Glutathione S-transferase derived from Antarctic krill as well as coding gene and application of glutathione S-transferase

Abstract

The invention relates to an euphausia superba-derived glutathione S-transferase and a coding gene and application thereof, and belongs to the field of enzymology, the amino acid sequence of the glutathione S-transferase is shown as SEQ ID NO.1, the amino acid sequence of the gene for coding the glutathione S-transferase is shown as SEQ ID NO.2, and the coding gene of the glutathione S-transferase has a nucleotide sequence shown as SEQ ID NO.1. The specific activity of the glutathione S-transferase disclosed by the invention can be as high as 6.01 [mu] moL / min / mg. The optimal temperature and the optimal pH are respectively 20 DEG C and 8. The glutathione S-transferase derived from the euphausia superba has good catalytic ability and substrate affinity ability at a lower temperature.

Description

Technical Field

[0001] The present invention belongs to the field of enzymology, and particularly relates to a glutathione S-transferase derived from Antarctic krill, its encoding gene, and applications thereof. Background Art

[0002] Antarctic krill is a key species in the Antarctic marine ecosystem, with important biological characteristics and ecological and economic values. Antarctic krill can live in extreme environments and have qualities such as freeze resistance and antioxidant properties, and have various active substances, such as lipids, proteins, and enzymes. However, due to its tendency of autoproteolysis, it is challenging to isolate and extract enzymes from Antarctic krill. Currently, the development and utilization of enzymes derived from Antarctic krill are still in the primary stage, and we need to conduct more in-depth exploration of enzymes derived from Antarctic krill.

[0003] Glutathione S-transferase (GST) (EC 2.5.1.18) belongs to a key detoxifying enzyme widely distributed in prokaryotes and eukaryotes. It belongs to the phase II cellular detoxification system enzymes and plays a crucial role in the detoxification of various organisms. They catalyze the conjugation of reduced glutathione (GSH) with electrophilic exogenous substances, making them more hydrophilic and easier to excrete. GST is involved in the elimination of potentially harmful chemical components, such as exogenous organisms, drugs, environmental pollutants, herbicides, insecticides, and chemical carcinogens. 1-Chloro-2,4-dinitrobenzene (CDNB) is a commonly used substrate for glutathione S-transferase, which reacts with GSH to form a complex (GS-SNB). In addition, they protect cells by inactivating quinones, epoxides, α,β-unsaturated ketones, and oxides generated during oxidative stress.

[0004] Currently, there are few reports on the heterologous expression of glutathione S-transferase. Therefore, exploring glutathione S-transferase genes with high activity, constructing genetically engineered bacteria through genetic recombination methods to highly heterologously express glutathione S-transferase, and exploring the enzymatic hydrolysis process of glutathione S-transferase have important industrial application values and potentials. Summary of the Invention

[0005] The objective of the present invention is to provide a glutathione S-transferase derived from Antarctic krill, its encoding gene, and applications thereof. The present invention mined the Antarctic krill GST gene ( Es GST) based on genomic analysis and carried out heterologous expression in Escherichia coli BL21(DE3) chaprone.

[0006] The present invention is achieved through the following technical solutions: A glutathione S-transferase derived from Antarctic krill, wherein the amino acid sequence of the glutathione S-transferase is as shown in SEQ ID NO.1.

[0007] The present invention also provides a gene encoding the glutathione S-transferase, and the nucleotide sequence of the gene is as shown in SEQ ID NO.2.

[0008] A recombinant plasmid PET28a-EsGSST, which contains the nucleotide sequence shown in SEQ ID NO.2.

[0009] A recombinant Escherichia coli engineering bacterium, which contains the recombinant plasmid PET28a-EsGST.

[0010] An enzyme preparation, which contains the glutathione S-transferase derived from Antarctic krill.

[0011] The present invention also provides an application of the glutathione S-transferase, and the application is to use the glutathione S-transferase to catalyze a substrate.

[0012] Furthermore, the application is to catalyze the substrate at a temperature of 20 °C.

[0013] Beneficial effects of the present invention compared with the prior art: The present invention provides a glutathione S-transferase from Antarctic krill. The gene sequence of glutathione S-transferase was obtained by using the bioinformatics method of Antarctic krill protein transcriptome. The target gene was amplified in vitro by polymerase chain reaction (PCR) and was expressed with solubility and activity in Escherichia coli.

