CN111394372B

CN111394372B - Phthalate degrading enzyme gene, its coding product and preparation method

Info

Publication number: CN111394372B
Application number: CN202010275465.3A
Authority: CN
Inventors: 李荷; 王春庆
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2020-04-09
Filing date: 2020-04-09
Publication date: 2022-04-26
Anticipated expiration: 2040-04-09
Also published as: CN111394372A

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

Phthalate degrading enzyme gene, its coding product and preparation method

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, PrimeSTAR^TMThe 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 BL21 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 as follows: the method comprises the steps of connecting the phthalate ester degrading enzyme gene of claim 1 with pET-32a (+) expression vector to obtain a recombinant plasmid pET-32a (+) -est1051, transforming the recombinant plasmid into Escherichia coli BL21 competent cells to form recombinant engineering bacteria, inoculating the recombinant engineering bacteria into a 50mL flask containing 100 μ g/mL concentration of ampicillin LB culture medium, culturing in a shaker at 37 ℃, and culturing when OD is OD₆₀₀When the value reaches about 0.8-1.0, adding IPTG to make the final concentration 0.6mmol/L, inducing expression at 35 deg.C for 16h, collecting thallus, and ultrasonically crushing to obtain crude enzyme solution. Crude enzyme solution adopts

Purification 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;

organic solvents DMSO, methanol, ethanol, isopropanol, Triton X-When the concentration of the solvent is increased, the promotion effect of the organic solvents ethanol and isopropanol is reduced when the concentration of the solvent is 30%, 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 Li⁺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 of the invention is from the wild asafetida distribution area of south mountain of south Boragite Hezi of quasi-Geer basin in Xinjiang and the soil 5-10cm below the earth surface. Weigh 1.25g soil sample into 5mL EP tube, each tube add 1.5mL DNA extract, reverse mixing, 37 degrees C under 130rpm conditions in the shaking table for 30 min. Adding 400ul of 10% SDS into each tube, mixing uniformly, shaking uniformly every 15min in 65 ℃ water bath for 2h, centrifuging at 10000rpm for 10min, taking the supernatant, adding 0.7 time volume of isopropanol, centrifuging, removing the supernatant, adding a proper amount of 70% ethanol for washing, and dissolving with a proper amount of TE buffer solution to obtain the total DNA.

2. The total DNA was purified and recovered by using the DNA Extraction Kit and stored at-20 ℃ for further use. The purity and quality of the total DNA was checked by agarose gel electrophoresis. Carrying out incomplete enzyme digestion on the total DNA by using a BamHI restriction enzyme, carrying out 1% agarose gel electrophoresis, carrying out ultraviolet observation on a cut DNA fragment of 2.5-10Kb, putting the cut DNA fragment into a 2ML centrifuge tube, and recovering a target fragment by using a DNA Extraction Kit, wherein the steps are referred to an instruction book. The recovered enzyme digestion fragment and a vector PUC118/BamHi (BAP) are connected for 3h at 22 ℃ under the action of T4DNA Ligase, and the connection product is electrically shocked into competent Escherichia coli DH5a after being purified, thereby constructing a metagenome library. Screening to obtain a positive clone. Shaking culture at 37 deg.C and 220r/min overnight, taking 4mL thallus, extracting plasmid, sequencing, and sequence analysis of inserted fragment by NCBI to find that the DNA consists of 1011 bases, contains 274 amino acids, and is named as est 1012.

