CN116926042A - Pseudomonas PET degrading enzyme and encoding gene and application thereof - Google Patents
Pseudomonas PET degrading enzyme and encoding gene and application thereof Download PDFInfo
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- CN116926042A CN116926042A CN202310655016.5A CN202310655016A CN116926042A CN 116926042 A CN116926042 A CN 116926042A CN 202310655016 A CN202310655016 A CN 202310655016A CN 116926042 A CN116926042 A CN 116926042A
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- 241000589774 Pseudomonas sp. Species 0.000 claims abstract description 27
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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Abstract
The invention discloses a Pseudomonas PET degrading enzyme, and a coding gene and application thereof, and solves the problems that the sequence of the existing PET degrading enzyme for transformation is too close and the sequence of the degrading enzyme is lacking. The Pseudomonas PET degrading enzyme is derived from Pseudomonas (Pseudomonas sp.) BC1815, the Pseudomonas PET degrading enzyme is a single-transmembrane protein displayed on the cell membrane surface of the Pseudomonas (Pseudomonas sp.) BC1815, and bacterial cells with the cell membrane surface displaying the Pseudomonas PET degrading enzyme are obtained by breeding and culturing the Pseudomonas (Pseudomonas sp.) BC1815, and are applied to catalyzing and degrading PET plastics to obtain degradation products MHET and TPA, catalyzing BHET to produce MHET or catalyzing MHET to produce TPA.
Description
Technical Field
The invention relates to the technical field of genetic engineering and the field of environmental protection, in particular to pseudomonas PET degrading enzyme, and a coding gene and application thereof.
Background
For plastic garbage in natural environment, three main treatment modes are incineration, landfill and recovery treatment. In the global scope, 79% of plastic garbage is buried into ocean or land garbage to enter the environment, 12% of plastic garbage is incinerated, and the recycled plastic garbage is only 9%, but the incineration treatment and the landfill treatment cannot well solve the problem of white pollution. The plastic garbage can generate a large amount of harmful gases such as hydrogen chloride, toluene and the like in the incineration process, and the harmful gases can enter the atmosphere to cause atmospheric pollution, so that the human body health is seriously endangered; the residues after the plastic garbage incineration are subjected to landfill treatment, and some of the residues are directly landfilled after the plastic garbage incineration, but the landfilled garbage can generate a large amount of pathogenic bacteria and can cause harm to soil because no harmless treatment is performed. Therefore, development of new methods for treating plastic wastes has become more and more urgent, and biodegradation, which is an environmentally friendly degradation means, can degrade or recover plastic wastes by microorganisms or enzymes, and thus has received a great deal of attention.
At present, researchers have separated a plurality of PET hydrolases from a plurality of eukaryotic and prokaryotic microorganisms, and the PET hydrolases can degrade PET into raw material components such as bis (2-hydroxyethyl) terephthalate (BHET), mono (2-hydroxyethyl) terephthalate (MHET), terephthalic acid (TPA), ethylene Glycol (EG) and the like, and the hydrolases belong to the subfamily of carboxyhydrolases, mainly cutinases, lipases, esterases, PETase and the like.
The cutinase is an extracellular serine esterase present in most plant pathogens and insect pathogens and has an alpha/beta hydrolase structure capable of hydrolyzing water-soluble esters, synthetic polyesters such as polyethylene terephthalate PET. The cutinase has an oxyanion hole at its active site for recognition and binding to a substrate, and its catalytic triad (serine-histidine-aspartic acid) is located in a surface groove surrounded by hydrophobic amino acids, which is advantageous for the accommodation of the substrate PET.
Lipases, also known as glyceride hydrolases, are generally considered to be capable of catalyzing the hydrolysis of long chain water insoluble triglycerides. Lipases are distinguished from cutinases in that they have a unique interfacial activity in terms of enzymatic kinetics: the catalytic activity of the enzyme in the single water phase or the oil phase is obviously lower than that of the enzyme at the water-oil interface. The lipase has an alpha/beta hydrolase structure like the cutinase, and meanwhile, the lipase also has a unique cover structure formed by an alpha-helix extra peptide segment, which covers the catalytic center of the enzyme, thereby preventing the enzyme from effectively catalyzing with PET substrates, and leading the lipase to have lower hydrolytic activity on PET.
