Carboxylesterase derived from euphausia superba, coding gene and application of carboxylesterase

Abstract

The invention relates to euphausia superba-sourced carboxylesterase, a coding gene and application thereof, and belongs to the field of enzymology, the amino acid sequence of the carboxylesterase is shown as SEQ ID NO.1, and the amino acid sequence of the gene for coding the carboxylesterase is shown as SEQ ID NO.2 or SEQ ID NO.3. The carboxylesterase can degrade a plasticizer PAEs, and the carboxylesterase can be used for degrading the plasticizer PAEs. And the plasticizer is at least one of dimethyl phthalate, diethyl phthalate, dipropyl phthalate and 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 a carboxylesterase derived from Antarctic krill, a coding gene and an application thereof. Background Art

[0002] Antarctic krill contains extremely rich nutritional value such as protein, phospholipids, unsaturated fatty acids, etc. If this resource can be used by humans, it will greatly promote the development of marine resources. 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 in-depth research on Antarctic krill-derived enzymes.

[0003] Carboxylesterase (EC 3.1.1.1) is a non-specific ester hydrolase that can act on the carboxylic acid ester bond in the substrate to generate alcohol and carboxylic acid. It is one of the important members of the α / β hydrolase family. Carboxylesterase is divided into eight families according to its sequence similarity and enzymatic characteristics. The amino acid sequence contains a conserved catalytic triad and pentapeptide domain. Carboxylesterase can be found in almost all organisms in nature. It can efficiently and gently catalyze the hydrolysis, synthesis and exchange of ester compounds and the hydrolysis of amides. Therefore, it has good application value in related fields such as food industry, agriculture, chemical synthesis and environmental remediation. Among them, carboxylesterase can degrade plasticizers containing ester bonds - phthalates (PAEs). 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 in the environment. Compared with physical and chemical methods, bioremediation is a way that can completely degrade, is efficient and environmentally friendly. Carboxylesterase can play a major role in the hydrolysis of ester bonds, the key step in the degradation of PAEs. The resulting products are monoalkylphthalate (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 increase the degradation rate. Therefore, using a fast and effective screening method to discover carboxylesterases that can efficiently degrade PAEs, and constructing genetically engineered bacteria through genetic recombination methods to efficiently heterologously express carboxylesterases can reduce the cost of remediation of PAEs-contaminated environments 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 a carboxylesterase from Antarctic krill, a coding gene and its application in degrading plasticizers. The present invention mines the Antarctic krill CaEs gene (EsCaEs) based on genome analysis and heterologously expresses it in Bacillus subtilis WB600-PMA5, which has higher enzyme activity to meet the needs of industrial production.

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

[0006] The gene encoding the carboxylesterase, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.2 or SEQ ID NO.3.

[0007] A recombinant plasmid PMA5-EsCaEs, wherein the recombinant plasmid contains the nucleotide sequence shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant Bacillus subtilis engineering bacterium, wherein the recombinant Bacillus subtilis engineering bacterium contains the recombinant plasmid PMA5-EsCaEs.

[0009] The present invention also provides the use of the carboxylesterase, the recombinant plasmid PMA5-EsCaEs, and the recombinant plasmid and recombinant Bacillus subtilis engineering bacteria in degrading plasticizer PAEs, wherein the plasticizer is at least one of dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPRP) and dibutyl phthalate (DBP).

[0010] An enzyme preparation, comprising the carboxylesterase.

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

[0012] The Antarctic krill carboxylesterase obtained by the present invention has degradation effects on four common plasticizers, namely dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPRP) and dibutyl phthalate (DBP). The degradation rate is quantitatively determined by liquid phase, and the degradation rates of 1 mM dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPRP) and dibutyl phthalate (DBP) are 28.9%, 53.4%, 82.4% and 95.2% respectively within 0.5 h; and the degradation rates of 1 mM dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPRP) and dibutyl phthalate (DBP) are 43.1%, 67.9%, 95.8% and 98.9% respectively within 5 h. It has very good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a line graph showing the effect of temperature on carboxylesterase activity; Figure 2 is a bar graph showing the effect of temperature on the stability of carboxylesterase; Figure 3 It is a line graph showing the effect of pH on the activity of carboxylesterase; Figure 4 is a bar graph showing the effect of pH on the stability of carboxylesterase; Figure 5 It is a bar graph showing the effect of metal ions on the activity of carboxylesterase; Figure 6 It is a bar graph showing the effect of enzyme inhibitors on carboxylesterase activity; Figure 7 It is a line graph of the degradation ability of carboxylesterase 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 carboxylesterase 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 carboxylesterases from other species were compared by using the Blast analysis tool in the NCBI database. A batch of unverified functional gene sequences predicted to be putative carboxylesterases or having potential carboxylesterase activity in bioinformatics were selected, and the homology of the amino acid sequence of Antarctic krill with the sequence of known sources 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 EsCaEs was obtained.

