CN115927411A - Esterase mutant gene, protein expressed by gene and application - Google Patents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/62—Plastics recycling; Rubber recycling
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses an esterase mutant gene, a protein expressed by the gene and application, wherein the nucleotide sequence of the esterase mutant gene is shown as SEQ ID NO. 2; experiments show that the protein expressed by the esterase mutant gene can be correctly folded and can be purified in a large amount in an escherichia coli system. Meanwhile, the mutant protein has the function of degrading PET by wild esterase, and compared with wild esterase, the thermal stability and catalytic efficiency of the protein expressed by the esterase mutant gene are obviously improved. The protein expressed by the esterase mutant gene has the activity of degrading PET within the pH range of 6-11. The protein expressed by the esterase mutant gene has activity and stability at the temperature of 30-70 ℃. Specifically, the degradation capability of PET at pH 9.0 and temperature 50 ℃ was improved by 58 times compared with wild-type esterase.
Description
Technical Field
The invention belongs to the field of genetic engineering of protein, and particularly relates to an esterase mutant gene, a protein expressed by the gene and application of the gene.
Technical Field
Plastics play an important role in modern society, are widely applied to aspects such as packaging, construction, textile, transportation, electronic equipment, industrial machinery and the like, and greatly change the life style of human beings. Among various plastics, polyethylene terephthalate (PET) is one of the most widely used polyester plastics because of its excellent properties such as light weight, good insulation, high strength and transparency, high thermal performance, and chemical resistance. With the great use and consumption of PET products, more and more plastic waste products are accumulated in the environment, which causes serious damage to the global ecological environment and also brings serious threat to human health.
Biological processes are a new type of PET degradation recovery technology that has emerged in recent years to break down such organic materials by the action of biological entities (such as microorganisms, i.e., bacteria, fungi, and marine microalgae) or enzymes. The advantages of biological processes, including mild process conditions, relatively low energy input, no need for hazardous chemical reagents and expensive machinery, make them a very promising option. Recently, japanese scientists reported that a bacterium (Enterobacter sakaiensis 201-F6 Osaka; ideonella sakaiensis) can produce an esterase that hydrolyzes PET. Compared with other PET hydrolases, the esterase can play a degradation function at normal temperature (30 ℃); and only degrades PET specifically, but not other types of polyester plastics; esterases are capable of degrading commercial high crystallinity plastic bottles. However, the catalytic efficiency and thermal stability of esterase are obviously insufficient, so that the industrial application cannot be realized at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an esterase mutant gene aiming at PET hydrolysis.
The second purpose of the invention is to provide a protein expressed by the esterase mutant gene.
The third purpose of the invention is to provide a plasmid containing the esterase mutant gene.
The fourth purpose of the invention is to provide a recombinant strain containing the plasmid.
A fifth object of the invention is to provide the use of the above protein in the hydrolysis of PET.
The technical scheme of the invention is summarized as follows:
an esterase mutant gene, which is called A gene for short, and the nucleotide sequence of the esterase mutant gene is shown as SEQ ID NO. 2.
The amino acid sequence of the protein is shown as SEQ ID NO. 3.
The plasmid containing the gene is constructed by the following method: taking a sequence shown in SEQ ID NO.2 as a template, F1 as a forward primer and R1 as a reverse primer, carrying out PCR to obtain a sequence containing an esterase mutant gene, carrying out enzyme digestion and T4DNA ligase connection, and constructing the esterase mutant gene into a pET-21b (+) vector to obtain a plasmid pET-21b-A containing the gene; the nucleotide sequence of F1 is shown in SEQ ID NO. 4; the nucleotide sequence of R1 is shown in SEQ ID NO. 5.
The recombinant strain containing the plasmid is constructed by the following method: the plasmid pET-21b-A is transformed into a strain of Escherichia coli BL21 (DE 3) to obtain a recombinant strain.
The use of the above protein in the hydrolysis of PET.
The invention has the advantages that:
experiments show that the protein expressed by the esterase mutant gene can be correctly folded and can be purified in a large amount in an escherichia coli system. Meanwhile, the mutant protein has the function of degrading PET by wild esterase, and the thermal stability and the catalytic efficiency of the protein expressed by the esterase mutant gene are obviously improved compared with wild esterase. The protein expressed by the esterase mutant gene has the activity of degrading PET within the pH range of 6-11. The protein expressed by the esterase mutant gene has activity and stability at the temperature of 30-70 ℃. Specifically, the degradation capacity of PET at pH 9.0 and 50 ℃ was improved by 58 times compared with the wild-type esterase.
