CN111394372A - Phthalate degrading enzyme gene, its coding product and preparation method - Google Patents
Phthalate degrading enzyme gene, its coding product and preparation method Download PDFInfo
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Abstract
The invention discloses a phthalate degrading enzyme gene, a coding product and a preparation method thereof, wherein the phthalate degrading enzyme gene is named as est1012, and the nucleotide sequence of the phthalate degrading enzyme gene is shown as SEQ ID NO. 1. The amino acid sequence of the polypeptide is shown as SEQ ID NO. 2. Also discloses a recombinant plasmid containing the phthalate degrading enzyme DNA, a preparation method of the phthalate degrading enzyme and application of the phthalate degrading enzyme in degrading phthalate compounds DEHP, DEP and DNP, wherein the phthalate degrading enzyme has efficient soluble expression in an escherichia coli expression system.
Description
Technical Field
The invention relates to the technical field of enzyme gene engineering, in particular to novel esterase extracted from soil and related application thereof.
Background
The phthalate ester compound is an important chemical raw material, is various in variety, is more than 30 common, is widely applied to industries such as industry, food packaging, cosmetics and the like as a plasticizer with the widest use and the largest use amount, about 800 ten thousand of plasticizers are used in the world every year, and among numerous plasticizers, the phthalate ester compound has the largest use amount which accounts for about 70% of the global use amount and accounts for the largest proportion of DEHP and DEP. Because the phthalate esters are not covalently bonded to the resin in plasticizers and other articles, but rather are bonded by van der waals forces or hydrogen bonds, the phthalate esters plasticizers readily diffuse into the surrounding environment during use, resulting in air, soil, water, etc., contamination. It is also of great concern that it can cause serious environmental and human health hazards due to its carcinogenic and harmful effects on the human reproductive system. Therefore, finding an effective way to reduce or eliminate phthalate residues in food and environment has become a problem that is currently urgently needed to be solved. Compared with the traditional physical and chemical treatment method, the microbial degradation is concerned by the characteristics of high efficiency, environmental protection, low price, safety and no secondary pollution.
The method for degrading the phthalate by microorganisms mostly depends on the enzymolysis of intracellular enzymes, the enzymolysis method has the advantages of environmental protection, no public nuisance and the like, and the method for cloning the phthalate degrading enzyme gene by utilizing the biotechnology and producing the phthalate degrading enzyme by utilizing engineering bacteria as a bioreactor is a main research direction in the future. The traditional wild degrading enzyme gene has the problems of difficult expression, low enzyme activity, poor stability and the like when being expressed in engineering bacteria, and can not achieve the aim of effectively degrading phthalate and meet the requirement of realizing industrial application.
Based on the above practical difficulties, there is an urgent need to find more potential phthalate degrading enzymes from nature. In recent years, Metagenomics (Metagenomics) has rapidly developed, and is a research strategy for researching the genetic composition and community functions of all microorganisms contained in an environmental sample by directly extracting DNA of all microorganisms from the environmental sample to construct a metagenomic library. Namely, total DNA of all microorganisms in a specific environment is cloned, and a new physiologically active substance is obtained by means of constructing a metagenome library, screening and the like. At present, scientists at home and abroad clone new genes such as lipid hydrolase, cellulase and the like by using the technology, and the enzymes have good properties and application values. So far, no research report for finding novel efficient phthalate degrading enzyme from soil by using metagenome technology is available at home and abroad.
Disclosure of Invention
The first purpose of the invention is to provide a gene of phthalate degrading enzyme and a preparation method thereof.
The second purpose of the invention is to provide a phthalate degrading enzyme and a preparation method thereof.
The third purpose of the invention is to provide a recombinant plasmid pET32a-est1012 of the phthalate ester degrading enzyme.
The fourth object of the present invention is to provide a recombinant engineered bacterium containing the above phthalate ester-degrading enzyme.
