVAKTYEWNLEYEL

[0080] The nucleotide fragment sequence SEQ ID NO. 2 is:

[0081] gcgaaatatgtgctggcgctggatcagggcaccaccagcagcaaagcggtgatttttgataaaaacggcaacattgtggcg

[0082] gaaggcgaacgcgaacataccctgattcatccggcgccgggcgtgctggaacgcaacccgcgcgaaattctggaaaaaa

[0083] cccgcgaagtgattcgcgaagcgctggaacgcggcggcctgaccgcggcggatattgcggcggtgggcattaccaacca

[0084] gcgcgaaaccgcggtggtgtttgataaaaacaccggcgaaccggtgaccaacgcgattgtgtggcaggatacccgcacc

[0085] gcggcgattgtgaaagaactgggcggcccgggcggcgcgatgaaatattatgatattaccggccgcccgctgagcaccta

[0086] ttttagcggcccgaaaattaaactgattctggataccgtgccgggcgcgaaagaacgcgcggaaaaaggcgaactgctgttt

[0087] ggcaccaccgataccctggtgctgtgggaactgaccggcggcaccgatggcggcgtgcatgcgaccgattataccaacg

[0088] cgagccgcaccatgtttatggatctggaaaccctgaaatggaacagcgaaattctgaaagattttaacattccggaaaaaatg

[0089] atgccggaaattaaaccgaacagcattgtgtttggcaccggccgcgcggatggcgcgctgccgggcgtgccgattgcggg

[0090] catggcgggcgatcagcaggcggcgaccgtgggcgcggcggcgtttgaagaaggcgaagcgtttaacgattatggcacc

[0091] ggcaactttctgctgctgaacaccggcaccaccattgtgcgcagcaaacatggcctgctgaccaccgtgctgtataaactgg

[0092] gcgatgaaccgccggtgtatgcgctggaaggcagcattgcggtgggcggcggcctgccgaaatggctgcgcgaacgcct

[0093] gggcatgtttgaagatgtggaaaacattgaagaactggcggcgaaagtgagcagcaccggcggcctggtggtggtggcg

[0094] ccgtatgcgaacctgtttgaaccgtatggcaaaccgaacccgattggcgcgctggtgggcattaccgcggcggtgaaccgc

[0095] aaccatctggcgcgcgcggcgctgcgcagcattgcgtttgaaaccaaagcggtggtggatgcgatgaacgcggatagcg

[0096] gcattaaagcgaaacgcctgattgtgggcggcggcctgagcaacaacgaactgctgatgcaggatctggcggataccctg

[0097] ggcattccggtggtggtgccggcgaacccggaactggcggcgctgggcgcggcgtatctggcgggcattgcggtgggct

[0098] tttatggcggcaaagaagatgtgaaaaaaaaatttaaaattaaacgcgaatttctgccgagccgcgatccggcgctggtgga

[0099] acgcgaactgcgcaactggcgcaccgcggtggcgaaaacctatgaatggaacctggaatatgaactg

[0100] II. Construction of strain expressing wild-type glycerol kinase

[0101] 1) The plasmid containing the nucleotide fragment with the sequence as shown in SEQ ID NO. 4 was synthesized by Nanjing Qikexi Biotechnology Co., Ltd. The synthetic gene contained Nde I and Xho I enzyme cutting sites, and the amino terminal of the wild-type glycerol kinase was fused with His tag; and the plasmid was obtained.

[0102] 2) The plasmid obtained in step 1) was transformed into E. coli BL21 (DE3) to obtain E. coli expressing wild-type glycerol kinase (the amino acid sequence is shown in SEQ ID NO. 3).

[0103] 3) The E. coli containing the plasmid obtained in step 1) was streaked on a solid LB plate containing 50 μg / mL kanamycin, and E. coli monoclonal was obtained after 37°C culture for 14 h. Then, the E. coli monoclonal was picked from the solid LB plate and cultured in 5 mL liquid LB medium at 37°C overnight.

