Leucine aminopeptidase derived from euphausia superba as well as coding gene and application of leucine aminopeptidase

Abstract

The invention relates to euphausia superba-derived leucine aminopeptidase as well as a coding gene and application thereof, and belongs to the field of enzymology, and the amino acid sequence of the leucine aminopeptidase is shown as SEQ ID NO.1. The gene for coding the leucine aminopeptidase is as shown in SEQ ID NO. 2. The invention also provides a gene recombinant plasmid containing the leucine aminopeptidase and a recombinant bacterium containing the leucine aminopeptidase. The obtained leucine aminopeptidase from the euphausia superba can be used for carrying out enzymolysis on Leu-pNA, Glu-pNA, Arg-pNA, Met-pNA and Lys-pNA, bitter polypeptides are degraded into single amino acids or dipeptides one by one from the head of the N end, and generation of bad flavors is reduced.

Description

Technical Field

[0001] The invention belongs to the field of enzymology, and specifically relates to a leucine aminopeptidase derived from Antarctic krill and a coding gene and application thereof. Background Art

[0002] Antarctic krill contains nutritional value and rich proteins, phospholipids, unsaturated fatty acids and other ingredients. If this resource can be used by humans, it will greatly promote the development of marine resources. Antarctic krill has a huge protease system. These highly active proteases enable the krill digestive system to quickly digest and utilize algae and other foods. They also cause the Antarctic krill itself to be rapidly degraded during storage, transportation and processing, causing autolysis. Therefore, this also provides us with the possibility of exploring highly active Antarctic krill-derived enzymes as industrial biocatalysts. At present, the development and utilization of Antarctic krill-derived enzymes is still in its early stages, and we need to conduct more in-depth research on Antarctic krill-derived enzymes.

[0003] Aminopeptidases (Aps) (EC 3.4.11) are a class of exoproteinases that hydrolyze amino acids sequentially from the N-terminus of a polypeptide chain or protein, and separate some amino acids one by one. Leucine aminopeptidase can be used to hydrolyze peptide molecules with leucine at the end. Aminopeptidase is a very important flavor protease that can dissociate the hydrophobic amino acids at the N-terminus and is used to debitter protein hydrolysates; it can also be used for the preparation of bioactive peptides and the determination and analysis of protein sequences.

[0004] At present, there are few reports on heterologous expression of leucine aminopeptidase. Therefore, it is of great industrial application value and potential to discover leucine aminopeptidase genes with high activity, construct genetically engineered bacteria through gene recombination, efficiently heterologously express leucine aminopeptidase, and explore the enzymatic hydrolysis process of leucine aminopeptidase. Summary of the invention

[0005] The purpose of the present invention is to provide a leucine aminopeptidase from Antarctic krill and its encoding gene and application. The present invention mines the Antarctic krill LAP gene (EsLAP) based on genome analysis and heterologously expresses it in Escherichia coli RTS-BL21 (DE3) chaprone. This competent cell contains a molecular chaperone protein plasmid, and its expression product can assist the recombinant protein to correctly fold into a soluble active protein, so that it has higher enzyme activity to meet the needs of industrial production.

[0006] The present invention is achieved through the following technical solutions: A leucine aminopeptidase derived from Antarctic krill, the amino acid sequence of the leucine aminopeptidase is shown as SEQ ID NO.1.

[0007] The present invention also provides a gene encoding the leucine aminopeptidase, and the nucleotide sequence of the gene is shown as SEQ ID NO.2.

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

[0009] A recombinant Escherichia coli engineered bacterium, wherein the recombinant Escherichia coli engineered bacterium contains the recombinant plasmid PET28a-EsLAP.

[0010] An enzyme preparation contains the Antarctic krill-derived leucine aminopeptidase.

[0011] The present invention also provides the application of the Antarctic krill-derived leucine aminopeptidase, whose substrates are Leu-pNA, Glu-pNA, Arg-pNA, Met-pNA and Lys-pNA, and which can degrade bitter peptides whose N-termini are leucine, glutamic acid, arginine, methionine and lysine, and degrade the bitter peptides from the N-terminal head one by one into the form of single amino acids or dipeptides, thereby reducing the generation of unpleasant flavors.