The first, third and fourth objects of the present invention are achieved by the following technical solutions: a gene of phthalate degrading enzyme is named as est1012, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1:
GGCCTTTGGGTGCGTTATACTGAAGGGGACGATCAAAAGCGTTGGCGTTCGTTAAATATTTACGAGTGCTGCCTCATGTCAAAGTCAGAAAAAATAGTATAGGAGGTAACATATGGGATCCGAACATCATCATCATCATCATGAATCGGACGCTACCGGTCCTGCAACATGGCATCCACTAGCCCTACAGACATCGTATCTCGCCTCCGGGAACGCTGCAGGTCAAACGACTGGTGAAGATTACCAACCGGAGCTGATGTCGTGGTATTTCCTTGCCGGTGTCGATGTCGTTCCGATGGATGACAGTACGAGTGCAATCGTCACACTGGGTGATTCGATTACTGATGGATACGCCTCAACGCCGGATACGAACCGTCGCTATCCCAATTATCTTGCCCAACGGCTCAACGATCAGTCCAATATTGACCGTTCAGTACTCAATGCCGGTATTTCGGGGAATCGTATCCTCCACGATTCGCTGTCAGAGGGATCGTTCGGCACGAACGCTCTTGAGCGTCTCAATCGGGACGTAATCGCTCAACCAGGTGCGACGGATGTAATCGTCTTAGAGGGGATCAATGACATCGGTCAGTATCCACCATCGGTGAGCGCTGAGCAGATCATTCAAGGGCTCAAACAGATCGCGACACAGTGTCATGCCCACGGTTTGAACGTCTATGCTGGGACGCTCACGCTGACTCGTGGGACGGGTGAACGGTATAGTTCGCCAACGGGCGAAGCAAAACGGGAAATGGTAAACGAATTCATCAGATCGAATGAATATTTCGATGACGTCATCGATTTCGATGCGGCCATCCAAGATCCGGATCAGCCAGATCGGATGGATCCGGACTTCGATAGTGGGGATCATCTCCATCCGAATGATGCTGTCTATGAGGCGATGGCGAATGCAATCGATCTTTCGCTGTTTGAGTAACTTGCGGCCGCACTCGAGGAGCACCACCACCACCACTGGAGATCCGGCTGCTAGCAAAGGCCGAAAAGAAGCTG
the preparation method of the phthalate degrading enzyme gene comprises the following steps:
(1) extracting total DNA from a sample, purifying the total DNA, and carrying out enzyme digestion on the total DNA;
(2) recovering the DNA fragment obtained in the step (1) after enzyme digestion, and connecting the DNA fragment with a pUC118 vector to obtain a plasmid;
(3) transforming the plasmid obtained in the step (2), screening a library and identifying a positive clone;
(4) sequencing, namely, naming the plasmid as pUC118-est1012, and designing PCR primers;
(5) carrying out PCR amplification by using a plasmid pUC118-est1012 as a template to clone a gene;
(6) and purifying and enzyme-cutting the PCR product to obtain the phthalate degrading enzyme gene.
Further, the PCR primers are:
est1012-HindIII-F:5’-CCCAAGCTTGGCCTTTGGGTGCGTTATACTGAAG-3' (HindIII cleavage site is underlined);
est1012-Bg1II-R:5’-GGAAGATCTCAGCTTCTTTTCGGCCTTTGCTAGCAG-3' underlined is the BglII cleavage site).
Further, the operation of the step (5) is: plasmid pUC118-est1012 as template, est1012-HindIII-F and est1012-Bg1II-R as primer, PrimeSTARTMThe MaxPremix carries out PCR amplification of est1012 gene segments, and the system is as follows:
the reaction conditions for PCR were:
the second purpose of the invention is realized by the following technical scheme: an acetyl xylan esterase, the amino acid sequence of which is shown as SEQ ID NO. 2:
MGSEHHHHHHESDATGPATWHPLALQTSYLASGNAAGQTTGEDYQPELMSWYFLAGVDVVPMDDSTSAIVTLGDSITDGYASTPDTNRRYPNYLAQRLNDQSNIDRSVLNAGISGNRILHDSLSEGSFGTNALERLNRDVIAQPGATDVIVLEGINDIGQYPPSVSAEQIIQGLKQIATQCHAHGLNVYAGTLTLTRGTGERYSSPTGEAKREMVNEFIRSNEYFDDVIDFDAAIQDPDQPDRMDPDFDSGDHLHPNDAVYEAMANAIDLSLFE
the preparation method of the phthalate degrading enzyme comprises the steps of connecting the phthalate degrading enzyme gene with a pET-32a (+) expression vector to obtain a connection product recombinant plasmid pET-32a (+) -est1012, transforming the recombinant plasmid into escherichia coli B L21 competent cells to form recombinant engineering bacteria, culturing the recombinant engineering bacteria, crushing to obtain a crude enzyme solution, and purifying to obtain the phthalate degrading enzyme.
