CN110038250B - Method for degrading phthalate containing metal ions or organic solvent - Google Patents

Abstract

The invention discloses a method for degrading phthalate containing metal ions or organic solvent, belonging to the field of enzyme engineering. The carboxylesterase BaCEs01 with an amino acid sequence shown as SEQ ID NO.1 is heterologously expressed in escherichia coli, the optimum reaction temperature of the obtained carboxylesterase BaCEs01 is 40 ℃, the enzyme activity is kept above 40% after the carboxylesterase BaCEs01 is placed at 40 ℃ for 1 hour, and the carboxylesterase has good temperature stability. The degradation rates of the purified carboxylesterase BaCEs01 on phthalate (diethyl phthalate, dibutyl phthalate and diisobutyl phthalate) are respectively as high as 84.2%, 89.6% and 81.3%, and the method for efficiently degrading the plasticizer phthalate is provided.

Description

Method for degrading phthalate containing metal ions or organic solvent

Technical Field

The invention relates to a method for degrading phthalate containing metal ions or organic solvent, belonging to the field of enzyme engineering.

Background

Carboxylesterase (EC 3.1.1.1) refers to a non-specific esterase capable of catalyzing hydrolysis of a carboxylic acid ester to produce a carboxylic acid and an alcohol, and is a B esterase, which is equivalent to a monoacylglycerol esterase, an arylamidase, and a cholinesterase, and is structurally related to a lipase, and is a family of α/β hydrolases.

Carboxylesterases exist widely in animals, plants and microorganisms, and current research mainly focuses on carboxylesterases participating in drug metabolism in animal livers and carboxylesterases having drug resistance in insect bodies, and relatively few researches on carboxylesterases derived from microorganisms. However, carboxylesterases derived from microbial sources have been attracting more and more attention because of their high yield, good stability and high catalytic efficiency.

Phthalate (PAEs) are used as plasticizers in a large amount in production and living and enter the environment, and the ecological safety and the human health are seriously threatened. Efficient degradation of plasticizer residues in the environment has become an urgent need and research focus for environmental protection. Bioremediation is one of important means for environmental management, the main method at present is to degrade by using microbial strains, and from the separated phthalate degradation bacteria, the phthalate degradation rate of different concentrations can reach about 50 percent by culturing for 0.5 to 10 days at the temperature of 25 to 30 ℃. Therefore, the degradation by using the microbial strains has the defects of low degradation efficiency, poor stability, certain pollution and the like, so researchers pay attention to the enzymatic degradation. Microorganisms are the most widely distributed and diverse organisms in nature, and therefore, enzymes derived from microorganisms are also very wide. The research on the degradation of environmental pollutants by enzymes produced by microorganisms has great development prospect.

At present, researches on degrading phthalate esters by esterase are relatively limited, mainly focus on the degradation mechanism and the toxicity of degradation products, and the discovered esterase has the defects of low degradation efficiency, strict requirements on reaction conditions and the like. For example, in 2015, the reaction temperature of esterases EstZ22, which are cloned from Bacillus by Wangfenna et al and can degrade phthalate, needs 70 ℃ or above, and is not suitable for practical application; in 2016, the esterase EstSP1 cloned by Do Kyung Hong et al has the degradation rate of only 30 percent on 1mM dibutyl phthalate within 1 h; in 2014, the esterase EstS1 cloned by Xiao-Yan Zhang et al has the optimal degradation condition of 60 ℃. However, most of the prior carboxylesterases have low tolerance to metal ions and organic solvents, and the application of the carboxylesterases is severely limited. Therefore, the obtained carboxylesterase from bacteria with low requirements on reaction conditions has very important value and significance in industrial application.

Disclosure of Invention

The invention aims to provide a method for degrading phthalate in a system containing metal ions or an organic solvent at a lower reaction temperature, which takes carboxylesterase with an amino acid sequence shown as SEQ ID NO.1 as a catalyst to degrade the phthalate.

In one embodiment, the reaction temperature is 15 to 50 ℃.

In one embodiment, the reaction temperature is preferably 30 to 50 ℃.

In one embodiment, the reaction temperature is preferably 40 ℃.

In one embodiment, the carboxylesterase is degraded under the following conditions: the pH value is 5.0-8.0.

In one embodiment, the carboxylesterase is preferably degraded under conditions selected from the group consisting of: the pH was 6.5.

In one embodiment, the degradation time is 1 to 6 hours.

In one embodiment, the metal ion is Na⁺、K⁺、Zn²⁺、NH₄ ⁺、Mg²⁺、Ca²⁺、Cu²⁺Or Fe³⁺。

In one embodiment, the concentration of the metal ion is 0.5 to 2 mM.

