Euphausia superba-derived trypsin, coding gene and application of trypsin to degradation of plasticizer

Abstract

The invention relates to trypsin derived from euphausia superba, a coding gene and application of the trypsin to degradation of plasticizers, and belongs to the field of enzymology, the amino acid sequence of the trypsin is shown as SEQ ID NO.1, and the amino acid sequence of the gene for coding the trypsin is shown as SEQ ID NO.2. The trypsin can degrade the plasticizers PAEs, and the trypsin can be used for degrading the plasticizers PAEs. And the plasticizer is dimethyl phthalate, diethyl phthalate, dipropyl phthalate and / or dibutyl phthalate, and has a very good application prospect.

Description

Technical Field

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

[0002] Antarctic krill is one of the single species with huge biomass in the Southern Ocean, belonging to the order Euphausiaceae planktonic crustaceans. In the Antarctic ecosystem, Antarctic krill not only has rich biological resources, but also has a special evolutionary status. Antarctic krill contains extremely nutritious 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. Among all biological characteristics, Antarctic krill is known for its unique and efficient protease degradation system in the body. After the death of Antarctic krill, the inhibition system fails and autolysis occurs rapidly. Antarctic krill lives in the cold Antarctic waters, and the protease in its body is a cold-adaptive enzyme with high catalytic activity at low temperatures. Many 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 is still in its early stages, so we need to conduct more and deeper research on Antarctic krill-derived enzymes.

[0003] Trypsin (EC 3.4.21.4) is a serine protease widely found in nature. It can specifically hydrolyze the carboxyl terminus of arginine or lysine and activate other proteases by participating in the enzyme digestion cascade reaction. It is an endopeptidase. Trypsin can also catalyze the formation of amide bonds and ester bonds formed by arginine or lysine, resulting in two methods for evaluating trypsin activity: amidase activity and esterase activity. Trypsin belongs to the serine protein family, which 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 gently catalyze the hydrolysis of ester compounds and amide substances. Therefore, it has good application value in related fields such as food industry, agriculture, chemical synthesis and environmental remediation. Among them, trypsin can degrade phthalates (PAEs), a plasticizer containing ester bonds. It is mainly synthesized and widely used in the plastics industry. It is easy to detach from the matrix and release into the environment. Excessive residues have been detected in many 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, be efficient and environmentally friendly. The temperature of different PAEs hydrolyzing enzymes is generally 45-60 ℃, and Antarctic krill trypsin can maintain a certain activity in low temperature environments through a special response mechanism. It has high catalytic efficiency at low temperatures and exhibits superior catalytic performance relative to medium-temperature enzymes. Trypsin can play a major role in the hydrolysis of ester bonds, the key step in the degradation of PAEs. The generated products are monoalkyl phthalate (MNP) or phthalic acid (PTH). PTH is less toxic and can be easily mineralized by microorganisms in the environment to generate H 2 O and CO 2 , which can effectively increase the degradation rate. Therefore, using a fast and effective screening method to discover trypsin that can efficiently degrade PAEs at low temperature, and constructing genetically engineered bacteria through gene recombination methods to efficiently heterologously express trypsin can reduce the cost of repairing the PAEs-contaminated environment and accelerate the degradation process, which has important industrial application value and potential. Summary of the invention

[0004] The purpose of the present invention is to provide an Antarctic krill-derived trypsin, a coding gene and its application in degrading plasticizers. The present invention mines the Antarctic krill TRY gene (EsTRY) based on genome analysis and heterologously expresses it in Escherichia coli BL21 (DE3)-pCold-SUMO, which has higher enzyme activity at low temperatures to meet industrial production needs.

[0005] The present invention is achieved through the following technical solutions: A trypsin derived from Antarctic krill, the amino acid sequence of the trypsin is shown as SEQ ID NO.1.

[0006] The gene encoding the trypsin, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0007] A recombinant plasmid pCold-SUMO-EsTRY contains the nucleotide sequence shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant Escherichia coli engineering bacterium, wherein the engineering bacterium contains the recombinant plasmid pCold-SUMO-EsTRY.

