PS06828 esterase for degrading phthalates and its application

Abstract

The present invention discloses a PS06828 esterase for degrading phthalates and its application. The nucleotide sequence of the gene encoding the PS06828 esterase is shown in SEQ ID NO: 1, and the amino acid sequence of the PS06828 esterase protein is shown in SEQ ID NO: 2. The PS06828 esterase belongs to the carboxylesterase in the VI class lipase family. The kinetic curve measurement results of the PS06828 esterase degrading DBP show that the enzyme has a very strong degradation ability within 5 minutes, and the degradation rate reaches 53.35%; in addition, the optimum temperature of the PS06828 esterase is 30°C, and the relative activity of the enzyme is above 45% in the range of 25°C to 35°C. The optimal pH value is 7, and it shows a higher activity (>50%) when the pH value is in the range of 7 to 8, and has high activity and stability in a wide range of temperature and pH values.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a PS06828 esterase for degrading phthalates and an application thereof. Background Art

[0002] Phthalate esters (PAEs) are a typical synthetic organic compound synthesized by the dehydration reaction of phthalic anhydride and ethanol. PAEs have been widely used in plastic products such as food packaging, children's toys, medical equipment and agricultural films, and are a common plasticizer for plastics. Long-term exposure to PAEs may cause dysfunction of human reproductive, nervous and immune systems. Studies have found that continuous exposure to certain PAEs pollution can lead to decreased male reproductive capacity, initiate apoptosis of neocortical neurons, and even cause childhood obesity, metabolic disorders and delayed language development. Among them, dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) are commonly used plasticizers in plastics, with the highest detection rates in pollution sources and related environmental matrices, at 90.28% and 93.75% respectively. In addition, the DBP content detected in most areas and the DEHP content in a few areas have exceeded the relevant control standards for PAEs in the United States. Given that microorganisms, especially bacteria, have the advantages of complete pollutant degradation and polluted environmental remediation, low treatment cost, high safety and little environmental disturbance, bacteria-driven biodegradation is considered to be the most promising remediation strategy.

[0003] In the initial stage of bacterial degradation of PAEs, the main concern is the catalytic effect of bacterial hydrolases on the ester bonds of PAEs. Bacterial acid esters include eight families: "true" lipase (Ⅰ), GDSL (Ⅱ), Family (Ⅲ), the hormone-sensitive lipase (HSL) (Ⅳ), Family Ⅴ~Ⅷ, of which true lipase includes six subfamilies, and each hydrolase has a unique conserved functional sequence module. The process of functional verification of degrading enzymes is the process of binding and interaction between the protein macromolecule of the degrading enzyme and the small molecule of the substrate (organic pollutant), which is also one of the hot topics in molecular biology research today.

[0004] So far, the whole genomes of many strains of PAEs-degrading bacteria have been sequenced, and the related functions have been analyzed and annotated, but the sequences and functions of these genes still need to be fully analyzed and verified. In recent years, the cloning and characterization of PAE hydrolase genes based on DNA library construction, genome sequencing and metagenomic mining have received widespread attention. The research on esterases related to the hydrolysis of PAEs ester bonds mainly focuses on gene cloning and recombinant expression, enzyme activity characterization, protein structure analysis and functional verification. However, the interaction mechanism between PAEs and related esterases is still unclear, so the research progress on artificial modification and evolution of esterases is hindered, which is not conducive to the application of esterases in environmental remediation. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a PS06828 esterase for degrading phthalates and its application.

[0006] The first object of the present invention is to provide a gene encoding PS06828 esterase protein.

[0007] The second object of the present invention is to provide a PS06828 esterase protein.

[0008] The third object of the present invention is to provide a recombinant vector.

[0009] The fourth object of the present invention is to provide a recombinant engineered bacterium.

[0010] The fifth object of the present invention is to provide the use of the gene encoding PS06828 esterase protein in degrading phthalates.

[0011] The sixth object of the present invention is to provide the use of the PS06828 esterase protein in degrading phthalates.

[0012] The seventh object of the present invention is to provide the use of the recombinant vector in degrading phthalates.

[0013] The eighth object of the present invention is to provide the use of the recombinant engineered bacteria in degrading phthalates.

[0014] The ninth object of the present invention is to provide a method for degrading phthalates.

[0015] In order to achieve the above object, the present invention is implemented by the following scheme:

[0016] A gene encoding PS06828 esterase protein, wherein the nucleotide sequence of the gene encoding PS06828 esterase is shown in SEQ ID NO: 1.

[0017] A PS06828 esterase protein, the amino acid sequence of the PS06828 esterase protein is shown in SEQ ID NO: 2.

[0018] A recombinant vector contains the gene encoding the PS06828 esterase protein or expresses the PS06828 esterase protein.

[0019] A recombinant engineered bacterium contains the recombinant vector.

[0020] The application of the gene encoding PS06828 esterase protein in degrading phthalates.

[0021] Preferably, the phthalate is dibutyl phthalate.

[0022] The PS06828 esterase protein is used for degrading phthalates.

[0023] Preferably, the phthalate is dibutyl phthalate.

[0024] Application of the recombinant vector in degrading phthalates.

[0025] Preferably, the phthalate is dibutyl phthalate.

[0026] The application of the recombinant engineered bacteria in the degradation of phthalates.

