Trypsin derived from Antarctic krill, its encoding gene and application in degrading plasticizers

Abstract

The present invention relates to a trypsin derived from Antarctic krill, its encoding gene and its application in degrading plasticizers, belonging to the field of enzymology. The amino acid sequence of the trypsin is shown in SEQ ID NO.1, and the amino acid sequence of the gene encoding the trypsin is shown in SEQ ID NO.2. The trypsin of the present invention can degrade plasticizers PAEs, and the plasticizers are dimethyl phthalate, diethyl phthalate, dipropyl phthalate and / or dibutyl phthalate, having very good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of enzymology, and particularly relates to a trypsin derived from Antarctic krill, a coding gene thereof, and an application in degrading plasticizers. Background Art

[0002] Antarctic krill is one of the single-species organisms with a huge biomass in the Southern Ocean and belongs to the zooplankton crustaceans of the order Euphausiacea. In the Antarctic ecosystem, Antarctic krill not only has rich biological resources but also has a special evolutionary status. Antarctic krill contains components such as extremely nutritious proteins, phospholipids, and unsaturated fatty acids. If this resource can be utilized by humans, it will greatly promote the development of marine resources. Among all biological characteristics, Antarctic krill is famous for its unique and efficient protease degradation system in vivo. After the death of Antarctic krill, the inhibition system fails and autolysis occurs rapidly. Antarctic krill lives in the cold Antarctic waters, and the proteases in its body are cold-adapted enzymes with high catalytic activity at low temperatures. Numerous studies have shown that the adaptability of Antarctic krill to extreme environments is closely related to the enzymes in its body, which also provides additional possibilities for Antarctic krill-derived enzymes as industrial biocatalysts. At present, the development and utilization of Antarctic krill-derived enzymes are still in the primary stage, so we need to conduct more in-depth exploration of Antarctic krill-derived enzymes.

[0003] Trypsin (EC 3.4.21.4) is a serine proteolytic enzyme widely present in nature. It can specifically hydrolyze the carboxyl terminus of arginine or lysine, activate other proteaseogens by participating in the enzyme digestion cascade reaction, and belongs to endopeptidase. Trypsin can also catalyze the amide bonds and ester bonds formed by arginine or lysine, thus resulting in two methods for evaluating trypsin enzyme activity: amidase enzyme activity and esterase enzyme activity. Trypsin belongs to the serine protein family, and this family has the same Ser active center, conserved His, Asp, Ser catalytic triad, and the same catalytic mechanism. Trypsin can be found in almost all organisms in nature. It can efficiently and mildly catalyze the hydrolysis of ester compounds and amide substances. Therefore, it has good application value in related fields such as the food industry, agriculture, chemical synthesis, and environmental remediation. Among them, trypsin can degrade phthalate esters (PAEs), which contain ester bonds. PAEs are mainly synthetic and are widely used in the plastics industry. They are easily detached from the matrix and released into the environment. Excessive residues have been detected in various environments. The continuous accumulation of PAEs not only pollutes the environment but also seriously threatens the health of humans and other organisms. Therefore, it is very necessary to remove PAEs from the environment. Compared with physical and chemical methods, bioremediation is a way that can completely degrade and is highly efficient and environmentally friendly. The temperature of different enzymes for hydrolyzing PAEs is generally between 45 - 60 °C, while trypsin from Antarctic krill can maintain a certain activity in a low-temperature environment through a special coping mechanism and has high catalytic efficiency at low temperature, showing more excellent catalytic performance compared with mesophilic enzymes. Trypsin can play a great role in the key step of PAEs degradation - the hydrolysis of ester bonds, and the products generated are monoalkyl phthalate (MNP) or phthalic acid (PTH). PTH has lower toxicity and is easily mineralized by microorganisms in the environment to generate H2O and CO2, which can effectively improve the degradation rate. Therefore, using a rapid and effective screening method to discover trypsin that can efficiently degrade PAEs at low temperature, and through genetic recombination methods, constructing genetically engineered bacteria to highly express trypsin heterologously can reduce the repair cost of PAEs-polluted environments and accelerate the degradation process, having important industrial application value and potential. Summary of the Invention

[0004] The object of the present invention is to provide a trypsin derived from Antarctic krill, its coding gene, and its application in degrading plasticizers. Based on genomic analysis, the present invention mined the TRY gene (EsTRY) of Antarctic krill and heterologously expressed it in Escherichia coli BL21(DE3)-pCold-SUMO, which has higher enzyme activity at low temperature to meet the requirements of industrial production.