Esterase is a biocatalyst which is widely existed in procaryotes and eukaryotes and can catalyze the hydrolysis and synthesis reaction of short-chain fatty acid and alcohol-composed ester, and structural research shows that esterase has a typical alpha/beta hydrolase folding structure.
In 2016, yoshida et al screened a strain Ideonella sakaiensis-F6 capable of utilizing low crystallinity (1.9%) PET from 250 PET chip contaminated environmental samples collected from PET bottle recovery plants, which resulted in a PETase capable of 60mg weight loss of PET film at 30℃over six weeks, the degradation products were bis (2-hydroxyethyl) terephthalate (BHET), mono (2-hydroxyethyl) terephthalate (MHET), terephthalic acid (TPA). The hydrolytic activity of PETase on PET film was 120, 5.5, 88 times that of TfH, LCC, fsC at 30 ℃ and ph=7.0, respectively. Further structural studies have shown that PETase belongs to the alpha/beta hydrolase family, with a core structure surrounded by 7 alpha-helices and 9 beta-sheets, with a conserved catalytic triplet S131-H208-D177 and oxyanion cavities (Y58, M132). The enzyme has a uniquely longer loop than other homologous enzymes, with three additional residues (Ser 245, asn246, gln 247). Studies have shown that the extended loop provides more room for PET to bind, and that the pocket at the PETase active site is relatively widened compared to TfH, making PETase more suitable for containing long chain macromolecular polymers such as PET. Although PETase has attracted considerable attention, PETase has poor stability compared to other PET degrading enzymes, and the degradation activity of PETase is substantially lost at 37 ℃ for 12 hours.
Although the research on PET degrading bacteria and degrading enzymes is the greatest at present, the reported degrading enzymes still cannot meet the industrial application requirements, so that more PET degrading bacteria and degrading enzyme resources need to be excavated from the nature, and a foundation is provided for the evolution and transformation of PET degrading enzymes.
Disclosure of Invention
Therefore, the invention aims to provide pseudomonas PET degrading enzyme, and a coding gene and application thereof, and solve the problems that the sequence of the existing PET degrading enzyme for modification is too close and the sequence of the degrading enzyme is lacking.
The Pseudomonas PET degrading enzyme in the invention is derived from Pseudomonas sp BC1815, is a Lipase, and is named as PET Lipase. The Pseudomonas is Pseudomonas BC1815, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: m2022905; the 16S rDNA sequence of the strain is shown as SEQ ID NO. 4:
SEQ ID NO:4：
ggatgagaggagcttgctccttgatttagcggcggacgggtgagtaatgcctaggaatctgcctggtagtgggggataacgttccgaaaggaacgctaataccgcatacgtcctacgggagaaagcaggggaccttcgggccttgcgctatcagatgagcctaggtcggattagctagttggtgaggtaatggctcaccaaggcgacgatccgtaactggtctgagaggatgatcagtcacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattggacaatgggcgaaagcctgatccagccatgccgcgtgtgtgaagaaggtcttcggattgtaaagcactttaagttgggaggaagggcattaacctaatacgttagtgttttgacgttaccgacagaataagcaccggctaacttcgtgccagcagccgcggtaatacgaagggtgcaagcgttaatcggaattactgggcgtaaagcgcgcgtaggtggttcgttaagttggatgtgaaagccccgggctcaacctgggaactgcatccaaaactggcgagctagagtacggtagagggtggtggaatttcctgtgtagcggtgaaatgcgtagatataggaaggaacaccagtggcgaaggcgaccacctggactgatactgacactgaggtgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgtcaactagccgttgggttccttgagaacttagtggcgcagctaacgcattaagttgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttacctggccttgacatgctgagaactttccagagatggattggtgccttcgggaactcagacacaggtgctgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgtaacgagcgcaacccttgtccttagttaccagcacgtaatggtgggcactctaaggagactgccggtgacaaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacggccagggctacacacgtgctacaatggtcggtacaaagggttgccaagccgcgaggtggagctaatcccataaaaccgatcgtagtccggatcgcagtctgcaactcgactgcgtgaagtcggaatcgctagtaatcgtgaatcagaatgtcacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgctccagaagtagctagtctaaccttcgg
the Pseudomonas PET degrading enzyme is a single-transmembrane protein displayed on the surface of a cell membrane of the Pseudomonas BC1815, and bacterial cells with the Pseudomonas BC1815 displayed on the surface of the cell membrane are obtained by breeding and culturing the Pseudomonas BC1815, and the bacterial cells are applied to the catalytic degradation of PET plastics to obtain degradation products of mono (2-hydroxyethyl) terephthalate and terephthalic acid, and catalyze bis (2-hydroxyethyl) terephthalate to produce mono (2-hydroxyethyl) terephthalate or catalyze mono (2-hydroxyethyl) terephthalate to produce terephthalic acid.
The invention adopts the following specific technical scheme:
the first aspect of the invention provides a Pseudomonas PET degrading enzyme, wherein the amino acid residue sequence of the Pseudomonas PET degrading enzyme is shown as SEQ ID NO:1 is shown as follows:
SEQ ID NO：1：
MKKLLLALLLLLLAASATLYFFPATQLTSLRLIEQQRAGLSHERLSVRDLNIHYYQGGPASGETLVLLHGFAADKDNWLRFSRHLTKDYRVIALDLPGFGDSDLPPGSYDVGTQAERLADILDAMGVQQAHLLGNSMGGHIAAIFAARYPDRVRSLALLANAGIDSPHKSELYRLLTGGAPNPLVVKQPQDFDNLLQFIFVEPPYLPESLKRYLGERSMAKAAYYERVFKQLVERAIPLEPELAKIQAPTLLLWGKQDRVLDVSSIEVMQPLLGKPSVVIMDNVGHAPMLERPEESALLYRQFLEGLK
the Pseudomonas PET degrading enzyme PET Lipase amino acid residue sequence and the published PET degrading enzyme sequence are subjected to phylogenetic tree construction, and the Pseudomonas PET degrading enzyme PET Lipase is singly located on one branch in the phylogenetic tree, so that the sequence is very strong in innovation.
Further, the catalytic triplet of the Pseudomonas PET degrading enzyme is S136-H286-D258, and the N-terminal transmembrane domain is MKKLLLALLLLLLAASATLYFFPATQL.
Further, the deoxynucleotide sequence of the encoding gene of the pseudomonas PET degrading enzyme is shown as SEQ ID NO:2 is shown as follows:
SEQ ID NO：2：
atgaaaaaactgctgctcgctttgctgctactgcttctcgctgcctcagcaacgctgtatttcttcccggccactcagttgaccagcctgcgcctgatagagcaacagcgcgccgggctcagtcacgagcggctatctgtccgcgatcttaacatccattactaccagggaggaccggcgagtggcgaaaccctggtgttgctccacggtttcgctgcagacaaggataactggttgcgtttttctcgccacctgaccaaggactatcgcgtcatcgcattggacctgcccggcttcggtgacagcgacctgccacctggcagctacgatgtcggcacccaggccgaacggctagcggatattctcgatgccatgggcgtccagcaggcgcatctgctgggcaattcgatgggtggacacatcgccgcaatcttcgcggcacgctacccggatcgtgttcgttcgctggcgctattggccaacgccggaatcgacagcccgcacaagagcgaactgtatcgattgctgaccggtggtgcgcccaatccactggtggtcaagcaaccgcaggatttcgacaatctgctgcaattcatcttcgtcgaaccaccctatctgcccgagtcgctcaagcgatatctgggcgagcgctccatggccaaggctgcgtactatgaacgggtattcaagcaactggtggagcgcgccatcccattggagcccgaactggcgaaaatccaggcccccaccttgctgctttggggcaaacaggaccgggtgctggacgtatccagcatcgaggtcatgcagccgctgctaggcaaacccagcgtggtgatcatggataacgtcggccacgcccccatgctcgaacgccccgaggaaagcgccctgctctaccggcagtttctagaaggcctgaagtga
the second aspect of the invention provides the application of the pseudomonas PET degrading enzyme in degrading PET plastics.