[0027] Preferably, the phthalate is dibutyl phthalate.

[0028] A method for degrading phthalates, using the PS06828 esterase protein for degradation.

[0029] Preferably, in the method for degrading phthalates, the degradation temperature is 25° C. to 35° C., the degradation pH is 7 to 8, and the degradation time is less than 5 minutes.

[0030] More preferably, in the method for degrading phthalates, the degradation temperature is 30° C., the degradation pH is 7, and the degradation time is 5 min.

[0031] Preferably, the phthalate is dibutyl phthalate.

[0032] PS06828 esterase protein has a certain degradation ability for other PAEs because the hydrolysis structures are consistent, but PAEs compounds with long side chains produce greater steric hindrance, so the degradation effect of PS06828 esterase protein on different PAEs will be different.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention discloses a PS06828 esterase for degrading phthalates and its application. The nucleotide sequence of the gene encoding the PS06828 esterase is shown in SEQ ID NO: 1, and the amino acid sequence of the PS06828 esterase protein is shown in SEQ ID NO: 2. The PS06828 esterase belongs to the carboxylesterase in the VI class lipase family. The kinetic curve measurement results of the PS06828 esterase degrading DBP show that the enzyme has a very strong degradation ability within 5 minutes, and the degradation rate reaches 53.35%; in addition, the optimum temperature of the PS06828 esterase is 30°C, and the relative activity of the enzyme in the range of 25°C to 35°C is above 45%, and the optimal pH value is 7, and it shows a higher activity (>50%) when the pH value ranges from 7 to 8, and has high activity and stability in a wide range of temperature and pH values, and is suitable for practical application in complex natural environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the map of the pET-28a(+) plasmid vector.

[0036] Figure 2 Kyte & Doolittle algorithm was used to predict the transmembrane region of PS06828 esterase amino acid sequence and Hidden Markov Model (HMM) method. A is the protein hydrophobicity analysis result based on Kyte & Doolittle algorithm (ProScale); B is the transmembrane region analysis of PS06828 esterase.

[0037] Figure 3 The secondary structure of PS06828 esterase.

[0038] Figure 4 Phylogenetic tree based on the amino acid sequence of PS06828 esterase.

[0039] Figure 5 Multiple amino acid sequence alignment of PS06828 esterase and other esterases of Family VI.

[0040] Figure 6 This is the homology modeling of PS06828 esterase. A is the tertiary structure of template PA3859 in the database, B is the tertiary structure modeling result of PS06828 esterase, and C is the Ramachandran evaluation diagram of PS06828 esterase.

[0041] Figure 7 : This is the electrophoresis diagram of PCR amplification. M is D2000 Marker; Lane 1 is pET28a plasmid; Lane 2 is PS06828 gene; Lane 3 is positive clone.

[0042] Figure 8Induction analysis of PS06828 esterase protein. A is the induction analysis of PS06828 esterase protein at different temperatures; B is the purified PS06828 esterase protein in the supernatant induced at 20°C; C is the Western Blot detection of the purified PS06828 esterase protein. Lane 1 is the total protein before induction; Lane 2 is the protein supernatant induced at 20°C; Lane 3 is the protein precipitate induced at 20°C; Lane 4 is the protein supernatant induced at 37°C; Lane 5 is the protein precipitate induced at 37°C; M is a protein marker.

[0043] Fig. 9 BSA standard curve

[0044] Fig.10 The figure is the time kinetics of PS06828 esterase degrading DBP. A is the time kinetics curve of PS06828 esterase protein degrading DBP; B is the effect of temperature on the activity of PS06828 esterase protein; C is the effect of pH on the activity of PS06828 esterase protein.

[0045] Fig.11 Modeling of the homodimer of PS06828 esterase. A is the homodimer modeling result of PS06828 esterase, and B is the Ramachandran evaluation diagram.

[0046] Fig.12 This is an Autodock docking simulation of the interaction between PS06828 esterase homodimer and DBP. Figures A and B show the location of DBP (red structure) in the protein structure; C shows the key amino acid residues around DBP in three-dimensional perspective.

[0047] Fig.13 The key amino acid residues around DBP in two-dimensional perspective, where the green dotted lines represent hydrogen bonds and their lengths.

[0048] Fig.14 The inference of the catalytic mechanism of the hydrolysis of DBP by PS06862 esterase mediated by the catalytic triad. The blue arrows in the catalytic triad of amino acids indicate the sites of nucleophilic attack.

[0049] Fig.15 It is the catalytic activity of wild-type PS06828 esterase and its PS06828-Ser113 mutant PS06828 esterase, PS06828-Asp166 mutant PS06828 esterase, PS06828-His197 mutant PS06828 esterase, PS06828-Asp167 mutant PS06828 esterase, and PS06828-Phe72 mutant PS06828 esterase on the substrate DBP. DETAILED DESCRIPTION

[0050] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0051] Bacterial strains and plasmid vectors: PAEs-degrading bacteria Pseudomonas aeruginosa PS1, the Pseudomonas aeruginosa PS1 was deposited in Guangdong Microbiological Culture Collection Center on October 27, 2021, and its deposit number is GDMCC No: 62009. E. coli DH5α and E. coli BL21 were purchased from Tiangen Biotechnology (Beijing) Co., Ltd.; the expression vector plasmid pET-28a (+) was preserved in the laboratory, and its vector map is as follows Figure 1 shown.