The detailed operation is that the phthalate degrading enzyme gene of claim 1 is connected with pET-32a (+) expression vector to obtain recombinant plasmid pET-32a (+) -est1051, the recombinant plasmid is transformed into Escherichia coli B L21 competent cell to form recombinant engineering bacteria, the recombinant engineering bacteria is inoculated into 50ml flask containing L B culture medium of 100 ug/m L percent of concentration of ampicillin and cultured in a shaker at 37 ℃, when OD is reached600When the value reaches about 0.8-1.0, adding IPTG to make its final concentration be 0.6 mmol/L, inducing expression at 35 deg.C for 16h, collecting thallus, ultrasonic breaking thallus to obtain crude enzyme solutionPurification was performed using Resin kit. Collecting the purified enzyme and storing in a refrigerator at the temperature of-20 ℃ for later use to obtain the phthalate degrading enzyme.
An application of phthalate ester degrading enzyme in degrading phthalate ester compounds.
An application of phthalate degrading enzyme in degrading DEHP, DEP and DNP.
In the present specification, the phthalate ester-degrading enzyme is also referred to as a recombinant esterase or esterase.
The advantages of the invention include: the phthalate degrading enzyme provided has efficient soluble expression in an escherichia coli expression system;
the optimal catalytic activity of p-nitrophenyl laurate, C2, was measured at a temperature ranging from 4 to 80 ℃. The optimal reaction temperature is 40 ℃, and the maximum activity is more than 40% at 4-50 ℃. The enzyme is proved to have higher adaptability to middle and low temperature. After the temperature is kept for 2 hours within the temperature range of 4-90 ℃, the esterase is stable at the temperature of 0-50 ℃. The relative enzyme activity remained at 40%. Even at 70 ℃, the enzyme activity is still maintained at about 20% after the temperature is maintained for 2 hours, which indicates that the enzyme has good thermal stability; the optimum pH value of the esterase is 8.95, and the relative activity is more than 60% at the pH value of 8.0-12.0. Thus indicating stability under both alkaline and neutral conditions;
the organic solvents DMSO, methanol, ethanol, isopropanol and Triton X-100 have strong promotion effect on enzyme activity at 1%, and the promotion effect of the organic solvents ethanol and isopropanol at 30% is reduced with the increase of solvent concentration, but the activities of the ethanol and isopropanol still are more than 200%. The esterase shows strong tolerance to organic solvents; the (1mM, 10mM) SDS and (1mM, 10mM) EDTA have inhibition and promotion effects on the protein, respectively; zn at a metal ion concentration of 1mM2+And L i+Has promoting effect on enzyme activity, K+、Na+、Mg2+、Mn2+、Ni2+、Ag+、Co2+Has great inhibition effect on enzyme activity. Zn at a metal ion concentration of 10mM2 +、Ni2+、Fe2+、Mg2+、Mn2+、Cu2+、Ag+The enzyme activity is greatly promoted and is increased to more than 2 times of the original activity of the enzyme;
the esterase is found to have strong degradation effect on 50mg/ml DEP and DEHP phthalate compounds when the enzymatic properties of the esterase are measured, and the result of the measurement of the degradation of DEP and DEHP by the recombinant esterase shows that the degradation rate of the esterase exceeds 90 percent, so that the esterase has wide application prospect in the aspect of degrading phthalate plasticizers.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention:
FIG. 1 is an SDS-PAGE electrophoresis chart in example 2;
wherein, M is a standard protein molecular weight marker, 1 is a recombinant protein crude extract, and 2 is a purified recombinant protein;
FIG. 2 is an experimental graph showing the reaction characteristics of esterase Est1012 with different substrates;
FIG. 3 is a standard curve for p-nitrophenol;
FIG. 4 is a line graph showing the results of optimum temperature and thermal stability of the recombinant esterase;
FIG. 5 is a line graph showing the results of pH optimum and pH stability of the recombinant esterase;
FIG. 6 shows the effect of different metal ions on esterase enzyme activity;
FIG. 7 shows the results of the influence of different organic solvents and compounds on esterase enzyme activity;
FIG. 8 is a GC-Ms analysis spectrum of the degradation of phthalate DEHP by the recombinant esterase;
FIG. 9 is a GC-Ms analysis spectrum of the degradation of phthalate DEP by the recombinant esterase.
Detailed Description
The present invention will be described in detail with reference to the drawings and specific embodiments, which are illustrative of the present invention and are not to be construed as limiting the present invention.
Example 1: cloning and transformation of esterase genes
1. The soil sample is from a wild asafetida distribution area of southern mountain of southern Boragite Hezi, Queguer basin, Xinjiang, and soil 5-10cm below the surface of the earth.1.25 g of the soil sample is weighed into 5m L EP tubes, 1.5m L DNA extracting solution is added into each tube, the tubes are inverted and mixed evenly, the tubes are shaken for 30min at 37 ℃ and 130rpm, 400ul of 10% SDS is added into each tube and mixed evenly, the tubes are shaken evenly in a water bath for 2h at 65 ℃ every 15min, the tubes are centrifuged at 10000rpm for 10min, isopropanol with the volume 0.7 time is added into the supernatant, the supernatant is centrifuged and discarded, an appropriate amount of 70% ethanol is added for washing, and an appropriate amount of TE buffer solution is used for dissolving to obtain the total DNA.