[0009] The present invention also provides the use of the trypsin, the recombinant plasmid pCold-SUMO-EsTRY, and the recombinant plasmid recombinant Escherichia coli engineering bacteria in degrading plasticizer PAEs. The plasticizers are dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPRP) and dibutyl phthalate (DBP).

[0010] An enzyme preparation contains the trypsin.

[0011] The beneficial effects of the present invention compared with the prior art are as follows: The present invention uses the Antarctic krill protein transcriptome bioinformatics method to obtain the gene sequence of trypsin, amplifies the target gene in vitro through polymerase chain reaction (PCR), and expresses the soluble active gene in Escherichia coli, laying a theoretical foundation for the application of trypsin in the field of bioremediation.

[0012] The Antarctic krill trypsin obtained by the invention has a degradation effect on four common plasticizers, namely dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPRP) and dibutyl phthalate (DBP). The degradation rates were quantitatively determined by liquid phase. The degradation rates of 1 mM dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPRP) and dibutyl phthalate (DBP) were 14.8%, 43.6%, 73.9% and 67.0% respectively within 0.5 h; the degradation rates of 1 mM dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPRP) and dibutyl phthalate (DBP) were 43.1%, 56.9%, 86.8% and 98.7% respectively within 5 h. It has very good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the spectrum of the recombinant plasmid pCold-SUMO-EsTRY; Figure 2 It is a line graph of the effect of temperature on trypsin activity; Figure 3 It is a line graph showing the effect of pH on trypsin activity; Figure 4 This is a line graph showing the degradation ability of trypsin on different plasticizers. DETAILED DESCRIPTION

[0014] 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.

[0015] Example 1: A batch of trypsin genomic sequences 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 trypsins 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 trypsins or have potential trypsin activity in bioinformatics were selected, and the homology of the Antarctic krill amino acid sequence to 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 candidate sequence was designed with front and rear primers, 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 EsTRY was obtained.

[0016] After large-scale screening and in-depth research, a new trypsin 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. Trypsin can also catalyze 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 temperatures and can be expressed in Escherichia coli cells. At the same time, the present invention also optimizes the recombinant expression method of the trypsin, so that it can be efficiently expressed in host cells.

[0017] The inventors first performed codon optimization according to the expression situation (the optimized sequence is shown in SEQ ID NO.3) to improve the expression efficiency and stability of the DNA fragment. The optimized sequence was inserted into the plasmid pCold-SUMO to obtain the recombinant plasmid pCold-SUMO-EsTRY, and then 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 shown in Figure 1 shown.

[0018] Example 2: This example provides a construction process of a recombinant Escherichia coli engineering bacterium of a recombinant expression vector, as follows: EsTRY gene was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The synthesized EsTRY gene was used as a template to amplify EsTRY with forward primer 5'- GATCCCATCATCACCATCATCA-3' (SEQ ID NO.4) and 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 after double-digestion with BamHI / HindIII.

[0019] The plasmid was recovered using a plasmid extraction kit. After gene sequencing, it was transformed into Escherichia coli 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, and the Escherichia coli bacterial solution was used as a template. pCold-F (ACGCCATATCGCCGAAAGG, SEQ ID NO.6) and pCold-R (GGCAGGGATCTTAGATTCTG, SEQ ID NO.7) were used as front and back primers. The annealing temperature was set to 53.9 °C for PCR, and 20 μL of the PCR product was sent to Bioengineering for sequencing to verify the sequence. After the sequencing was correct, the bacterial solution was transferred to fresh LB medium and cultured at 37 °C for shaking until the OD 600 Reach 0.6-0.8. Add 0.55 M IPTG and carry out induction culture at 15 ℃ for 24 h, with an oscillation speed of 200 r / min, and then centrifuge at 8000 r / min for 10 minutes to collect the bacteria, with an oscillation speed of 200 r / min. After 20 h of induction culture, centrifuge at 8000 r / min for 10 minutes 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.

