Recombinant glycerol kinase as well as production method and application thereof

Abstract

The invention provides recombinant glycerol kinase as well as a production method and application thereof. 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 provided by the invention has the characteristics of high expression quantity, high enzyme activity and good thermal stability, the production method provided by the invention has the characteristic of good enzyme production stability, and the recombinant glycerol kinase provided by the invention has good application prospects in preparation of triglyceride detection reagents and / or kits.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant glycerol kinase and a production method and application thereof. Background Art

[0002] Triglycerides are widely present in nature and are usually stored in non-hydrated form in animals and plants. They are one of the main forms of energy acquisition and storage for animals and humans, and are also the main component of fat in food. Increased triglycerides in serum are common in familial lipid metabolism disorders, nephrotic syndrome, diabetes, hypothyroidism, etc. The determination of triglycerides is helpful for understanding blood lipid metabolism, liver function (the liver is one of the organs that synthesize triglycerides), and the diagnosis of diseases caused by atherosclerosis. Common methods for detecting triglycerides include enzymatic / dry chemical methods and electrochemical methods. Compared with other detection methods, the glycerol kinase method for detecting triglycerides is simple to operate, fast and accurate, and can be automatically batch operated on a biochemical analyzer. It is a routine method for determining triglycerides in clinical laboratories.

[0003] The basic principle of enzymatic detection of triglycerides is to use lipoprotein lipase (LPL) to hydrolyze triglycerides (TG) in serum into glycerol and fatty acids, and phosphorylate the generated glycerol with glycerol kinase (GK) and adenosine triphosphate (ATP). Use phosphoglycerol oxidase (GPO) to oxidize 3-phosphoglycerol (G-3-P), and then use peroxidase (POD), 4-aminoantipyrine (4-AAP) and 4-chlorophenol (the three are collectively called PAP) to react and develop color. Monitor the absorbance value at a specific wavelength and calculate the triglyceride content. Glycerol kinase (EC 2.7.1.30) is the key enzyme for enzymatic detection of TG, which is mainly derived from microorganisms such as Escherichia coli and Bacillus that 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 overall performance and cannot compete with similar foreign products. Summary of the invention

[0004] The object of the present invention is to provide a recombinant glycerol kinase and a production method and application thereof. The recombinant glycerol kinase of the present invention has the characteristics of high expression level, high enzyme activity and good thermal stability, and the production method of the present invention has the characteristics of good enzyme production stability. The recombinant glycerol kinase of the present invention has good application prospects in the preparation of triglyceride detection reagents and / or kits.

[0005] The above objects of the present invention are achieved through the following technical solutions:

[0006] In a first aspect, the present invention provides a recombinant glycerol kinase having 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 a wild glycerol kinase having an amino acid sequence as shown in SEQ ID NO.3, preferably, the wild glycerol kinase amino acid sequence (such as the sequence shown in SEQ ID NO.3) is used as a parent, and is designed 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 a 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] In a third aspect, 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 the expression vector and the nucleic acid molecule 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] In a fourth aspect, the present invention provides a recombinant host cell, wherein the recombinant host cell comprises 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 invention, the host cell comprises a prokaryotic cell.

[0016] In a fifth aspect, the present invention provides a recombinant strain, wherein the recombinant strain comprises the recombinant host cell described in the fourth aspect.

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

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

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

[0020] In a sixth aspect, the present invention provides a method for producing the recombinant glycerol kinase according to the first aspect, the production method comprising the following steps:

[0021] The recombinant host cell described in the fourth aspect or the recombinant strain described in the fifth aspect is cultured, and the recombinant glycerol kinase is isolated from the obtained culture product.

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

[0023] In some embodiments of the present invention, the separation comprises disrupting cells containing recombinant glycerol kinase to release the recombinant glycerol kinase; preferably, the disruption is performed in a high-pressure homogenizer.

[0024] In some embodiments of the present invention, the separation includes removing the precipitate after centrifugation, and the centrifugation is performed at 10,000 to 20,000 rpm for 30 to 50 minutes.