In one embodiment, the organic solvent is methanol, ethanol, acetonitrile, acetone, n-hexane, or isopropanol.

In one embodiment, the concentration of the organic solvent is 0.5-2% (v/v).

In one embodiment, the phthalate esters are: diethyl phthalate, dibutyl phthalate or diisobutyl phthalate.

The second purpose of the invention is to provide a recombinant bacterium for producing carboxylesterase, which takes escherichia coli as a host and expresses a gene with an amino acid sequence shown as SEQ ID NO. 1.

In one embodiment, the expression is in escherichia coli using pColdII as an expression vector.

The third purpose of the invention is to provide a method for producing carboxylesterase, which is to use the recombinant bacterium for fermentation production.

The invention also provides application of the recombinant bacterium or the produced carboxylesterase thereof in the field of environmental protection.

The invention has the beneficial effects that:

(1) the carboxylesterase BaCEs01 with an amino acid sequence shown as SEQ ID NO.1 is heterologously expressed in escherichia coli, the optimum reaction temperature of the obtained carboxylesterase BaCEs01 is 40 ℃, the enzyme activity is kept above 40% after the carboxylesterase BaCEs01 is placed at 40 ℃ for 1 hour, and the carboxylesterase has good temperature stability.

(2) After the carboxylesterase BaCEs01 is placed in a buffer solution containing ethanol and acetonitrile for 1 hour, the residual enzyme activity is 76.9 percent and 71.6 percent respectively, and organic solvents (methanol, acetone, normal hexane and isopropanol) have no obvious influence on the enzyme activity.

(3) The degradation rates of the purified carboxylesterase BaCEs01 on phthalate (diethyl phthalate, dibutyl phthalate and diisobutyl phthalate) are respectively as high as 84.2%, 89.6% and 81.3%, and the method for efficiently degrading the plasticizer phthalate is provided.

Drawings

FIG. 1 shows the optimum reaction temperature.

FIG. 2 is temperature stability.

FIG. 3 shows the optimum reaction pH.

FIG. 4 is pH stability.

FIG. 5 is a graph of 1-naphthyl acetate enzymatic reaction rates.

FIG. 6 is a graph of the rate of 2-naphthyl acetate enzymatic reaction.

Detailed Description

(1) The method for determining the enzyme activity of carboxylesterase by using 1-naphthyl acetate or 2-naphthyl acetate as a substrate comprises the following steps:

1% fast blue B salt: weighing 1g of fast blue B salt, dissolving in distilled water to a constant volume of 100mL, and storing in dark.

5% SDS: 5g of SDS is weighed and dissolved in distilled water, water bath is carried out for 1 hour at 37 ℃, after the SDS is completely dissolved, the volume is determined to be 100mL, and the mixture is stored in a refrigerator.

0.6M of naphthyl 1-acetate or naphthyl 2-acetate: 11.17g of 1-naphthyl acetate or 2-naphthyl acetate is weighed and dissolved in 95 percent ethanol, the volume is determined to be 100mL, and the mixture is stored in a dark place.

Disodium hydrogen phosphate-potassium dihydrogen phosphate buffer: 1/15M disodium hydrogen phosphate was mixed with 1/15M potassium dihydrogen phosphate and the mixture was adjusted to pH 7.0.

mu.L of substrate 1-naphthyl acetate or 2-naphthyl acetate was added to 1.5mL of disodium hydrogenphosphate-potassium dihydrogenphosphate buffer (pH 7.0) and incubated in a water bath at 37 ℃ for 5 minutes, 250. mu.L of purified enzyme solution was added, reaction was carried out for 5 minutes, 0.5mL of stop color developing solution DBLS (1% fast blue B salt mixed with 5% SDS at 2: 5) was added, shaking was carried out, standing was carried out for 10 minutes, and the absorbance at 595/555nm was measured.

Definition of enzyme activity: under the optimal reaction condition, the enzyme quantity required for releasing 1 mu M of 1-naphthol or 2-naphthol from 0.6M of 1-naphthyl acetate or 2-naphthyl acetate solution within 1min is one enzyme activity unit.

(2) Determination of carboxylesterase protein concentration: according to the method of the Bradford protein quantitative kit, enzyme liquid diluted by a certain time is mixed with G250 staining solution, the light absorption value at 595nm is measured by an enzyme-labeling instrument, and the protein concentration is calculated according to the protein concentration standard. Specific activity (U. mg)^-1) Enzyme activity (U.mL)^-1) X [ protein concentration (mg. mL)^-1)]^-1。

(3) The detection conditions of the phthalate high performance liquid chromatography are as follows: c18 column (Agilent 4.6X 250mm), wavelength 254nm, mobile phase ratio methanol: water 80:20, detection temperature 30 ℃.