[0020] Trypsin can catalyze the formation of amide and ester bonds by the carboxyl groups of basic amino acids, and the sensitivity of the catalytic hydrolysis activity to the carboxyl side of basic amino acids is as follows: ester bond > amide bond > peptide bond. Therefore, trypsin activity is usually esterase and amidase activity with synthetic substrates Nα-benzoyl-l-arginine ethyl ester (BAEE) and Nα-benzoyl-debutyryl-p-nitroanilide (BAPAN). The esterase activity of the sample is determined as follows: 100 µL of sample solution and 900 µL of substrate solution (67 mM, pH 7.6 sodium phosphate buffer containing 0.25 mM BAEE) are immediately mixed in a quartz cuvette at 25 °C. The change in absorbance at 253 nm after 5 min is monitored by spectrophotometry. One unit (U) of esterase is defined as the amount of esterase that makes ∆A 253 The amount of enzyme was increased by 0.001 per minute. The amidase activity of the samples was determined as follows: 100 µL of sample solution was mixed with 800 µL of assay 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 spectrophotometrically after 5 min. One unit (U) of amidase was defined as the amount of enzyme increased by ∆A per minute. 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%.

[0021] (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%.

[0022] (3) Degradation ability of trypsin on four plasticizers: 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.

[0023] 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.

[0024] 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.

[0025] like Figure 4As shown in the figure, in four 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 recombinant enzyme EsTRY at 25 ℃ for 1-5 h, the concentration of each plasticizer was reduced to varying degrees, that is, the degradation rate gradually increased with time. EsTRY had the highest degradation rate for DBP, 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 were 14.8%, 43.6%, and 73.9%, respectively, and within 5 h, the degradation rates were 39.4%, 56.9%, and 86.8%, respectively. The degradation rates of Camelimonas sp.M11 for 0.1 mM DEP and DBP were 72% and 56% respectively within 72 h; the degradation rates of the esterase from Sulfobacillus acidophilus after reacting with DEP, DMP and DPRP for 1 h were 6, 10 and 8% respectively; the degradation rates of PAEs hydrolase DphB from esterase family IV were less than 5% after reacting with 0.1 mM DMP and DEP for 8 h at 37 ℃; the degradation rates of carboxylesterase from Bacillus bestensis after reacting with 1 mM DMP, DEP, DPRP and DBP at 45 ℃ for 5 h were 25, 50, 94.8 and 88.4% respectively.

[0026] In contrast, the trypsin obtained in this example has a higher degradation rate for DMP, DEP, DPRP and DBP at low temperature, which proves that the recombinant enzyme EsTRY has a good application potential in repairing PAEs-contaminated environments.

[0027] The amino acid sequence of Antarctic krill trypsin is as follows (SEQ ID NO.1): MSAAVVPCCVLATATALPDGFSVQPRPDFTIQPRIPHFKLPAFVPRSTISCSRKLVKTTLAPGESATFTSPNYPSDYSSKSVCKWRFTSTTDITITCSDFLLQSANKKGNCKDKLTIDGTKFCGSSLSSYSGDKSINVVFKSNKKKNFDGFSCTAIAESDTTVTTAAPETTAAPETTAAPETTAAPGTTAAPGSCKCGQANPGTRIVGGTETEVLEYPWQAAMVYSGSTSVFCGASVIGSKHILTAAHCTQAVTDYSINYQVLVGAHSRTSAASSQQLLDASTFIQNSGYSDSTYDNDIAIIVLSSSIDFTSTDIRPVCLPTSDADDYDSVIATVSGWGALEYQGDQPDVLMEVDVPTMTNTKCDSYYGGSITSNMLCAGFQAGGKDSCQGDSGGPLIYNSGSGYTQIGVVSWGNGCAWANYPGVYARVTEYLSWISANSASSTTCPAA。

[0028] The nucleotide sequence of Antarctic krill trypsin is as follows (SEQ ID NO.2): The optimized nucleotide sequence of Antarctic krill trypsin is as follows (SEQ ID NO.3):

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 pCold-SUMO-EsTRY 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 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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