[0025] In some embodiments of the present invention, the production method further comprises purifying the crude enzyme solution containing recombinant glycerol kinase separated from the obtained 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 pH = 7.5 for elution.

[0026] In some embodiments of the present invention, the purification further comprises washing the sample loaded into the purification column with 20 mM PBbuffer at pH=7.5 before elution.

[0027] In some embodiments of the present invention, the purification further comprises collecting the protein fraction after elution and dialyzing to remove excess imidazole.

[0028] In some embodiments of the present invention, the purification is performed in a HisCap 6FF prepacked column produced by Changzhou Tiandirenhe Biotechnology Co., Ltd.

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

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

[0031] In some embodiments of the present invention, 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 invention, the kit comprises the following reagents:

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

[0034] The beneficial effects of the present invention are as follows:

[0035] The recombinant glycerol kinase of the present invention has the characteristics of high expression level, high enzyme activity and good thermal stability. It is used to prepare detection reagents or prepare kits in triglyceride enzymatic detection, which is beneficial to improve the stability of the detection reagents or kits. At the same time, it can also reduce the amount of glycerol kinase in the triglyceride detection reagents or kits, thereby reducing the detection cost or the kit production cost.

[0036] The recombinant glycerol kinase of the present invention can be efficiently expressed in recombinant strains such as Escherichia coli, the recombinant strain culture method is simple, the enzyme expression amount is high, and the enzyme production stability is good and the enzyme activity is high. The production method of the present invention is simple to operate, low in cost, and can be mass-produced to obtain high-purity glycerol kinase to meet the demand for glycerol kinase in the production of triglyceride detection reagents and kits. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0039] Figure 3These are the results of enzyme activity loss determination in thermal stability analysis. The spotting volumes of the GK samples in the figure are 4μL, 6μL, 8μL, and 12μL, respectively.

[0040] Figure 4 is a linear relationship graph for enzymatic determination of triglycerides using Roche glycerol kinase.

[0041] Figure 5 It is a linear relationship diagram of enzymatic determination of triglyceride using the recombinant glycerol kinase of the present invention. DETAILED DESCRIPTION

[0042] The following examples further illustrate the technology of the present invention. These examples are for illustration and example of the present invention and are not intended to limit the scope of the present invention in any form.

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

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

[0045] Solution A: 0.3M glycerol;

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

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

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

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

[0050] Solution F: 200 mM HEPES, pH = 7.9, containing 20 mM MgCl 2 and 40 mM ATP.

[0051] (2) Preparation of reaction mixture:

[0052] Solution A: 1.67 mL; Solution B: 40 mL; Solution C: 400 μL; Solution D: 3 mL; Solution E: 3 mL; Solution F: 10 mL; make up to 100 mL with deionized water.

[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 with pH = 7.5.

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

[0055] (4-1) The reaction mixture was heated 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 diluted enzyme solution into the cuvette and mix with the reaction mixture;

[0058] (4-4) Place the cuvette in a spectrophotometer and monitor the absorbance change (ΔAs) at a wavelength of 555 nm within 1 min.

[0059] (5) Using the enzyme diluent as a blank control, that is, following the detection procedure of step (4), replacing the diluted enzyme solution in step (4-3) with the enzyme diluent, measuring the blank absorbance change (ΔAb), and then calculating ΔA=ΔAs-ΔAb.

[0060] The calculation formula of glycerol kinase activity is:

[0061] GK (μmoL / min / mg

[0062] prot)=[(ΔA+0.0041)÷0.1326×V1]÷(V1×Cpr)÷T=1.51×(ΔA+0.0041)÷CprIn the formula, Cpr represents the protein concentration in the sample, mg / mL;

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

[0064] Example 1

[0065] 1. Construction of strain expressing recombinant glycerol kinase

[0066] 1. The wild-type sequence SEQ ID NO.3 of glycerol kinase derived from Cellulomonas sp. strain was obtained from databases such as NCBI, and recombinant glycerol kinase was designed by the Protein Design (ProteinMPNN) module using the Wemol biomedical digital intelligent computing platform. 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. The plasmid containing the nucleotide fragment shown in SEQ ID NO.2 was delivered to Nanjing Qingke Biotechnology Co., Ltd. for synthesis. The synthetic gene contained NdeI and XhoI restriction sites, and the amino terminus of the recombinant glycerol kinase was fused with a His tag; the recombinant plasmid pET-28a-GK was obtained.