2. The method comprises the steps of purifying and recovering total DNA by using a DNA Extraction Kit, storing the total DNA at-20 ℃ for later use, detecting the purity and quality of the total DNA by using agarose gel electrophoresis, carrying out incomplete enzyme digestion on the total DNA by using BamHi restriction enzyme, carrying out electrophoresis on 1% agarose gel, carrying out ultraviolet observation on a cut DNA fragment of 2.5-10Kb, putting the cut DNA fragment into a 2M L centrifuge tube, recovering a target fragment by using the DNA Extraction Kit, referring to the instruction, connecting the recovered enzyme digestion fragment with a vector PUC118/BamHi (BAP) at 22 ℃ for 3h under the action of T4DNA L igase, purifying a connection product, and then carrying out electric shock on the connection product into competent Escherichia coli DH5a, thereby constructing a metagenome library, obtaining a positive clone by screening, culturing the positive clone at 37 ℃, 220r/min for overnight, taking 4M L bacteria for plasmid Extraction and sequencing, and finding that the DNA consists of 1011 bases and contains 274 amino acids by using NCEST as a shaking table BI.
Example 2: amplification and expression of esterase genes
And comparing and designing a PCR primer according to a sequencing result and NCBI, wherein HindIII and BglII restriction enzyme sites are introduced into the primer, and the sequence of the primer is as follows:
est1012-HindIII-F:5’-CCCAAGCTTGGCCTTTGGGTGCGTTATACTGAAG-3' (HindIII cleavage site is underlined);
est1012-Bg1II-R:5’-GGAAGATCTCAGCTTCTTTTCGGCCTTTGCTAGCAG-3' underlined is the BglII cleavage site).
Using a plasmid containing pUC118-est1012 as a template, Prime STAR was usedTMMax Premix is used for PCR amplification,
the system is as follows:
TABLE 1
The amplification conditions were as follows:
TABLE 2
The purified PCR product and the vector pET-32a (+) are subjected to double enzyme digestion treatment by HindIII and BglII respectively, and the enzyme digestion reaction is carried out for 20min at 37 ℃. The enzyme digestion reaction system is as follows:
TABLE 3 enzyme digestion System
Table The system of digestion
Subjecting the digested product to agarose gel electrophoresis, and detecting with OMEGA corporation (GelExtraction Kit) Kit for purifying and recovering the enzyme digestion product, and the specific steps refer to the instruction. And (4) storing the recovered enzyme digestion product at-20 ℃ for later use.
The est1012 gene is linked to a vector
The two-enzyme-digested and purified est1012 gene and a vector pET-32a (+) are subjected to ligation reaction for 1h under the action of T4DNA L igase of Fermentas under the reaction condition of 22 ℃, and the ligation reaction system is shown in Table 4:
TABLE 4 connection System
Table 4 The system for ligation
Conversion of ligation products
Competent cells were prepared and transformed according to the molecular cloning guidelines.
Plasmid est1012-pET-32a (+) transformation competence E.coli B L21 (DE 3). transformants were randomly picked, after culturing, the plasmids were extracted and double-restriction verified, transformants with correct restriction verification were inoculated into flasks containing 50ml L B medium (100. mu.g/m L conjugation of ampicillin) and cultured in a shaker at 37 ℃ when OD was OD600When the value reaches about 0.8-1.0, IPTG is added to make the final concentration be 0.6mmol/L, inducing expression at 35 deg.C for 16h, collecting thallus, and ultrasonically crushing thallus to obtain crude enzyme solutionPurification was performed using Resin kit. The purified enzyme was collected and stored in a-20 ℃ freezer for future use.