Further, the Pseudomonas PET degrading enzyme obtained by heterologous expression in the escherichia coli is applied to catalytic degradation of PET plastics.
Further, the degradation products of the catalytic degradation of PET plastic are terephthalic acid and mono (2-hydroxyethyl) terephthalate, which are applied to the catalysis of bis (2-hydroxyethyl) terephthalate to produce mono (2-hydroxyethyl) terephthalate, and which are applied to the catalysis of mono (2-hydroxyethyl) terephthalate to produce terephthalic acid.
Further, the method for heterologous Pseudomonas PET degrading enzyme in Escherichia coli comprises the following steps:
(1) According to the amino acid residue sequence of the Pseudomonas PET degrading enzyme, removing the N-terminal transmembrane domain, reasonably designing a cloning primer, cloning the coding gene of the Pseudomonas PET degrading enzyme, connecting the cloning gene fragment into an E.coli pET series expression vector, constructing an expression plasmid, adding a6 xHis tag at the C-terminal of the Pseudomonas PET degrading enzyme by using the 6 xHis tag sequence of the pET series expression vector, and then introducing the E.coli BL21 (DE 3) for culture propagation;
(2) After the culture is completed, the pseudomonas PET degrading enzyme is obtained through separation and purification after the expression of isopropyl-beta-D-thiogalactoside induced enzyme.
Further, the pseudomonas PET degrading enzyme containing the 6 xhis tag has an amino acid sequence shown in SEQ ID NO:3, shown in the following:
SEQ ID NO：3：
TSLRLIEQQRAGLSHERLSVRDLNIHYYQGGPASGETLVLLHGFAADKDNWLRFSRHLTKDYRVIALDLPGFGDSDLPPGSYDVGTQAERLADILDAMGVQQAHLLGNSMGGHIAAIFAARYPDRVRSLALLANAGIDSPHKSELYRLLTGGAPNPLVVKQPQDFDNLLQFIFVEPPYLPESLKRYLGERSMAKAAYYERVFKQLVERAIPLEPELAKIQAPTLLLWGKQDRVLDVSSIEVMQPLLGKPSVVIMDNVGHAPMLERPEESALLY RQFLEGLKLEHHHHHH
further, in the step (1), the culture was propagated to OD in LB medium containing kanamycin resistance 600 And (3) performing induction expression in the step (2) at 0.3-0.8.
Further, the separation and purification process comprises the following steps: and centrifugally collecting thalli, ultrasonically crushing cells, and separating and purifying by nickel ion affinity chromatography to obtain the pseudomonas PET degrading enzyme.
The Pseudomonas PET degrading enzyme PET Lipase can be obtained through heterologous expression, can also be directly obtained through fermenting and culturing Pseudomonas sp BC1815, has the advantages of novel sequence, high catalytic activity and the like, has catalytic activity on various substrates such as PET, BHET, MHET and the like, and has good application prospect.
