CN110982803B

CN110982803B - Novel phthalate hydrolase EstJ6, and coding gene and application thereof

Info

Publication number: CN110982803B
Application number: CN201911359278.7A
Authority: CN
Inventors: 辛志宏; 邱佳容; 张月琦; 姜俊伟; 吴盛露; 李龙祥; 邵玉庭
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2022-09-27
Anticipated expiration: 2039-12-25
Also published as: CN110982803A

Abstract

The invention provides a novel phthalate hydrolase gene derived from a soil metagenome library, and the nucleotide sequence and the amino acid sequence of the novel phthalate hydrolase gene are shown as SEQ ID NO.l and SEQ ID N0.2. After the esterase gene is connected to an expression vector pET28a (+), the esterase gene is transformed into escherichia coli BL21(DE3) to realize heterologous expression. The purified recombinase (EstJ6) has a molecular weight of 33.31 kDa. EstJ6 has broad substrate specificity for phthalates, and EstJ6 can hydrolyze both phthalates having simple side chains and also diethylhexyl phthalate and monoethylhexyl phthalate having complex and longer side chains. In addition, site-directed mutagenesis experiments show that the catalytic triad residue of EstJ6 is S146-E240-H270, and the catalytic capability of EstJ6 is lost due to mutation of any amino acid in the three. The novel phthalate ester hydrolase of the present invention can be applied to the fields of food industry, agriculture, biotechnology, and the like due to its specific activity and enzymatic properties.

Description

Novel phthalate ester hydrolase EstJ6, and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a novel phthalate ester hydrolase obtained by screening a soil metagenome library, and a coding gene and application thereof.

Background

Phthalates (PAEs) are a class of toxic organic compounds that are ubiquitous in the environment as they are widely used as additives or plasticizers in plastics manufacture. Six of these (dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), dioctyl phthalate (DNOP), diethylhexyl phthalate (DEHP) and Butyl Benzyl Phthalate (BBP)) have been the priority pollutants of the U.S. environmental protection agency and some of its international co-ordinates. Among them, DEHP accounts for a high proportion of the environment (about 31%). The use of PAEs in large quantities leads to serious environmental problems and various diseases including respiratory diseases, childhood obesity, neuropsychological disorders, and the like.

With the overall understanding of the potential hazards of PAEs, the elimination of PAEs has attracted a great deal of attention. Microbial degradation of PAEs is currently the most common method used, although many PAEs degrading microorganisms have been isolated and identified, such as Enterobacter sp. However, enzymes having DEHP degrading activity in these microorganisms are still unknown. Therefore, the search for effective novel phthalate degrading enzymes is very critical, especially for the degradation of DEHP.

Research has shown that the efficiency of finding novel esterases by traditional culture techniques is very low, since only a very small number of microorganisms can be cultured under the existing experimental conditions, and over 99% of microorganisms are unculturable, greatly limiting the development and utilization of novel enzymes. Metagenomics, a culture-independent technique that avoids natural deficiencies, is considered one of the best methods for obtaining and studying microbial resources. By directly extracting all DNA of culturable and non-culturable microorganisms in the environment and constructing a metagenome library for screening novel enzymes, the utilization space of microorganism resources is greatly expanded, and the method is an effective method for researching and developing the non-culturable microorganisms. Metagenomic strategies based on functional screening have been successfully applied to the discovery of novel enzymes, such as feruloyl esterase, xylanase, protease, and the like, which have enhanced enzymatic properties.

In the research, a new phthalate hydrolase gene EstJ6 is separated from a soil metagenome library through functional screening, the gene is subjected to heterologous expression, and a recombinant enzyme EstJ6 is subjected to purification, enzymatic characterization, homologous modeling and site-directed mutagenesis. EstJ6 would be an interesting candidate for biomass degradation and environmental protection, illustrating the advantages of the macrogenomic strategy in developing novel enzyme genes in agricultural, food industry and biotechnology applications.

Disclosure of Invention

The invention screens a novel phthalate hydrolase gene from a soil metagenome library, performs heterologous expression on the gene, and the molecular weight of the purified recombinase (EstJ6) is 33.31 kDa. Phylogenetic analysis indicated that EstJ6 is a new member of family IV. Biochemical characterization showed that EstJ6 showed the highest activity (128U/mg) against DBP at 40 ℃ and pH 7.5. EstJ6 has wide substrate specificity to (C1-C9) phthalate compounds, and EstJ6 can hydrolyze phthalate with simple side chains and degrade diethylhexyl phthalate and monoethylhexyl phthalate with complex and longer side chains. Site-directed mutagenesis experiments indicated that the putative catalytic triad residue of EstJ6 was S146-E240-H270.

The first object of the present invention is to provide a novel phthalate ester hydrolase.

The second object of the present invention is to provide a gene encoding the above novel phthalate hydrolase.

The third purpose of the invention is to provide a metagenomic screening method of the gene.

The fourth object of the present invention is to provide an expression vector containing the above-mentioned novel phthalate hydrolase gene.

The fifth object of the present invention is to provide a recombinant microorganism containing the above expression vector.

The sixth purpose of the invention is to provide a preparation method of the recombinant phthalate ester hydrolase.

The seventh object of the present invention is to provide specific amplification primers for the gene of a novel phthalate ester hydrolase.

An eighth object of the present invention is to provide primers for mutating key amino acid residues of the novel phthalate ester hydrolase.