[0052] Main kits and enzymes: Bacterial genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.); Plasmid DNA rapid extraction and purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.); EcoR family and Hind family restriction endonucleases (NEB Biotechnology (Beijing) Co., Ltd.); II One Step Cloning Kit (Novozymes Biotech (Nanjing) Co., Ltd.); Ni-NAT protein purification kit (C600332) (Sangon Biotech (Shanghai) Co., Ltd.); non-interfering protein concentration determination kit (Sangon Biotech (Shanghai) Co., Ltd.); site-directed mutagenesis kit (FastMutagenesis System) (Beijing Quanshijin Co., Ltd.).

[0053] Main reagents: isopropyl-β-D-thiogalactoside (IPTG) (Biosharp Biotechnology Co., Ltd.); acrylamide, methylene acrylamide, various amino acids, etc. were all from Sigma-Aldrich Company, USA, and other analytical grade inorganic reagents were purchased from Tianjin Damao Chemical Reagent Company.

[0054] Main instruments: PCR instrument (T100 TMThermal Cycler, BIO-RAD); gel imager (UniversalHoodⅡ, BIO-RAD); gel electrophoresis instrument (Power Pac Basic, BIO-RAD); ultra-micro-spectrophotometer (ND-ONE-W, Thermo Scientific); ultrasonic cell disruptor (JY92-IIDN, Ningbo); ultraviolet spectrophotometer (UV-2550, Japan).

[0055] Example 1 Informatics Analysis of PS06828 Esterase

[0056] 1. Experimental Methods

[0057] The whole genome and transcriptome of the PAEs-degrading bacterium Pseudomonas aeruginosa PS1 were sequenced and analyzed, and the complete gene sequence of a key esterase was excavated, which has 648 bases and can encode 215 amino acids. The esterase was named PS06828. The specific nucleotide sequence and amino acid sequence of the esterase are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The Pseudomonas aeruginosa PS1 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 27, 2021, and its preservation number is GDMCC No: 62009.

[0058] By using online servers or bioinformatics related analysis programs, the basic physicochemical properties of PS06828 esterase are predicted and the corresponding structural information is obtained.

[0059] (1) Prediction of the primary, secondary and tertiary structures of PS06828 esterase

[0060] The amino acid sequence of PS06828 esterase was input into the ProtParam tool online server, PSIPRED online server and SWISS MODEL server respectively, and the primary, secondary and tertiary structures and physicochemical properties of PS06828 esterase were predicted and analyzed, and a suitable template was found to finally model and generate the tertiary structure.

[0061] (2) Classification and identification of PS06828 esterase

[0062] The amino acid sequence of PS06828 esterase was uploaded to the ClustalX program for multiple alignment with the classical esterase and hydrolase sequences previously reported in the literature, and the alignment results were uploaded to the MEGA 5.05 program to construct a phylogenetic tree and analyze the sequence characteristics of the esterase and its family classification.

[0063] 2. Experimental Results

[0064] 1. Primary structure of PS06828 esterase

[0065] The molecular formula of PS06828 esterase is C 1051 H 1638 N 292 O 306 S5, composed of 215 amino acids, has a molecular weight (M) of 23.420 KDa and a theoretical isoelectric point of 5.02. The amino acid composition of the esterase is shown in Table 1:

[0066] Table 1 Amino acid composition of PS06828 esterase

[0067]

[0068]

[0069] The hydrophobicity analysis of the esterase amino acid sequence was performed using the Kyte & Doolittle algorithm, and the results are as follows Figure 2 As shown in A, the amino acid distribution in the hydrophobic and hydrophilic regions is relatively uniform, but the protein GRAVY result is 0.017, which is greater than zero, indicating that PS06828 esterase is a hydrophobic protein. This result shows that although PS06828 esterase is hydrophobic, its hydrophobicity is weak. In addition, the online software TMHMM Server v.2.0 was used to predict the transmembrane region of the esterase based on the hidden Markov model (HMM). Figure 2 As shown in B, no transmembrane structure was found.

[0070] 2. PS06828 esterase secondary structure

[0071] The secondary structure of PS06828 esterase protein is predicted to be Figure 3 As shown, the contents of α-helix, β-sheet and random coil in the structure are 39.07%, 14.88% and 46.05%, respectively.

[0072] 3. Classification and identification of PS06828 esterase

[0073] Microbial degradation of PAEs usually starts with the hydrolysis of ester bonds, and esterases are involved as key enzymes. In general, bacterial esterases are divided into eight families based on basic biological characteristics and amino acid sequence alignment. To find out which lipase family PS06828 esterase belongs to, the amino acid sequences of selected lipases from eight different families and reported PAEs hydrolases were compared with the esterase, and a phylogenetic tree analysis was performed ( Figure 4), the results showed that PS06828 esterase belongs to the VI class lipase family. The molecular weight of the enzyme protein in this family is between 23 and 26 kDa, which is the smallest esterase family known so far. The active form of this type of enzyme is generally a dimer, and its subunit has a typical α / β folding structure and a classic Ser-Asp-His catalytic triad. Its substrate range is very wide, but the research on this type of esterase is still very scarce, and its protein structure characteristics and catalytic mechanism have not yet been elucidated.