Example 3: reaction characteristics of esterases with different substrates
In order to determine the enzyme activity of esterase hydrolyzing different carbon chains, 8 types of reaction groups are arranged according to different enzymatic reaction substrates, and each type of reaction group is internally provided with three parallel experiment groups and a control experiment group without enzyme liquid; each sample was 200. mu.l enzyme reaction per parallel group: mu.l of the enzyme solution, p-nitrophenol ester (substrate for enzymatic reaction) at a final concentration of 1mM, and the balance supplemented with 0.04M B-R triacid buffer solution (pH7), mixed well and reacted at 40 ℃ and pH7 in a water bath for 20 min. P-nitrophenylacetate (C2), p-nitrophenylbutyrate (C4), p-nitrophenylacetate (C6), p-nitrophenylacetate (C8), p-nitrophenyldecanoate (C10), p-nitrophenylsulfate (C12), p-nitrophenylacetate (C14) and p-nitrophenylacetate (C16) were added to the enzymatic reaction system of each parallel group, respectively, at a final concentration of 1 mM; the absorbance at a wavelength of 405nm was measured with a microplate reader. The highest value of the enzyme activity is 100 percent, the relative enzyme activity is taken as a graph, and one enzyme activity unit is defined as the enzyme amount required by hydrolyzing a substrate per minute to generate 1 mu mol of p-nitrophenol.
As shown in fig. 2: the best substrate for the recombinant esterase is C2.
EXAMPLE 4 determination of the p-nitrophenol (pNP) Standard Curve
The absorbance at OD405nm was measured, and the absorbance at OD405nm at a concentration of 0.00mM of p-nitrophenol (pNP) was used as a blank. The pNP concentration was plotted on the abscissa, as shown in Table 5 below, and the OD405nm value was plotted on the ordinate, as shown in FIG. 3.
TABLE 5 determination of pNP Standard Curve
Table 5 Drawing of pNP Standard Curve
Example 5: optimum temperature and temperature stability of esterase
Setting 8 types of experimental groups according to different experimental temperatures, setting three parallel experimental groups and a control group for each type of experimental group, wherein each parallel experimental group is a 200 mu l enzyme reaction system which comprises 10 mu l enzyme solution and the optimal substrate of enzyme with the final concentration of 1mM, supplementing the rest with 0.04mM B-R triacid buffer solution (pH7), placing each type of experimental group in water bath with the temperature of 4 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ respectively for reaction for 20min, and measuring OD405And (4) determining the optimal temperature for the hydrolysis reaction of the esterase. Respectively carrying out water bath on the enzyme solution at 4 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ and 90 ℃ for 2h, determining the residual enzyme activity of the esterase under the condition of pH7 according to the method, drawing a curve by using the relative enzyme activity to the temperature with the highest enzyme activity as 100 percent, and determining the influence of the temperature on the stability of the enzyme activity of the recombinant esterase.
As shown in fig. 4: the maximum activity is shown at 40 ℃, and the maximum activity is shown above 40% at 4-50 ℃. The enzyme is proved to have higher adaptability to middle and low temperature. The esterase is stable at 0-50 ℃. The activity exceeds 40% of the maximum. The enzyme activity is maintained at about 20% even after culturing at 70 ℃ for 2h, which indicates that the enzyme has good thermal stability.
Example 6: optimum pH and pH stability of esterase
Setting 6 types of experimental groups, respectively adopting 0.04mM B-R triacid buffer solutions with different pH values, wherein the pH values of the buffer solutions are respectively set to be 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0, and each type of experimental group is provided with three parallel experimental groups and a blank control experimental group.
Each parallel group was 200. mu.l of an enzyme reaction system containing 10. mu.l of an enzyme solution and the optimum substrate C2 for the enzyme at a final concentration of 1mM, the remainder being supplemented with a corresponding 0.04mM B-R triacid buffer at a pH to give an enzyme solution under different pH conditions. Performing water bath at the optimum reaction temperature of 40 deg.C for 20min, and measuring OD405A value; after standing at 40 ℃ for 2h, the solution was brought to pH8 as above.And measuring the residual enzyme activity of the esterase under the condition of 95. The highest enzyme activity was defined as 100%, and the effect of pH on the stability of the enzyme activity of the recombinant esterase was determined by plotting the relative enzyme activity against pH, as shown in fig. 5: the esterase has an optimum pH value of 8.95 and relative activity at pH 8.0-12.0>60 percent. In terms of pH stability, the activity of the enzyme is maintained at 30% or more at a pH of 10-12.0. Thus indicating stability under both alkaline and neutral conditions.
Example 7: influence of different metal ions on esterase enzyme activity.
Under the conditions of the optimal reaction temperature and the optimal reaction pH value of the esterase, different metal ions are correspondingly added into an enzyme reaction system containing enzyme liquid of each parallel experiment group, namely Fe2+ with the final concentration of 1mM, L i + with the final concentration of 1mM, Ag + with the final concentration of 1mM, Cu2+ with the final concentration of 1mM, Ni2+ with the final concentration of 1mM, Zn2+ with the final concentration of 1mM, Co2+ with the final concentration of 1mM, Mg2+ with the final concentration of 1mM, Mn2+ with the final concentration of 1mM, K + with the final concentration of 1mM, Na + with the final concentration of 1mM, Fe2+ with the final concentration of 10mM, L i + with the final concentration of 10mM, Ag + with the final concentration of 10mM, Cu2+ with the final concentration of 10mM, Ni2+ with the final concentration of 10mM, Zn + with the final concentration of 10mM, and blank enzyme activity of 10mM, and the relative concentration of the enzyme activity of the metal ions is determined by plotting the relative influence of the metal ions on the enzyme activity of the metal ions, namely the enzyme activity of 10mM, namely the metal ions, namely the enzyme activity of the blank metal ions, namely the blank ion added into the enzyme activity of the metal ions, namely.