The ninth object of the present invention is to provide the novel phthalate hydrolase EstJ6, the gene EstJ6 encoding the novel phthalate hydrolase EstJ6, the expression vector, the recombinant microorganism containing the expression vector, the preparation method of the recombinant phthalate hydrolase EstJ6, or the application of the primer for specific amplification of the gene EstJ6 in the degradation of phthalate compounds.

The technical scheme adopted by the invention is as follows:

a novel phthalate ester hydrolase EstJ6, wherein the amino acid sequence of the phthalate ester hydrolase EstJ6 is shown as SEQ ID No. 2:

MASPQLQMALDAFKTMGEKMAQAGNDVKALRAVMEEMSGFPSAGETKCTPVNAGGVPAEWISGPGAADDRVILYVHGGGYVMGSIATHRETVARLSKASGARGLALDYRLAPEHPFPAAVDDATAAYRWLLSQNIKPAHIVIAGDSAGGGLTLATLIALRDAKVPLPAAGVCISPWTDMEGAGESMTTRAKADPVVQKQGLLGMAQLYLGGKDPKSPLAAPLHANLAGLPPLLIQVGDAETLLDDSIRVAEKAKKAGVKVDLEVWPEMPHVWHLFAPFLPEGQQAIDKIGKYVRQITA。

further, the key amino acids of the phthalate ester hydrolase EstJ6 were site-directed mutated and activity-verified, and it was assumed that the catalytic triad residue of the phthalate ester hydrolase EstJ6 was serine (S146) -glutamic acid (E240) -histidine (H270).

The gene EstJ6 of the novel phthalate hydrolase EstJ6 is coded, and the nucleotide sequence of the gene EstJ6 is shown as SEQ ID No. 1:

ATGGCAAGTCCGCAACTACAGATGGCCCTTGATGCGTTCAAGACGATGGGCGAGAAAATGGCGCAGGCGGGAAATGACGTGAAAGCCTTGCGCGCTGTCATGGAAGAGATGTCTGGCTTTCCCTCAGCAGGGGAGACGAAGTGTACGCCGGTAAATGCTGGCGGCGTTCCTGCCGAATGGATTTCCGGTCCTGGTGCCGCGGATGATCGCGTGATCCTGTACGTACACGGCGGTGGCTATGTGATGGGTTCTATCGCTACTCACCGCGAGACGGTTGCTCGTCTGTCGAAAGCCTCGGGAGCGCGTGGTCTGGCGTTAGATTACCGCCTGGCCCCGGAGCATCCATTCCCCGCCGCGGTTGATGACGCGACGGCAGCGTATCGCTGGCTGCTCTCGCAAAATATTAAACCTGCCCACATTGTCATTGCCGGTGACTCTGCGGGCGGAGGGCTTACGCTGGCGACTCTCATCGCGTTACGGGACGCGAAGGTTCCCCTTCCCGCCGCGGGTGTGTGTATTTCACCGTGGACGGACATGGAAGGGGCTGGGGAGTCAATGACGACCAGGGCGAAGGCCGATCCCGTCGTGCAAAAGCAAGGACTGCTGGGTATGGCACAGCTCTACCTCGGCGGCAAAGATCCGAAGTCGCCGCTCGCCGCTCCACTGCACGCCAATCTCGCGGGACTCCCGCCGCTCTTGATTCAAGTGGGAGACGCCGAGACCTTGCTCGACGACTCCATTCGTGTTGCCGAAAAAGCCAAGAAAGCGGGCGTCAAAGTCGATCTCGAGGTTTGGCCGGAGATGCCCCACGTGTGGCACCTGTTCGCCCCGTTCCTGCCGGAAGGCCAGCAAGCGATCGACAAGATCGGGAAGTACGTCCGGCAGATCACCGCGTAA。

the metagenomic screening method of the gene EstJ6 for encoding the novel phthalate ester hydrolase EstJ6 comprises the following steps: the gene estj6 for coding phthalate ester hydrolase is obtained by a functional screening method and a subcloning strategy.

Further, the metagenome screening method comprises the specific steps of,

(1) screening positive clones by using a substrate plate;

(2) high Performance Liquid Chromatography (HPLC) is used for verifying the activity of the phthalate ester hydrolase;

(3) extracting positive clone plasmid DNA, performing partial enzyme digestion by utilizing Sau3AI, connecting to a vector pUC118 and transforming to Escherichia coli E.coli DH5 a;

(4) the positive subclones were screened using the same substrate plate, sequenced and Blast compared to screen for the gene estj6 encoding phthalate hydrolase.

Further, the metagenomic screening method of the gene encoding the phthalate ester hydrolase comprises the following steps:

(1) positive clones were screened using substrate plates: dibutyl phthalate (DBP) was added to the medium at a final concentration of 1mM after the addition of the membrane as a substrate for screening. And (3) properly diluting and coating the duplicated library, observing whether a corresponding transparent ring appears on a screening plate after culturing, and selecting a clone capable of generating the transparent ring, namely the screened positive clone.

(2) HPLC verification of the enzymatic activity of the phthalate ester hydrolase: it was inoculated into LB liquid medium containing 0.1mM DBP overnight and shaken, and a part of the fermentation broth was extracted with an equal volume of n-hexane and redissolved with methanol for HPLC analysis. Whether the clone has the phthalate degradation enzyme activity or not is judged by the DBP residual quantity.