(4) The phthalate degradation rate calculation formula: percent (%) degradation rate (%) remaining substrate concentration/initial substrate concentration

Example 1: construction of engineered Strain

Artificially synthesizing a carboxylesterase BaCEs01 gene sequence with a nucleotide sequence shown as SEQ ID NO.2 (an amino acid sequence shown as SEQ ID NO. 1). The carboxylesterase BaCEs01 gene sequence and the plasmid vector pColdII are subjected to double digestion by restriction enzymes SacI and XbaI and then are connected and transformed into E.coli BL21(DE3) competent cells to obtain the recombinant bacterium E.coli BL21-pColdII-BaCEs 01.

Example 2: expression and purification of carboxylesterase (BaCEs01)

LB medium g/L: sodium chloride 10, tryptone 10, Yeast Extract 5, pH7.

Recombinant Escherichia coli E.coli BL21-pColdII-BaCEs01 was inoculated in a medium containing 100 mg/mL^-1Ampicillin-containing LB liquid medium, starting strain E.coli BL21(DE3) and an empty-loading strain (pClodII plasmid transferred into E.coli BL21(DE 3)) were used as controls, and cultured at 37 ℃ and 200rmp for 12 hours, and then 500. mu.L of the above seed solution was inoculated into 50mL of LB medium containing 50. mu.L of ampicillin and cultured at 37 ℃ for 2.5 hours to OD₆₀₀At 0.6, the shaking table was cooled to 15 ℃ and allowed to stand for 30 min. 40 μ L of IPTG with a final concentration of 0.4mol/L was added to each flask as an inducer and no inducer was added as a control and cultured at 15 ℃ at 200rmp for 24 h.

Collecting bacterial liquid, centrifuging at 4 deg.C and 8000rmp for 10min to obtain thallus, adding 5mL phosphate buffer (0.02mol/L, pH7.0) to resuspend the thallus, crushing with ultrasonic crusher, centrifuging, and collecting supernatant to obtain crude enzyme solution. And (3) carrying out nickel column purification on the obtained crude enzyme solution by adopting an AKTA avant 150 protein purification system to obtain BaCEs01 enzyme solution.

Example 3: determination of the enzymatic Activity of carboxylesterase (BaCEs01)

In a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 7), 2-naphthyl acetate is taken as a substrate, the enzyme activity of the carboxylesterase BaCEs01 is measured at intervals of 5 ℃ within the range of 20-50 ℃, and the optimum temperature of the carboxylesterase BaCEs01 is known to be 40 ℃. And under the condition that the optimal reaction temperature is 40 ℃, the pH value is 5.0-9.0, the enzyme activity is measured every 0.5, and the optimal reaction pH value is determined to be 6.5.

Under the optimum reaction conditions, namely disodium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 6.5), the specific enzyme activities of the carboxylesterase BaCEs01 purified in example 2 were measured to be 5.20U/mg and 1.20U/mg respectively at 40 ℃ using 0.6M 1-naphthyl acetate and 2-naphthyl acetate as substrates.

Temperature stability: and (3) storing 250 mu L of carboxylesterase (BaCEs01) enzyme solution at pH 6.5 at 10,20,30,40,50 and 60 ℃ for 1h, and determining residual enzyme activity, wherein the highest enzyme activity is set as 100%. The results show that: after the carboxylesterase BaCEs01 is placed at 40 ℃ for 1 hour, the enzyme activity is kept above 40 percent, and the carboxylesterase BaCEs01 has good temperature stability.

pH stability: and (3) storing 250 mu L of carboxylesterase BaCEs01 enzyme solution at 40 ℃ for 1h at pH5.0,5.5,6.0,6.5,7.0,7.5 and 8.0 respectively, and determining the residual enzyme activity, wherein the highest enzyme activity is set as 100%. The results show that: the carboxylesterase has the enzyme activity of over 40 percent at pH5.5-7.5, and has good pH stability.

Example 4: effect of Metal ions on Carboxylic esterases (BaCEs01)

To 1.5mL of disodium hydrogenphosphate-potassium dihydrogenphosphate buffer (pH 6.5), 250. mu.L of BaCEs01 enzyme solution, 15. mu.L of 0.6M 2-naphthyl acetate, and 1mM of different metal ions (Na) were added at 40 ℃⁺,K⁺,Zn⁺,NH₄ ⁺,Mg²⁺,Ca²⁺,Cu²⁺,Fe³⁺) And determining the influence of the metal ions on the enzyme activity of the carboxylesterase BaCEs 01. As a result, K is shown in Table 1⁺，NH₄ ⁺，Mg²⁺Has slight promotion effect on enzyme activity, and the rest metal ions have inhibition effect on enzyme to different degrees.