[0014] In the soluble expression of the recombinant Escherichia coli obtained in the present invention, after detecting the supernatant of the bacterial cells after centrifugation, the specific activity of the glutathione S-transferase can be as high as 6.01 μmoL / min / mg. Through the basic enzymological property research on it, the optimal temperature and the optimal pH are 20 °C and 8 respectively, and the protein molecular weight is 24 kDa. The optimal temperature of the glutathione S-transferase in the prior art is about 25 °C - 40 °C. The glutathione S-transferase derived from Antarctic krill provided by the present invention has good catalytic ability and substrate affinity at a lower temperature. Description of the Drawings

[0015] Figure 1 It is the map of the recombinant plasmid PET28a-EsGST; Figure 2 It is the recombinant expressed protein purified by nickel column Es The SDS-PAGE diagram of GST; Figure 3 It is the line graph of the influence of temperature on the enzyme activity of glutathione S-transferase; Figure 4 Line graph of the effect of temperature on the stability of glutathione S-transferase; Figure 5 Line graph of the effect of pH on the enzyme activity of glutathione S-transferase; Figure 6 Line graph of the effect of pH on the stability of glutathione S-transferase; Figure 7 Bar graph of the effect of metal ions on the activity of glutathione S-transferase; Figure 8 Bar graph of the effect of inhibitors on the activity of glutathione S-transferase. Detailed implementation manners

[0016] The technical solutions of the present invention will be further prepared below through examples, but the protection scope of the present invention is not limited by any form of the examples.

[0017] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0018] Example 1: Through a previously explored Antarctic krill genome database (not publicly available), a batch of genomic sequences of glutathione S-transferase were discovered and analyzed. After aligning the amino acid sequences of this batch of sequences, the Blast analysis tool in the NCBI database was used to compare the confidence and homology with glutathione S-transferase from other species sources. A batch of unvalidated functional gene sequences predicted to be putative glutathione S-transferase or having potential glutathione S-transferase activity in bioinformatics were selected. The homology between the Antarctic krill amino acid sequence and the known source sequence was between 70-90%. After further analyzing the domain and protein family classification, the candidate sequences were determined. Subsequently, forward and reverse primers were designed for the candidate sequences, and using Antarctic krill cDNA as a template, it was verified by PCR. After the PCR product was sequenced correctly, the candidate gene Es GST.

[0019] After large-scale screening and in-depth research, a new glutathione S-transferase was identified and isolated from Antarctic krill, and its amino acid and nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2. The glutathione S-transferase of the present invention has good adaptability to low temperature and can be expressed in Escherichia coli cells.

[0020] The inventors first optimized the codons according to the expression situation, and the optimized sequence is shown in SEQ ID NO.3 to improve the expression efficiency and the stability of the DNA fragment.

[0021] Example 2: This example provides the construction of a recombinant Escherichia coli engineering bacterium of a recombinant expression vector and the process of recombinant protein expression, which is as follows: Es The GST gene was synthesized by Suzhou Hongxun Biotechnology Co., Ltd. (Suzhou, China). Using the synthesized Es GST gene as a template, the forward primer 5'-ATGGACTTCTACTACATGTCTC-3' (SEQ ID NO.4) and the reverse primer 5'-GTTAAAGACCAAGCTTAGG-3' (SEQ ID NO.5) were used to amplify Es GST. The amplified product was purified and double-digested with NdeI / XhoI, and then ligated into the PET28a vector double-digested with NdeI / XhoI. As Figure 1 shown, the PET28a-EsGST fusion protein expression plasmid with a 6×His tag at the C-terminus was successfully constructed by gene cloning technology. After the recombinant plasmid was sequenced and confirmed to be correct, the optimized sequence was inserted into the plasmid PET28a to obtain the recombinant plasmid PET28a-EsGST.

[0022] The plasmid was recovered using a plasmid extraction kit. After gene sequencing, the recombinant plasmid PET28a-EsGST was transformed into BL21(DE3) chaprone expression competent cells, and the transformants were transferred to LB medium for culture (this medium consists of 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and 50 μg / mL kanamycin). The temperature was 37 °C and the oscillation speed was 200 r / min. Using the Escherichia coli bacterial solution as a template, T7-F (5'-TAATACGACTCACTATAGGG-3') and T7-R (5'-GCTAGTTATTGCTCAGCGG-3') were used as the forward and reverse primers, and the annealing temperature was set at 50 °C for PCR. 20 μL of the PCR product was sent to Sangon for sequencing to verify the sequence. After the sequencing was correct, the bacterial solution was transferred to fresh LB medium and cultured with shaking at 37 °C until the OD600 value reached 0.6 - 0.8. Then, 0.6 mM IPTG was added and the culture was induced at 16 °C on a shaker for 20 h with an oscillation speed of 200 r / min. After that, the cells were collected by centrifugation at 8000 r / min for 10 min, and the collected cells were resuspended three times in buffer. After resuspension, the cells were sonicated on ice, and the centrifuged supernatant was the crude enzyme solution.