[0074] 4. Homology analysis of PS06828 esterase within the family

[0075] From the above phylogenetic tree analysis, we know that PS06828 esterase belongs to esterase family VI, so we selected the typical esterases in the traditional family VI for homology analysis. Figure 5 As shown, the amino acid sequences of PS06828 esterase and the other five esterases from the sixth family all have a highly conserved pentapeptide motif of "GFSQG" and a catalytic triad of "Ser-Asp-His".

[0076] 5. Tertiary structure prediction and homology modeling of PS06828 esterase

[0077] The amino acid sequence of PS06828 esterase was uploaded to the SWISS-MODEL protein structure homology modeling server. Through sequence alignment, the sequence with higher similarity was selected as the template of the protein to be tested, and the tertiary structure of PS06828 esterase was predicted by automatic modeling. In general, the higher the similarity between the target sequence and the template sequence, the more accurate the structural model obtained by homology modeling. Therefore, according to the Blast search results of the template, the amino acid sequence similarity between PS06828 esterase and the intracellular carboxylesterase PA3859 from Pseudomonas aeruginosa is 61%. PA3859 carboxylesterase has been reported to catalyze the hydrolysis of aliphatic and aromatic esters and has a wide range of substrate specificity, including p-nitrophenyl ester and α-naphthyl ester, but there is no report that this esterase hydrolyzes PAEs. The crystal structure 3CN7.1.A of carboxylesterase PA3859 was selected as the structural prediction template of PS06828 esterase ( Figure 6 A), the tertiary structure of PS06828 esterase was obtained using the SWISS-MODEL automatic modeling method ( Figure 6 B). Ramachandran evaluation of the modeled structure ( Figure 6 C), 96.1% of the dihedral angles of amino acid residues are located in reasonable regions, which means that the model is reliable.

[0078] Example 2 Heterologous expression and enzymatic properties of PS06828 esterase

[0079] 1. Experimental Methods

[0080] (1) Primer design

[0081] Select appropriate restriction sites on the pET-28a(+) vector and design primers. The primer sequences are shown in Table 2.

[0082] Table 2 Primer sequences

[0083]

[0084] (2) PCR amplification

[0085] The PAEs-degrading bacteria Pseudomonas aeruginosa PS1 of Example 1 was cultured in an expanded manner. DNA of the Pseudomonas aeruginosa PS1 was extracted using an Omega genomic DNA extraction kit (EZNA™ Bacterial DNA Kit), and used as a PCR template to amplify the PS06828 esterase gene sequence according to the PCR system shown in Table 3. The primer sequences of PS6838-F and PS06828-R are shown in Table 2.

[0086] Table 3 PCR amplification reaction system

[0087]

[0088] The PCR amplification procedure is as follows:

[0089]

[0090] The PCR product is then subjected to gel electrophoresis for detection and purified and recovered to obtain a purified PCR product.

[0091] (3) Construction of recombinant vector

[0092] The pET28a plasmid was extracted using a plasmid DNA rapid extraction and purification kit, and was digested for 4 hours using endonucleases EcoR I and Not I. The digestion system (Table 4) was provided by the endonuclease instructions to obtain a linear plasmid.

[0093] Table 4 Plasmid double restriction enzyme digestion system

[0094]

[0095] The purified PCR product of step (2) was connected with the linear plasmid of step (3) by the one-step infusion method. After adding and mixing according to the system shown in Table 5, the mixture was incubated at 37°C for 30 minutes, and then the temperature was adjusted to 80°C and reacted for 20 minutes to completely inactivate the endonuclease in the system. The ligation product (i.e., the recombinant vector) was stored in a -20°C refrigerator for future use.

[0096] Table 5 Infusion ligation reaction system

[0097]

[0098] Note: The volume of the linearized plasmid and target fragment is determined according to their concentration, usually 2 μL.

[0099] (4) Screening of positive colonies

[0100] The ligation product of step (3) and the empty vector (pET28a plasmid) were respectively transferred into E. coli DH5α by heat shock method, wherein the empty vector was set as blank control; 1 mL LB culture medium was added and incubated in a constant temperature shaker (37°C, 200 rpm) for 45 min to revive the E. coli DH5α cells; the bacterial solution was centrifuged and the precipitate was spread on an LB plate (containing 50 μg / mL ampicillin), inverted and cultured at 37°C for 12 to 16 h; a positive single colony was picked for bacterial solution PCR verification, and the system is shown in Table 6.

[0101] Table 6 Bacterial liquid PCR system

[0102]

[0103] Note: The sequences of primers T7 and T7t are taatacgactcactatagg and gctagttattgctcagagg respectively.

[0104] After the reaction is completed, gel electrophoresis is performed, and the product band is aligned with the marker to check whether the position is correct. The E. coli DH5α colony transferred with the ligation product of step (3) is further expanded and cultured, and the plasmid is extracted and sequenced. The E. coli DH5α strain transferred with the ligation product of step (3) that is correctly sequenced is expanded and cultured and placed in a -80°C refrigerator for use.