TABLE 1 Effect of different Metal ions on carboxylesterase enzyme Activity

Example 5: effect of organic solvents on Carboxylic esterases (BaCEs01)

In 1.5mL of disodium hydrogenphosphate-potassium dihydrogenphosphate buffer (pH 6.5), 250. mu.L of BaCEs01 enzyme solution, 15. mu.L of 0.6M 2-naphthyl acetate, and 1% (v/v) of various organic solvents (methanol, ethanol, acetonitrile, acetone, n-hexane, and isopropanol) were added at 40 ℃ and left for 1 hour, and then DBLS solution was added to terminate the reaction, and the effect of various organic solvents on the carboxylesterase activity was measured. The results are shown in table 2, and it is known that the enzyme activity of BaCEs01 remained after 1 hour of storage in the buffer containing ethanol and acetonitrile is 76.9% and 71.6%, respectively, and the remaining organic solvent has no significant effect on the enzyme activity.

TABLE 2 Effect of different organic solvents on carboxylesterase enzyme Activity

Example 6: substrate specificity of carboxylesterase (BaCEs01)

In 1.5mL of disodium hydrogenphosphate-potassium dihydrogenphosphate buffer (pH 6.5), 250. mu.L of BaCEs01 enzyme solution was added at 40 ℃ to determine that carboxylesterase BaCEs01 catalyzes 0.2 to 3.4 mmol.L^-1And 0.1-3.2 mmol. multidot.L of 1-naphthyl acetate^-1The reaction rate of 2-naphthyl acetate is calculated by utilizing Origin software to perform nonlinear fitting curve to obtain Vmax and Km values, and then the Kcat/Km value is calculated (see figure 5 and figure 6). As shown in Table 3, the carboxylesterase has a greater affinity for naphthyl 1-acetate than for naphthyl 2-acetate (K)_mLower, greater affinity), catalytic efficiency (K) for naphthyl 1-acetate_cat/K_m) More preferably, it reaches 0.042.

TABLE 3 kinetic parameters of different substrates

Example 7: use of carboxylesterase (BaCEs01)

50 mu L of purified carboxylesterase (BaCEs01) enzyme solution is added into phthalate (diethyl phthalate, dibutyl phthalate and diisobutyl phthalate) with the final concentration of 1mM, in 1.5mL of disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 6.5), the carboxylesterase (BaCEs01) enzyme solution is not added as a control group, after water bath at 40 ℃ for 1h, 100 mu L of HCl solution with the concentration of 1M is added into the reaction system to terminate the reaction, and then the reaction is extracted by ethyl acetate with the same volume, and three parallel experiments are set for each group of experiments. The degree of hydrolysis was judged by measuring the amount of the remaining substrate by high performance liquid chromatography. The degradation rates of the carboxylesterase BaCEs01 on the phthalic acid esters (diethyl phthalate, dibutyl phthalate and diisobutyl phthalate) as 3 plasticizers were calculated to be 74.5%, 80.3% and 70.2%, respectively. Therefore, the carboxylesterase has a degradation rate of more than 70% on three plasticizers with low concentration, and has great application value in environmental remediation.

TABLE 4 degradation of low concentrations of phthalates by BaCEs01

Example 8 use of a carboxylesterase (BaCEs01)

The same procedure as in example 7 was followed, except that the final concentration of the phthalate ester in the system was 10 mM.

mu.L of the carboxylesterase (BaCEs01) enzyme solution purified in example 2 was added to phthalate esters (diethyl phthalate, dibutyl phthalate, diisobutyl phthalate) having a final concentration of 10mM, and 100. mu.L of a 1M HCl solution was added to the reaction system after 1 hour of water bath at 40 ℃ to terminate the reaction in 1.5mL of a disodium hydrogenphosphate-potassium dihydrogenphosphate buffer solution (pH 6.5) without addition of the carboxylesterase (BaCEs01), followed by extraction with an equal volume of ethyl acetate. The degree of hydrolysis was determined by measuring the amount of the remaining substrate by high performance liquid chromatography, and the degradation rates of the carboxylesterase to phthalic acid esters (diethyl phthalate, dibutyl phthalate, diisobutyl phthalate) as 3 plasticizers were calculated to be 44.6%, 50.4%, and 40.7%, respectively. From these results, it was found that the carboxylesterase showed 40% or more degradation rate of all of the 3 plasticizers at high concentration without increasing the amount of the enzyme.