[0005] The present invention is achieved by the following technical solutions:

[0006] A trypsin derived from Antarctic krill, wherein the amino acid sequence of the trypsin is as shown in SEQ ID NO.1.

[0007] A gene encoding the trypsin, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO.2.

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

[0009] The present invention also provides a recombinant Escherichia coli engineering bacterium, which contains the recombinant plasmid pCold-SUMO-EsTRY.

[0010] The present invention also provides the application of the trypsin, the recombinant plasmid pCold-SUMO-EsTRY, and the recombinant Escherichia coli engineering bacterium containing the recombinant plasmid in degrading phthalate esters (PAEs). The phthalate esters are dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPRP), and dibutyl phthalate (DBP).

[0011] An enzyme preparation, which contains the trypsin.

[0012] The beneficial effects of the present invention compared with the prior art:

[0013] The present invention obtains the gene sequence of trypsin by using the bioinformatics method of Antarctic krill protein transcriptome, amplifies the target gene in vitro by polymerase chain reaction (PCR), and performs soluble active expression in Escherichia coli, laying a theoretical foundation for the application of trypsin in the field of bioremediation.

[0014] The degradation effect of Antarctic krill trypsin obtained in the present invention on four common plasticizers - dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPRP), and dibutyl phthalate (DBP) was studied. The degradation rate was quantitatively determined by liquid phase, and the degradation rates of 1 mM DMP, DEP, DPRP, and DBP within 0.5 h were 14.8%, 43.6%, 73.9%, and 67.0% respectively; the degradation rates of 1 mM DMP, DEP, DPRP, and DBP within 5 h were 43.1%, 56.9%, 86.8%, and 98.7% respectively. It has very good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the spectrum of recombinant plasmid pCold - SUMO - EsTRY;

[0016] Figure 2 It is the line graph of the effect of temperature on the enzyme activity of trypsin;

[0017] Figure 3 It is the line graph of the effect of pH on the enzyme activity of trypsin;

[0018] Figure 4 It is the line graph of the degradation ability of trypsin on different plasticizers. DETAILED DESCRIPTION OF THE INVENTION

[0019] Unless otherwise specified, 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 all be obtained from commercial channels.

[0020] Example 1: Through the previously explored Antarctic krill genome database (not publicly available), a batch of genomic sequences of trypsin 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 trypsin from other species. A batch of unvalidated functional gene sequences predicted to be putative trypsin or having potential trypsin activity in bioinformatics were selected, and the homology between the amino acid sequences of Antarctic krill and the known source sequences was between 70 - 90%. After further analyzing the domains 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 EsTRY was obtained.

[0021] After large-scale screening and in-depth research, a new trypsin was identified and isolated from Antarctic krill. Its amino acid and nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2. The trypsin can also catalyze the amide bonds and ester bonds formed by arginine or lysine, and has very good application prospects. The trypsin 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 optimized the recombinant expression method of the trypsin, so that it was highly expressed in host cells.

[0022] The inventors first optimized the codons according to the expression situation (the optimized sequence is shown in SEQ ID NO.3) to improve the expression efficiency and the stability of the DNA fragment. The optimized sequence was inserted into the plasmid pCold-SUMO to obtain the recombinant plasmid pCold-SUMO-EsTRY. Subsequently, the recombinant plasmid pCold-SUMO-EsTRY was transformed into Escherichia coli BL21(DE3) competent cells for heterologous expression. The spectrum of the recombinant plasmid pCold-SUMO-EsTRY is as Figure 1 shown.

[0023] Example 2: This example provides the construction process of a recombinant Escherichia coli engineering bacterium with a recombinant expression vector, which is specifically as follows:

[0024] The EsTRY gene was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Using the synthesized EsTRY gene as a template, the EsTRY was amplified with the forward primer 5’- GATCCCATCATCACCATCATCA-3'(SEQ ID NO.4) and the reverse primer 5’-AGCTTTTAATGGTGATGATGATGG-3'(SEQ ID NO.5). The amplified product was purified and double digested with BamHI / HindIII, and then ligated into the pCold-SUMO vector double digested with BamHI / HindIII.