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:

Using a plasmid containing pUC118-est1012 as a template, Prime STAR was used^TMMax 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 (

Gel Extraction 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 Ligase 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 (+) was transformed into competent E.coli BL21(DE 3). Randomly selecting transformants, culturing, extracting plasmids of the transformants and carrying out double enzyme digestion verification. The transformants verified to be correct by digestion were inoculated into 50mL of LB medium (100. mu.g/mL of concentration of ampicillin) in flasks and cultured on a shaker at 37 ℃ when OD was measured₆₀₀When the value reaches about 0.8-1.0, adding IPTG to make the final concentration 0.6mmol/L, inducing expression at 35 deg.C for 16h, collecting thallus, and ultrasonically crushing to obtain crude enzyme solution. Crude enzyme solution adopts

Purification 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 experimental groups according to different experimental temperatures, wherein each experimental group is provided with three parallel experimental groups and a control group, each parallel experimental group is a 200 mu l enzyme reaction system which comprises 10 mu l enzyme solution and enzyme with the final concentration of 1mMOptimizing substrate, supplementing the rest with 0.04mM B-R triacid buffer solution (pH7), reacting in water bath at 4 deg.C, 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, and 80 deg.C for 20min, and determining 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 being placed at 40 ℃ for 2h, the esterase residual activity was measured according to the above method at pH 8.95. 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.

22 types of reaction experimental groups are arranged according to different metal ions, and each type of reaction experimental group is provided with three parallel experimental groups and one control group. Under the conditions of the optimal reaction temperature and the optimal reaction pH value of esterase, different metal ions are respectively and correspondingly added into the enzymatic reaction systems containing enzyme solutions of each parallel experimental group: fe2+ at a final concentration of 1mM, Li + at a final concentration of 1mM, Ag + at a final concentration of 1mM, Cu2+ at a final concentration of 1mM, Ni2+ at a final concentration of 1mM, Zn2+ at a final concentration of 1mM, Co2+ at a final concentration of 1mM, Mg2+ at a final concentration of 1mM, Mn2+ at a final concentration of 1mM, K + at a final concentration of 1mM, Na + at a final concentration of 10mM, Fe2+ at a final concentration of 10mM, Li + at a final concentration of 10mM, Ag + at a final concentration of 10mM, Cu2+ at a final concentration of 10mM, Ni2+ at a final concentration of 10mM, Zn2+ at a final concentration of 10mM, Co2+ at a final concentration of 10mM, Mg2+ at a final concentration of 10mM, Mn2+ at a final concentration of 10mM, and Na + at a final concentration of 10mM to determine the influence of different metal ions on the enzyme activity; and defining the enzyme activity of enzyme solution of a blank control group without adding metal ions as 100%, mapping each metal ion by using relative enzyme activity, and detecting the influence of different metal ions on the enzyme activity of Est1012 by measuring residual enzyme activity.

The results are shown in FIG. 6: zn at a metal ion concentration of 1mM²⁺And Li⁺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 activity of a control group to which no compound and no 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/mL DEHP standard solution and 200ul of 50mg/mL DEP standard solution, respectively mixing, reacting at 40 ℃ for 6h, then adding concentrated HCL to terminate the reaction, adding equal volume of ethyl acetate for extraction, adding a proper amount of anhydrous sodium sulfate to remove all water, carrying out high-speed centrifugation to transfer ethyl acetate to a new centrifuge tube, evaporating at 50 ℃ until the ethyl acetate is completely volatilized, taking 1 ul of the mixture according to the following table conditions to carry out GC-Ms determination, and analyzing by using GC-MS, wherein GC-MS determination maps are shown in figures 8 and 9.

TABLE 6

And the DNP has no map because the residual amount of the sample is too small and no instrument displays, and peaks are obtained according to the map and correspond to the ordinate concentration of the standard curve, namely the residual sample concentration. According to the formula: the degradation rate is (A0-A1)/A0 multiplied by 100% (A0 represents the total amount of the added DEHP and the DEP and the residual amount of the A1 after degradation), and the DEHP and DEP degradation rates 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

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Met Gly Ser Glu His His His His His His Glu Ser Asp Ala Thr Gly

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Pro Ala Thr Trp His Pro Leu Ala Leu Gln Thr Ser Tyr Leu Ala Ser

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Gly Asn Ala Ala Gly Gln Thr Thr Gly Glu Asp Tyr Gln Pro Glu Leu