[0012] The beneficial effects of the present invention compared with the prior art are as follows: The present invention provides an Antarctic krill leucine aminopeptidase. The gene sequence of the leucine aminopeptidase was obtained by using the Antarctic krill protein transcriptome bioinformatics method, the target gene was amplified in vitro by polymerase chain reaction (PCR), and soluble active expression was performed in Escherichia coli, laying a theoretical foundation for the application of aminopeptidase in hydrolyzing polypeptides and debittering food.

[0013] In the soluble expression of the recombinant Escherichia coli obtained by the present invention, the activity of leucine aminopeptidase in the supernatant after centrifugation was detected to be as high as 265.6 U / mL. Through the study of its basic enzymatic properties, the optimal temperature and optimal pH were 60°C and 8.5 respectively, and the protein molecular weight was 56 kDa. Concentration 1 mM Co 2+ It has a strong promoting effect on the enzymatic activity of leucine aminopeptidase, with a promotion multiple of 2.96 times. This highly active soluble leucine aminopeptidase and its enzymatic characteristics lay a good foundation for the preparation of biologically active peptides, deep protein hydrolysis and debittering of hydrolyzates. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the map of the recombinant plasmid PET28a-EsLAP; Figure 2 This is the SDS-PAGE image of the recombinant protein EsLAP purified by nickel column; Figure 3 It is a line graph showing the effect of temperature on the activity of leucine aminopeptidase; Figure 4 is a line graph showing the effect of temperature on the stability of leucine aminopeptidase; Figure 5 It is a line graph showing the effect of pH on the activity of leucine aminopeptidase; Figure 6 is a line graph showing the effect of pH on the stability of leucine aminopeptidase; Figure 7 This is a bar graph showing the effect of metal ions on leucine aminopeptidase activity; Figure 8 is a bar graph showing the effect of inhibitors on leucine aminopeptidase activity; Fig. 9 It is a bar graph showing the decomposition ability of leucine aminopeptidase on different substrates; Fig.10 A diagram showing the catalytic decomposition of leucine p-nitroanilide by the recombinant expressed protein EsLAP. DETAILED DESCRIPTION

[0015] The technical solution of the present invention is further described below through embodiments, but the protection scope of the present invention is not limited in any form by the embodiments.

[0016] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0017] Example 1: A batch of leucine aminopeptidases were discovered and analyzed through the previously discovered Antarctic krill genome database (unpublished). After the amino acid sequence of these sequences was aligned, the confidence and homology of leucine aminopeptidases from other species were compared by using the Blast analysis tool in the NCBI database. A batch of unverified functional gene sequences that were predicted to be hypothetical leucine aminopeptidases or have potential leucine aminopeptidase activity in bioinformatics were selected, and the homology of the Antarctic krill amino acid sequence with the known source sequence was between 70-90%. After further classification and analysis of the domain and protein family, the candidate sequence was determined. Subsequently, the front and rear primers were designed for the candidate sequence, and the Antarctic krill cDNA was used as a template to verify it by PCR. After the PCR product was sequenced correctly, the candidate gene EsLAP was obtained.

[0018] After large-scale screening and in-depth research, a new leucine aminopeptidase was identified and separated from Antarctic krill, and its amino acid and nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2. It can not only catalyze the hydrolysis of polypeptides, but also has specificity for different substrates, and has very good application prospects. The leucine aminopeptidase of the present invention has good adaptability to low temperature and can be expressed in Escherichia coli cells. At the same time, the present invention also optimizes the recombinant expression method of the leucine aminopeptidase, so that it can be efficiently expressed in host cells.

[0019] The inventors firstly performed codon optimization 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.

[0020] Example 2: This example provides a recombinant expression vector of the construction of recombinant Escherichia coli engineering bacteria and the recombinant protein expression process, as follows: The EsLAP gene was synthesized by Suzhou Hongxun Biotechnology Co., Ltd. (Suzhou, China). The synthesized EsLAP gene was used as a template to amplify EsLAP using the forward primer 5'-ATGGGTGGTTCCAAGATCAAC-3' (SEQ ID NO.4) and the reverse primer 5'-TCAATGATGATGATGATGAT-3' (SEQ ID NO.5). The amplified product was purified and double-digested with EcoRI / NotI, and then ligated into the PET28a vector double-digested with EcoRI / NotI. Figure 1 As shown in the figure, the PET28a-EsLAP 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-EsLAP.

