Antarctic krill-derived glutathione s-transferase and its encoding gene and application

Abstract

This invention relates to a glutathione S-transferase derived from Antarctic krill, its encoding gene, and its applications, belonging to the field of enzymology. The amino acid sequence of the glutathione S-transferase is shown in SEQ ID NO.1, and the amino acid sequence of the gene encoding the glutathione S-transferase is shown in SEQ ID NO.2. The specific activity of the glutathione S-transferase of this invention can reach up to 6.01 μmol / min / mg. The optimal temperature and optimal pH are 20 °C and 8, respectively. The Antarctic krill-derived glutathione S-transferase of this invention exhibits good catalytic activity and substrate affinity at relatively low temperatures.

Description

Technical Field

[0001] This invention belongs to the field of enzymology, specifically relating to a glutathione S-transferase derived from Antarctic krill, its encoding gene, and its applications. Background Technology

[0002] Antarctic krill is a key species in the Antarctic marine ecosystem, possessing significant biological characteristics and ecoeconomic value. Antarctic krill can live in extreme environments and exhibits qualities such as freeze resistance and antioxidant capacity, containing various bioactive substances, including lipids, proteins, and enzymes. However, due to its tendency to hydrolyze its own proteins, isolating and extracting enzymes from Antarctic krill is challenging. Currently, the development and utilization of enzymes derived from Antarctic krill are still in their early stages, and further in-depth research is needed.

[0003] Glutathione S-transferase (GST) (EC 2.5.1.18) is a key detoxification enzyme widely distributed in prokaryotes and eukaryotes. Belonging to the phase II cellular detoxification system, it plays a crucial role in detoxification in various organisms. It catalyzes the binding of reduced glutathione (GSH) to electrophilic exogenous substances, making them more hydrophilic and easier to excrete. GST participates in the elimination of potentially harmful chemical components, such as exogenous organisms, drugs, environmental pollutants, herbicides, pesticides, and chemical carcinogens. 1-Chloro-2,4-dinitrobenzene (CDNB) is a common substrate for glutathione S-transferase, which reacts with GSH to form a complex (GS-SNB). Furthermore, they exert cellular protective effects by inactivating quinones, epoxides, α,β-unsaturated ketones, and oxidants produced during oxidative stress.

[0004] Currently, there are few reports on heterologous expression of glutathione S-transferase. Therefore, discovering genes with high glutathione S-transferase activity, constructing genetically engineered bacteria through gene recombination, efficiently heterologously expressing glutathione S-transferase, and exploring enzymatic hydrolysis processes of glutathione S-transferase have significant industrial application value and potential. Summary of the Invention

[0005] The objective of this invention is to provide a glutathione S-transferase derived from Antarctic krill, its encoding gene, and its applications. This invention is based on genomic analysis and the discovery of the Antarctic krill GST gene (GST). Es GST was heterologously expressed in Escherichia coli BL21(DE3) chaprone.

[0006] This invention is achieved through the following technical solution:

[0007] A glutathione S-transferase derived from Antarctic krill, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also provides a gene encoding the glutathione S-transferase, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0009] A recombinant plasmid PET28a-EsGSST, wherein the recombinant plasmid contains the nucleotide sequence shown in SEQ ID NO.2.

[0010] A recombinant engineered Escherichia coli strain containing the recombinant plasmid PET28a-EsGST.

[0011] An enzyme preparation comprising the Antarctic krill-derived glutathione S-transferase.

[0012] The present invention also provides the application of the glutathione S-transferase, wherein the application is to utilize the glutathione S-transferase to catalyze a substrate.

[0013] Furthermore, the application described herein involves catalyzing the substrate at a temperature of 20°C.

[0014] The beneficial effects of this invention compared to existing technologies are as follows: This invention provides an Antarctic krill glutathione S-transferase. The gene sequence of glutathione S-transferase was obtained using Antarctic krill protein transcriptome bioinformatics methods. The target gene was amplified in vitro by polymerase chain reaction (PCR) and expressed in soluble activity in Escherichia coli.

[0015] In the recombinant *E. coli* soluble expression obtained in this invention, the specific activity of glutathione S-transferase in the supernatant after centrifugation reached as high as 6.01 μmol / min / mg. Basic enzymatic studies revealed that the optimal temperature and pH were 20 °C and 8, respectively, and the protein molecular weight was 24 kDa. The optimal temperature for existing glutathione S-transferases is approximately 25 °C to 40 °C. The glutathione S-transferase derived from Antarctic krill described in this invention exhibits good catalytic activity and substrate affinity at lower temperatures. Attached Figure Description

[0016] Figure 1 The spectrum of recombinant plasmid PET28a-EsGST;

[0017] Figure 2 Purification of recombinant expressed protein using nickel column Es GST's SDS-PAGE plot;