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Met Ser Trp Tyr Phe Leu Ala Gly Val Asp Val Val Pro Met Asp Asp

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Ser Thr Ser Ala Ile Val Thr Leu Gly Asp Ser Ile Thr Asp Gly Tyr

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Ala Ser Thr Pro Asp Thr Asn Arg Arg Tyr Pro Asn Tyr Leu Ala Gln

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Arg Leu Asn Asp Gln Ser Asn Ile Asp Arg Ser Val Leu Asn Ala Gly

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Ile Ser Gly Asn Arg Ile Leu His Asp Ser Leu Ser Glu Gly Ser Phe

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Gly Thr Asn Ala Leu Glu Arg Leu Asn Arg Asp Val Ile Ala Gln Pro

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Gly Ala Thr Asp Val Ile Val Leu Glu Gly Ile Asn Asp Ile Gly Gln

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Tyr Pro Pro Ser Val Ser Ala Glu Gln Ile Ile Gln Gly Leu Lys Gln

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Ile Ala Thr Gln Cys His Ala His Gly Leu Asn Val Tyr Ala Gly Thr

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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

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Pro Asp Arg Met Asp Pro Asp Phe Asp Ser Gly Asp His Leu His Pro

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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

1. An application of phthalate degrading enzyme in degrading phthalate compounds, wherein the amino acid sequence of the phthalate degrading enzyme is shown as SEQ ID number 2, and the phthalate compounds are selected from DEHP, DEP and DNP.

2. The use of the phthalate ester-degrading enzyme according to claim 1 for degrading a phthalate ester compound, wherein:

the gene for coding the phthalate degrading enzyme is named asest1012，The nucleotide sequence is shown in SEQ ID NO. 1.

3. The use of the phthalate ester-degrading enzyme according to claim 1 for degrading a phthalate ester compound, wherein:

also comprises a recombinant plasmid pET-32a (+) -est1012。

4. The use of the phthalate ester-degrading enzyme according to claim 1 for degrading a phthalate ester compound, wherein:

also comprises a recombinant engineering bacterium containing the phthalate degrading enzyme gene.

5. The use of the phthalate ester-degrading enzyme according to claim 2 for degrading a phthalate ester compound, wherein:

the PCR primer of the phthalate degrading enzyme gene is as follows:

est1012-HindIII-F:5’-CCCAAGCTTGGCCTTTGGGTGCGTTATACTGAAg-3', the underlined part is the HindIII restriction site;

est1012-Bg1II-R:5’-GGAAGATCTCAGCTTCTTTTCGGCCTTTGCTAGCAG-3', the underlined part is the BglII cleavage site.

6. The use of a phthalate ester degrading enzyme according to claim 2 in degrading phthalate ester compounds, wherein:

the preparation method of the phthalate degrading enzyme comprises the following steps: the phthalate degradation enzyme gene is connected with a pET-32a (+) expression vector to obtain a recombinant plasmid pET-32a (+) -est1012，And (3) transforming the recombinant plasmid into escherichia coli BL21 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.

7. The use of the phthalate ester degrading enzyme according to claim 6, wherein:

the detailed operation is as follows: the phthalate degradation enzyme gene is connected with a pET-32a (+) expression vector to obtain a recombinant plasmid pET-32a (+) -est1012，Transforming the recombinant plasmid into escherichia coli BL21 competent cells to form recombinant engineering bacteria, inoculating the recombinant engineering bacteria into a 50mL flask containing LB culture medium with 100 mug/mL ampicillin concentration, culturing in a shaker at 37 ℃, and culturing when OD is OD₆₀₀When the value reaches 0.8-1.0, adding IPTG to make the final concentration 0.6mmol/L, inducing expression at 35 deg.C for 16h, collecting thallus, and ultrasonically crushing thallus to obtain crude enzyme solution; purifying the crude enzyme solution by using a His.bind 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.