[0068] 3. The recombinant plasmid pET-28a-GK was transformed into Escherichia coli BL21 (DE3) to obtain recombinant Escherichia 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 culture at 37°C for 14 hours to obtain a recombinant E. coli monoclone. Then pick the recombinant E. coli monoclone from the solid LB plate and culture it in 5 mL of liquid LB medium at 37°C overnight. The amino acid sequence SEQ ID NO.1 is:

[0070] AKYVLALDQGTTSSKAVIFDKNGNIVAEGEREHTLIHPAPGVLERNPREILEKT

[0071] REVIREALERGGLTAADIAAVGITNQRETAVVFDKNTGEPVTNAIVWQDTRTA

[0072] AIVKELGGPGGAMKYYDITGRPLSTYFSGPKIKLILDTVPGAKERAEKGELLFG

[0073] TTDTLVLWELTGGTDGGVHATDYTNASRTMFMDLETLKWNSEILKDFNIPEK

[0074] MMPEIKPNSIVFGTGRADGALPGVPIAGMAGDQQAATVGAAAFEEGEAFNDY

[0075] GTGNFLLLNTGTTIVRSKHGLLTTVLYKLGDEPPVYALEGSIAVGGGLPKWLR

[0076] ERLGMFEDVENIEELAAKVSSTGGLVVVAPYANLFEPYGKPPNPIGALVGITAA

[0077] VNRNHLARAALRSIAFETKAVVDAMNADSGIKAKRLIVGGGLSNNELLMQDL

[0078] ADTLGIPVVVPANPELAALGAAYLAGIAVGFYGGKEDVKKKFKIKREFLPSRD

[0079] PALVERELRNWRTAVAKTYEWNLEYEL

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

[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] 2. Construction of strains expressing wild-type glycerol kinase

[0101] 1) Submitting a plasmid containing a nucleotide fragment as shown in SEQ ID NO.4 to Nanjing Qingke Biotechnology Co., Ltd. for synthesis, wherein the synthetic gene contains NdeI and XhoI restriction sites, and the amino terminus of the wild glycerol kinase is fused with a His tag; obtaining a plasmid.

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

[0103] 3) Streak the E. coli containing the plasmid obtained in step 1) on a solid LB plate containing 50 μg / mL kanamycin, and culture at 37°C for 14 hours to obtain a single clone of E. coli. Then pick a single clone of E. coli from the solid LB plate and culture it in 5 mL of 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 production method of recombinant glycerol kinase specifically comprises the following steps:

[0140] a. The bacterial solution cultured overnight in step 4 of Example 1 was inoculated into 2L LB medium at a 1% inoculum, cultured at 37°C until OD600 was 0.6-0.8, IPTG inducer was added to a final concentration of 0.1mM, and cultured at 16°C for 18h. The cells were collected by centrifugation at 6000rpm, suspended in 100mL of Lysis buffer (20mM PB pH=7.5, 10mM Mimidazol, 300mM NaCl), broken by a high-pressure homogenizer, and then centrifuged at 14000rpm for 40min to remove the precipitate.

[0141] b. The supernatant obtained in step a was purified using a HisCap6FF pre-packed column produced by Changzhou Tiandi Renhe Biotechnology Co., Ltd.: 5 column volumes of 20 mM PB buffer (pH = 7.5) were used to equilibrate the purification column, and the sample was loaded at a flow rate of 2 mL / min. After loading, 20 mM PB buffer (pH = 7.5) was used for washing, and finally 20 mM PB buffer (pH = 7.5) containing 250 mM imidazole was used for elution. The protein components were collected and dialyzed to remove excess imidazole to obtain purified recombinant glycerol kinase.