The beneficial effects of the invention are as follows:
the invention provides an amino acid residue sequence of a Pseudomonas PET degrading enzyme, wherein the primary sequence of the Pseudomonas PET degrading enzyme has high novelty; the pseudomonas PET degrading enzyme PET Lipase has catalytic activity on substrates PET plastic, BHET and MHET; the Pseudomonas PET degrading enzyme is a single-transmembrane protein displayed on a Pseudomonas (Pseudomonas sp.) BC1815 cell membrane, so that the Pseudomonas (Pseudomonas sp.) BC1815 cell can be used for carrying out catalytic degradation on PET plastic, BHET and MHET, a large amount of Pseudomonas PET degrading enzyme can be obtained only by fermenting and culturing Pseudomonas (Pseudomonas sp.) BC1815, and the method has the characteristics of simplicity and convenience in obtaining, and meanwhile, a large amount of Pseudomonas PET degrading enzyme can be obtained through heterologous expression.
Drawings
FIG. 1 is a phylogenetic tree of the PET degrading enzyme PET Lipase of Pseudomonas sp.BC 1815 with the PET degrading enzyme that has been reported.
FIG. 2 is a PET degrading enzyme PET Lipase of Pseudomonas sp.) BC1815 aligned with the Lipase 2OCG sequence.
FIG. 3 shows SDS-PAGE electrophoresis after separation and purification of a heterologously expressed Pseudomonas (Pseudomonas sp.) BC1815 PET degrading enzyme PET Lipase in Escherichia coli, wherein the M lane is a protein molecular weight standard, and the 1-2 lanes are the separated and purified Pseudomonas PET degrading enzyme PET Lipase.
FIG. 4 shows the results of activity detection of a heterologously expressed Pseudomonas sp. BC1815 PET degrading enzyme PET Lipase to degrade PET plastic films at different temperatures.
FIG. 5 shows the results of activity detection of a heterologously expressed Pseudomonas sp. BC1815 PET degrading enzyme PET Lipase to degrade PET plastic films at different pH.
FIG. 6 is a liquid chromatogram of the detection of a heterologously expressed Pseudomonas sp. BC1815 PET degrading enzyme PET Lipase at pH 7.0 degrading PET plastic film. a is a control group control without adding PET degrading enzyme PET Lipase; b is adding PET degrading enzyme PET Lipase for the experimental group; c is MHET standard.
FIG. 7 shows the results of activity detection of a heterologously expressed Pseudomonas sp. BC1815 PET degrading enzyme PET Lipase to degrade BHET at different pH values.
FIG. 8 is a liquid chromatogram of the detection of the degradation of BHET by a heterologously expressed Pseudomonas sp. BC1815 PET degrading enzyme PET Lipase at pH 7.5. a is a control group control without adding PET degrading enzyme PET Lipase; b is adding PET degrading enzyme PET Lipase for the experimental group; c is MHET standard.
FIG. 9 shows the results of activity detection of the heterogeneously expressed Pseudomonas sp. BC1815 PET degrading enzyme PET Lipase to degrade MHET at different pH values.
FIG. 10 is a liquid chromatogram of the detection of the degradation of MHET by a heterologously expressed Pseudomonas sp. a is a control group control without adding PET degrading enzyme PET Lipase; b is adding PET degrading enzyme PET Lipase for the experimental group; c is TPA standard.
FIG. 11 shows the results of activity detection of Pseudomonas sp. BC1815 bacterial cells to degrade PET plastic films at different pH values.
FIG. 12 is a liquid chromatogram of the detection of Pseudomonas sp. BC1815 bacterial cells at pH7.5 and pH8.0 in degraded PET plastic films. a is MHET standard; b is pH7.5 control group without PET degrading enzyme PET Lipase; c is the PET degrading enzyme PET Lipase added for the pH7.5 experimental group; d is pH8.0, and the control group is not added with PET degrading enzyme PET Lipase; e is the PET degrading enzyme PET Lipase added to the pH8.0 experimental group.
Detailed Description
In order to describe the technical content, constructional features, achieved objects and effects of the technical solution in detail, the following description is made in connection with the specific embodiments in conjunction with the accompanying drawings.
The experimental methods used in the following examples are all conventional.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
And (5) constructing a Pseudomonas PET degrading enzyme PET Lipase phylogenetic tree.