[0021] The plasmid was recovered using a plasmid extraction kit. After gene sequencing, the recombinant plasmid PET28a-EsLAP was transformed into RTS-BL21(DE3) chaprone lytic expression competent cells, and the transformants were transferred to LB medium for culture (the medium consisted of 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and 50 μg / mL penicillin). The temperature was 37°C, the shaking speed was 200 r / min, Escherichia coli liquid was used as a template, T7-F (5'-TAATACGACTCACTATAGGG-3', SEQ ID NO.6) and T7-R (5'-GCTAGTTATTGCTCAGCGG-3'SEQ ID NO.7) were used as front and back primers, the annealing temperature was set to 50°C, PCR was performed, and 20 μL of PCR product was sent to Biotech for sequencing verification. After sequencing is correct, transfer the bacterial solution to fresh LB medium and shake culture at 37 ℃ until the OD600 value reaches 0.6-0.8. Then add 0.6 mM IPTG and place it in a 16 ℃ shaker for 20 h of induction culture at a shaking speed of 200 r / min. Then centrifuge at 8000 r / min for 10 min to collect the bacteria, and add the collected bacteria to the buffer and resuspend them three times. After resuspension, add PMSF with a final concentration of 1 mM, place it on ice for ultrasonic disruption, and the supernatant of centrifugation is the crude enzyme solution.

[0022] Nickel column purification of recombinant protein: The recombinantly expressed EsLAP gene carries a 6×His tag at the C-terminus, which can bind to the Ni of the HisTrap HP column filler, and Ni can bind to imidazole. Therefore, elution with different concentrations of imidazole can achieve the purpose of purification, thereby obtaining a single protein. Centrifuge the fermentation broth at 10,000 r / min for 30 min to obtain the supernatant, which is then refrigerated at 4°C for later use. First, rinse the A and B pumps and system of the AKTA protein rapid purification instrument with ddH2O, and then rinse the A and B pumps separately with the equilibrium buffer; secondly, adjust the flow rate to 1 mL / min, and adjust the flow rate to 2 mL / min after connecting the nickel column, and load the crude enzyme solution after passing through a 0.45μm filter membrane, with a loading flow rate of 1 mL / min; after the penetration peak runs flat, rinse with washing buffer to remove impurities; finally, after the baseline is stable, elute the target protein with elution buffer, sample the peak, and stop sampling when the peak runs flat. Figure 2 As shown in the figure, EsLAP was purified by one-step method using HisTrap™ HP nickel column. SDS-PAGE analysis showed that a single electrophoretic pure band with a relative molecular weight of 56 kDa appeared in the electrophoretic pattern, which was completely consistent with the predicted value of ExPASy, further verifying the purity and accuracy of the target protein.

[0023] The equilibration buffer was: 50 mM Tris-HCl buffer; 500 Mm NaCl; pH 8.5; The washing buffer was: 50 mM Tris-HCl buffer; 500 mM NaCl; 10 mM imidazole; pH 8.5; The elution buffer was: 50 mM Tris-HCl buffer; 500 mM NaCl; 500 mM imidazole; pH 8.5.

[0024] Determination of enzyme activity: The maximum absorption peak of the product of the catalytic reaction, p-nitroanilide, at a wavelength of 405 nm is used to characterize the enzyme activity of the recombinant leucine aminopeptidase. The release of 1 µmol p-nitroanilide per minute is one unit of enzyme activity (U). Dilute the enzyme solution appropriately with pH 8.5 Tris-HCl buffer, then take 20 μl of the diluted enzyme solution, add 160 μl of buffer, preheat at 60 ℃, add 20 μl of 10 mM Leu-pNA ethanol solution (control plus inactivated enzyme), and react in a 60 ℃ water bath. After the reaction is completed, take it out immediately, add 200 μl of acetic acid immediately to terminate the reaction, take 200 μl of the reaction mixture to the wells of the ELISA plate, and measure the absorbance at a wavelength of 405 nm.