TABLE 5 degradation of high concentrations of phthalates by BaCEs01

Example 9 use of a carboxylesterase (BaCEs01)

The same procedure as in example 7 was followed, except that the hydrolysis time was extended to 6 h.

50 mu L of the purified enzyme solution is added into phthalate (diethyl phthalate, dibutyl phthalate and diisobutyl phthalate) with the final concentration of 1mM, the enzyme solution is not added as a control group, 1.5mL of disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 6.5) is added into water bath at 40 ℃ for 6h, 100 mu L of HCl solution with the concentration of 1M is added to stop the reaction, the solution is extracted by ethyl acetate with the same volume, and three parallel experiments are set in each group of experiments. The degree of hydrolysis was determined by measuring the amount of the remaining substrate by high performance liquid chromatography, and the degradation rates of the carboxylesterase to 3 plasticizers were calculated to be 75.6%, 84.7%, and 72.3%, respectively. The results show that the carboxylesterase has a degradation rate of more than 75% on 3 plasticizers after the reaction time is prolonged.

TABLE 6 degradation of low concentrations of phthalates by BaCEs01

Example 10 use of a carboxylesterase (BaCEs01)

The procedure of example 7 was repeated, except that the amount of the carboxylesterase (BaCEs01) enzyme solution was increased to 100. mu.L.

100 mu L of the purified enzyme solution is added into phthalate (diethyl phthalate, dibutyl phthalate and diisobutyl phthalate) with the final concentration of 1mM, the enzyme solution is not added as a control group, 1.5mL of disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 6.5) is added into water bath at 40 ℃ for 1h, 100 mu L of HCl solution with the concentration of 1M is added to stop the reaction, the solution is extracted by ethyl acetate with the same volume, and three parallel experiments are set in each group of experiments. The degree of hydrolysis was judged by measuring the amount of the remaining substrate by high performance liquid chromatography. The degradation rates of the carboxylesterase on 3 plasticizers are respectively calculated to be 84.2%, 89.6% and 81.3% by measuring and analyzing the amount of the residual substrate through high performance liquid chromatography. This result indicates that increasing the amount of enzyme increases the degradation rate.

TABLE 7 degradation of low concentrations of phthalates by BaCEs01

Example 11: recombinant bacterium E.coli BL21-pColdII-BaCEs01 whole cell catalytic reaction

The recombinant bacterium E.coli BL21-pColdII-BaCEs01 obtained in example 1 was collected and then resuspended and diluted to OD by using disodium hydrogenphosphate-potassium dihydrogenphosphate buffer solution (pH 6.5)₆₀₀At 1.0, 100 mul of bacterial liquid is respectively added into 1mM phthalate (diethyl phthalate, dibutyl phthalate and diisobutyl phthalate) with final concentration, 100 mul of HCl solution with concentration of 1M is added to stop reaction after 1h of water bath at 40 ℃, then ethyl acetate with equal volume is used for extraction, and three parallel experiments are set for each group of experiments. The hydrolysis degree of the residual substrate is judged by measuring the amount of the residual substrate through high performance liquid chromatography, and the degradation rates of the recombinant strain on 3 plasticizers are calculated to be 19.8%, 15.4% and 21.6% respectively.

TABLE 8 degradation of phthalates by recombinant bacteria

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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<120> a method for degrading phthalic acid esters containing metal ions or organic solvents

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taa 603

Claims (4)

1. A method for degrading phthalic acid esters in a system containing metal ions or organic solvents at a relatively low reaction temperature is characterized in thatDegrading phthalic acid esters by using carboxylesterase with an amino acid sequence shown as SEQ ID NO.1 as a catalyst, wherein the phthalic acid esters are as follows: diethyl phthalate, dibutyl phthalate or diisobutyl phthalate; the reaction temperature is 15-50 ℃, and the metal ions are Na⁺、K⁺、Zn²⁺、NH₄ ⁺、Mg²⁺、Ca²⁺、Cu^{2 +}Or Fe³⁺(ii) a The organic solvent is methanol, ethanol, acetonitrile, acetone, n-hexane or isopropanol.

2. The method according to claim 1, wherein the concentration of the metal ion is 0.5 to 2 mM.

3. The method according to claim 1, wherein the concentration of the organic solvent is 0.5 to 2% (v/v).

4. A method for producing carboxylesterase is characterized in that recombinant bacteria are used for fermentation production, wherein the recombinant bacteria take escherichia coli as a host and express a gene with an amino acid sequence shown as SEQ ID No. 1.

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