The results are shown in FIG. 6: zn at a metal ion concentration of 1mM2+And L i+Has promoting effect on enzyme activity, K+、Na+、Fe2+、Mg2+、Mn2+、Ni2+、Ag+、Co2+Has great inhibition effect on enzyme activity. Zn at a metal ion concentration of 10mM2+、Ni2+、Fe2 +、Mg2+、Mn2+、Cu2+、Ag+Has great promotion effect on enzyme activity, and the activity is increased to more than 2 times of the original activity of the enzyme.
Example 8: influence of different organic reagents and compounds on esterase enzyme activity.
According to different organic reagents and compounds, 19 types of reaction experimental groups are arranged, and each type of reaction experimental group is provided with three parallel experimental groups and a control group. Under the conditions of the optimal reaction temperature and the optimal reaction pH value of esterase, different reagents and compounds are respectively and correspondingly added into an enzymatic reaction system containing enzyme liquid of each parallel experimental group: 1% methanol, 15% methanol, 30% methanol, 1% acetone, 15% acetone, 30% acetone, 1% isopropanol, 15% isopropanol, 30% isopropanol, 1% Triton X-100, 15% Triton X-100, 30% Triton X-100, 1% DMSO, 15% DMSO, 30% DMSO, 1mM EDTA, 10mM EDTA and 1mM SDS, 10mM SDS to determine the effect of organic solvents and compounds on esterase enzyme activity, and enzyme-solution enzyme activity of a control group to which no compound and organic reagent were added was defined as 100%. The relative enzyme activity was plotted for each compound,
as shown in fig. 7: the organic solvents DMSO, methanol, ethanol, isopropanol and Triton X-100 have strong promotion effect on enzyme activity at 1%, and the promotion effect of the organic solvents ethanol and isopropanol at 30% is reduced with the increase of solvent concentration, but the activities of the ethanol and isopropanol still are more than 200%. The esterase shows strong tolerance to organic solvents; the enzyme activity is inhibited by (1mM, 10mM) SDS, and the enzyme activity is promoted by (1mM, 10mM) EDTA.
Example 9: application of esterase in degrading phthalate compounds
Respectively adding 400ul esterase enzyme solution into 200ul of 50mg/m L DEHP standard solution and 200ul of 50mg/m L DEP standard solution, respectively mixing, reacting at 40 ℃ for 6 hours, adding concentrated HC L to terminate the reaction, adding equal volume of ethyl acetate for extraction, adding a proper amount of anhydrous sodium sulfate to remove all water, centrifuging at a high speed to transfer ethyl acetate to a new centrifuge tube, evaporating at 50 ℃ until all the ethyl acetate is volatilized, taking 1 mu l of the mixture according to the following table conditions to perform GC-Ms determination, and analyzing by using GC-MS, wherein GC-MS determination maps are shown in figures 8 and 9.
TABLE 6
The DNP has no map because the instrument for detecting the residual amount of the sample does not display, peaks are obtained according to the map, and the peak corresponds to the ordinate concentration of the standard curve, namely the residual sample concentration, according to the formula, the degradation rate is (A0-A1)/A0 × 100% (A0 represents the total amount of the added DEHP and the DEP, and the residual amount of the A1 after degradation), the DEHP and the DEP degradation rate can reach more than 90%.
The technical solutions provided by the embodiments of the present invention are described in detail above, and the principles and embodiments of the present invention are explained herein by using specific examples, and the descriptions of the embodiments are only used to help understanding the principles of the embodiments of the present invention; meanwhile, for a person skilled in the art, according to the embodiments of the present invention, there may be variations in the specific implementation manners and application ranges, and in summary, the content of the present description should not be construed as a limitation to the present invention.