(3) Plasmid DNA of the positive clones was extracted, partially digested with Sau3AI, electrophoresed, and a DNA fragment of 1-5kb in size was recovered, ligated to the vector pUC118 and transformed into E.coli DH5 a.

(4) Positive clones were screened using the same substrate screening plate. And (3) determining a positive subcloned sequence to obtain a phthalate ester hydrolase coding gene estj 6.

An expression vector containing a gene estj6 encoding the novel phthalate ester hydrolase.

Furthermore, the expression vector is obtained by cloning a gene EstJ6 of the novel phthalate hydrolase EstJ6 into pET28a (+).

A recombinant microorganism containing the above expression vector;

further, the recombinant microorganism takes Escherichia coli BL21(DE3) as a host cell.

A preparation method of a recombinant phthalate ester hydrolase EstJ6 comprises the following steps: preparing the expression vector, transforming host cells by using the expression vector, culturing a transformant, and separating the recombinant phthalate hydrolase EstJ6 from a culture.

Further, the specific steps are that the primer:

an upstream primer: 5' -CATGCCATGGGCATGGCAAGTCCGCAACTA-3' (SEQ ID No. 3); and

a downstream primer: 5' -CCCAAGCTTCGCGGTGATCTGCCGGAC-3' (SEQ ID No.4) amplified gene estj6, with the primers underlined at the upstream and downstream representing the restriction sites for NcoI and HindIII, respectively. Carrying out double enzyme digestion on the PCR amplification product of the gene EstJ6 by NcoI and HindIII, connecting the PCR amplification product to a pET28a (+) vector subjected to the same enzyme digestion to obtain a pET28a (+) -etsj6 connection product, namely the expression vector, transforming the expression vector into escherichia coli BL21(DE3), culturing the transformant, inducing by IPTG, and separating and purifying from a culture to obtain a recombinant phthalate hydrolase EstJ 6;

the specific amplification primer for specifically amplifying the gene estj6 encoding the novel phthalate ester hydrolase comprises the following two primer sequences:

an upstream primer: 5' -CATGCCATGGGCATGGCAAGTCCGCAACTA-3’(SEQ ID No.3)；

A downstream primer: 5' -CCCAAGCTTCGCGGTGATCTGCCGGAC-3’(SEQ ID No.4)。

The above-mentioned novel phthalate ester hydrolase EstJ6, or

The above gene EstJ6 encoding a novel phthalate hydrolase EstJ6, or

The above expression vector containing gene estj6 encoding a novel phthalate ester hydrolase, or

A recombinant microorganism containing the above expression vector, or

The preparation method of the recombinant phthalate ester hydrolase EstJ6, or

The primer for specific amplification of the specific amplification gene estj6 is applied to hydrolysis of phthalate compounds;

preferably, the phthalate ester compound is a phthalate diester compound with a chain of C1-C9;

further preferably, the phthalate-based compound is any one selected from the group consisting of dibutyl phthalate (DBP), dipentyl phthalate (DPP), dipropyl phthalate (DPRP), diethyl phthalate (DEP), dihexyl phthalate (DHP), dimethyl phthalate (DMP), diethylhexyl phthalate (DEHP), monomethyl phthalate (MMP), monoethyl phthalate (MEP), monobutyl phthalate (MBP), monohexyl phthalate (MHP) and monoethylhexyl phthalate (MEHP).

The recombinant phthalate ester hydrolase has the following enzymological properties:

after purifying the recombinase obtained by heterologous expression, carrying out enzyme catalysis kinetic analysis, including the determination of Km, Vmax, Kcat and Kcat/Km values, and the characterization of conventional enzymological properties, including the influence of substrate specificity, optimal temperature and thermal stability, optimal pH, different metal ions, organic solvents and surfactants, and the like.

The invention has the beneficial effects that:

the invention constructs a metagenome library for screening novel enzymes by directly extracting all DNA of culturable and non-culturable microorganisms in the environment, greatly expands the utilization space of microorganism resources, and provides a novel phthalate hydrolase gene estj6 derived from a soil metagenome library, wherein the nucleotide sequence and the amino acid sequence of the novel phthalate hydrolase gene estj6 are respectively shown as SEQ ID NO. l and SEQ ID NO. 2. The gene is expressed in escherichia coli BL21(DE3) in a heterologous manner, and the molecular weight of purified recombinase (EstJ6) is 33.31 kDa. EstJ6 showed the highest activity (128U/mg) against dibutyl phthalate at 40 ℃ and pH 7.5. EstJ6 has wide substrate specificity to (C1-C9) phthalate compounds, and EstJ6 not only can hydrolyze phthalate with simple side chains, but also can degrade diethylhexyl phthalate and monoethylhexyl phthalate with complex and longer side chains. Site-directed mutagenesis experiments showed that the putative catalytic triad residue of EstJ6 was S146-E240-H270. The novel phthalate ester hydrolase EstJ6 and the gene EstJ6 thereof provided by the invention provide a new effective means for biomass degradation and environmental protection.

Drawings

FIG. 1 Primary screening of the phthalate hydrolase active clones in example 1.

FIG. 2 is a high performance liquid chromatogram of the fermentation broth of the clone in example 1.

a: a control group containing no target gene; b: and (4) experimental groups.

