CN116478958A - High-temperature-resistant plastic degrading enzyme mutant and encoding gene and application thereof - Google Patents

High-temperature-resistant plastic degrading enzyme mutant and encoding gene and application thereof Download PDF

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CN116478958A
CN116478958A CN202310461082.9A CN202310461082A CN116478958A CN 116478958 A CN116478958 A CN 116478958A CN 202310461082 A CN202310461082 A CN 202310461082A CN 116478958 A CN116478958 A CN 116478958A
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吴培均
史聪慧
罗建杰
李富伟
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Department Of Inner Mongolia Bo Biological Technology Co ltd
Beijing Crvab Bio Tech Co ltd
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Beijing Crvab Bio Tech Co ltd
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Abstract

The invention belongs to the field of protein engineering and aims to overcome the defects of low degradation efficiency, poor thermal stability and the like of the existing plastic degrading enzyme. The invention provides a high-temperature-resistant plastic degrading enzyme mutant, and a coding gene and application thereof, wherein the mutant comprises the following amino acid substitutions compared with the amino acid sequence of a wild plastic degrading enzyme SEQ ID NO. 1: bit 233, 282, 122, 186, 280 and 207. The invention also provides a coding gene of the mutant, and the nucleotide sequence of the mutant is shown as SEQ ID NO. 4. The mutant has better stability, good pH stability and better thermal stability. The optimum temperature is 60 ℃, the melting temperature is up to 86.5 ℃, and the enzyme activity of more than 90 percent can be maintained after the treatment for 2 hours at 75 ℃; the degradation efficiency is high, and is improved by 3.5 times compared with the wild type. The high temperature resistant characteristic of the mutant can meet the requirement of the high temperature environment of industrial production.