Drawings
FIG. 1 is a gel chromatography assay of wild-type esterase protein.
FIG. 2 is a protein gel chromatography assay of esterase mutant gene expression.
FIG. 3 is an SDS-PAGE gel of proteins expressed by esterase mutant genes.
FIG. 4 is a graph showing the degradation activity of PET at 50 ℃ by a protein expressed from an esterase mutant gene.
Detailed Description
The expression vector pET-21b (+) is commercially available, and E.coli DH 5. Alpha. And BL21 (DE 3) are commercially available.
Experimental materials:
(1) LB liquid medium: 10g of peptone, 5g of yeast powder and 10g of NaCl, adding double distilled water to the volume of the mixture to be 1L, and sterilizing the mixture for 20 minutes at the temperature of 121 ℃ and under the pressure of 0.1 MPa. LB solid medium 10g peptone, 5g yeast powder, 10g NaCl, 15g agar powder, adding double distilled water to fix volume to 1L, sterilizing at 121 ℃ and 0.1MPa for 20 minutes.
(2) Ampicillin (100 mg/mL): 2g of ampicillin solid powder is added into 20mL of sterilized deionized water, and the solution is shaken gently to be dissolved fully, and filtered and sterilized by using a 0.22 mu m filter;
(3) Isopropylthio- β -D-galactoside (IPTG): under aseptic conditions, 10g of IPTG powder was dissolved in 42mL of sterile double distilled water and mixed well.
(4) SDS-PAGE (Polyacrylamide gel electrophoresis)
10% APS (ammonium persulfate) solution: 1g of APS solid powder was dissolved thoroughly by adding sterile water to 10 mL.
10% SDS (sodium dodecyl sulfate) solution: 1g SDS solid powder, and sterile water was added to 10mL to dissolve it sufficiently.
30% acrylamide solution (100 mL): 30g of acrylamide and 0.8g of methylene bisacrylamide are added with double distilled water to be 100mL.
1.5M Tris-HCl pH =8.8 buffer (1L): 181.71g Tris is weighed and dissolved in 800mL pure water, the pH is adjusted to 8.8, and the volume is adjusted to 1L.
0.5M Tris-HCl pH =6.8 buffer (500 mL): 60.57g Tris was weighed and dissolved in 400mL pure water, pH adjusted to 6.8 and made up to 500mL.
TABLE 1 12% SDS-PAGE gels
TABLE 2SDS-PAGE condensables
(5) 5 × Tris-glycine electrophoresis buffer (5L): 75.5g of Tris,470g of glycine and 25g of SDS were dissolved in purified water to a constant volume of 5L.
(6) pH 2.5 phosphate buffer: configuration of 0.02M NaH 2 PO 4 By H 3 PO 4 The pH was adjusted to 2.5 and finally the volume was 1L.
(7) Stopping liquid: 75mL pH 2.5 phosphate buffer, 25mL methanol.
The present invention will be further described with reference to the following examples, which are only some preferred embodiments of the present invention and are not intended to limit the present invention in other forms. Any modifications or equivalent variations of the embodiments according to the technical essence of the present invention are within the scope of the present invention, unless they depart from the technical essence of the present invention.
Example 1: construction of pET-21b-A plasmid
A sequence containing the A gene is obtained by PCR with an artificially synthesized A gene SEQ ID NO.2 as a template, F1 as a forward primer and R1 as a reverse primer, and the A gene is constructed on a pET-21b (+) carrier by enzyme digestion and T4DNA ligase connection to obtain a plasmid pET-21b-A containing the A gene.
The method comprises the following specific steps:
after a gene A (SEQ ID NO. 2) sequence is designed and obtained through gene synthesis, firstly, primers are designed for PCR, a forward primer and a reverse primer are respectively named as F1 (SEQ ID NO. 4) and R1 (SEQ ID NO. 5), a target gene is amplified, and a PCR system is shown in Table 3.