FIG. 3 SDS-PAGE detection of purified phthalate hydrolytic esterase EstJ6 pattern in example 3

M: a protein Marker; 1: no-load comparison; 2: crude enzyme solution; 3: 5ug of purified enzyme EstJ 6.

FIG. 4 Radar chart for enzymatic characterization of phthalate ester hydrolases in example 4.

a: effect of temperature on the activity of EstJ 6; b: thermal stability of EstJ 6; c: effect of pH on EstJ6 activity; d: substrate specificity of EstJ 6; e: effect of organic solvents on EstJ6 activity; f: effect of metal ions on the activity of EstJ 6; g: effect of surfactant on EstJ6 activity.

FIG. 5. example 4 the degradation pathway of DEHP by the phthalate ester hydrolase EstJ6 is postulated.

a: total ion flow chromatogram of DEHP and its degradation products (MEHP and PA);

b-d: mass spectra of DEHP and its degradation products (MEHP and PA);

e: the putative pathway of eshp degradation by EstJ 6.

FIG. 6 phylogenetic tree of the phthalate ester hydrolase EstJ6 in example 4.

FIG. 7 alignment of the amino acid sequence of the phthalate ester hydrolase EstJ6 with similar proteins in example 4. Wherein conserved motifs are marked with rectangular boxes, conserved amino acid residues are marked with circles, and putative catalytic triad residues are marked with filled circles.

FIG. 8. homologous modeling of the phthalate ester hydrolase EstJ6 in example 5.

Detailed Description

The method of operation of the present invention is further illustrated below with reference to specific examples. However, these examples are only for illustrating the present invention in detail and are not intended to limit the present invention.

Example 1 screening of phthalate hydrolase genes in soil metagenomic library

1. Primary screening of positive clones by substrate plate method

Preparing a primary screening culture medium, sterilizing an LB solid culture medium at high temperature and high pressure, cooling the culture medium to a proper temperature (60 ℃), adding substrate 1mM dibutyl phthalate (dissolved in DMSO) and 100 mu g/mL ampicillin after membrane sterilization, shaking uniformly, and pouring the mixture into a flat plate. The cosmid library bacterial liquid is coated on a screening plate after being diluted properly, cultured for 1-2 days at 37 ℃, and the formation of a transparent ring around a colony is observed, so that the positive clone is obtained (figure 1).

2. Rescreening, and HPLC verifying the enzymatic activity of phthalate ester hydrolase

The primary screening monoclonal obtained was inoculated in liquid LB medium (containing 100. mu.g/mL ampicillin) and incubated overnight (12h) at 37 ℃, centrifuged at 12,000 Xg for 8min at room temperature, the supernatant was discarded, the cells were resuspended in an equal volume of sterile deionized water, inoculated at 1% (v/v) in liquid LB medium (containing 0.1mM DBP and 100. mu.g/mL Amp), and incubated at 37 ℃ for 12h with shaking at 180rpm in the dark. Taking out 5mL of culture solution, adding equal volume of n-hexane, shaking vigorously for 2min, and mixing well for 30min at 180r/min in a shaking table. Standing for layering, taking out an organic phase, taking 1mL of the organic phase, drying in an ultra-clean bench, and then fixing the volume with 1mL of chromatographic grade methanol. After passing through a 0.22 μm organic membrane, the DBP residue was analyzed by HPLC. Liquid chromatography conditions: the mobile phase was methanol/water (95/5 (containing 0.1% formic acid), V/V), the flow rate was 0.5mL/min, and the column was Zorbax SB-C ₁₈ Chromatography column (4.6 × 150mm, 5 μm) with UV detector wavelength of 242nm, the column temperature is 35 ℃, and the sample injection amount is 20 mu L.

The fermentation broth detected the degradation of DBP and the production of new substances indicating that clones with phthalate hydrolase activity were screened (fig. 2).

3. Subcloning of

The positive clones obtained by rescreening were cultured overnight to extract plasmid DNA, partially digested with Sau3AI, electrophoresed, gel-cut to recover DNA fragments of 1-5kb in size, ligated into BamHI digested vector pUC118 and transformed into e.coli DH5a, and positive subclones were screened using the same substrate screening plate.

4. Positive subclone sequence determination and analysis

Sequencing was performed using the M13 primer sequence carried by the vector pUC118, open reading frame prediction using the on-line analysis tool ORFFinder (https:// www.ncbi.nlm.nih.gov/ORFFinder /), and the BlastP program searched for homologous sequences of the predicted protein (https:// blast. The obtained phthalate-degrading enzyme gene was designated as estj 6.

Example 2 cloning of phthalate ester hydrolase

PCR amplification

Using a primer:

an upstream primer: 5' -CATGCCATGGGCATGGCAAGTCCGCAACTA-3' (SEQ ID No. 3); and

a downstream primer: 5' -CCCAAGCTTCGCGGTGATCTGCCGGAC-3' (SEQ ID No.4) amplified phthalate hydrolase gene estj6, with the upstream and downstream primers underlined for NcoI and HindIII, respectively.

PCR reaction (25. mu.L): 9.5. mu.L of ultrapure water, 12.5. mu.L of 2 XTaq Master Mix, 1uL of each of the upstream and downstream primers, and L. mu.L of positive subclone plasmid DNA. And (3) PCR reaction conditions: 3min at 94 ℃; 30s at 94 ℃, 45s at 63 ℃, lmin at 72 ℃ and 35 cycles; 10min at 72 ℃. And (4) carrying out electrophoresis on the PCR product, and recovering tapping rubber to obtain a purified PCR product.