[0105] (5) Inducible expression of PS06828 esterase gene

[0106] The E. coli DH5α strain with the correct sequencing and the ligation product of step (3) was inoculated into 50 mL LB liquid culture medium (1:100) and cultured at 37°C, 220 rpm for about 3 h until the OD of the fermentation liquid reached 600When the value reaches 0.6-0.8, immediately add isopropyl-β-D-thiogalactoside (IPTG) solution to a final concentration of 0.5mM, 220rpm, 20℃ induction overnight, and the treatment without IPTG addition is used as a blank control. The next day, the bacteria were collected, washed three times with PBS (pH, 7.4) and resuspended; the bacterial solution was placed on ice and ultrasonically broken and centrifuged, and the supernatant and precipitate were retained. Prepare the polyacrylamide gel listed in Table 7 in advance for SDS-PAGE gel electrophoresis test, and then immerse the gel in Coomassie Brilliant Blue dye for staining for 20 minutes, decolorize it, take pictures and observe, and save the pictures.

[0107] Table 7 Polyacrylamide gel formula

[0108]

[0109] (6) Purification and Western Blot Detection of PS06828 Esterase

[0110] The E. coli DH5α strain cells transferred with the ligation product of step (3) were dissolved with PBS, ultrasonically broken in an ice bath, and the supernatant was collected by centrifugation. The supernatant was purified by a nickel column, and the crude protein, the wash outflow, and the elution outflow were processed and sampled separately to obtain the purified PS06828 esterase protein, and SDS-PAGE detection and Western Blot detection were prepared. The Western Blot detection steps refer to the literature (Zeng X, Du H, Zhao H, et al. Insights into the binding interaction of substrate with catechol 2,3-dioxygenase from biophysics point of view. Journal of Hazardous Materials, 2020 391: 122211.).

[0111] (7) Determination of PS06828 esterase concentration

[0112] The concentration of purified PS06828 esterase protein was determined according to the instructions of the SK3071 non-interference protein quantification kit.

[0113] (8) PS06828 esterase activity assay

[0114] 712 μL Tris-Hcl (10 m Mol / L) reaction buffer solution, 278 μL dibutyl phthalate (DBP) standard mother solution (100 mg / L) and 10 μL PS06828 esterase protein solution purified in step (6) were mixed evenly to obtain a reaction mixture; the reaction mixture was placed in a constant temperature water bath and reacted at 30°C for 5 min. After the reaction was completed, 100 μL trichloroacetic acid (TCA) (1 mol / L) solution was added to terminate the reaction. The obtained product was used as the experimental group. The purified PS06828 esterase protein solution treated with boiling at 100°C was added as the blank control group, and each treatment was repeated three times. The extraction method and GC-MS determination conditions of DBP in the samples of the experimental group and the blank control group are as follows:

[0115] 1) Extraction of DBP

[0116] The specific extraction method for sample pretreatment is as follows: add 20mL of dichloromethane to the culture bottle containing the culture medium, shake at 120r / min for 10min at room temperature to fully mix the extractant dichloromethane with the remaining DBP in the culture bottle; transfer to a separatory funnel, let it stand for 10min, collect the organic dichloromethane in the lower layer, pour the upper aqueous phase back into the original culture bottle, and extract twice (add 20mL and 10mL of dichloromethane respectively). The final organic dichloromethane is passed through a 15cm anhydrous Na2SO4 column (dried in a muffle furnace at 400m℃) to dry the residual water, collect it in a volumetric flask and adjust the volume to 50mL to obtain the sample extract. Dilute the sample extract 50 times, take 1mL and filter it, collect it in an injection bottle, and determine its residual amount by gas chromatography-mass spectrometry (GC-MS).

[0117] 2) GC-MS determination

[0118] GC-MS detection conditions: chromatographic column, DB-5ms; carrier gas is high-purity helium, injection port temperature is 280℃, automatic sampler injection; initial pressure is 33.6kPa, flow rate is 1.0mL / min; non-split injection is used, and the injection volume is 1.0μL. Temperature program: initial temperature is 60℃ and maintained for 1min; increase to 280℃ at 60℃ / min and maintain for 2min, then increase to 290℃ at 10℃ / min and maintain for 1min. Mass spectrometry qualitative and quantitative: mass spectrometry uses EI ion source, temperature is 200℃; DBP target ion is 149m / z, reference ions are 41 and 150m / z, retention time is 5.393min; DBP is qualitatively analyzed by comparing the sample with the standard solution with the target ion, reference ion and retention time, and the concentration of DBP in the sample is quantitatively analyzed using the drawn standard curve.

[0119] The enzyme activity unit (U) was defined as the amount of enzyme required to hydrolyze 1 μmol of DBP per minute at 30 °C.

[0120] (9) Determination of the time kinetic curve of PS06828 esterase degradation of DBP

[0121] The reaction mixture of step (8) was prepared, mixed evenly, placed in a 30°C water bath, and samples were taken at 0, 1, 3, 5, 10, 20, 30 and 40 min, and the DBP content in the samples was determined. The determination method was the same as step (8) of Example 2, and three repetitions were set for each time point. Finally, the time (min) was used as the abscissa and the DBP residual content (mg / L) in the solution was used as the ordinate to draw the time kinetic curve of the degradation of the substrate DBP by PS06828 esterase.