[0016] After large-scale screening and in-depth research, a new carboxylesterase 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, which can catalyze the hydrolysis of carboxylic acid ester bonds to generate alcohol and acid, and has very good application prospects. The carboxylesterase of the present invention has good adaptability to low temperatures and can be expressed in subtilis spore cells. At the same time, the present invention also optimizes the recombinant expression method of the carboxylesterase, so that it can be efficiently expressed in host cells.

[0017] The inventors firstly performed codon optimization according to the expression situation to improve the expression efficiency and the stability of the DNA fragment. The optimized nucleotide sequence is shown in SEQ ID NO.3.

[0018] The optimized sequence was inserted into plasmid PMA5 to obtain recombinant plasmid PMA5-EsCaEs, which was then transformed into Bacillus subtilis WB600 competent cells for heterologous expression.

[0019] Example 2: This example provides a process for constructing a recombinant Bacillus subtilis engineering bacterium of a recombinant expression vector, as follows: The EsCaEs gene was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The synthesized EsCaEs gene was used as a template to amplify EsCaEs using forward primer 5'-GATCCATGCATCACCATCATC-3' (SEQ ID NO. 4) and reverse primer 5'-CTAGCTTAATGATGATGGTGATGATGGC-3' (SEQ ID NO. 5). The amplified product was purified and double-digested with BamHI / NheI, and then ligated into the PMA5 vector after double-digestion with BamHI / NheI.

[0020] The plasmid was recovered using a plasmid extraction kit. After gene sequencing, it was transformed into Escherichia coli competent cells, and the transformant was transferred to LB medium for culture (the medium consisted of 10g / L peptone, 5g / L yeast extract, 10g / L NaCl, and 50 μg / mL penicillin). The temperature was 37°C, the shaking speed was 200 r / min, Bacillus subtilis liquid was used as a template, PMA5-7164F (AATGCCTCACATTTGTGCCACCT, SEQ ID NO.6) and PMA5-253R (GGCTCCAAAAGGAGCCTTTAATTGTATCGGT, SEQ ID NO.7) were used as front and back primers, the annealing temperature was set to 59.8°C, PCR was performed, and 20 μL of PCR product was sent to Biotech for sequencing to verify the sequence. After sequencing is correct, transfer the bacterial solution to fresh LB medium and culture at 37℃ with shaking speed of 200 r / min. After 20 hours of induction culture, centrifuge at 8000 r / min for 10 minutes to collect the bacteria, and resuspend the collected bacteria in buffer three times. After resuspension, add PMSF with a final concentration of 1 mM, place on ice for ultrasonic disruption, and the supernatant of centrifugation is the crude enzyme solution.

[0021] The enzyme activity of recombinant carboxylesterase was determined by the α-naphthol method. Carboxylesterase catalyzes α-naphthyl acetate to generate naphthol, which further reacts with the fast blue color developer to generate a colored substance. The enzyme activity of the recombinant carboxylesterase was characterized by detecting the rate of increase in light absorption of the colored substance at 600 nm. 1 mL of 50 mM Tris-HCl buffer (pH: 8.0) reaction system contained 5ul enzyme solution and 3 mM α-naphthyl acetate, and reacted at 37 ℃ for 5 min. 150 ul DBLS (1% Fast Blue B salt: 5% SDS = 2:5 mixture) was added to terminate the reaction. The reaction was allowed to react at room temperature for 5 min. The absorbance value was read at a wavelength of 600 nm. The yield of α-naphthol produced by the reaction was calculated using the α-naphthol standard curve. Enzyme activity unit definition: The amount of enzyme required to catalyze the production of 1 µmol of α-naphthol in 1 min was defined as one enzyme activity unit. All tests were repeated three times at 37 ℃.