[0018] Figure 3Line graph showing the effect of temperature on glutathione S-transferase activity;

[0019] Figure 4 Line graph showing the effect of temperature on the stability of glutathione S-transferase;

[0020] Figure 5 Line graph showing the effect of pH on glutathione S-transferase activity;

[0021] Figure 6 Line graph showing the effect of pH on the stability of glutathione S-transferase;

[0022] Figure 7 Bar chart showing the effect of metal ions on glutathione S-transferase activity;

[0023] Figure 8 The bar chart shows the effect of inhibitors on glutathione S-transferase activity. Detailed Implementation

[0024] The technical solution of the present invention will be further prepared through the following embodiments, but the scope of protection of the present invention is not limited in any way by the embodiments.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0026] Example 1: Using a previously explored Antarctic krill genome database (not publicly available), a batch of glutathione S-transferase genomic sequences were discovered and analyzed. After amino acid sequence alignment, these sequences were compared with glutathione S-transferases from other species using the BLAST analysis tool in the NCBI database for confidence and homology. A batch of candidate sequences were identified as presumed glutathione S-transferases or unverified functional gene sequences with potential glutathione S-transferase activity in bioinformatics. The Antarctic krill amino acid sequences showed 70-90% homology with known source sequences. Further analysis of domains and protein families confirmed the candidate sequences. Primers were then designed for the candidate sequences, and PCR was used to verify them using Antarctic krill cDNA as a template. After successful sequencing of the PCR products, the candidate genes were obtained. Es GST.

[0027] Through large-scale screening and in-depth research, a novel glutathione S-transferase has been identified and isolated from Antarctic krill, with its amino acid and nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2. The glutathione S-transferase of this invention exhibits good adaptability to low temperatures and can be expressed in Escherichia coli cells.

[0028] The inventors first optimized the codons based on the expression situation. The optimized sequence is shown in SEQ ID NO.3, in order to improve the expression efficiency and the stability of the DNA fragment.

[0029] Example 2: This example provides a process for constructing a recombinant Escherichia coli engineered bacterium using a recombinant expression vector and expressing the recombinant protein, as detailed below:

[0030] Es The GST gene was synthesized by Suzhou Hongxun Biotechnology Co., Ltd. (Suzhou, China). The synthesized GST gene... Es Using the GST gene as a template, amplification was performed using the forward primer 5'-ATGGACTTCTACTACATGTCTC-3' (SEQ ID NO.4) and the reverse primer 5'-GTTAAAGACCAAGCTTAGG-3' (SEQ ID NO.5). Es GST. The amplification product was purified and double-digested with NdeI / XhoI, then ligated into the NdeI / XhoI double-digested PET28a vector. (See figure) Figure 1 As shown, a PET28a-EsGST fusion protein expression plasmid with a 6×His marker at the C-terminus was successfully constructed using gene cloning technology. After confirming the recombinant plasmid with sequencing, the optimized sequence was inserted into plasmid PET28a to obtain the recombinant plasmid PET28a-EsGST.

[0031] The plasmid was recovered using a plasmid extraction kit. After gene sequencing, the recombinant plasmid PET28a-EsGST was transformed into BL21(DE3) chaprone competent cells. The transformants were then transferred to LB medium (composed of 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and 50 μg / mL carbolic acid) for culture. The temperature was 37 °C, the shaking speed was 200 r / min, and PCR was performed using *E. coli* culture as a template, with T7-F (5'-TAATACGACTCACTATAGGG-3') and T7-R (5'-GCTAGTTATTGCTCAGCGG-3') as primers and an annealing temperature of 50 °C. 20 μL of the PCR product was sent for sequencing verification. After successful sequencing, the culture was transferred to fresh LB medium and cultured at 37 °C with shaking until the OD600 value reached 0.6-0.8. After adding 0.6 mM IPTG, the cells were incubated in a shaker at 16 ℃ for 20 h at a shaking speed of 200 r / min. Then, the cells were collected by centrifugation at 8000 r / min for 10 min. The collected cells were resuspended three times in buffer. After resuspending, the cells were sonicated on ice, and the supernatant was obtained by centrifugation.