[0142] Test Case

[0143] 1. Investigation of the expression level of glycerol kinase

[0144] The bacterial solution cultured overnight in step 4 of Example 1 and the bacterial solution cultured overnight in step 3 of Example 1 were inoculated into 5 mL of liquid LB medium at a 1% inoculation rate, and cultured at 37°C until the biomass OD600 reached 0.6-0.8, and then the inducer isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 0.1 mM, and the culture was continued at 16°C for 18 hours, 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 disrupted (power 450 W, ultrasonic 5 s, interval 10 s) for 30 minutes, and then centrifuged at 15000 rpm at 4°C for 15 minutes to collect the supernatant, and the enzyme expression level was measured by the supernatant, and the results are shown as follows. Figure 1 As shown. Figure 1 It can be seen that, compared with the strain expressing wild glycerol kinase, the recombinant E. coli of the present invention expresses a greater amount of glycerol kinase.

[0145] 2. Purity investigation of glycerol kinase

[0146] The purified recombinant glycerol kinase obtained in Example 2 was identified for purity using SDS-PAGE. The results are as follows: 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] 3. Investigation of the enzyme activity of glycerol kinase

[0148] The enzyme activity of the purified recombinant glycerol kinase obtained in Example 2 was determined by a coupled enzyme method. The result showed that the enzyme activity of the recombinant glycerol kinase was 104.9 U / mg.

[0149] 4. 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 days, the treated enzyme solution was centrifuged at 12000 rpm for 2 min, and the supernatant solution was taken 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 analyzed by grayscale using ImageJ. Figure 3 shown.

[0153] The concentration of the recombinant glycerol kinase solution before water bath treatment was 6.64 mg / mL, and the concentration of the centrifugal supernatant after water bath treatment was 6.36 mg / mL. Compared with the recombinant glycerol kinase solution before water bath treatment, the loss of recombinant glycerol kinase in the recombinant glycerol kinase solution after 7 days of 37°C water bath treatment was only 4.2%.

[0154] The enzyme activity of the recombinant glycerol kinase solution before water bath treatment was 104.9 U / mg, and the enzyme activity of the supernatant solution after water bath treatment was 99.33 U / mg. The enzyme activity loss rate of the recombinant glycerol kinase after 37°C water bath treatment for 7 days was 5.3%.

[0155] 5. Investigation of enzyme stability of glycerol kinase

[0156] Three batches of recombinant glycerol kinase were produced according to the production method of Example 2, the yields of the three batches of glycerol kinase were measured and the enzyme production rates (mg / L fermentation liquid) were calculated; the results showed that the enzyme production rates of the three batches of recombinant glycerol kinase were 42.3, 38.5, and 40.43 mg / L fermentation liquid, respectively.

[0157] Should be implemented

[0158] Prepare the reagents of the kit according to the following amounts:

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

[0160] The recombinant glycerol kinase produced in Example 2 was used to prepare the experimental group reagents, and the glycerol kinase produced by Roche was used as a control. The accuracy, precision and linear relationship indicators of the obtained kit were investigated.

[0161] Among them, the accuracy test is to use the kit to measure 16 samples containing triglycerides respectively, and the results of using two kits containing Roche glycerol kinase or the recombinant glycerol kinase of the present invention to measure 16 samples are shown in Table 1. As can be seen from Table 1, the relative deviations of the two kits containing Roche glycerol kinase or the recombinant glycerol kinase of the present invention are within the range of ±5%, and the correlation coefficient r≥0.990.