Sequence listing
<110> university of Guangdong department of pharmacy
<120> phthalate ester degrading enzyme gene, its coding product and preparation method
<130>2020
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cgaacatcat catcatcatc atgaatcgga cgctaccggt cctgcaacat ggcatccact 180
agccctacag acatcgtatc tcgcctccgg gaacgctgca ggtcaaacga ctggtgaaga 240
ttaccaaccg gagctgatgt cgtggtattt ccttgccggt gtcgatgtcg ttccgatgga 300
tgacagtacg agtgcaatcg tcacactggg tgattcgatt actgatggat acgcctcaac 360
gccggatacg aaccgtcgct atcccaatta tcttgcccaa cggctcaacg atcagtccaa 420
tattgaccgt tcagtactca atgccggtat ttcggggaat cgtatcctcc acgattcgct 480
gtcagaggga tcgttcggca cgaacgctct tgagcgtctc aatcgggacg taatcgctca 540
accaggtgcg acggatgtaa tcgtcttaga ggggatcaat gacatcggtc agtatccacc 600
atcggtgagc gctgagcaga tcattcaagg gctcaaacag atcgcgacac agtgtcatgc 660
ccacggtttg aacgtctatg ctgggacgct cacgctgact cgtgggacgg gtgaacggta 720
tagttcgcca acgggcgaag caaaacggga aatggtaaac gaattcatca gatcgaatga 780
atatttcgat gacgtcatcg atttcgatgc ggccatccaa gatccggatc agccagatcg 840
gatggatccg gacttcgata gtggggatca tctccatccg aatgatgctg tctatgaggc 900
gatggcgaat gcaatcgatc tttcgctgtt tgagtaactt gcggccgcac tcgaggagca 960
ccaccaccac cactggagat ccggctgcta gcaaaggccg aaaagaagct g 1011
<210>2
<211>274
<212>PRT
<213> unknown (Unkwon)
<400>2
Met Gly Ser Glu His His His His His His Glu Ser Asp Ala Thr Gly
1 5 10 15
Pro Ala Thr Trp His Pro Leu Ala Leu Gln Thr Ser Tyr Leu Ala Ser
20 25 30
Gly Asn Ala Ala Gly Gln Thr Thr Gly Glu Asp Tyr Gln Pro Glu Leu
35 40 45
Met Ser Trp Tyr Phe Leu Ala Gly Val Asp Val Val Pro Met Asp Asp
50 55 60
Ser Thr Ser Ala Ile Val Thr Leu Gly Asp Ser Ile Thr Asp Gly Tyr
65 70 75 80
Ala Ser Thr Pro Asp Thr Asn Arg Arg Tyr Pro Asn Tyr Leu Ala Gln
85 90 95
Arg Leu Asn Asp Gln Ser Asn Ile Asp Arg Ser Val Leu Asn Ala Gly
100 105 110
Ile Ser Gly Asn Arg Ile Leu His Asp Ser Leu Ser Glu Gly Ser Phe
115 120 125
Gly Thr Asn Ala Leu Glu Arg Leu Asn Arg Asp Val Ile Ala Gln Pro
130 135 140
Gly Ala Thr Asp Val Ile Val Leu Glu Gly Ile Asn Asp Ile Gly Gln
145 150 155 160
Tyr Pro Pro Ser Val Ser Ala Glu Gln Ile Ile Gln Gly Leu Lys Gln
165 170 175
Ile Ala Thr Gln Cys His Ala His Gly Leu Asn Val Tyr Ala Gly Thr
180185 190
Leu Thr Leu Thr Arg Gly Thr Gly Glu Arg Tyr Ser Ser Pro Thr Gly
195 200 205
Glu Ala Lys Arg Glu Met Val Asn Glu Phe Ile Arg Ser Asn Glu Tyr
210 215 220
Phe Asp Asp Val Ile Asp Phe Asp Ala Ala Ile Gln Asp Pro Asp Gln
225 230 235 240
Pro Asp Arg Met Asp Pro Asp Phe Asp Ser Gly Asp His Leu His Pro
245 250 255
Asn Asp Ala Val Tyr Glu Ala Met Ala Asn Ala Ile Asp Leu Ser Leu
260 265 270
Phe Glu
<210>3
<211>34
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>3
cccaagcttg gcctttgggt gcgttatact gaag 34
<210>4
<211>36
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>4
ggaagatctc agcttctttt cggcctttgc tagcag 36
Claims (10)
1. A phthalate ester degrading enzyme gene, which is characterized in that:
is named as est1012, and the nucleotide sequence is shown as SEQ ID NO. 1.
2. A phthalate degradation enzyme encoded by the phthalate degradation enzyme gene according to claim 1, wherein:
the amino acid sequence is shown in SEQ ID NO. 2.