[0032] Nickel column purification of recombinant proteins: recombinant expression Es The GST gene carries a 6*His tag at its C-terminus, which can bind to Ni in the HisTrap HP column packing material. Ni can then bind to imidazole. Therefore, elution with different concentrations of imidazole can achieve purification, yielding a single protein. The fermentation broth was centrifuged at 10000 r / min for 30 min to obtain the supernatant, which was then stored at 4℃ for later use. First, the A and B pumps and system of the AKTA protein rapid purification instrument were rinsed with ddH2O, followed by rinsing with equilibration buffer. Next, the flow rate was adjusted to 1 mL / min, and after connecting the nickel column, the flow rate was adjusted to 2 mL / min. The crude enzyme solution was filtered through a 0.45 μm filter and loaded at a flow rate of 1 mL / min. After the breakthrough peak leveled out, the sample was washed with washing buffer to remove impurities. Finally, after the baseline stabilized, the target protein was eluted with elution buffer, and the eluted peak was inoculated until it leveled out. Figure 2 As shown, purification was achieved using a one-step HisTrap™ HP nickel column. Es GST, through SDS-PAGE analysis, showed a single band with a relative molecular weight of 24 kDa in the electrophoretic pattern, which was completely consistent with the predicted value of ExPASy, further verifying the purity and accuracy of the target protein.

[0033] The equilibration buffer was: 50 mM Tris-HCl buffer; 500 Mm NaCl; pH 8.

[0034] The washing buffer was: 50 mM Tris-HCl buffer; 500 mM NaCl; 10 mM imidazole; pH 8.

[0035] The elution buffer was: 50 mM Tris-HCl buffer; 500 mM NaCl; 500 mM imidazole; pH 8.

[0036] Enzyme activity assay: Recombinant Es The activity of GST was measured using the 1-chloro-2,4-dinitrobenzene (CDNB) method. 200 μL of the reaction mixture contained 0.2 μg of GST. 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 clear 96-well plate and incubate at 20°C for 10 minutes. Then measure the absorbance at 340 nm over 0–2 minutes. The heat-inactivated... Es GST (treated in boiling water for 10 minutes) as an alternative EsGST was added to the same reaction mixture and used as a negative control. The experimental samples and the negative control were subjected to three technical replicates and three biological replicates.

[0037] Es The specific activity of GST is calculated as (ΔOD340 × V) / (ε × T × L × E), where ΔOD340 is the change in absorbance at 340 nm over 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 length 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 milligram per minute at 20 °C.

[0038] Enzymatic properties (1) Determination of optimal temperature: The optimal temperature was determined at different temperatures of 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃ and 70 ℃. Es GST enzyme activity was measured. Three replicates were performed for each temperature group, and the relative enzyme activity was determined, with the highest enzyme activity measured at 20 °C being taken as 100%.

[0039] (2) Determination of 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 ℃ water bath for 10 min, and three replicates were performed for each temperature group. The relative enzyme activity was measured, and the highest enzyme activity at pH 8 was taken as 100%.

[0040] (3) Temperature stability test: The purified enzyme solution was placed in metal baths at 4 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃ and 50 ℃, 60 ℃ for 420 min. During this period, it was taken out every 60 min and the enzyme activity was measured under pH=8. Each time group was performed in 3 parallels to measure the relative enzyme activity. The highest enzyme activity under the condition of 4 ℃ was taken as 100%.

[0041] (4) pH stability test: The purified enzyme solution was placed in a buffer solution at 4 ℃ and pH 5.0-10.0 for 240 min. During this period, it was taken out every 60 min and the enzyme activity was measured at 20 ℃. Each pH group was performed in triplicate to measure the relative enzyme activity. The highest enzyme activity at pH 9 was taken as 100%.

[0042] (5) Metal ion pair recombination EsEffects on GST enzyme activity: A 100 mM solution of various metal ions (Na+) was prepared using ddH2O. + K + Mg 2+ Ba 2+ Ca 2+ Cu 2+ Fe 2+ Ni 2+ Fe 3+ Zn 2+ Mn 2+ Co 2+ The final concentration of metal ions was set to 10 mM. The purified enzyme solution was placed in a metal ion system of different concentrations at 4 ℃ for 30 min. The enzyme activity was then measured at pH=8 and 20 ℃. Three replicates were performed for each temperature group to measure the relative enzyme activity. The highest enzyme activity under ddH2O conditions was taken as 100%.

[0043] (6) Inhibitors on recombinant Es Effect of GST enzyme activity: 100 mM SDS, β-mercaptoethanol, NaBH, DTT, and EDTA were prepared using ddH2O, with a final concentration of 10 mM. The purified enzyme solution was placed in different concentrations of protease inhibitor systems at 4 ℃ for 30 min, and then enzyme activity was measured at pH=8 and 20 ℃. Each temperature group was performed in triplicate, and the relative enzyme activity was measured. The highest enzyme activity under standard buffer conditions was taken as 100%.

[0044] The results of the above reaction are shown in the figure. Figure 3 , Figure 4 The results show that the optimal reaction temperature for the enzyme is 20 °C. After the enzyme solution was kept at 4 °C, 10 °C, 20 °C, and 30 °C for 7 hours, the enzyme activity retained 86%, 83%, 67%, and 57% of the initial enzyme activity, respectively. However, when the temperature was increased to 40 °C, 50 °C, and 60 °C, the relative enzyme activity was less than 10% after 7 hours. This indicates that the remaining enzyme activity gradually decreases with increasing temperature, but it has good stability under low-temperature conditions.