[0023] Purification of the recombinant protein by nickel column: The recombinantly expressed EsThe GST gene carries a 6*His tag at the C-terminus, which can bind to Ni in the HisTrap HP column packing, and Ni can bind to imidazole. Therefore, elution with different concentrations of imidazole can achieve the purpose of purification, and thus a single protein can be obtained. Centrifuge the fermentation broth at 10,000 r / min for 30 min to obtain the supernatant, which is refrigerated at 4°C for later use. First, rinse the A and B pumps and the system of the AKTA protein rapid purification instrument with ddH2O, and then rinse the A and B pumps with the equilibration buffer respectively; secondly, adjust the flow rate to 1 mL / min, and after connecting the nickel column, adjust the flow rate to 2 mL / min. Filter the crude enzyme solution through a 0.45 μm filter membrane and then load the sample, and the sample loading flow rate is 1 mL / min; after the breakthrough peak runs flat, rinse with the washing buffer to remove the miscellaneous proteins; finally, after the baseline is stable, elute the target protein with the elution buffer, collect the eluted peak until the peak runs flat, and stop collecting the sample. As Figure 2 shown, one-step purification of Es GST was achieved by using a HisTrap™ HP nickel column. Through SDS-PAGE analysis, a single electrophoretic band with a relative molecular weight of 24 kDa appeared in the electrophoretogram, which was completely consistent with the predicted value of ExPASy, further verifying the purity and accuracy of the target protein.

[0024] The equilibration buffer is: 50 mM Tris-HCl buffer; 500 mM NaCl; pH 8; The washing buffer is: 50 mM Tris-HCl buffer; 500 mM NaCl; 10 mM imidazole; pH 8; The elution buffer is: 50 mM Tris-HCl buffer; 500 mM NaCl; 500 mM imidazole; pH 8.

[0025] Determination of enzyme activity: The activity of recombinant Es GST was measured by the 1-chloro-2,4-dinitrobenzene (CDNB) method. The 200 μL reaction mixture contained 0.2 μg of Es GST, 10 mM GSH, 1 mM CDNB, and the remaining 50 mM Tris-HCl buffer (pH 8.0). Add the reaction mixture to a transparent 96-well plate and incubate at 20°C for 10 minutes. Then measure the absorbance at 340 nm within 0 - 2 minutes. Use heat-inactivated Es GST (treated in boiling water for 10 minutes) to replace Es GST and add it to the same reaction mixture as a negative control. The experimental samples and negative controls were performed with three technical replicates and three biological replicates.

[0026] EsSpecific activity of GST = (ΔOD340 × V) / (ε × T × L × E), where ΔOD340 is the change in absorbance at 340 nm within 2 minutes, V is the volume of the reaction mixture (200 μL), ε is the extinction coefficient of the product (ε = 9.6 L / mol / cm), L is the optical path of the reaction mixture (1 cm), T is the reaction time (2 minutes), and E is the amount of enzyme added to the reaction system (0.2 μg). One unit of enzyme activity is defined as the amount of CDNB-coupled product synthesized per minute per milligram of enzyme at 20 °C.

[0027] Enzymatic properties (1) Determination of the optimal temperature: The enzyme activity of GST was measured at different temperatures of 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 40 °C, 50 °C, 60 °C, and 70 °C respectively. Es Three parallel experiments were conducted at each temperature to measure the relative enzyme activity, with the highest enzyme activity measured at 20 °C set as 100%.

[0028] (2) Determination of the optimal pH: To analyze the effect of pH on enzyme activity, sodium citrate buffer (pH 4.0 - 5.0), phosphate buffer (pH 5.0 - 7.0), Tris-HCl buffer (pH 7.0 - 9.0), and glycine-sodium hydroxide buffer (pH 9.0 - 10.0) were prepared. The reaction system was placed in a 20 °C water bath for 10 min. Three parallel experiments were conducted at each pH to measure the relative enzyme activity, with the highest enzyme activity at pH 8 set as 100%.

[0029] (3) Temperature stability determination: The purified enzyme solution was placed in a metal bath at 4 °C, 10 °C, 20 °C, 30 °C, 40 °C, 50 °C, and 60 °C for 420 min. Samples were taken every 60 min and the enzyme activity was measured at pH = 8. Three parallel experiments were conducted at each time point to measure the relative enzyme activity, with the highest enzyme activity at 4 °C set as 100%.