[0025] Enzyme properties (1) Optimum temperature determination: The enzyme activity of EsLAP was measured at different temperatures of 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃ and 80 ℃. Three parallel experiments were performed for each temperature group to determine the relative enzyme activity, with the highest enzyme activity measured at 60 ℃ being 100%.

[0026] (2) Determination of optimal pH: The effect of pH on enzyme activity was analyzed from the change of pH. 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 60 ℃ water bath for 10 min. Three parallels were made for each temperature group. The relative enzyme activity was measured, and the highest enzyme activity at pH 8.5 was set as 100%.

[0027] (3) Temperature stability test: The purified enzyme solution was placed in a metal bath at 20, 30, 40, 50, 60, 70, and 80 °C for 240 min. The solution was taken out every 60 min and the enzyme activity was measured at pH 8.5. Three replicates were performed for each time period and the relative enzyme activity was measured. The highest enzyme activity at 4 °C was taken as 100%.

[0028] (4) pH stability determination: The purified enzyme solution was placed in a buffer solution at 4°C and pH 4.0-9.0 for 240 min. The enzyme solution was taken out every 60 min and the enzyme activity was determined at 60°C. Three parallel assays were performed for each pH group and the relative enzyme activity was determined, with the highest enzyme activity at pH 8.5 being set as 100%.

[0029] (5) Effect of metal ions on the activity of recombinant EsLAP: Various metal ions Na2O were prepared at a concentration of 10 mM. + , K + Mg 2+ , Ba 2+ , Ca 2+ , Cu 2+ 、Ni 2+ , Fe 3+ 、Zn 2+ , Mn 2+ 、Co 2+ The final concentration of metal ions was configured to 1 mM and 10 mM. The purified enzyme solution was placed in a metal ion system with different concentrations at 4 °C for 30 min, and then reacted in a water bath at pH = 8.5 and 60 °C for 10 min. Three parallel reactions were performed for each temperature group, and the relative enzyme activity was determined, with the highest enzyme activity under ddH2O conditions being 100%.

[0030] (6) Effect of inhibitors on the activity of recombinant EsLAP: SDS, PMSF, β-mercaptoethanol, NaBH, DTT and EDTA were prepared with ddH2O at a concentration of 10 mM and a final concentration of 1 mM and 10 mM, respectively. The purified enzyme solution was placed in a protease inhibitor system with different concentrations at 4 °C for 30 min and then reacted in a water bath at pH = 8.5 and 60 °C for 10 min. Three parallel experiments were performed for each temperature group and the relative enzyme activity was measured. The highest enzyme activity under standard buffer conditions was taken as 100%.

[0031] The above reaction results are shown in the figure. Figure 3 , Figure 4 It can be seen that the optimal reaction temperature of the enzyme is 60 ℃. When the temperature is kept in the range of 30 ℃~50 ℃ for 4 h, the relative enzyme activity remains above 90%. When kept at 60 ℃ for 4 h, the enzyme activity only retains 31.22%. After being kept at 70 ℃ and 80 ℃ for 1 h, the enzyme activity drops rapidly, only about 20% of the original. This shows that the residual enzyme activity gradually decreases with the increase of temperature, but it has good stability under low temperature conditions.

[0032] Compared with aminopeptidases from other sources, the optimum temperature is about 60 ℃, but the enzyme activity of aminopeptidases from Antarctic krill can reach half of the maximum enzyme activity at 25 ℃, and the enzyme activity can reach 65% of the maximum enzyme activity at 35 ℃, while the enzyme activity of aminopeptidases from other sources is only 30% of the maximum enzyme activity at 35 ℃. This shows that the leucine aminopeptidase from Antarctic krill has good catalytic ability and substrate affinity at lower temperatures.

[0033] like Figure 5 , Figure 6 As shown in the figure, the optimum pH of the enzyme is 8.5, and it has good stability in the pH range of 8.0-9.0. After incubation in pH 8.5 and pH 9.0 buffers for 4 h, the relative activity of the enzyme is greater than 90%.