The whole genome sequencing OF the Pseudomonas (Pseudomonas sp.) BC1815 is completed, the gene number OF the encoding gene OF the Pseudomonas PET degrading enzyme PET Lipase in the genome is OF 113-10420, and the published sequence OF the PET plastic degrading enzyme and the Pseudomonas PET degrading enzyme PET Lipase OF the invention are selected to carry out the phylogenetic tree construction by using software MEGA 6. From FIG. 1, it can be seen that the Pseudomonas PET degrading enzyme PET Lipase is singly located on one branch in the evolutionary tree, which proves that the Pseudomonas PET degrading enzyme PET Lipase primary sequence has strong novelty.
Example 2
Identification of the active site of the Pseudomonas PET degrading enzyme PET Lipase
The Pseudomonas PET degrading enzyme PET Lipase (No. OF 113-10420) was compared with the Lipase No. 2OCG, the catalytic triad OF Lipase 2OCG was S102-H235-D207, and the Pseudomonas sp. PET degrading enzyme PET Lipase OF Pseudomonas BC1815 was S136-H286-D258 (FIG. 2).
Example 3
The Pseudomonas PET degrading enzyme PET Lipase is heterologously expressed in colibacillus, separated and purified.
According to the amino acid residue sequence OF the Pseudomonas PET degrading enzyme PET Lipase, a transmembrane domain at the N end is removed, a cloning primer is reasonably designed, an enzyme cutting site is introduced, the primer sequences are OF 113_10420_NdeI:GGGAATTCCATATGACCAGCCTGCTGATA and OF 113_10420_XhoI:GCCCTCCGTCAGGCTCGAGCTTCTAAACTGCCG, a cloning gene fragment is connected to an Escherichia coli pET series expression vector pET-30b to construct an expression plasmid, a6 XHis tag sequence OF the pET-30b expression vector is utilized to add a6 XHis tag at the C end OF the Pseudomonas PET degrading enzyme, the purification OF a target protein is facilitated, and the Pseudomonas PET degrading enzyme containing the 6 XHis tag has the amino acid sequence shown in SEQ ID NO:3, the plasmid pET-30b is used as a carrier to heterologously express the pseudomonas PET degrading enzyme PET Lipase in the escherichia coli.
E.coli BL21 (DE 3) was transformed with the vector pET-30b-PETLipase containing the gene sequence of the Pseudomonas PET degrading enzyme PET Lipase, and cultured to OD in LB medium containing kanamycin resistance 600 At 0.3-0.8, the OD is selected in this example 600 The next induction is carried out at 0.6, after 0.5mM inducer IPTG (isopropyl-beta-D-thiogalactoside) is added to induce the expression of enzyme during the induction and the induction is carried out for 4 hours at 37 ℃, thalli are collected by centrifugation, cells are broken by ultrasound, the protein is expressed in the form of inclusion bodies in sediment, the sediment is dissolved by urea, and the pseudomonas PET degrading enzyme PET Lipase with the size of 32kDa is obtained by separating and purifying by nickel ion affinity chromatography, as shown in figure 3. Other examples include, in the case of the KarenamycinCulturing in LB culture medium with resistance to element to OD 600 0.3 or 0.8, and then performing induced expression.
Example 4
Heterologously expressing the optimal temperature of the Pseudomonas PET degrading enzyme PET Lipase degrading PET plastic.
The Pseudomonas PET degrading enzyme PET Lipase prepared by the separation and purification of the example 3 is used for degrading PET plastics. The reaction system was 300. Mu.l of 350mM phosphate buffer solution (pH 6.0), the Pseudomonas PET degrading enzyme PET Lipase was 0.2. Mu.M, the reaction temperature was 20℃and 25℃and 30℃and 35℃and the reaction time was 40℃and 30 hours, the reaction system contained 5 pieces of1 cm. Times.1 cm (thickness: 0.013 mM) PET plastic film, and after the reaction was completed, the PET plastic film was removed, and the degradation products were measured by HPLC, as shown in FIG. 4, the Pseudomonas PET degrading enzyme PET Lipase was able to hydrolyze to produce 0.08. Mu.M MHET on PET plastic at 30 ℃.