[0104] The amino acid sequence SEQ ID NO. 3 is:

[0105] ADYVLAIDQGTTSSRAIVFDHSGEIYSTGQLEHDQIFPRAGWVEHNPEQIWNN

[0106] VREVVGLALTRGNLTHEDIAAVGITNQRETAVVWDKTTGKPVYNAIVWQDT

[0107] RTQKIVDELGGDEGAEKYKSIVGLPLATYFSGPKIKWILDNVEGAREKAEKGD

[0108] LLFGNTDTWVLWNMTGGTEGGVHVTDVTNASRTMLMDLDTLSWREDIAAD

[0109] MGIPLSMLPDIRSSSEVYGHGRPRGLVPGVPIAGILGDQQAATFGQACFEVGQA

[0110] KNTYGTGNFLLLNTGTEKVMSKNGLLTTVCYKIGDAPAVYALEGSIAVTGSL

[0111] VQWLRDNLGMFEDAPDVEWLAGKVQDNGGAYFVPAFSGLFAPYWRPDARG

[0112] ALVGLTRYVNRNHIARAALEATAFQSREVVDAMNADSGVDLTELRVDGGMV

[0113] ANELLMQFQADQLGVDVVRPKVAETTALGAAYAAGIAVGFWKGEQDVIDN

[0114] WAEDKRWSPSMESGERERLYRNWKKAVTKTMEWVDEDVE

[0115] The nucleotide fragment sequence SEQ ID NO. 4 is:

[0116] gcggattatgtgctggcgattgatcagggcaccaccagcagccgcgcgattgtgtttgatcatagcggc

[0117] gaaatttatagcaccggccagctggaacatgatcagatttttccgcgcgcgggctgggtggaacataac

[0118] ccggaacagatttggaacaacgtgcgcgaagtggtgggcctggcgctgacccgcggcaacctgacccat

[0119] gaagatattgcggcggtgggcattaccaaccagcgcgaaaccgcggtggtgtgggataaaaccaccggc

[0120] aaaccggtgtataacgcgattgtgtggcaggatacccgcacccagaaaattgtggatgaactgggcggc

[0121] gatgaaggcgcggaaaaatataaaagcattgtgggcctgccgctggcgacctattttagcggcccgaaa

[0122] attaaatggattctggataacgtggaaggcgcgcgcgaaaaagcggaaaaaggcgatctgctgtttggc

[0123] aacaccgatacctgggtgctgtggaacatgaccggcggcaccgaaggcggcgtgcatgtgaccgatgtg

[0124] accaacgcgagccgcaccatgctgatggatctggataccctgagctggcgcgaagatattgcggcggat

[0125] atgggcattccgctgagcatgctgccggatattcgcagcagcagcgaagtgtatggccatggccgcccg

[0126] cgcggcctggtgccgggcgtgccgattgcgggcattctgggcgatcagcaggcggcgacctttggccag

[0127] gcgtgctttgaagtgggccaggcgaaaaacacctatggcaccggcaactttctgctgctgaacaccggc

[0128] accgaaaaagtgatgagcaaaaacggcctgctgaccaccgtgtgctataaaattggcgatgcgccggcg

[0129] gtgtatgcgctggaaggcagcattgcggtgaccggcagcctggtgcagtggctgcgcgataacctgggc

[0130] atgtttgaagatgcgccggatgtggaatggctggcgggcaaagtgcaggataacggcggcgcgtatttt

[0131] gtgccggcgtttagcggcctgtttgcgccgtattggcgcccggatgcgcgcggcgcgctggtgggcctg

[0132] acccgctatgtgaaccgcaaccatattgcgcgcgcggcgctggaagcgaccgcgtttcagagccgcgaa

[0133] gtggtggatgcgatgaacgcggatagcggcgtggatctgaccgaactgcgcgtggatggcggcatggtg

[0134] gcgaacgaactgctgatgcagtttcaggcggatcagctgggcgtggatgtggtgcgcccgaaagtggcg

[0135] gaaaccaccgcgctgggcgcggcgtatgcggcgggcattgcggtgggcttttggaaaggcgaacaggat

[0136] gtgattgataactgggcggaagataaacgctggagcccgagcatggaaagcggcgaacgcgaacgcctg

[0137] tatcgcaactggaaaaaagcggtgaccaaaaccatggaatgggtggatgaagatgtggaa

[0138] Example 2

[0139] The method for producing recombinant glycerol kinase specifically comprises the following steps:

[0140] a. The bacteria solution of step 4 of Example 1 is inoculated into 2L LB culture medium at an inoculation amount of 1% and cultured at 37°C until the OD600 is 0.6-0.8, then IPTG inducer is added to a final concentration of 0.1mM, and the culture is continued at 16°C for 18h. The bacteria are collected by centrifugation at 6000rpm, suspended with 100mL of Lysis buffer (20mM PB pH=7.5, 10mM Imidazol, 300mM NaCl), broken by using a high-pressure homogenizer, then centrifuged at 14000rpm for 40min, and the precipitate is removed.

[0141] b. The supernatant obtained in step a is purified using a HisCap6FF pre-packed column produced by Changzhou Tiandirenhe Biotechnology Co., Ltd.: the purification column is equilibrated with 5 times the column volume of 20mM PB buffer (pH=7.5), loaded at a flow rate of 2mL / min, washed with 20mM PB buffer (pH=7.5) after loading is completed, and finally eluted with 20mM PB buffer (pH=7.5) containing 250mM imidazole, and the protein component is collected and dialyzed to remove excess imidazole, to obtain purified recombinant glycerol kinase.

[0142] Test Example

[0143] I. Investigation of the expression amount of glycerol kinase

[0144] The overnight culture of the bacteria solution of Example 1 Step 4 and the overnight culture of the bacteria solution of Example 1 Step 3) were inoculated into 5 mL of liquid LB medium at an inoculation amount of 1%, and cultured at 37°C until the biomass OD600 reached 0.6-0.8, and then 0.1 mM of inducer isopropyl-β-D-thiogalactoside (IPTG) was added to continue the culture at 16°C for 18 h, and the bacteria were collected by centrifugation at 4°C. The collected bacteria were resuspended in Lysis buffer (20 mM PB pH 7.5, 10 mM Imidazol, 300 mM NaCl), and ultrasonically broken (power 450 W, ultrasonic 5 s, interval 10 s) for 30 min, and then the supernatant was collected by centrifugation at 15000 rpm at 4°C for 15 min, and the enzyme expression amount was determined by the supernatant, and the results are shown in Figure 1 It can be seen that, compared with the strain expressing wild-type glycerol kinase, the recombinant E. coli of the present application expresses more glycerol kinase. Figure 1

[0145] II. Investigation of the purity of glycerol kinase

[0146] The purified recombinant glycerol kinase obtained in Example 2 was subjected to purity identification by SDS-PAGE, and the results are shown in Figure 2 The molecular weight of the recombinant glycerol kinase is about 60 kDa, and the purity is greater than or equal to 90%.

[0147] III. Investigation of the enzyme activity of glycerol kinase

[0148] The purified recombinant glycerol kinase obtained in Example 2 was subjected to enzyme activity determination by coupling enzyme method, and the results showed that the enzyme activity of the recombinant glycerol kinase was 104.9 U / mg.

[0149] IV. Investigation of the thermal stability of glycerol kinase

[0150] The purified recombinant glycerol kinase obtained in Example 2 was subjected to SDS-PAGE gel identification and enzyme activity detection;

[0151] 1 mL of the purified recombinant glycerol kinase obtained in Example 2 was placed in a 37°C water bath for 7 d, and the treated enzyme solution was centrifuged at 12000 rpm for 2 min to take the supernatant solution for SDS-PAGE gel identification and enzyme activity detection;

[0152] The SDS-PAGE gel identification results obtained after water bath treatment and the SDS-PAGE gel identification results obtained before water bath treatment were subjected to gray scale analysis using ImageJ. The results are shown in​Figure 3 The concentration of the recombinant glycerokinase solution before water bath treatment was 6.64 mg / mL, and the concentration of the supernatant after water bath treatment was 6.36 mg / mL. The loss of the recombinant glycerokinase in the recombinant glycerokinase solution after 7 days of water bath treatment at 37°C was only 4.2% compared to the recombinant glycerokinase solution before water bath treatment.