[0034] like Figure 7 As shown, Co 2+ It has an activating effect on leucine aminopeptidase EsLAP. When its concentration is only 1 mM, the remaining enzyme activity is 296%. When the working concentration of metal ions is 10 mM, Zn 2+ , Mn 2+ It has a slight activation effect on EsLAP, and the remaining enzyme activities are 114.32% and 109.65% respectively; Cu 2+ 、Ni 2+ , Fe 3+ The residual enzyme activities of the two compounds were 53.67%, 65.32% and 68.44% respectively. + , K + Mg 2+ , Ba 2+ , Ca 2+ The activity of the enzyme was not significantly affected, and the remaining enzyme activity was greater than 90%.

[0035] like Figure 8 As shown in the figure, SDS has almost no effect on the activity of leucine aminopeptidase, and PMSF has little effect on the activity of recombinant leucine aminopeptidase. However, β-mercaptoethanol, NaBH, DTT and EDTA have a very significant inhibitory effect on enzyme activity. When the working concentration of the inhibitor is 10 mM, β-mercaptoethanol, NaBH and DTT can reduce the remaining enzyme activity to less than 10%.

[0036] Example 3 Decomposition ability of leucine aminopeptidase on different substrates Different substrates with a concentration of 10 mM were prepared respectively, including Leu-pNA, Glu-pNA, Arg-pNA, Met-pNA, Lys-pNA, Ile-pNA, Val-pNA, Ala-pNA, and Pro-pNA. 20 μl of each was added to a 200 μl reaction system, and then reacted in a water bath at pH = 8.5 and 60 ℃ for 10 min. Three parallel experiments were performed for each group of substrates, and the relative enzyme activity was determined. The highest enzyme activity under the condition of Leu-pNA as the substrate was 100%.

[0037] like Fig. 9 As shown in the figure, among the 9 aminopeptidase substrates (working concentration 10 mM), the recombinant aminopeptidase has the strongest relative decomposition ability for Leu-pNA (100%), followed by Glu-pNA (42.59%), Arg-pNA (18.32%), Met-pNA (12.96%), Lys-pNA (9.25%), and has no decomposition effect on the remaining 4 substrates, indicating that the aminopeptidase has strong substrate specificity. In addition, it can also be verified that EsLAP has a degradative effect on peptides with leucine, glutamic acid, arginine, methionine, and lysine at the N-terminus, degrading bitter peptides from the N-terminal head one by one into single amino acids or dipeptides, reducing the generation of unpleasant flavors.

[0038] like Fig.10 As shown in the figure, EsLAP uses Leu-pNA as a substrate and catalyzes its decomposition into Leu (leucine) and pNA (p-nitroaniline, yellow), which can verify that EsLAP has a degradation effect on peptides with leucine at the N-terminus. In addition, the enzymatic degradation of these large molecular peptides is more conducive to the body's absorption.

[0039] The amino acid sequence of Antarctic krill leucine aminopeptidase is SEQ ID NO.1. The details are as follows: .

[0040] The nucleotide sequence of Antarctic krill leucine aminopeptidase is SEQ ID NO.2, which is as follows:

[0041] The nucleotide sequence of Antarctic krill leucine aminopeptidase after codon optimization is SEQ ID NO.3, which is as follows:

Claims

1. A leucine aminopeptidase derived from Antarctic krill, characterized in that: The amino acid sequence of the leucine aminopeptidase is shown as SEQ ID NO.

1.

2. The gene encoding the leucine aminopeptidase according to claim 1, characterized in that The nucleotide sequence of the gene is shown as SEQ ID NO.

2.

3. A recombinant plasmid PET28a-EsLAP, characterized in that: The recombinant plasmid contains the gene according to claim 2.

4. A recombinant Escherichia coli engineered bacterium, characterized in that: The recombinant Escherichia coli engineered bacteria contains the recombinant plasmid PET28a-EsLAP according to claim 3.

5. An enzyme preparation, characterized in that The enzyme preparation contains the leucine aminopeptidase according to claim 1.

6. The use of the Antarctic krill-derived leucine aminopeptidase according to claim 1, characterized in that: The application is to use the leucine aminopeptidase to hydrolyze Leu-pNA, Glu-pNA, Arg-pNA, Met-pNA and Lys-pNA, degrade bitter peptides with leucine, glutamic acid, arginine, methionine and lysine at the N-terminus, and degrade the bitter peptides from the N-terminal head one by one into the form of single amino acids or dipeptides, thereby reducing the generation of unpleasant flavors.

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