Example 5
And (3) determining the optimal pH of the PET plastic degraded by the heterologously expressed pseudomonas PET degrading enzyme PET Lipase.
The Pseudomonas PET degrading enzyme PET Lipase prepared by the separation and purification of the example 3 is used for degrading PET plastics. The reaction system was 300. Mu.l of 350mM phosphate buffer solution (pH 3.0-11.0), the Pseudomonas PET degrading enzyme PET Lipase was 0.6. Mu.M, the reaction temperature was 30℃and the reaction time was 72 hours, the reaction system contained 5 pieces of1 cm. Times.1 cm (thickness: 0.013 mM) PET plastic film, after the reaction was completed, the PET plastic film was removed, and the degradation products were measured by HPLC, as shown in FIG. 5, the Pseudomonas PET degrading enzyme PET Lipase had an optimum pH of 8.0 for PET plastic, and could hydrolyze to produce 0.62. Mu.M MHET. In a reaction system having a pH of 8.5 to 11.0, TPA can be detected as a degradation product. The liquid chromatography detection chromatogram of the Pseudomonas PET degrading enzyme PET Lipase degrading PET plastic at pH 7.0 is shown in FIG. 6.
Example 6
Determination of optimal pH for heterologous expression of Pseudomonas PET degrading enzyme PET Lipase to degrade BHET.
BHET was degraded by using the Pseudomonas PET degrading enzyme PET Lipase prepared by the separation and purification of example 3. The reaction system was 300. Mu.l of 350mM phosphate buffer (pH 3.0-8.0), the Pseudomonas PET degrading enzyme PET Lipase was 0.4. Mu.M, the reaction temperature was 30℃and the reaction time was 5 hours, the reaction system contained about 1.0mM BHET, and after the completion of the reaction, the degradation products were measured by HPLC. As shown in fig. 7, at an optimum pH of 7.5, at 30 ℃, ph=7.5, hydrolyzing BHET produced 0.78mM MHET and 2.0 μm TPA. The liquid chromatography detection chromatogram of the Pseudomonas PET degrading enzyme PET Lipase degrading BHET at pH7.5 is shown in FIG. 8.
Example 7
And (3) determining the optimal pH of the heterologous expression pseudomonas PET degrading enzyme PET Lipase degrading MHET.
The Pseudomonas PET degrading enzyme PET Lipase prepared by separation and purification in example 3 is used for degrading MHET. The reaction system was 300. Mu.l of 350mM phosphate buffer (pH 3.0-8.0), the Pseudomonas PET degrading enzyme PET Lipase was 0.4. Mu.M, the reaction temperature was 30℃and the reaction time was 48 hours, the reaction system contained about 1.0mM MHET, and after the completion of the reaction, the degradation products were measured by HPLC. As shown in FIG. 9, the optimum pH was 8.0 and hydrolysis of MHET produced 0.04mM TPA at 30 ℃. The liquid chromatography detection chromatogram of the Pseudomonas PET degrading enzyme PET Lipase degrading MHET at pH7.5 is shown in FIG. 10.
Example 8
PET plastic was degraded with Pseudomonas sp BC 1815.
The PET plastic was degraded with Pseudomonas PET degrading enzymes displayed on the surface of the BC1815 cell membrane. Fermenting and culturing Pseudomonas sp BC1815 in LB medium, centrifuging to obtain thallus, and re-suspending with 350mM phosphate buffer solution (pH 3.0-11.0) to make the OD of Pseudomonas sp BC1815 thallus in the reaction system 600 =1.0, and 5 pieces of PET film each having a length and width of 1cm were added to each reaction system, hydrolyzed at 30 ℃ for 72 hours in a shaking table of 150 rpm, and after the completion of the reaction, the plastic film was removed, and the cells were removed by centrifugation, and the degradation products were measured by HPLC. As a result, it was found that the cell had the highest hydrolysis ability for PET film at the optimum pH of 9.5, and the amount of MHET produced was 6.2. Mu.M (FIG. 11). Pseudomonas sp BC1815 degrades P under pH7.5 and pH8.0 reaction conditionsThe liquid chromatography detection chromatogram of ET plastic film is shown in fig. 12.