[0025] The recovered plasmid was extracted using a plasmid extraction kit. After gene sequencing, it was transformed into competent Escherichia coli cells, and the transformants were transferred to LB medium (which consists of 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and 50 μg / mL carbenicillin) for culture. The temperature was 37 °C and the oscillation speed was 200 r / min. Using the Escherichia coli bacterial solution as a template, pCold-F (ACGCCATATCGCCGAAAGG, SEQ ID NO.6) and pCold-R (GGCAGGGATCTTAGATTCTG, SEQ ID NO.7) were used as forward and reverse primers, and the annealing temperature was set at 53.9 °C for PCR. 20 μL of the PCR product was sent to Sangon for sequencing verification of the sequence. After correct sequencing, the bacterial solution was transferred to fresh LB medium and cultured with shaking at 37 °C until 600 the OD reached 0.6 - 0.8. 0.55 M IPTG was added for induction culture at 15 °C for 24 h with an oscillation speed of 200 r / min. Then, the cells were collected by centrifugation at 8000 r / min for 10 minutes. After induction culture at 200 r / min for 20 h, the cells were collected by centrifugation at 8000 r / min for 10 minutes. The collected cells were resuspended three times in buffer. After resuspension, PMSF with a final concentration of 1 mM was added, and the mixture was sonicated on ice. The centrifuged supernatant was the crude enzyme solution.

[0026] Trypsin can catalyze the amide bonds and ester bonds formed by the carboxyl groups of basic amino acids, showing selectivity for the carboxyl side of basic amino acids. The sensitivity of the catalytic hydrolysis activity of these chemical bonds is: ester bond > amide bond > peptide bond. Therefore, trypsin activity is usually related to the esterase and amidase activities of the synthetic substrates Nα-benzoyl-l-arginine ethyl ester (BAEE) and Nα-benzoyl-decylalanine-p-nitroanilide (BAPAN). The esterase activity of the sample was measured as follows: 100 μL of the sample solution was immediately mixed with 900 μL of the substrate solution (67 mM, pH 7.6 sodium phosphate buffer containing 0.25 mM BAEE) in a quartz test tube at 25 °C. The change in absorbance at 253 nm was monitored by spectrophotometry for 5 min. One unit (U) of esterase was defined as the amount of enzyme that caused ∆A 253 to increase by 0.001 per minute. The amidase activity of the sample was measured as follows: 100 μL of the sample solution was mixed with 800 μL of the detection buffer (50 mM, pH 8.0 Tris-HCl, 40 °C) and 100 μL of 10 mM BAPAN (in DMSO). The change in absorbance at 410 nm was monitored by spectrophotometry for 5 min. One unit (U) of amidase was defined as the amount of enzyme that caused ∆A 410Increase the amount of enzyme by 0.1. Enzyme properties: (1) Optimum temperature determination: The enzyme activity of EsTRY was measured at different temperatures of 15 ℃, 20 ℃, 22 ℃, 25 ℃, 27 ℃, 30 ℃, 35 ℃, and 40 ℃. Three parallels were made for each temperature group to measure the relative enzyme activity, with the highest enzyme activity measured at 25 ℃ being 100%.

[0027] (2) Determination of optimal pH: The effect of pH on enzyme activity was analyzed from the change of pH. Acetic acid-sodium acetate buffer (pH 4.0-6.0), phosphate buffer (pH 6.5-7.5), tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 8.0-9.0) and glycine sodium hydroxide buffer (pH 9.5-11.0) were prepared. The reaction system was placed at 25°C for 5 min. Three parallels were made for each temperature group. The relative enzyme activity was measured, and the highest enzyme activity at pH 7.0 was taken as 100%.

[0028] (3) Degradation ability of trypsin on four plasticizers:

[0029] Four PAEs with a concentration of 100 mM were prepared, namely dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPRP) and dibutyl phthalate (DBP). 10 μL of each was added to 1 ml reaction system, and then reacted in a water bath at pH = 7.0 and 25 ℃ for 5 h. Samples were taken every 1 h to detect the concentration of PAEs. Three parallel experiments were performed for each group of substrates to determine the degradation rate. The reaction with the addition of inactivated enzyme solution and other conditions unchanged was used as the control group.

[0030] The above reaction results are shown in the figure. Figure 2 It can be seen that the optimum reaction temperature of the enzyme is 25℃. As the reaction temperature increases from 15℃ to 25℃, the enzyme activity of EsTRY increases all the time. When the temperature is 25℃, the enzyme activity reaches the highest value. Then, as the temperature gradually increases, the catalytic activity of EsTRY decreases significantly. The optimum temperature of trypsin from different sources is generally 45℃, indicating that trypsin from Antarctic krill has higher activity at low temperatures.