TABLE 3PCR System
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 50-60 ℃ by setting a temperature gradient for 30s, extension at 72 ℃ for 30s, setting 33 cycles, and finally extension for 5min. After the reaction is finished, electrophoresis is carried out by using 1% agarose gel electrophoresis, and then gel recovery is carried out by using a gel recovery kit to obtain a PCR product.
The PCR product obtained above and pET-21b (+) no-load were subjected to double digestion at 37 ℃ (see Table 4 for digestion system) for 30min using NdeI and XhoI enzymes. After the completion of the enzyme digestion, electrophoresis was performed by 1% agarose gel electrophoresis, and then gel recovery was performed by using a gel recovery kit to obtain a gene containing a cohesive end and a pET-21b (+) vector. Ligation was then carried out using T4DNA ligase at 22 ℃ (ligation system see Table 5) for 1h ligation reaction time.
TABLE 4 double enzyme digestion System
TABLE 5 connection System
After completion of the ligation reaction, 20. Mu.L of the ligation product was added to 100. Mu.L of E.coli DH 5. Alpha. Competence, allowed to stand on ice for 30min, then heat-shocked in a water bath at 42 ℃ for 90s, then rapidly inserted into ice, allowed to stand for 5min, added to 500. Mu.L of LB liquid medium, and thawed on a shaker at 37 ℃ for 45min. The recovered cells were centrifuged at 3000rpm for 4min, the supernatant was aspirated, and about 100. Mu.L of the supernatant was retained to gently mix the cells, and the mixture was spread on LB solid medium containing 100. Mu.g/mL ampicillin using a spreading bar, and placed in an incubator at 37 ℃ overnight for culture. On the next day, a single colony was picked into 5mL of LB containing 100. Mu.g/mL ampicillin, and shake-cultured at 37 ℃ for 10-12 hours. Extracting plasmids by using a Tiangen plasmid miniprep kit, then carrying out double digestion verification on the extracted plasmids, wherein a double digestion verification system is shown in table 6, placing the reaction system in a water bath at 37 ℃ for reaction for 30min, carrying out agarose gel electrophoresis after the reaction is finished, and sending the plasmids with correct digestion verification to a sequencing company for sequencing verification.
TABLE 6 double restriction enzyme validation System
Wild-type esterase gene WT, pET-21b-WT plasmid construction reference is made to this example.
The wild-type esterase is derived from enterobacter sakazakii: ideonella sakaiensis 201-F6, amino acid sequence accession No.: uniProtKB A0A0K8P6T7.1, which is artificially synthesized, and has the amino acid sequence of SEQ ID NO.6 (WT) obtained after codon optimization of Escherichia coli.
Example 2 expression and purification of esterase mutant genes
Transforming the recombinant plasmid pET-21b-A into E.coli BL21 (DE 3), culturing overnight, picking out a single colony to 5mL LB liquid culture medium containing 100 mug/mL ampicillin, and shake culturing for 6-8h at 37 ℃ by a shaker (obtaining a recombinant strain containing pET-21 b-A); then, transferring the bacterial liquid into 1L LB liquid culture medium containing 100 mug/mL ampicillin, shaking and culturing for 6-8h at 37 ℃ by a shaking table, and culturing the bacterial liquid to OD 600 Cooling to about 0.6 deg.C, adding IPTG to final concentration of 400 μ M, and performing induced culture for 16-18h. Then, the cells were collected by centrifugation, and the collected cells were resuspended using a suspension buffer (20 mM Tris-HCl,300mM NaCl,10% glycerol, pH 7.5), followed by disruption using a high-pressure disrupter. The disrupted product was centrifuged at high speed (18000rpm, 4 ℃,45 min), the supernatant was collected and added to a nickel affinity chromatography packing equilibrated with suspended bacteria buffer in advance, and incubated at 4 ℃ for 1h. After the incubation was completed, a wash buffer (20 mM Tris-HCl,300mM NaCl,20mM imidazole, 10% glycerol, pH 7.5), and washing the hetero protein non-specifically bound to the Ni column. After washing, the target protein bound to the Ni column was eluted using about 30mL of an eluent (20 mM Tris-HCl,300mM NaCl,300mM imidazole, 10% glycerol, pH 7.5). The eluate was collected and concentrated using a 10kDa concentration tube.