[0122] (10) Effects of temperature and pH on enzyme activity

[0123] Effect of temperature on enzyme activity: The reaction mixture of step (8) was placed in a water bath at 20, 25, 30, 35, 40 and 45°C, respectively. After 5 minutes of reaction, the DBP content in the solution was determined. The determination method was the same as step (8) of Example 2, and each treatment was repeated three times. The enzyme activity at different temperatures was calculated. The enzyme activity calculation method was the same as step (8) of Example 2. The temperature treatment group with the highest enzyme activity was taken as 100%, and the relative enzyme activity was used to analyze the effect of temperature on the enzyme activity of PS06828 esterase.

[0124] Effect of pH on enzyme activity: The pH value of 10mM / L Tris-Hcl buffer solution was adjusted to 5.0, 6.0, 6.5, 7.0, 7.5, 8.0 and 9.0 respectively, and the reaction mixture of step (8) was prepared and placed in a 30°C water bath. After reacting for 5 minutes, the DBP content in the solution was detected. The determination method was the same as step (8) of Example 2, and each treatment was repeated three times. The enzyme activity was calculated under different pH conditions. The enzyme activity calculation method was the same as step (8) of Example 2. The temperature treatment group with the highest enzyme activity was taken as 100%, and the relative enzyme activity was used to analyze the effect of pH on the enzyme activity of PS06828 esterase.

[0125] 2. Experimental Results

[0126] 1. Construction of recombinant vector

[0127] The positive colony PCR verification results are as follows Figure 7 As shown, the PCR amplification product bands are clear and single. Compared with the DNA Marker, it can be seen that the length of the inserted target fragment in the recombinant vector is consistent with the size of the PS06828 esterase gene. The PCR product was sequenced, and the result was that the inserted target fragment sequence was the PS06828 esterase gene sequence, proving that the connection product (i.e., the recombinant vector) was successfully constructed.

[0128] 2. Expression and purification of PS06828 esterase

[0129] The SDS-PAGE electrophoresis of the induced expression and purification of PS06828 esterase protein is shown in Figure 8 As shown, under IPTG induction overnight, PS06828 esterase protein was expressed in the supernatant, and the size of the produced recombinant protein was about 29 kDa, which was roughly consistent with the theoretically calculated molecular weight of the recombinant protein of 23.420 KDa. Figure 8 A is the induction analysis of PS06828 esterase protein at different temperatures; Figure 8 B is the PS06828 esterase protein purified from the supernatant induced at 20°C. In addition, Western Blot detection ( Figure 8 C) It was also verified that the PS06828 esterase protein was expressed and purified.

[0130] 3. Enzyme concentration and activity determination

[0131] According to the instructions of the non-interference protein quantification kit, the absorbance values ​​of the standard and the sample to be tested were determined by spectrophotometry, and a standard curve was drawn ( Fig. 9 ), the concentration of pure PS06828 esterase protein was calculated to be 1.08 mg / mL.

[0132] 4. Characterization of Enzymatic Properties

[0133] (1) Determination of the kinetic curve of enzyme degradation of DBP

[0134] Fig.10 A shows that PS06828 esterase has a very strong ability to degrade DBP within 0 to 5 minutes. The DBP in the solution quickly dropped from the initial concentration to 14.83 (±0.84) mg / L, and the degradation rate reached 53.35%. However, after 5 minutes, the DBP concentration in the solution did not change significantly and tended to a stable state, indicating that the enzyme has a very weak degradation ability after 5 minutes, with almost no degradation ability.

[0135] (2) Effects of temperature and pH on enzyme activity

[0136] Fig.10 B shows that the optimum temperature range of PS06828 esterase is 30℃, and the relative activity of the enzyme is above 45% in the range of 25℃ to 35℃; in the range of 20℃ to 30℃, the relative activity of the enzyme increases with the increase of temperature, and then decreases rapidly. At 45℃, the relative activity of the enzyme is only 20.08%. Fig.10C shows that PS06828 esterase exhibits high activity (>50%) in the pH range of 7-8, with the optimal pH being 7; while the relative activity of the enzyme decreases rapidly as the pH moves further toward the acidic or alkaline end. PS06828 esterase has high activity and stability in a wide range of temperature and pH values, and is suitable for practical use in complex natural environments.

[0137] Example 3 Molecular simulation and site-directed mutagenesis to study the catalytic mechanism of PS06828 esterase

[0138] 1. Experimental Methods

[0139] (1) Molecular docking of PS06828 esterase and DBP

[0140] AutoDockTools was used to simulate the molecular docking of PS06828 esterase in the process of binding to dibutyl phthalate (DBP). The 3D structure of the small molecule DBP can be downloaded from the Protein Data Bank (PDB) online website. The amino acid sequence of PS06828 esterase (SEQ ID NO: 1) was input into the Swiss-Model server for homology modeling. The obtained monomer structure of the esterase was 61% similar to the carboxylesterase PA3859 from the strain Pseudomonas aeruginosa reported on the PDB website (template 3CN7.1.A), and the dimer structure was 48% similar to the carboxylesterase from the strain Pseudomonas fluorescens (template 1AUO1.B).