[0031] like Figure 3 As shown in the figure, the optimum pH is 7.0. Between pH 4.0 and 7.0, the catalytic activity of the enzyme increases with the increase of pH, and reaches the maximum at pH 7.0. As the pH further increases from 7.0 to 11.0, the catalytic activity of EsTRY decreases, indicating that the enzyme belongs to a neutral protease.

[0032] like Figure 4As shown in the figure, among the 4 PAEs substrates (working concentration 1 mM, the substrates are dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPRP) and dibutyl phthalate (DBP)), after being treated with the recombinant enzyme EsTRY at 25 °C for 1 - 5 h respectively, the concentrations of each plasticizer decreased to varying degrees, that is, the degradation rate gradually increased with the extension of time. The degradation rate of EsTRY for DBP was the highest, reaching 67.0% within 30 min, 79.9% within 1 h, and 98.7% within 5 h. Within 30 min, the degradation rates of EsTRY for DMP, DEP, and DPRP reached 14.8%, 43.6%, and 73.9% respectively, and the degradation rates within 5 h reached 39.4%, 56.9%, and 86.8% respectively. Camelimonas sp. M11 had degradation rates of 72% and 56% for 0.1 mM DEP and DBP within 72 h; the esterase from Sulfobacillus acidophilus had degradation rates of 6%, 10%, and 8% after reacting with DEP, DMP, and DPRP for 1 h; the PAEs hydrolase DphB from the esterase family IV had degradation rates of less than 5% after reacting with 0.1 mM DMP and DEP at 37 °C for 8 h; the carboxylesterase from Bacillus bestii had degradation rates of 25%, 50%, 94.8%, and 88.4% after reacting with 1 mM DMP, DEP, DPRP, and DBP at 45 °C for 5 h.

[0033] In contrast, the trypsin obtained in this example had higher degradation rates for DMP, DEP, DPRP, and DBP at low temperature, proving that the recombinant enzyme EsTRY has good application potential in repairing PAEs - polluted environments.

[0034] The amino acid sequence of Antarctic krill trypsin is as follows (SEQ ID NO.1):

[0035] MSAAVVPCCVLATATALPDGFSVQPRPDFTIQPRIPHFKLPAFVPRSTISCSRKLVKTTLAPGESATFTSPNYPSDYSSKSVCKWRFTSTTDITITCSDFLLQSANKKGNCKDKLTIDGTKFCGSSLSSYSGDKSINVVFKSNKKKNFDGFSCTAIAESDTTVTTAAPETTAAPETTAAPETTAAPGTTAAPGSCKCGQANPGTRIVGGTETEVLEYPWQAAMVYSGSTSVFCGASVIGSKHILTAAHCTQAVTDYSINYQVLVGAHSRTSAASSQQLLDASTFIQNSGYSDSTYDNDIAIIVLSSSIDFTSTDIRPVCLPTSDADDYDSVIATVSGWGALEYQGDQPDVLMEVDVPTMTNTKCDSYYGGSITSNMLCAGFQAGGKDSCQGDSGGPLIYNSGSGYTQIGVVSWGNGCAWANYPGVYARVTEYLSWISANSASSTTCPAA。

[0036] The nucleotide sequence of Antarctic krill trypsin is as follows (SEQ ID NO.2):

[0037]

[0038] The optimized nucleotide sequence of Antarctic krill trypsin is as follows (SEQ ID NO.3):

[0039]

Claims

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

1.

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

2.

3. A recombinant plasmid pCold-SUMO-EsTRY, characterized in that: The recombinant plasmid is obtained by inserting the gene according to claim 2 into pCold-SUMO.

4. A recombinant Escherichia coli engineered bacterium, characterized in that: The recombinant Escherichia coli engineered bacteria contains the recombinant plasmid pCold-SUMO-EsTRY described in claim 3.

5. The use of trypsin according to claim 1 in degrading plasticizer PAEs, characterized in that: The plasticizer is dimethyl phthalate, diethyl phthalate, dipropyl phthalate and / or dibutyl phthalate.

6. An enzyme preparation, characterized in that The enzyme preparation contains the trypsin according to claim 1.

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