[0045] Compared to glutathione S-transferases from other sources, the optimal temperature is around 25 ℃ to 40 ℃. This indicates that glutathione S-transferases from Antarctic krill exhibit good catalytic activity and substrate affinity at lower temperatures.

[0046] like Figure 5 , Figure 6As shown, the optimal pH for the enzyme is 8, and it exhibits good stability within the pH range of 8.0–10.0. Furthermore, after incubation for 7 hours in buffers at pH 9 and pH 10.0, the relative activity of the enzyme is greater than 90%.

[0047] like Figure 7 As shown, K + Na + Glutathione S-transferase Es GST has an activating effect, with relative enzyme activity of 108% and 110%, while Zn 2+ Ni + Al 3+ Co 2+ Fe 2+ Cu 2+ Fe 3+ It has a significant inhibitory effect on enzyme activity, with relative enzyme activities of 37%, 20%, 15%, 16%, 12%, 21%, and 13%. Mn 2+ Ca 2+ Mg 2+ It has a slight inhibitory effect on enzyme activity, with relative enzyme activity of 77%, 80%, and 95%.

[0048] like Figure 8 As shown, DTT activated the enzyme activity of glutathione S-transferase, with a relative enzyme activity of 131%; SDS significantly inhibited the enzyme activity, with a relative enzyme activity of 20%; EDTA, urea, NaBH4, and β-mercaptoethanol had slight inhibitory effects on the enzyme activity, with relative enzyme activities of 92%, 92%, 88%, and 87%, respectively.

[0049] The amino acid sequence of Antarctic krill glutathione S-transferase is SEQ ID NO. 1. Details are as follows:

[0050] MDFYYMSLSAPCRAPMLTAKAVGVELNMKSLNLFTGEQNTPEFIAINPQHVVPTLVDGDLKLWESRAICTYLASQYGKDDSLYPNDPKKRALVDRFLYFDMGTLYHR FGEYAYPVMFRGQDKPDPEKLEKLNEAFGWLNGWLEGKKYITGDKVTVADHSLVASVSTFVAGGIDISKYPNIVAWLEVCKKLPGYDEVNAPGAEEFGKMAKPKLGL.

[0051] The nucleotide sequence of Antarctic krill glutathione S-transferase is SEQ ID NO.2, as follows:

[0052] ATGGACTTCTACTACATGTCTCTCTCGGCTCCATGTCGTGCACCAATGCTCACAGCTAAAGCAGTTGGCGTGGAGTTAAATATGAAATCTTTAAATCTATTTACCGGAGAGCAGAATACGCCAGAATTTATTGCCATTAACCCTCAGCATGTTGTTCCTACTCTGGTTGATGGAGACCTGAAACTCTGGGAAAGTCGGGCCATATGCACCTACTTGGCTTCTCAATATGGAAAAGATGACTCCTTATATCCAAATGATCCTAAGAAAAGAGCACTTGTTGATAGATTCCTCTACTTCGATATGGGAACATTGTATCACCGCTTTGGAGAATATGCTTATCCAGTGATGTTCAGAGGACAAGACAAACCAGATCCAGAGAAGTTGGAGAAGCTCAATGAAGCCTTCGGTTGGCTAAATGGATGGCTTGAAGGGAAGAAATACATTACTGGAGACAAAGTGACCGTTGCTGACCATTCTCTTGTAGCATCAGTATCAACTTTTGTAGCTGGTGGTATTGATATCTCTAAGTACCCAAATATTGTGGCCTGGTTAGAGGTCTGTAAGAAACTCCCTGGATATGATGAGGTCAATGCTCCAGGTGCTGAAGAATTTGGAAAAATGGCAAAACCTAAGCTTGGTCTTTAA。

[0053] The nucleotide sequence of the Antarctic krill glutathione S-transferase after codon optimization is SEQ ID NO. 3, as follows:

[0054] .

[0055] The nucleotide sequence of the forward primer for PCR verification of the Antarctic krill glutathione S-transferase is SEQ ID NO. 4. Specifically:

[0056] 5'-ATGGACTTCTACTACATGTCTC-3';

[0057] The reverse primer nucleotide sequence for the Antarctic krill glutathione S-transferase PCR verification is SEQ ID NO. 5. Details are as follows:

[0058] 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. The gene encoding the glutathione S-transferase of 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 engineered Escherichia coli strain, characterized in that, The recombinant Escherichia coli engineered bacteria contains the recombinant plasmid PET28a-EsGST as described in claim 3.

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

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

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

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