[0141] The two templates (template 3CN7.1.A and template 1AUO1.B) were used to perform homology modeling on PS06828 esterase, and then molecular simulation docking experiments were performed: AutoDock software was used to pre-process the constructed protein model, including removing water molecules, adding hydrogen atoms and Gasteiger charges, etc. to obtain the optimal configuration of PS06828 esterase. The ligand DBP was optimized using Gaussian 03W software. Finally, the optimal configuration of PS06828 esterase was docked with the optimized ligand DBP. During the process, the coordinates of the grid x, y, and z surrounding the PS06828 esterase were set to The grid spacing is The number of dockings was 100, and the binding sites and force types of the interaction between the small molecule ligand DBP and PS06828 esterase were calculated by the relevant genetic algorithm. The conformation with the lowest binding energy was finally selected for observation in the PyMOL program, and the DBP ligand binding domain and hydrophobic residues of PS06828 esterase were analyzed using the LigPlot+ program.

[0142] (2) Site-directed mutagenesis

[0143] A site-directed mutagenesis kit was used to perform site-directed mutagenesis experiments on the PS06828 esterase gene. The five amino acid residues, Ser113, Asp166, His 197, Asp167 and Phe72, predicted in the molecular simulation results, were mutated to simple alanine (GCC). Because alanine has the characteristics of being uncharged, hydrophobic and having less steric hindrance, the mutants were named PS06828-Ser113, PS06828-Asp166, PS06828-His197, PS06828-Asp167, and PS06828-Phe72 according to the different amino acids at the mutation sites. Primers were designed according to the operating instructions of the mutation kit and the selected mutation sites. The primer sequences are shown in Table 8. Finally, the recombinant vector of step (3) of Example 2 was used as a PCR amplification template for site-directed mutagenesis PCR. After amplification, DMT enzyme was added and incubated at 37° C. for 1 h to digest the non-mutated vector and sequenced. After confirming that the mutation is successful, the wild-type and mutant positive vectors are transformed into competent E. coli (E. Coli BL21) to prepare PS06828 esterase wild-type and mutant recombinant bacteria, respectively. The mutant recombinant bacteria are induced to express by IPTG, and the bacterial cells are ultrasonically disrupted to obtain the target protein for SDS-PAGE electrophoresis analysis.

[0144] Table 8 Site-directed mutation sites and related primers of PS06828 esterase gene

[0145]

[0146]

[0147] (3) Study on the catalytic performance of wild-type PS06828 esterase and mutant enzymes

[0148] After induced expression, the mutant recombinant bacteria and the wild-type recombinant bacteria were ultrasonically disrupted to obtain crude enzyme proteins, and then the PS06828 esterase activities of the wild-type and each mutant recombinant bacteria were measured. The enzyme activity determination method was the same as in Example 2, and the wild-type PS06828 esterase activity was 100%, and the mutant PS06828 esterase activity was the relative enzyme activity.

[0149] 2. Experimental Results

[0150] The structural information and conformational changes of proteins during catalysis are the basis for understanding the catalytic mechanism of enzymes. When a substrate binds to an enzyme, it becomes a component of a macromolecule (enzyme-substrate complex). The subsequent dynamics of the conformational changes of the macromolecule is the catalytic process itself, which will directly affect the catalytic efficacy of the enzyme. In addition, this interaction can not only provide some valuable information for the catalytic and recognition processes of the enzyme, but also can be used to guide the modification of the catalytic properties of the enzyme, and can even be used to design and synthesize artificial enzymes.

[0151] 1. Molecular docking simulation analysis of PS06828 esterase and DBP

[0152] PS06828 esterase belongs to the esterase VI family. The active form of this type of enzyme is generally a dimer, and its subunit has a typical α / β folding structure and a classic Ser-Asp-His catalytic triad. The model constructed by the crystal structure 3CN7.1.A of the best template carboxylesterase PA3859 is a monomer structure. After 100 docking runs of DBP and the PS06828 esterase monomer model using Auto dock software, the binding conformation with the lowest free energy was selected from the energy cluster distribution map as the best binding conformation for the prediction and analysis of the interaction between PS06828 esterase and DBP. The results showed that although DBP can bind to the monomer model, the binding sites are found to be far away from the catalytic triad or no catalytic triad is formed, which indicates that the monomer of this esterase (carboxylesterase PA3859 from the strain Pseudomonas aeruginosa) may not be able to catalyze DBP. Based on this, the homodimer model of PS06828 esterase was re-modeled using 1AUO.1.B as a template (sequence similarity 48%). Fig.11 As shown in A, Ramachandran evaluated ( Fig.11 B) 97.89% of the dihedral angles of amino acid residues are located in the reasonable region, indicating that the model is very reliable. The dimer model of the esterase (carboxylesterase from the bacterium Pseudomonas fluorescens) was used for molecular docking simulation with DBP, and the optimal binding result showed that the two can be well combined, and the binding energy is -4.03 kcal / mol.