[0153] The enzyme activity of the recombinant glycerokinase solution before water bath treatment was 104.9 U / mg, and the enzyme activity of the supernatant after water bath treatment was 99.33 U / mg. The loss of the enzyme activity of the recombinant glycerokinase after 7 days of water bath treatment at 37°C was 5.3%.

[0154] Five, investigation of the enzyme production stability of glycerokinase

[0155] Five, investigation of the enzyme production stability of glycerokinase

[0156] Three batches of recombinant glycerokinase were produced according to the production method of Example 2, and the yields of the three batches of glycerokinase were determined and the enzyme production rate (mg / L of fermentation broth) was calculated. The enzyme production rates of the three batches of recombinant glycerokinase were 42.3, 38.5, and 40.43 mg / L of fermentation broth, respectively.

[0157] Example

[0158] The reagents of the kit were prepared according to the following reagent amounts:

[0159] Good's buffer (pH = 7.2) with a concentration of 50 mmol / L; 4-chloro-phenol with a concentration of 4 mmol / L; Mg 2+ with a concentration of 15 mmol / L; ATP with a concentration of 2 mmol / L; triglyceride kinase with a concentration of ≥0.4 KU / L; peroxidase with a concentration of ≥2 KU / L; lipoprotein lipase with a concentration of ≥2 KU / L; 4-amino-antipyrine with a concentration of 0.5 mmol / L; glycerol-3-phosphate oxidase with a concentration of ≥0.5 KU / L.

[0160] The recombinant glycerokinase produced in Example 2 was used to prepare the reagents of the experimental group, and the glycerokinase of Roche was used as a control. The accuracy, precision, and linearity of the obtained kits were investigated.

[0161] The accuracy investigation was performed by using the kit to determine 16 samples containing triglycerides. The results of using two kinds of kits containing Roche glycerokinase or the recombinant glycerokinase of the present application to determine 16 samples are shown in Table 1. As can be seen from Table 1, the relative deviations of the two kinds of kits containing Roche glycerokinase or the recombinant glycerokinase of the present application are within ±5%, and the correlation coefficient r is ≥0.990.

[0162] Table 1 Kit accuracy determination results

[0163] Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Roche 0.96 1.05 0.82 0.8 0.43 1.25 0.82 1.62 The invention 0.96 1.05 0.82 0.81 0.43 1.24 0.81 1.61 Relative bias 0 0 0 0.0125 0 -0.008 -0.0122 -0.0062 Sample 9 Sample 10 Sample 11 Sample 12 Sample 13 Sample 14 Sample 15 Sample 16 Roche 1.16 3.92 0.59 0.85 2.88 0.58 1.46 2.93 The invention 1.17 3.93 0.6 0.87 2.87 0.57 1.48 2.96 Relative bias 0.0086 0.0026 0.0169 0.0235 -0.0035 -0.0172 0.0137 0.0102

[0164] The precision test is repeated 10 times for the same sample containing triglyceride using the kit. Two samples containing different concentrations of triglyceride are each repeatedly measured 10 times using two kits containing Roche glycerol kinase or the recombinant glycerol kinase of the present application, and the results are shown in Table 2. As shown in Table 2, the CV values of the two kits containing Roche glycerol kinase or the recombinant glycerol kinase of the present application are all within 1%, which meets the requirements.