2. Enzyme digestion

And carrying out double enzyme digestion on the purified and recovered PCR product for 3-4 h. The enzyme cutting system is as follows: NcoI 5uL, HindIII 5uL, l0XK Buffer 10uL, 0.1% BSA 10uL, PCR product 5ug, sterile water to 100 uL. And (4) tapping and recovering to obtain a purified PCR product subjected to double enzyme digestion.

pET28a (+) plasmid DNA was double digested for 3-4h in NcoI 5uL, HindIII 5uL, l0XK Buffer 10uL, 0.1% BSA 10uL, pET28a (+) plasmid DNA5ug, sterile water added to 100 uL. The purified double-digested pET28a (+) vector is obtained after tapping and recovery.

3. Connection of

The double-digested PCR product was ligated with pET28a (+) vector at a molar ratio of 10: 1. The ligation temperature was l6 ℃ and the ligation time was 12 h.

4. Transformation and selection

Adding 10uL of the ligation product into 100uL of Escherichia coli DH5a competent cells, incubating on ice for 30min, performing heat shock in a water bath at 42 ℃ for 45s, performing ice bath for 2min, adding 900 μ L of LB liquid medium, performing shake culture at 180rpm and 37 ℃ for 1 h. The bacterial suspension was spread on LB plates containing kanamycin, cultured overnight at 37 ℃ and then single colonies were picked. After single colony is cultured in 5ml LB culture medium overnight, plasmid is extracted, double enzyme digestion verification is carried out, and positive clone is obtained if the size of enzyme digestion fragment is the same as that of gene. Sequencing the obtained positive clone, comparing the sequencing result with the nucleotide sequence (shown as SEQ ID NO. l) of EstJ6, and confirming that the result is completely correct, thereby obtaining pET28a (+) plasmid with EstJ6 gene, which is named as pET28a-EstJ 6.

Example 3: heterologous expression and purification of phthalate ester hydrolysis esterase EstJ6

1. Transformation of

10uL of the pET28a-esjt6 plasmid obtained in example 2 was added to 100uL of competent cells of Escherichia coli BL21(DE3), incubated on ice for 30min, heat-shocked in a water bath at 42 ℃ for 90s, ice-cooled for 2min, and then added with 900uL of LB liquid medium, cultured at 180rpm and 37 ℃ for 1h with shaking. The cells were resuspended and plated on LB plates containing kanamycin, cultured overnight at 37 ℃ and single colonies were picked.

2. Inducible expression

The recombinant strain was inoculated into 5ml of LB liquid medium containing kanamycin and cultured at 37 ℃ and 180rpm for 12 hours. Inoculating 1ml of the strain into 100ml of fresh LB medium, culturing at 37 deg.C and 200rpm until OD 600nm is about 0.6, adding IPTG until its final concentration is 0.5mM, and further culturing at 16 deg.C and 180rpm for 20 h.

3. Purification of

Centrifuging the bacterial solution for about 20h under the conditions of 4 ℃ and 15000rpm to collect thalli, resuspending in PBS buffer solution, ultrasonically crushing (ice water bath, ultrasonic l s interval 2s, 30min), centrifuging at 4 ℃ and 15000rpm for 30min, and taking the supernatant to obtain the crude enzyme solution. The obtained crude enzyme solution was purified by passing through a Ni-NTA purification resin pre-packed column, washed with 5 to 10 volumes of an eluent (PBS, pH 8.0, containing 20mM, 50mM, 100mM, 250mM, and 500mM of imidazole), and then the protein was eluted from the column. The purified protein was detected by polyacrylamide gel electrophoresis (FIG. 3).

Example 4 characterization of the enzymatic Properties of the phthalate ester hydrolase EstJ6

1. Determination of phthalate ester hydrolase EstJ6 Activity

Definition of enzyme activity units: under optimum conditions, the amount of enzyme required to convert 1uM substrate (DBP) per minute is defined as one unit of enzyme activity.

2. Optimum temperature and thermal stability

The optimal temperature of EstJ6 in the range of 16-80 ℃ was studied by incubating the enzyme (0.25ug/ml protein) in 10mM PBS (pH7.5) containing 1mM DBP as substrate for 8min using 10mM Phosphate (PBS) pH7.5 as buffer and dibutyl phthalate (DBP) as substrate, and the results are shown in FIG. 4 a. The thermostability of EstJ6 was analyzed by preincubating the enzyme at a temperature of 30-60 ℃ for 10-60min under the same reaction system, and the results are shown in FIG. 4 b. The enzyme has the highest activity at 40 ℃, and the initial enzyme activity of the enzyme can be kept to be close to 90% after the enzyme reacts for 1 hour at 40 ℃; the activity of the enzyme is kept above 65% at 25-50 ℃; when it exceeds 50 ℃, the enzyme activity decreases rapidly, while when the temperature reaches 80 ℃ the enzyme activity is completely lost.