[0030] (4) pH stability determination: The purified enzyme solution was placed in a buffer at 4 °C and pH 5.0 - 10.0 for 240 min. Samples were taken every 60 min and the enzyme activity was measured at 20 °C. Three parallel experiments were conducted at each pH to measure the relative enzyme activity, with the highest enzyme activity at pH 9 set as 100%.

[0031] (5) Effect of metal ions on the activity of recombinant Es GST enzyme: Various metal ions with a concentration of 100 mM, namely Na + , K + , Mg 2+ , Ba 2+, Ca 2+ , Cu 2+ , Fe 2+ , Ni 2+ , Fe 3+ , Zn 2+ , Mn 2+ , Co 2+ , and configure the final concentration of its metal ions to 10 mM. Place the purified enzyme solution in metal ion systems with different concentrations at 4 °C for 30 min, and then measure the enzyme activity at pH = 8 and 20 °C. Do 3 parallels for each temperature, measure the relative enzyme activity, and take the highest enzyme activity under the condition of ddH2O as 100%.

[0032] (6) Effect of inhibitors on the activity of recombinant Es GST: Prepare 100 mM SDS, β-mercaptoethanol, NaBH, DTT, and EDTA with ddH2O, and configure their final concentration to 10 mM. Place the purified enzyme solution in protease inhibitor systems with different concentrations at 4 °C for 30 min, and then measure the enzyme activity at pH = 8 and 20 °C. Do 3 parallels for each temperature, measure the relative enzyme activity, and take the highest enzyme activity under the condition of standard buffer as 100%.

[0033] The above reaction results are shown in the figure. It can be seen from Figure 3 , Figure 4 that the optimal reaction temperature of the enzyme is 20 °C. After the enzyme solution is kept at 4 °C, 10 °C, 20 °C, and 30 °C for 7 hours, the enzyme activities are 86%, 83%, 67%, and 57% of the initial enzyme activity respectively. However, when the temperature reaches 40 °C, 50 °C, and 60 °C, the relative enzyme activity after 7 hours is less than 10%. This shows that the remaining enzyme activity gradually decreases with the increase of temperature, but it has good stability under low temperature conditions.

[0034] Compared with glutathione S-transferases from other sources, the optimal temperature is about 25 °C - 40 °C. This shows that glutathione S-transferase from Antarctic krill has good catalytic ability and substrate affinity at lower temperatures.

[0035] As Figure 5 , Figure 6 shows, the optimal pH of the enzyme is 8, and it has good stability in the range of pH 8.0 - 10.0. After incubating in buffers with pH 9 and pH 10.0 for 7 h, the relative activity of the enzyme is greater than 90%.

[0036] As Figure 7 shows, K + , Na + on glutathione S-transferaseEs GST has an activating effect, with relative enzyme activities of 108% and 110%, while Zn 2+ , Ni + , Al 3+ , Co 2+ , Fe 2+ , Cu 2+ , Fe 3+ have obvious inhibitory effects on enzyme activity, with relative enzyme activities of 37%, 20%, 15%, 16%, 12%, 21%, and 13% respectively. Mn 2+ , Ca 2+ , Mg 2+ have slight inhibitory effects on enzyme activity, with relative enzyme activities of 77%, 80%, and 95% respectively.

[0037] As Figure 8 shown, DTT has an activating effect on the enzyme activity of glutathione S-transferase, with a relative enzyme activity of 131%; SDS has an obvious inhibitory effect on enzyme activity, with a relative enzyme activity of 20%; EDTA, urea, NaBH4, and β-mercaptoethanol have slight inhibitory effects on enzyme activity, with relative enzyme activities of 92%, 92%, 88%, and 87% respectively.

[0038] The amino acid sequence of Antarctic krill glutathione S-transferase is SEQ ID NO.1. Specifically as follows: MDFYYMSLSAPCRAPMLTAKAVGVELNMKSLNLFTGEQNTPEFIAINPQHVVPTLVDGDLKLWESRAICTYLASQYGKDDSLYPNDPKKRALVDRFLYFDMGTLYHRFGEYAYPVMFRGQDKPDPEKLEKLNEAFGWLNGWLEGKKYITGDKVTVADHSLVASVSTFVAGGIDISKYPNIVAWLEVCKKLPGYDEVNAPGAEEFGKMAKPKLGL.