[0162] Table 1 Kit accuracy test 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 present invention 0.96 1.05 0.82 0.81 0.43 1.24 0.81 1.61 Relative deviation 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 present invention 1.17 3.93 0.6 0.87 2.87 0.57 1.48 2.96 Relative deviation 0.0086 0.0026 0.0169 0.0235 -0.0035 -0.0172 0.0137 0.0102

[0164] Among them, the precision study is to repeat the measurement 10 times for the same triglyceride-containing sample using the kit; and to repeat the measurement 10 times for two samples containing different concentrations of triglycerides using two kits containing Roche glycerol kinase or the recombinant glycerol kinase of the present invention, respectively, and the results are shown in Table 2. As can be seen from Table 2, the CV values ​​of the two kits containing Roche glycerol kinase or the recombinant glycerol kinase of the present invention are within 1%, which meets the requirements.

[0165] Table 2 Kit precision determination results

[0166]

[0167]

[0168] The linear relationship was investigated by diluting a sample with a triglyceride content of 11.0 mmol / L at multiples of 1X, 0.5X, 0.25X, 0.125X, 0.0625X, 0.0313X and 0.0156X to obtain 7 samples, and then using two kits containing Roche glycerol kinase and the recombinant glycerol kinase of the present invention to measure the 7 samples respectively and perform regression analysis on the measured values ​​and the theoretical values. The results are as follows: Figure 4 and Figure 5 As shown. 2 ≥0.995 is considered qualified. Figure 5 It can be seen that the linear relationship R 2 =0.9985, which meets the requirements.

[0169] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation of the present invention. The present invention has been described with reference to the embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A recombinant glycerol kinase, characterized in that The recombinant glycerol kinase has an amino acid sequence as shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleotide fragment encoding the recombinant glycerol kinase according to claim 1; preferably, the nucleotide fragment has a base sequence as shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule described in claim 2; preferably, the recombinant expression vector is recombined by an expression vector and the nucleic acid molecule described in claim 2; more preferably, the expression vector is selected from the pET series expression vectors, preferably pET-32a plasmid, pET-28a plasmid or pET-41a plasmid.

4. A recombinant host cell, characterized in that The recombinant host cell comprises the nucleic acid molecule of claim 2 or the recombinant expression vector of claim 3; preferably, the host cell comprises a prokaryotic cell.

5. A recombinant strain, characterized in that: The recombinant strain comprises the recombinant host cell of claim 4; preferably, the recombinant strain is prepared by transforming the recombinant expression vector of claim 3 into a host bacterial species; more preferably, the host bacterial species is selected from Escherichia coli or Bacillus, preferably Escherichia coli.

6. A method for producing recombinant glycerol kinase according to claim 1, characterized in that: The production method comprises the following steps: Cultivate the recombinant host cell according to claim 4 or the recombinant strain according to claim 5, and isolate the recombinant glycerol kinase from the obtained culture product.

7. The production method according to claim 6, characterized in that: The culture conditions include: the culture medium is selected from LB culture medium, preferably liquid LB culture medium; the culture temperature is 36-40°C; the inoculation amount is 0.5%-1.5%; And / or, the separation comprises disrupting cells containing recombinant glycerol kinase to release the recombinant glycerol kinase; preferably, the disrupting is performed in a high-pressure homogenizer; And / or, the separation includes removing the precipitate after centrifugation, and the centrifugation is performed at 10,000 to 20,000 rpm for 30 to 50 minutes.

8. The production method according to claim 6 or 7, characterized in that: The production method further comprises purifying the crude enzyme solution containing the recombinant glycerol kinase separated from the obtained culture product; preferably, the purification is performed by column purification; more preferably, the purification process is eluted using 20 mM PB buffer containing 250 mM imidazole and pH = 7.

5.

9. Use of the recombinant glycerol kinase according to claim 1 or the recombinant glycerol kinase produced by the production method according to any one of claims 6 to 8 in the preparation of a triglyceride detection reagent and / or kit.

10. A triglyceride detection kit, characterized in that: The glycerol kinase used in the kit is the recombinant glycerol kinase according to claim 1 and / or the recombinant glycerol kinase produced by the production method of any one of claims 6 to 8; preferably, the working concentration of the recombinant glycerol kinase in the kit is ≥0.4 KU / L, preferably 1.0 to 2.0 kU / L.

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