[0022] Enzyme properties: (1) Optimum temperature determination: The enzyme activity of EsCaEs was measured at different temperatures of 10°C, 20°C, 30°C, 40°C, 45°C, 50°C, 55°C, 60°C, and 70°C. Three replicates were performed for each temperature group, and the relative enzyme activity was measured, with the highest enzyme activity measured at 45°C being 100%.

[0023] (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 5.0-7.0), tris(hydroxymethylaminomethane) hydrochloride buffer (pH 7.5-8.5) and glycine sodium hydroxide buffer (pH 9.0-10.0) were prepared. The reaction system was placed in a 45°C water bath for 30 min. Three parallels were made for each temperature group. The relative enzyme activity was measured, and the highest enzyme activity at pH 9 was set as 100%.

[0024] (3) Temperature stability determination: The purified enzyme solution was placed in a metal bath at 0°C, 10°C, 20°C, 30°C and 40°C for 180 min. The enzyme activity was determined at pH 9. Three parallel experiments were performed for each time group. The relative enzyme activity was determined, with the highest enzyme activity without temperature treatment being 100%.

[0025] (4) pH stability determination: The purified enzyme solution was placed in a buffer solution at 0°C and pH 5-9 for 180 min. The enzyme activity was determined at 45°C. Three parallel assays were performed for each pH group. The relative enzyme activity was determined, with the highest enzyme activity without pH treatment being taken as 100%.

[0026] (5) Effect of metal ions on the enzyme activity of recombinant EsCaEs: Various metal ions K⁺, Ca²⁺, Na⁺, Mg²⁺, AI³⁺, Zn²⁺, Fe²⁺, Fe³⁺, Mn²⁺, Cu²⁺, Ni²⁺, and Co²⁺ were prepared with ddH2O at a concentration of 1 M. The enzyme solution was placed in a 10 mM metal ion system at 4°C for 180 min, and then reacted in a water bath at pH = 9 and 45°C for 5 min. Three parallel reactions 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%.

[0027] (6) Effects of enzyme inhibitors on the activity of recombinant EsCaEs Use ddH2O to prepare 100mM urea, EDTA, DTT, SDS, PMSF, NaBH4 and β-mercaptoethanol. Place the enzyme solution in a 10mM inhibitor system at 4℃ for 180 min, then react in a water bath at pH=9 and 45℃ for 5 min. Perform 3 parallels for each temperature group and measure the relative enzyme activity. The highest enzyme activity under standard buffer conditions is taken as 100%.

[0028] (7) Degradation ability of carboxylesterase on four plasticizers: Four kinds of PAEs with a concentration of 100 mM were prepared respectively, 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 = 9 and 45°C 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.

[0029] The above reaction results are shown in the figure. Figure 1 , 2 It can be seen that the optimum reaction temperature of the enzyme is 45℃. As the reaction temperature increases from 10℃ to 70℃, the enzyme activity of EsCaEs has been increasing. When the temperature is 45℃, the enzyme activity reaches the highest value. Then, as the temperature gradually increases, the catalytic activity of EsCaEs decreases significantly. The temperature stability of EsCaEs was tested and it was found that the enzyme was highly stable at 0℃ and could maintain more than 98% of the original activity after incubation for 3 hours. After incubation for 4 hours at 10℃, 20℃, 30℃ and 40℃, the enzyme activity was 93%, 85%, 69% and 33% of the original activity, respectively, indicating that the enzyme has low stability in an environment greater than 20℃, but can maintain high stability at low temperatures.

[0030] like Figure 3 , 4 As shown, the optimum pH is 9. Between pH 5.0 and 9.0, the catalytic activity of the enzyme increases with increasing pH. At pH 9.0, the enzyme activity reaches a maximum value of 3794.6 U / mg. As the pH further increases from 9.0 to 10.0, the catalytic activity of EsCaEs decreases. Figure 4 The stability of EsCaEs under different pH conditions was shown. After incubation at pH 9.0 for 4 h, the enzyme activity could be maintained at 96% of its original activity. At pH 5.0, 6.0, 7.0, 7.5 and 8.0, the enzyme retained activity after incubation for 3 h was 0%, 8%, 2%, 30%, 70% and 49%, respectively.