While the embodiments have been described above, other variations and modifications will occur to those skilled in the art once the basic inventive concepts are known, and it is therefore intended that the foregoing description and drawings illustrate only embodiments of the invention and not limit the scope of the invention, and it is therefore intended that the invention not be limited to the specific embodiments described, but that the invention may be practiced with their equivalent structures or with their equivalent processes or with their use directly or indirectly in other related fields.
Claims (10)
1. The pseudomonas PET degrading enzyme is characterized in that the amino acid residue sequence of the pseudomonas PET degrading enzyme is shown as SEQ ID NO: 1.
2. The pseudomonas PET degrading enzyme of claim 1, wherein the catalytic triplet for the pseudomonas PET degrading enzyme is S136-H286-D258 and the N-terminal transmembrane domain is MKKLLLALLLLLLAASATLYFFPATQL.
3. The pseudomonas PET degrading enzyme encoding gene according to claim 2, wherein the deoxynucleotide sequence of the encoding gene is shown in SEQ ID NO: 2.
4. Use of a pseudomonas PET degrading enzyme according to claim 3 for degrading PET plastics.
5. The use according to claim 4, wherein the Pseudomonas PET degrading enzyme obtained by heterologous expression in E.coli is used for catalytic degradation of PET plastics.
6. The use according to claim 5, wherein the degradation products of the catalytic degradation of PET plastic are terephthalic acid and mono (2-hydroxyethyl) terephthalate, for the catalysis of bis (2-hydroxyethyl) terephthalate to produce mono (2-hydroxyethyl) terephthalate, and for the catalysis of mono (2-hydroxyethyl) terephthalate to produce terephthalic acid.
7. The use according to claim 5, wherein the method for heterologous expression of the pseudomonas PET degrading enzyme in e.coli comprises the steps of:
(1) The method comprises the steps of (1) removing a transmembrane domain at the N end of an amino acid residue sequence of a pseudomonas PET degrading enzyme according to claim 1, reasonably designing a cloning primer, cloning a coding gene of the pseudomonas PET degrading enzyme according to claim 3, connecting a cloned gene fragment into an escherichia coli pET series expression vector, constructing an expression plasmid, adding a6 xHis tag at the C end of the pseudomonas PET degrading enzyme by utilizing a6 xHis tag sequence of the pET series expression vector, and then introducing the expression plasmid into escherichia coli BL21 (DE 3) for culture propagation;
(2) After the culture is completed, the pseudomonas PET degrading enzyme is obtained through separation and purification after the expression of isopropyl-beta-D-thiogalactoside induced enzyme.
8. The use according to claim 7, characterized in that the pseudomonas PET degrading enzyme containing a6 xhis tag has the amino acid sequence as set forth in SEQ ID NO: 3.
9. The use according to claim 7, wherein in step (1), the culture is propagated to OD in LB medium containing kanamycin resistance 600 0.3-0.8, and then carrying out the induction expression of the step (2); the separation and purification process comprises the following steps: and centrifugally collecting thalli, ultrasonically crushing cells, and separating and purifying by nickel ion affinity chromatography to obtain the pseudomonas PET degrading enzyme.
10. The use according to claim 4, wherein the pseudomonas PET degrading enzyme is directly obtained by fermenting and culturing pseudomonas (pseudomonas sp.) BC1815, wherein the pseudomonas PET degrading enzyme is a single transmembrane protein displayed on the cell membrane surface of the pseudomonas (pseudomonas sp.) BC1815, and the bacterial cell with the cell membrane surface displaying the pseudomonas PET degrading enzyme is obtained by reproducing and culturing pseudomonas (pseudomonas sp.) BC 1815.
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