The protein of interest was subsequently further purified by AKTA pure system using a gel filtration chromatography column (Superdex 200Increate 10/300GL, GE). The buffer used was 20mM Tris-HCl,300mM NaCl, pH 7.5, and the fractions eluted at the peak positions were collected (see FIG. 2) and verified by protein gel electrophoresis (see FIG. 3), and the target protein was collected according to the molecular weight.
Expression purification of wild-type esterase the expression and purification of the reference esterase mutant genes are shown in FIG. 1.
Example 3 protein degradation of esterase mutant Gene expression PET assay
(1) Firstly, a PET circular sheet (diameter of 6 mm) is punched by a puncher; putting the PET wafer into an EP tube, adding 0.5% Triton X-100 aqueous solution by volume concentration, incubating in a metal bath at 50 ℃ for 30min, and clamping the wafer; then adding the mixture to 10mmol/L Na 2 CO 3 Incubating the aqueous solution in a metal bath at 50 ℃ for 30min; the wafer is clamped out and put into ddH 2 Soaking in oxygen, and incubating in a metal bath at 50 ℃ for 30min; taking out, and blowing the mixture to dry by nitrogen for later use.
(2) An aqueous protein solution (obtained in example 2, wild-type esterase as a control) having a final concentration of 500nM and the PET sheet obtained in step (1) were incubated in 600. Mu.L of a 50mM glycine-sodium hydroxide buffer solution having a pH of 50mM =9.0, and a PET hydrolysis reaction was carried out at a reaction temperature of 50 ℃ for 18 hours, the reaction was terminated by diluting the aqueous solution with a terminator solution prepared from 25% by volume of a phosphate buffer solution having a pH of 2.5 and 75% by volume of methanol, heat-treated at 85 ℃ for 10min, and then centrifuged at 12000rpm for 10min to remove inactivated protein. The supernatant was aspirated for HPLC analysis. All experiments were performed in triplicate.
HPLC was carried out on a water e2695 chromatograph equipped with a HyPURITY C18 column (4.6X 250 mm) in which the mobile phase A was phosphate buffer (pH 2.5) and B was methanol, the flow rate was set at 0.5mL/min and monitored at a wavelength of 240 nm. The setting procedure is as follows: 0-5min,25% V/V B; 5-25min,25-100% V/V B liquid linear gradient. The results are shown in FIG. 4.
Experiments show that, compared with wild esterase (SEQ ID NO.1, the nucleotide sequence of the protein expressed by the esterase mutant gene is optimized by escherichia coli codons and is shown by SEQ ID NO. 6), the protein expressed by the esterase mutant gene has six amino acid mutations, namely: S94A, D, H, N, 206C, S T, N219D, S C. The mutant protein can be correctly folded and can be purified in a large amount in an Escherichia coli system. Meanwhile, the mutant protein has the function of wild esterase, and the thermal stability and the catalytic efficiency of the protein expressed by the esterase mutant gene are obviously improved compared with wild esterase. The degradation capability of the PET at the pH of 9.0 and the temperature of 50 ℃ is improved by 58 times compared with that of the wild esterase.
Claims (5)
1. An esterase mutant gene, which is called A gene for short, and the nucleotide sequence of the esterase mutant gene is shown as SEQID No. 2.
2. An esterase mutant gene expressed protein according to claim 1, characterized in that the amino acid sequence of said protein is as shown in SEQ id No. 3.
3. A plasmid containing the gene of claim 1, which is constructed by the following method: taking a sequence shown in SEQ ID NO.2 as a template, F1 as a forward primer and R1 as a reverse primer, carrying out PCR to obtain a sequence containing an esterase mutant gene, carrying out enzyme digestion and T4DNA ligase connection, and constructing the esterase mutant gene into a pET-21b (+) vector to obtain a plasmid pET-21b-A containing the gene of claim 1; the nucleotide sequence of F1 is shown in SEQ ID NO. 4; the nucleotide sequence of R1 is shown in SEQ ID NO. 5.
4. A recombinant strain comprising the plasmid of claim 3, wherein said recombinant strain is constructed by: the plasmid pET-21b-A of claim 3 is transformed into Escherichia coli BL21 (DE 3) strain to obtain a recombinant strain.
5. Use of the protein of claim 2 in the hydrolysis of PET.
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