3. A recombinant plasmid pET-32a (+) -est1012 comprising the phthalate ester-degrading enzyme gene according to claim 1.
4. A recombinant engineered bacterium comprising the phthalate ester-degrading enzyme gene according to claim 1.
5. The method for producing the phthalate ester-degrading enzyme gene according to claim 1, wherein:
the method comprises the following steps:
(1) extracting total DNA from a sample, purifying the total DNA, and carrying out enzyme digestion on the total DNA;
(2) recovering the DNA fragment obtained in the step (1) after enzyme digestion, and connecting the DNA fragment with a pUC118 vector to obtain a plasmid;
(3) transforming the plasmid obtained in the step (2), screening a library and identifying a positive clone;
(4) sequencing, namely, naming the plasmid as pUC118-est1012, and designing PCR primers;
(5) carrying out PCR amplification by using a plasmid pUC118-est1012 as a template to clone a gene;
(6) and purifying and enzyme-cutting the PCR product to obtain the phthalate degrading enzyme gene.
6. The method for producing the phthalate ester-degrading enzyme gene according to claim 5, wherein:
the PCR primers are:
est1012-HindIII-F:5’-CCCAAGCTTGGCCTTTGGGTGCGTTATACTGAAG-3' (HindIII cleavage site is underlined);
est1012-Bg1II-R:5’-GGAAGATCTCAGCTTCTTTTCGGCCTTTGCTAGCAG-3' underlined is the BglII cleavage site).
7. The method for producing the phthalate ester-degrading enzyme gene according to claim 5, wherein:
the operation of the step (5) is as follows: plasmid pUC118-est1012 is used as a template, est1012-HindIII-F and est1012-Bg1II-R are used as primers, and Prime STAR is adoptedTMMax Premix is used for carrying out PCR amplification on est1012 gene segments, and the system is as follows:
the reaction conditions for PCR were:
8. the method for producing the phthalate ester-degrading enzyme according to claim 2, wherein:
connecting the phthalate degrading enzyme gene with a pET-32a (+) expression vector to obtain a connection product recombinant plasmid pET-32a (+) -est1012, converting the recombinant plasmid into escherichia coli B L21 competent cells to form recombinant engineering bacteria, culturing the recombinant engineering bacteria, crushing to obtain a crude enzyme solution, and purifying to obtain the phthalate degrading enzyme.
9. The method for producing a phthalate ester-degrading enzyme according to claim 8, wherein:
the detailed operation is that the phthalate degrading enzyme gene is connected with pET-32a (+) expression vector to obtain the recombinant plasmid pET-32a (+) -est1012 of the connection product, the recombinant plasmid is transformed into escherichia coli B L21 competent cell to form recombinant engineering bacteria, the recombinant engineering bacteria is inoculated into 50ml flask containing L B culture medium of 100 mug/m L percent of concentration of ampicillin to be cultured in a shaker at 37 ℃, when OD is reached600When the value reaches about 0.8-1.0, adding IPTG to make its final concentration be 0.6 mmol/L, inducing expression at 35 deg.C for 16h, collecting thallus, and ultrasonically crushing to obtain crude enzyme solutionPurifying with a Resin kit; collecting the purified enzyme and storing in a refrigerator at the temperature of-20 ℃ for later use to obtain the phthalate degrading enzyme.
10. Use of the phthalate ester-degrading enzyme according to claim 2 for degrading phthalate ester compounds.
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CN110982803A (en) * | 2019-12-25 | 2020-04-10 | 南京农业大学 | Novel phthalate ester hydrolase EstJ6, and coding gene and application thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101985627A (en) * | 2010-11-17 | 2011-03-16 | 中山大学 | Novel esterase and application thereof |
-
2020
- 2020-04-09 CN CN202010275465.3A patent/CN111394372B/en active Active
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CN101985627A (en) * | 2010-11-17 | 2011-03-16 | 中山大学 | Novel esterase and application thereof |
Non-Patent Citations (3)
Title |
---|
HAN HUANG等: "Biodegradation of Structurally Diverse Phthalate Esters by a Newly Identified Esterase with Catalytic Activity toward Di(2-ethylhexyl) Phthalate", 《J. AGRIC. FOOD CHEM》 * |
LI,H.等: "登录号MK524484.1", 《NCBI_GENBANK》 * |
沈思等: "细菌降解邻苯二甲酸酯的研究进展", 《生物工程学报》 * |
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CN110982803A (en) * | 2019-12-25 | 2020-04-10 | 南京农业大学 | Novel phthalate ester hydrolase EstJ6, and coding gene and application thereof |
CN110982803B (en) * | 2019-12-25 | 2022-09-27 | 南京农业大学 | Novel phthalate hydrolase EstJ6, and coding gene and application thereof |
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