[0039] The nucleotide sequence of Antarctic krill glutathione S-transferase is SEQ ID NO.2. Specifically as follows: ATGGACTTCTACTACATGTCTCTCTCGGCTCCATGTCGTGCACCAATGCTCACAGCTAAAGCAGTTGGCGTGGAGTTAAATATGAAATCTTTAAATCTATTTACCGGAGAGCAGAATACGCCAGAATTTATTGCCATTAACCCTCAGCATGTTGTTCCTACTCTGGTTGATGGAGACCTGAAACTCTGGGAAAGTCGGGCCATATGCACCTACTTGGCTTCTCAATATGGAAAAGATGACTCCTTATATCCAAATGATCCTAAGAAAAGAGCACTTGTTGATAGATTCCTCTACTTCGATATGGGAACATTGTATCACCGCTTTGGAGAATATGCTTATCCAGTGATGTTCAGAGGACAAGACAAACCAGATCCAGAGAAGTTGGAGAAGCTCAATGAAGCCTTCGGTTGGCTAAATGGATGGCTTGAAGGGAAGAAATACATTACTGGAGACAAAGTGACCGTTGCTGACCATTCTCTTGTAGCATCAGTATCAACTTTTGTAGCTGGTGGTATTGATATCTCTAAGTACCCAAATATTGTGGCCTGGTTAGAGGTCTGTAAGAAACTCCCTGGATATGATGAGGTCAATGCTCCAGGTGCTGAAGAATTTGGAAAAATGGCAAAACCTAAGCTTGGTCTTTAA。

[0040] The nucleotide sequence of the Antarctic krill glutathione S-transferase after codon optimization is SEQ ID NO. 3, which is as follows: ATGGATTTCTACTACATGTCTCTGAGCGCGCCGTGCCGTGCACCGATGCTGACCGCGAAAGCGGTTGGTGTTGAACTGAACATGAAATCTCTGAACCTGTTCACCGGTGAACAGAACACCCCGGAATTTATCGCTATCAACCCGCAGCACGTTGTTCCGACCCTGGTTGATGGTGATCTGAAACTGTGGGAATCTCGTGCGATCTGCACCTACCTGGCTTCTCAGTACGGTAAAGATGATAGCCTGTACCCGAACGATCCGAAAAAACGTGCGCTGGTTGATCGTTTCCTGTATTTCGATATGGGTACCCTGTACCACCGTTTCGGTGAATACGCGTACCCGGTTATGTTCCGTGGTCAGGATAAACCGGATCCGGAAAAACTGGAAAAACTGAACGAAGCGTTCGGTTGGCTGAACGGTTGGCTGGAAGGTAAAAAATACATCACCGGTGATAAAGTTACCGTTGCGGATCACTCTCTGGTTGCGAGCGTTTCTACCTTCGTTGCGGGTGGTATCGATATCTCTAAATACCCGAACATCGTTGCGTGGCTGGAAGTTTGCAAAAAACTGCCGGGTTACGATGAAGTTAACGCGCCGGGTGCGGAAGAATTCGGTAAAATGGCTAAACCGAAACTGGGTCTGCACCACCACCACCACCACTAA。

[0041] The forward primer nucleotide sequence for PCR verification of Antarctic krill glutathione S-transferase is SEQ ID NO. 4. Specifically as follows: 5'-ATGGACTTCTACTACATGTCTC-3'; The reverse primer nucleotide sequence for PCR verification of Antarctic krill glutathione S-transferase is SEQ ID NO. 5. Specifically as follows: 5'-GTTAAAGACCAAGCTTAGG-3'.

Claims

1. A glutathione S-transferase derived from Antarctic krill, characterized in that, The amino acid sequence of the glutathione S-transferase is shown in SEQ ID NO.

1.

2. A gene encoding the glutathione S-transferase according to claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A recombinant plasmid pET28a-EsGSST, characterized in that, The recombinant plasmid PET28a-EsGSST contains the gene described in claim 2.

4. A recombinant Escherichia coli engineering bacterium, characterized in that, The recombinant Escherichia coli engineering bacteria contain the recombinant plasmid PET28a-EsGST described in claim 3.

5. An enzyme preparation, characterized in that, The enzyme preparation contains the glutathione S-transferase derived from Euphausia superba described in claim 1.

6. Use of the glutathione S-transferase according to claim 1, characterized in that, The application is to use the glutathione S-transferase to catalyze the substrate.

7. The application according to claim 6, characterized in that, The application is to catalyze the substrate at a temperature of 20°C.

Images