3. Optimum pH

The activity of EstJ6 in the pH range of 3.0-11.0 was studied by incubating the enzyme (0.25ug/ml protein) in 10mM PBS (pH7.5) containing 1mM DBP as substrate for 8min at 40 ℃ with dibutyl phthalate (DBP) as substrate. The results are shown in FIG. 4 c. The enzyme has maximal enzyme activity at pH 7.5; the activity of EstJ6 gradually increased when the pH was 3.0-7.5; whereas the activity of EstJ6 decreases rapidly above pH 7.5.

4. Substrate specificity

The results of comparing the hydrolysis capacities of EstJ6 to different substrates according to the above enzyme activity assay method are shown in FIG. 4 d. EstJ6 is capable of hydrolyzing phthalic diesters having a chain C1-C9. Of these, EstJ6 was the most catalytically active towards DBP at 40 ℃ and pH7.5, up to 95%, followed by DPP (82%), DPRP (75.68%) and DEP (68.81%). In particular, EstJ6 also had some activity on DEHP, a long and complex side chain (18%). The kinetic parameters of EstJ6 are shown in Table 1, where the lower the Km value, the higher the Vmax value, and the higher the Kcat/Km value, the higher the catalytic efficiency. The EstJ6 hydrolysis rate trend was: DBP > DPP > DPRP > DEP > DHP > DMP. In addition, the substrate specificity of EstJ6 for the phthalic monoester compound was analyzed by HPLC, and the results showed that EstJ6 was also able to hydrolyze MMP, MEP, MBP, MHP and MEHP.

Table 1 kinetic parameters of EstJ6

5. Effect of organic solvents, Metal ions and surfactants on EstJ6 Activity

The reaction system was charged with 25% and 50% organic solvents (EDTA, SDS, acetone, DMSO, methanol, acetonitrile, DMF, cyclohexane, isopropanol, ethanol), 1mM and 5mM metal ions (Mn), respectively ²⁺ 、Ca ²⁺ 、Fe ³⁺ 、Mg ²⁺ 、Cr ²⁺ 、Cu ²⁺ 、Ag ⁺ 、Zn ² ⁺ ) 0.5% of surfactant (Tween20, Tween40, Tween60, Tween80, Triton-100, SDS and CTAB), and DBP is used as a substrate to measure the enzyme activity of EstJ6 under the optimal condition.3M HCl (10%) is added to terminate the reaction, 2 times the volume of chromatographic grade methanol is added after sufficient shaking, the organic phase is taken and subjected to membrane filtration to remove impurities, and the residual amount of the substrate is quantified by HPLC. The results are shown in FIGS. 4e-g, with the relative activity of EstJ6 defined as 100% without the addition of metal ions or chemicals. 1mM metal ion (Mn) ²⁺ ，Mg ²⁺ ，Cu ² ⁺ ) 25% ethanol, 0.5% (Tween20, Tween40 and Triton-100) had little effect on EstJ6 activity; 50% organic solvent (EDTA, acetone, DMSO, methanol, acetonitrile, DMF, isopropanol and ethanol), 1mM metal ion (Cr) ⁺ And Ag ⁺ ) 5mM metal ion (Mn) ²⁺ ，Fe ³⁺ ，Cr ²⁺ ，Cu ²⁺ ，Ag ⁺ ，Zn ²⁺ ) SDS and CTAB strongly inhibit the activity of EstJ 6; 1mM metal ion (Ca) ²⁺ ，Fe ³⁺ And Zn ²⁺ ) 5mM metal ion (Ca) ²⁺ And Mg ²⁺ ) Has slight inhibition effect on EstJ6 activity; while Tween60 and Tween80 had a slightly promoting effect on the activity of EstJ 6.

ESTJ6 presumption of the DEHP biodegradation pathway

The ability of purified EstJ6 to degrade phthalate was determined using DEHP as a substrate. To a 1mL 10mM PBS (pH7.5) reaction was added 12ug/mL purified EstJ6 and 1mM DEHP, mixed well and incubated at 40 ℃ for 12h, and the sample was blown dry on a clean bench and redissolved in chromatographic grade methanol. The organic phase was taken through a 0.22 μm organic membrane and analyzed by GC-MS.

The conditions for GC-MS were as follows: the sample volume is 1uL, and the split mode is 20: 1, the flow rate is 1 mL/min. The operation method comprises the following steps: firstly, keeping the temperature at 60 ℃ for 1 min; then raising the temperature to 180 ℃ at the speed of 10 ℃/min and keeping the temperature for 10 min; finally, the temperature is increased to 220 ℃ at the speed of 15 ℃/min and kept for 5 min.

The degradation of DEHP by EstJ6 was analyzed using GC-MS, and EstJ6 efficiently hydrolyzed DEHP into the corresponding MEHP and PA by cleaving the ester bond, as shown in fig. 5a-d, where 5a is the total ion flux chromatogram of the remaining DEHP and its degradation products (MEHP and PA) after DEHP degradation, and 5b-d is the mass spectrum of the remaining DEHP and its degradation products (MEHP and PA) after DEHP degradation. The pathway of depp degradation by EstJ6 was predicted based on the degradation products, as shown in fig. 5 e. During DEHP degradation, EstJ6 mediates the hydrolysis of DEHP to PA via the intermediate MEHP.

EstJ6 phylogenetic Tree analysis

The amino acid sequence of EstJ6 and other known types of esterases were used to construct a phylogenetic tree using MEGA6.0 software (figure 6). EstJ6 contains three characteristic motifs of this family (YXLPPE, HGGG and GDSAGG) and it also contains a conserved motif EXLLD, which is different from the motif DPLXD of the other members of family IV. The results indicate that EstJ6 belongs to a new member of the Family IV of esterases.