[0031] like Figure 5 , Figure 6 The results show the effects of 10 mmol / L metal ions and enzyme inhibitors on the activity of EsCaEs enzyme. After 3 hours of treatment with 10 mmol / L metal ions, K + , Ca 2+ 、Na + , Fe 3+The relative enzyme activities of Al 3+ , Fe 2+ , Mn 2+ , Cu 2+ 、Ni 2+ 、Co 2+ The enzyme activity was significantly inhibited by Mg and Mg. The relative retained enzyme activities were 4%, 3%, 67%, 0%, 32% and 28%, respectively. 2+ It has a slight inhibitory effect on enzyme activity, with a relative retained enzyme activity of 85%.

[0032] like Figure 6 As shown in the figure, after treatment with 10 mmol / L enzyme inhibitor for 3 hours, 10mM EDTA, DTT, SDS, PMSF, and β-mercaptoethanol had a significant inhibitory effect on the enzyme activity, with the relative retained enzyme activities being 30%, 0%, 47%, 7%, and 7%; 10mM urea and NaBH4 had a slight inhibitory effect on the enzyme activity, with the relative retained enzyme activities being 69% and 83%.

[0033] like Figure 7 As shown in the figure, among the four PAEs substrates (working concentration 1 mM, the substrates were dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPRP) and dibutyl phthalate (DBP)), after being treated with the recombinant enzyme EsCaEs for 1-5 h, the concentration of each plasticizer was reduced to varying degrees, that is, the degradation rate gradually increased with time. EsCaEs had the highest degradation rate for DBP, reaching 89.3% within 15 min, 95.7% within 1 h, and 98.9% within 5 h. Within 15 min, the degradation rates of EsCaEs for DMP, DEP, and DPRP were 19.1, 49.1, and 75.3%, respectively, and within 5 h, the degradation rates were 43.1, 67.9, and 95.8%, 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 were 6, 10 and 8% respectively after reacting with DEP, DMP and DPRP for 1 h; 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°C; the degradation rates of carboxylesterase from Bacillus bestensis were 25, 50, 94.8 and 88.4% respectively after reacting with 1 mM DMP, DEP, DPRP and DBP for 5 h.

[0034] In contrast, the carboxylesterase obtained in this paper has higher degradation rates and degradation rates for DMP, DEP, DPRP and DBP, proving that the recombinant enzyme EsCaEs has great application potential in remediating PAEs-contaminated environments.

[0035] The amino acid sequence of Antarctic krill carboxylesterase is as follows (SEQ ID NO.1): MGLKDQTLALKWVKNNIHAFGGDNDRITIFGESAGSAVHFHLLTPYSKGLFHGAIMQSGSALSPWATGRDFNWTCTEIAKRFGCPVKPSEDLVRCLQRVDH RRLDEMYYYFMEWNTQPFYFAPRIDGDFLPPDEPATLLKNGRYKNVPVIMGINRDEGALESAEMYTFPFAIKSLQNHFKENGPKSLLLLEDEDPVNTAAKVYGR YLNGVFITEKNADNLTQLFSDRLFLVPLDWSAELMASHGPVFTYELHHKGQYGYTSLMHLLGLRLPQAEHWISHGDDLMYVMDILGRYWLKINTPEDLAVREIFTTTWTNFARTRIPTPDSSLGVNWTVTGGKDALCHLHIVPTPYMENDQRKSTRTFWESLPLRINKLINGKYPCYDKQRPKNNIIDINSNKEVSLNESISTLC.

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

[0037] The optimized nucleotide sequence of Antarctic krill carboxylesterase is as follows (SEQ ID NO.3):

Claims

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

1.

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

3.

3. A recombinant plasmid PMA5-EsCaEs, characterized in that: The recombinant plasmid PMA5-EsCaEs contains the gene according to claim 2.

4. A recombinant Bacillus subtilis engineered bacterium, characterized in that: The recombinant Bacillus subtilis engineered bacteria contains the recombinant plasmid PMA5-EsCaEs described in claim 3.

5. The use of the carboxylesterase according to claim 1 in degrading plasticizer PAEs, characterized in that: The plasticizer is at least one of dimethyl phthalate, diethyl phthalate, dipropyl phthalate and dibutyl phthalate.

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

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