[0165] Table 2 Kit precision test results

[0166]

[0167]

[0168] The linear relationship test is performed by diluting a sample containing 11.0 mmol / L of triglyceride by 1X, 0.5X, 0.25X, 0.125X, 0.0625X, 0.0313X and 0.0156X to obtain 7 samples, and then the 7 samples are measured using two kits containing Roche glycerol kinase and the recombinant glycerol kinase of the present application, respectively, and the measured values are regressed with the theoretical values, and the results are shown in Table 3 and Table 4. Figure 4 and Figure 5 R 2 ≥ 0.995 is considered to be qualified, and Figure 5 As shown in Table 3 and Table 4, the linear relationship R 2 = 0.9985 of the kit of the present application meets the requirements.

[0169] It should be noted that the above-described examples are only used to explain the present application and do not constitute any limitation on the present application. The present application is described by referring to the examples, but it should be understood that the words used therein are descriptive and explanatory words, rather than limiting words. The present application can be modified within the scope of the claims, and the present application can be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and examples, it does not mean that the present application is limited to the specific examples disclosed therein, on the contrary, the present application can be extended to all other methods and applications having the same function.

Claims

1. A recombinant glycerol kinase, characterized in that, The amino acid sequence of the recombinant glycerokinase is shown as SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant glycerokinase of claim 1.

3. The nucleic acid molecule of claim 2, wherein, The base sequence of the nucleic acid molecule is shown as SEQ ID NO.

2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 2 or 3.

5. The recombinant expression vector of claim 4, wherein, The recombinant expression vector is recombined from an expression vector and the nucleic acid molecule of claim 2 or 3.

6. The recombinant expression vector of claim 5, wherein, The expression vector is selected from the pET series expression vectors.

7. The recombinant expression vector of claim 6, wherein, The expression vector is pET 32a plasmid, pET 28a plasmid or pET 41a plasmid.

8. A recombinant bacterial strain, characterized in that, The recombinant strain comprises the nucleic acid molecule of claim 2 or 3 or the recombinant expression vector of any one of claims 4 to 7.

9. The recombinant bacterial strain of claim 8, wherein, The recombinant strain is prepared by transforming the recombinant expression vector of any one of claims 4 to 7 into a host strain.

10. The recombinant bacterial strain of claim 9, wherein, The host strain is Escherichia coli.

11. A method for producing the recombinant glycerol kinase according to claim 1, characterized by, The production method comprises the following steps: culturing the recombinant strain of any one of claims 8 to 10 and isolating the recombinant glycerokinase from the resulting culture product.

12. The production method according to claim 11, characterized by, The culture conditions include that the culture medium is selected from LB medium, the culture temperature is 36 to 40℃, and the inoculation amount is 0.5% to 1.5%; and / or, the isolation comprises subjecting the cells containing the recombinant glycerokinase to a crushing treatment to release the recombinant glycerokinase.

13. The production method according to claim 12, characterized by, The isolation comprises, after the crushing treatment of the cells containing the recombinant glycerokinase, removing the precipitate after centrifugation, and the centrifugation is performed at 10,000 to 20,000 rpm for 30 to 50 min.

14. The production method according to claim 12, characterized by, The culture medium is liquid LB medium. and / or, the crushing treatment is performed in a high-pressure homogenizer.

15. The production method according to any one of claims 11 to 14, characterized by, The production method further comprises purifying the crude enzyme solution containing the recombinant glycerokinase isolated from the resulting culture product.

16. The production method according to claim 15, characterized by, The purification is performed by column purification.

17. The production method according to claim 16, characterized by, The purification process uses 20mM PB buffer containing 250mM imidazole and pH=7.5 for elution.

18. Use of the recombinant glycerokinase of claim 1 or produced by the production method of any one of claims 11 to 17 in the preparation of a triglyceride detection reagent and / or kit.

19. A triglyceride detection kit, characterized by, The glycerokinase used in the kit is the recombinant glycerokinase of claim 1 and / or produced by the production method of any one of claims 11 to 17.

20. The triglyceride detection kit according to claim 19, characterized in that, The working concentration of the recombinant glycerokinase in the kit is ≥0.4 KU / L.

21. The triglyceride detection kit according to claim 19, characterized in that, The working concentration of the recombinant glycerokinase in the kit is 1.0 to 2.0 kU / L.

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