Multiple sequence alignment of EstJ6

Multiple sequence alignment of EstJ6 and its homologous sequence was performed using the online tool Clustal Omega (FIG. 7). The EstJ6 sequence contained three conserved motifs, HGGG (76-79), YXLAPE (108-113) and GDSAG (144-148). Among them, HGGG is close to active sites, and helps to form oxygen anion holes to participate in catalytic process. The yxlpae motif has been previously reported, but its function is still unknown. For GX1SX2G, which is a well-known pentapeptide motif in PAEs hydrolases, a serine residue (S) involved in the catalytic triad is identified in the GX1SX2G motif. These three conserved regions are very common in esterases/lipases. In addition, EstJ6 also contains a conserved motif EXLLD (240-244), which is different from the conserved motif DPLXD of other PAEs hydrolases, where D and E may be subunits that make up the catalytic triad.

Example 5 EstJ6 homologous modeling

The PDB database was searched for a homologous template of EstJ6 for modeling by NCBI BLAST. The crystal structure of a microbial esterase (PBD: 4XVC-A) from the bacterial Hormone Sensitive Lipase (HSL) family was selected as a template with sequence identity of 53% and query coverage of 98%.

As shown in FIG. 8, the superposition of the three-dimensional structures of EstJ6 and microbial esterase (PBD: 4XVC-A) indicates that they belong to the serine hydrolase superfamily. They have an α/β hydrolase fold in which two domains can be recognized, a catalytic domain and a screw cap domain covering the entry to the active site.

As shown in fig. 8, docking studies were performed using DBP as the ligand and EstJ6 as the receptor. DBP is located between the catalytic domain and the cap domain, indicating the position of the substrate binding pocket and the amino acid residues S146-E240-H270 found in the vicinity of the substrate with which it interacts.

Example 6: site-directed mutagenesis of conserved amino acid residue of EstJ6

The conserved amino acid of EstJ6 was site-directed mutated (S146, E240, D244 and H270) using Treief TM SoSoSoso cloning kit (Ongki technologies, Inc., China), and the mutation primers are shown in Table 2. The activity experiment result shows that the activities of S146A, E240A and H270A are completely lost, and the activity of D244A is obviously inhibited. Furthermore, kinetic parameters of EstJ6 and mutants were determined using DBP as substrate (table 3). The D244A mutant has a DBP catalytic activity of 55% of that of the wild-type enzyme, and has Km and Vmax values of 0.756mM and 27.549umol/min/mg, respectively. To further verify that one of the bases of the catalytic triad consists of E instead of D, E240 was further mutated to D, indicating that the activity of mutant E240D (7%) was almost completely lost. Thus, the catalytic triad residue of EstJ6 is presumed to be S146-E240-H270, which is different from the classical catalytic triad (SDH) of the other members of the phthalate degradation capability in family IV.

TABLE 2 primers for site-directed mutagenesis of EstJ6

TABLE 3 kinetic constants of wild type (EstJ6) and mutant EstJ6

Sequence listing

<110> Nanjing university of agriculture

<120> novel phthalate ester hydrolase EstJ6, and coding gene and application thereof

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atggcaagtc cgcaactaca gatggccctt gatgcgttca agacgatggg cgagaaaatg 60

gcgcaggcgg gaaatgacgt gaaagccttg cgcgctgtca tggaagagat gtctggcttt 120

ccctcagcag gggagacgaa gtgtacgccg gtaaatgctg gcggcgttcc tgccgaatgg 180

atttccggtc ctggtgccgc ggatgatcgc gtgatcctgt acgtacacgg cggtggctat 240

gtgatgggtt ctatcgctac tcaccgcgag acggttgctc gtctgtcgaa agcctcggga 300

gcgcgtggtc tggcgttaga ttaccgcctg gccccggagc atccattccc cgccgcggtt 360

gatgacgcga cggcagcgta tcgctggctg ctctcgcaaa atattaaacc tgcccacatt 420

gtcattgccg gtgactctgc gggcggaggg cttacgctgg cgactctcat cgcgttacgg 480

gacgcgaagg ttccccttcc cgccgcgggt gtgtgtattt caccgtggac ggacatggaa 540

ggggctgggg agtcaatgac gaccagggcg aaggccgatc ccgtcgtgca aaagcaagga 600

ctgctgggta tggcacagct ctacctcggc ggcaaagatc cgaagtcgcc gctcgccgct 660

ccactgcacg ccaatctcgc gggactcccg ccgctcttga ttcaagtggg agacgccgag 720

accttgctcg acgactccat tcgtgttgcc gaaaaagcca agaaagcggg cgtcaaagtc 780

gatctcgagg tttggccgga gatgccccac gtgtggcacc tgttcgcccc gttcctgccg 840

gaaggccagc aagcgatcga caagatcggg aagtacgtcc ggcagatcac cgcgtaa 897

<210> 2

<211> 298

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Met Ala Ser Pro Gln Leu Gln Met Ala Leu Asp Ala Phe Lys Thr Met