[0153] From the structural diagram of DBP binding to PS06828 esterase ( Fig.12 AB) It can be seen that when the DBP molecule binds to the PS06828 esterase, it is completely enclosed in a cavity in the middle of the protein dimer. Fig.12C shows the key amino acid residues around DBP. The cavity is formed by 10 amino acid residues of PS06828 esterase, namely Leu22, Val57, Ile69, Phe72, Ser73, Ser113, Asp166, Asp167, Val168 and His197. Among them, there are 5 hydrophobic amino acids, namely Leu22, Val157, Ile69, Phe72 and Val168, accounting for 50% of the total number of amino acid residues. Further two-dimensional processing using LIGPLOT+ software revealed that DBP is in the core position of the catalytic triad (Ser113-Asp166-His197) and forms hydrogen bonds with two catalytic active residues, Ser113 and His197, with bond lengths of 2.60 and 2.96, respectively. ( Fig.13 ), studies have shown that the formation of hydrogen bonds can reduce the hydrophilicity of DBP and increase its hydrophobicity, thereby stabilizing the binding force between DBP and esterase. In addition, the catalytic residue His197 forms hydrogen bonds with the catalytic active residue Asp166 and the surrounding residue Asp167, with bond lengths of The above key amino acid residues around DBP all come from a monomer in the dimer, but the catalytic triad cannot be found when only the monomer model is used for docking simulation. This result indicates that the esterase is in the form of a dimer during the catalytic hydrolysis of DBP, and the monomer has no catalytic activity.

[0154] 2. Inference of the mechanism of DBP hydrolysis catalyzed by PS06828 esterase protein

[0155] Mechanism of DBP hydrolysis catalyzed by PS06828 esterase Fig.14 ):

[0156] (1) In the Ser113-Asp166-His197 catalytic triad, Asp166 and His197 are hydrogen bonded, increasing the pKa of the imidazole nitrogen in His197, allowing His197 to further deprotonate Ser113 and transfer the proton hydrogen on its hydroxyl group to the imidazole ring of His197;

[0157] (2) The activated Ser113-Oγ acts as a nucleophile to attack the carbonyl carbon atom of one of the ester bonds of DBP, and causes the oxygen atom on the carbonyl group to accept electrons, forming a tetrahedral intermediate complex;

[0158] (3) The proton hydrogen obtained from the hydroxyl group of Ser113 on the imidazole ring of His197 is transferred to the alcoholic hydroxyl group of the ester bond, resulting in the cleavage of the ester bond and the release of the alcohol part. At this time, the protonated Ser113 re-forms an ester bond with the carbonyl group to form an acyl-enzyme intermediate;

[0159] (4) The water molecule enters the active center, and the imidazole ring of His197, which has lost proton hydrogen, captures the proton hydrogen in the water molecule (deprotonation), allowing its oxygen atom to attack the carbonyl carbon atom of the acyl-enzyme intermediate;

[0160] (5) The carbonyl carbon atom of the acyl-enzyme intermediate is forced to accept electrons and form another tetrahedral intermediate complex;

[0161] (6) The proton hydrogen obtained from the water molecule on the imidazole ring of His197 is transferred to the anionic oxygen of Ser113, restoring Ser113 to its original state and releasing the carboxyl-containing MBP.

[0162] 3. Site-directed mutagenesis verification

[0163] The sequencing results show that a total of five amino acid residues, Ser113, Asp166, His197, Asp167 and Phe72, in the active site predicted in the molecular simulation results were successfully mutated, and thus the corresponding five PS06828 mutant recombinant plasmids were successfully obtained.

[0164] The catalytic activities of wild-type and mutant PS06828 esterases on substrate DBP were further compared, and it was found that the catalytic activities of each enzyme were significantly different ( Fig.15 ). Compared with the wild-type PS06828 esterase activity (100%), the relative enzyme activity of the PS06828-Ser113 mutant PS06828 esterase was 0, and the catalytic ability was completely lost; the catalytic activities of the other four mutant PS06828 esterase substrates were reduced to varying degrees, among which the relative enzyme activities of the PS06828-Asp166 mutant PS06828 esterase (5.16828%) and the PS06828-His197 mutant PS06828 esterase (12.5828%) were extremely low, while the relative enzyme activities of the PS06828-Asp167 mutant PS06828 esterase (46.3828%) and the PS06828-Phe72 mutant PS06828 esterase (72.9828%) were both greater than 45%. This result confirmed the results of molecular docking simulation predictions, that the three active site residues Ser113, Asp166 and His197 play a decisive role in the catalytic process, while residues Asp167 and Phe72 are involved in the process of binding to the substrate. Finally, the catalytic mechanism of PS06828 esterase for DBP was successfully revealed and verified.

[0165] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. The use of a gene encoding PS06828 esterase protein in the degradation of phthalates, characterized in that: The nucleotide sequence of the gene encoding PS06828 esterase is shown in SEQ ID NO: 1, and the phthalate is dibutyl phthalate.

2. The use of PS06828 esterase protein in the degradation of phthalates, characterized in that: The amino acid sequence of the PS06828 esterase protein is shown in SEQ ID NO: 2, and the phthalate is dibutyl phthalate.

3. The use of the recombinant vector in the degradation of phthalates, characterized in that: The recombinant vector contains the gene encoding the PS06828 esterase protein described in claim 1 or expresses the PS06828 esterase protein described in claim 2, and the phthalate is dibutyl phthalate.

4. The use of recombinant engineered bacteria in the degradation of phthalates, characterized in that: The recombinant engineered bacteria contains the recombinant vector described in claim 3, and the phthalate is dibutyl phthalate.

5. A method for degrading phthalates, characterized in that: The degradation is carried out using the PS06828 esterase protein described in claim 2, wherein the phthalate is dibutyl phthalate.

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