1 5 10 15

Gly Glu Lys Met Ala Gln Ala Gly Asn Asp Val Lys Ala Leu Arg Ala

20 25 30

Val Met Glu Glu Met Ser Gly Phe Pro Ser Ala Gly Glu Thr Lys Cys

35 40 45

Thr Pro Val Asn Ala Gly Gly Val Pro Ala Glu Trp Ile Ser Gly Pro

50 55 60

Gly Ala Ala Asp Asp Arg Val Ile Leu Tyr Val His Gly Gly Gly Tyr

65 70 75 80

Val Met Gly Ser Ile Ala Thr His Arg Glu Thr Val Ala Arg Leu Ser

85 90 95

Lys Ala Ser Gly Ala Arg Gly Leu Ala Leu Asp Tyr Arg Leu Ala Pro

100 105 110

Glu His Pro Phe Pro Ala Ala Val Asp Asp Ala Thr Ala Ala Tyr Arg

115 120 125

Trp Leu Leu Ser Gln Asn Ile Lys Pro Ala His Ile Val Ile Ala Gly

130 135 140

Asp Ser Ala Gly Gly Gly Leu Thr Leu Ala Thr Leu Ile Ala Leu Arg

145 150 155 160

Asp Ala Lys Val Pro Leu Pro Ala Ala Gly Val Cys Ile Ser Pro Trp

165 170 175

Thr Asp Met Glu Gly Ala Gly Glu Ser Met Thr Thr Arg Ala Lys Ala

180 185 190

Asp Pro Val Val Gln Lys Gln Gly Leu Leu Gly Met Ala Gln Leu Tyr

195 200 205

Leu Gly Gly Lys Asp Pro Lys Ser Pro Leu Ala Ala Pro Leu His Ala

210 215 220

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

225 230 235 240

Thr Leu Leu Asp Asp Ser Ile Arg Val Ala Glu Lys Ala Lys Lys Ala

245 250 255

Gly Val Lys Val Asp Leu Glu Val Trp Pro Glu Met Pro His Val Trp

260 265 270

His Leu Phe Ala Pro Phe Leu Pro Glu Gly Gln Gln Ala Ile Asp Lys

275 280 285

Ile Gly Lys Tyr Val Arg Gln Ile Thr Ala

290 295

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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catgccatgg gcatggcaag tccgcaacta 30

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cccaagcttc gcggtgatct gccggac 27

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<212> DNA

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gtcaccggca atgacaatgt gggcaggttt aatatt 36

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attgtcattg ccggtgacgc tgcgggcgga gggcttac 38

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ggcgtctccc acttgaatca agagcggcgg gagtcc 36

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<211> 39

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attcaagtgg gagacgccgc gaccttgctc gacgactcc 39

<210> 9

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gagcaaggtc tcggcgtctc ccacttgaat caagag 36

<210> 10

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

acgccgagac cttgctcgcc gactccattc gtgttgccg 39

<210> 11

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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catctccggc caaacctcga gatcgacttt gacgcc 36

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<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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aggtttggcc ggagatgccc gccgtgtggc acctgttcgc 40

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ggcgtctccc acttgaatca agagcggcgg gagtcc 36

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attcaagtgg gagacgccga caccttgctc gacgactcc 39

Claims

1. Phthalate ester hydrolase EstJ6 or gene for coding phthalate ester hydrolase EstJ6estj6Or comprising said geneestj6The expression vector or the recombinant microorganism containing the expression vector or the preparation method of the recombinant phthalate ester hydrolase EstJ6 is applied to hydrolysis of phthalate ester compounds;

the phthalate ester compound is selected from dibutyl phthalate, dipentyl phthalate, dipropyl phthalate, diethyl phthalate, dihexyl phthalate, dimethyl phthalate, diethylhexyl phthalate, monomethyl phthalate, monoethyl phthalate, monobutyl phthalate, monohexyl phthalate and monoethylhexyl phthalate;

the amino acid sequence of the phthalate ester hydrolase EstJ6 is shown as SEQID number 2.

2. Use according to claim 1, characterized in that the gene coding for the phthalate ester hydrolase EstJ6estj6The nucleotide sequence of (A) is shown as SEQ ID number 1.

3. The use of claim 1, wherein said gene is includedestj6The expression vector of (a) is obtained by introducing the gene of claim 2estj6Cloning into pET28a (+).

4. The use according to claim 1, wherein the recombinant microorganism comprising the aforementioned expression vector uses E.coli BL21(DE3) as host cell.

5. The use according to claim 1, characterized in that the recombinant phthalate ester hydrolase EstJ6 is prepared by a method comprising the steps of: preparation of a Gene comprising saidestj6The recombinant phthalate hydrolase EstJ6 is obtained by transforming a host cell with the expression vector, culturing a transformant and separating from a culture.

6. The application of claim 5, wherein the preparation method comprises the following specific steps: the gene is introduced intoestj6Performing PCR amplification, and subjecting the amplification product toNcoI andHindIII double digestion, and the fragment is connected with a pET28a (+) vector to obtain pET28a (+) -etsj6The expression vector is transformed into escherichia coli BL21(DE3), a transformant is cultured, IPTG induction is carried out, and separation and purification are carried out on the culture, so as to obtain the recombinant phthalate hydrolase EstJ 6.

7. Use according to claim 1 for the specific amplification of genesestj6The primer comprises the following two primer sequences:

the upstream primer is shown as SEQ ID number 3;

the downstream primer is shown as SEQ ID No. 4.