Description

High-temperature-resistant plastic degrading enzyme mutant and encoding gene and application thereof
Technical Field
The invention belongs to the field of protein engineering, and particularly relates to a high-temperature-resistant plastic degrading enzyme mutant HotPETase, and a coding gene and application thereof.
Background
The plastic is a high molecular compound polymerized by polyaddition or polycondensation reaction with monomer as raw material, and its deformation resistance consists of synthetic resin and additives such as filler, plasticizer, stabilizer, lubricant, pigment, etc., and its main component is resin. Mainly comprises Polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), polyethylene terephthalate (PET), polyurethane (PU), polylactic acid (PLA), polyhydroxyalkanoate (PHA) and the like. Wherein PET is a high molecular compound formed by connecting terephthalic acid and glycol ester through ester bonds, and is mainly used for packaging materials such as beverage bottles and the like.
The massive accumulation of plastic waste presents a series of environmental problems, hundreds of thousands of marine mammals and turtles and millions of seabirds die from plastic pollution every year, which presents a great threat to the marine ecosystem, with PET plastic accounting for 18% of the total polymer worldwide. The current treatment mode of PET plastic waste mainly comprises: physical, chemical and biocatalytic methods. However, the physical method and the chemical method can only realize degradation recovery of PET plastics, the purity of the obtained monomer substances is lower, and only about 9% of PET plastics can be recycled, and the rest plastics are still buried or burned. In addition, the physical method and the chemical method for recycling the plastic need harsh reaction conditions, which not only increases the recycling cost, but also generates other extra pollutants and causes secondary pollution to the environment. In contrast, the biocatalysis method for recycling PET plastic has the advantages of mild conditions, environmental protection, sustainability and the like. The method can obtain monomer substances with extremely high purity, and can still synthesize products with the same properties as the plastics from which the monomer substances are derived after being recovered.
The plastic biological recovery method mainly relies on a degrading enzyme system in microorganisms, and a plastic degrading enzyme leaf and branch composting cutinase which can decompose PET plastic into terephthalate and ethylene glycol is found in a composting metagenome by a learner of the university of osaka in 2012, but the degradation efficiency is slow. Japanese scholars in 2016 found a bacterium capable of "eating" plastic and named as I.Sakaiensis, the bacterium can grow and reproduce with PET plastic as the only carbon source and energy source and PET as the substrate, the bacterium can adsorb on the surface of plastic to secrete PET hydrolase, PET is decomposed into monomer ethylene terephthalate in the presence of water, the monomer enters cytoplasm through cell membrane and then is decomposed into terephthalic acid and ethylene glycol by MHETase, and the two can enter the bacterial metabolic network to be further decomposed into small-molecule harmless substances such as carbon dioxide, methane and water, so as to realize complete degradation of plastic.
Chinese patent application 202211188452.8 discloses a rhodococcus picolinae PET degrading enzyme and a coding gene and application thereof, and the rhodococcus picolinae PET degrading enzyme PET Esterase provided by the application has catalytic activity on substrates PET plastics, BHET and MHET. The application also provides application of the rhodococcus picolinae P23, the rhodococcus picolinae PET degrading enzyme is displayed on the cell membrane surface of the rhodococcus picolinae P23 as single transmembrane protein, bacterial cells of which the cell membrane surface is displayed are obtained by breeding and culturing the rhodococcus picolinae P23, and the bacterial cells are applied to catalytic degradation of PET plastics to obtain degradation products of terephthalic acid and mono (2-hydroxyethyl) terephthalate, catalytic production of mono (2-hydroxyethyl) terephthalate from bis (2-hydroxyethyl) terephthalate or catalytic production of terephthalic acid from mono (2-hydroxyethyl) terephthalate under acidic conditions.
Chinese patent application 202110651807.1 discloses a method for the combined degradation of polyethylene terephthalate by bacterial enzymes, which provides the method: the preparation method comprises the steps of performing degradation reaction by using a cell suspension obtained by expanding and culturing Klebsiella variabilis SY1 (Klebsiella variicola SY 1) and candida antarctica cutinase as catalysts, using polyethylene terephthalate as a substrate and using a buffer solution with the pH of 7.0-9.0 as a reaction medium to form a reaction system, adding the substrate and candida antarctica cutinase into the buffer solution for degradation reaction for 5 days at 60 ℃, adding the Klebsiella variabilis SY1 cell suspension after degradation for 1 day at 30 ℃, and continuously degrading the polyethylene terephthalate high polymer to a monomer compound after heating to 60 ℃ for 5 days.
The existing plastic degrading enzyme has the defects of low degradation efficiency, poor thermal stability and the like, and is not suitable for industrial production. The combination of synthetic biology and protein engineering can improve the performance of the enzyme by rationally designing the existing enzyme in nature.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a high-temperature-resistant plastic degrading enzyme mutant, and a coding gene and application thereof. The high-temperature-resistant plastic degrading enzyme mutant provided by the invention still maintains higher enzyme activity at high temperature, and has the capability of efficiently degrading plastic.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first aspect of the invention provides a high temperature resistant plastic degrading enzyme mutant HotPETase, which has the following amino acid substitution compared with a wild plastic degrading enzyme PETase with an amino acid sequence shown as SEQ ID NO. 1:
bit 233, 282, 122, 186, 280 and 207.
SEQ ID NO:1：
MNFPRASRLMQAAVLGGLMAVSAAATAQTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNDSLKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS。
In some embodiments, the mutant amino acid substitution sites are N233C and S282C.
In some embodiments, the amino acid substitution sites of the mutants further include S121D, D186K, R280I and S207K.
In some embodiments, the amino acid sequence of the mutant is as shown in SEQ ID NO. 2:
QTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPDSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNDSLKAAAPQAPWKSSTNFSSVTVPTLIFACENDKIAPVNSSALPIYDSMSRNAKQFLEICGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTIVCDFRTANCS。
the gene sequence of the amino acid sequence of the coding SEQ ID NO. 2 is shown as SEQ ID NO. 3:
ATGCAGACTAACCCCTATGCTCGCGGGCCGAATCCTACAGCGGCCTCGTTGGAAGCCAGTGCAGGTCCCTTCACCGTACGTTCGTTTACTGTTTCCCGTCCATCTGGATATGGGGCTGGCACCGTCTATTATCCGACTAACGCCGGTGGTACTGTGGGCGCAATCGCCATTGTCCCCGGCTACACTGCACGCCAATCCTCGATTAAATGGTGGGGACCACGCTTGGCTAGCCACGGGTTTGTTGTGATCACCATCGATACTAACAGTACGTTGGACCAGCCAGATAGTCGCAGTTCTCAGCAGATGGCGGCATTACGCCAGGTGGCGAGCTTAAATGGGACGAGTTCAAGTCCAATTTATGGCAAGGTCGATACCGCCCGTATGGGAGTAATGGGCTGGAGTATGGGTGGCGGCGGATCTCTTATCTCGGCAGCGAATAATGATAGCCTGAAGGCAGCAGCCCCGCAGGCACCCTGGAAAAGTAGTACGAACTTTTCGTCTGTTACGGTTCCTACGCTTATCTTTGCCTGTGAGAATGATAAAATCGCACCAGTGAACTCATCAGCCTTGCCAATTTACGACTCGATGTCGcgtAACGCTAAACAGTTTCTTGAGATTTGTGGGGGCTCTCACTCTTGTGCCAACAGTGGGAATTCCAATCAAGCCCTTATCGGAAAAAAGGGCGTTGCTTGGATGAAGCGCTTCATGGACAATGACACTCGTTATTCAACTTTCGCTTGCGAAAACCCAAACTCAACCATAGTATGTGATTTTCGCACTGCTAACTGCAGC。
in some embodiments, the mutant has more than 85% homology with SEQ ID NO. 1. In some embodiments, the mutant has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO. 1.
In some embodiments, the mutant has more than 90% homology to SEQ ID NO. 1. In some embodiments, the mutant has more than 95% homology to SEQ ID NO. 1. In some embodiments, the mutant has greater than 99% homology with SEQ ID NO. 1.
In some embodiments, the plastic may be PET or a derivative thereof, such as APET, RPET, PETG, and the like.
In a second aspect the present invention provides a method of mutant degradation of plastics according to the first aspect, the method comprising one or more of the following steps:
(1) Lysing a host cell expressing the mutant to obtain a fermentation broth comprising the mutant having enzymatic activity and/or a purified enzyme broth;
(2) The fermentation broth comprising mutants having enzymatic activity and/or the purified enzyme broth is incubated with the substrate plastic for 12-36h, preferably 24h, at 20-95 ℃, preferably 30-85 ℃, more preferably 65 ℃, pH 2.0-12.0, preferably pH 4.0-10.0, more preferably pH 9.0.
In some embodiments, the fermentation broth may be a fermentation broth supernatant; and/or
The plastic is PET and/or derivatives thereof, such as APET, RPET, PETG and the like.
In a third aspect the invention provides a polynucleotide encoding a mutant according to the first aspect.
In some embodiments, the polynucleotide has a sequence as set forth in SEQ ID NO. 4.
In a fourth aspect the invention provides an expression vector comprising a polynucleotide according to the second aspect.
In some embodiments, the expression vector may be a pichia vector including, but not limited to, ppic3.5, pPIC9, ppiczα A, pPICZ αb, or ppiczαc.
In some embodiments, the expression vector may be pPIC9.
In some embodiments, the expression vector may comprise a signal peptide. In some embodiments, the amino acid sequence is set forth in SEQ ID NO. 4:
MAIPRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSN STNNGLLEEAEAEAEPKFINTTIASIAAKEEGVSLEKREAEANFPRASRLMQAAVLGGLMA VSAAATA。
the gene sequence of the coding signal peptide amino acid sequence is shown in SEQ ID NO. 5:
ATGGCTATTCCAAGATTCCCATCTATCTTCACTGCTGTTTTGTTCGCTGCTTCCTCCGCTTTGGCTGCTCCAGTCAACACTACTACCGAGGACGAAACTGCTCAAATTCCAGCTGAGGCTGTCATCGGTTACTCTGACCTGGAGGGTGACTTCGACGTTGCTGTCTTGCCATTCTCCAACTCCACCAACAACGGTTTGTTGGAGGAGGCTGAAGCTGAAGCTGAACCTAAATTCATCAACACTACTATCGCTTCTATCGCTGCTAAGGAGGAGGGTGTTTCCCTCGAGAAAAGAGAGGCTGAAGCT。
the amino acid sequence of the full-length high-temperature-resistant plastic degrading enzyme mutant containing the signal peptide is shown as SEQ ID NO. 6, which codes 289 amino acids, and 128 amino acids at the N end are the predicted signal peptide sequences.
SEQ ID NO:6：
MAIPRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLEEAEAEAEPKFINTTIASIAAKEEGVSLEKREAEAPKFINTTIASIAAKEEGVSLEKREAEANFPRASRLMQAAVLGGLMAVSAAATAQTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTL DQPDSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNDSLKAAAPQAPWKSSTNFSSVTVPTLIFACENDKIAPVNSSALPIYDSMSRNAKQFLEICGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTIVCDFRTANCS
The gene sequence for encoding the amino acid sequence is shown in SEQ ID NO. 7:
ATGGCTATTCCAAGATTCCCATCTATCTTCACTGCTGTTTTGTTCGCTGCTTCCTCCGCTTTGGCTGCTCCAGTCAACACTACTACCGAGGACGAAACTGCTCAAATTCCAGCTGAGGCTGTCATCGGTTACTCTGACCTGGAGGGTGACTTCGACGTTGCTGTCTTGCCATTCTCCAACTCCACCAACAACGGTTTGTTGGAGGAGGCTGAAGCTGAAGCTGAACCTAAATTCATCAACACTACTATCGCTTCTATCGCTGCTAAGGAGGAGGGTGTTTCCCTCGAGAAAAGAGAGGCTGAAGCTATGCAGACTAACCCCTATGCTCGCGGGCCGAATCCTACAGCGGCCTCGTTGGAAGCCAGTGCAGGTCCCTTCACCGTACGTTCGTTTACTGTTTCCCGTCCATCTGGATATGGGGCTGGCACCGTCTATTATCCGACTAACGCCGGTGGTACTGTGGGCGCAATCGCCATTGTCCCCGGCTACACTGCACGCCAATCCTCGATTAAATGGTGGGGACCACGCTTGGCTAGCCACGGGTTTGTTGTGATCACCATCGATACTAACAGTACGTTGGACCAGCCAGATAGTCGCAGTTCTCAGCAGATGGCGGCATTACGCCAGGTGGCGAGCTTAAATGGGACGAGTTCAAGTCCAATTTATGGCAAGGTCGATACCGCCCGTATGGGAGTAATGGGCTGGAGTATGGGTGGCGGCGGATCTCTTATCTCGGCAGCGAATAATGATAGCCTGAAGGCAGCAGCCCCGCAGGCACCCTGGAAAAGTAGTACGAACTTTTCGTCTGTTACGGTTCCTACGCTTATCTTTGCCTGTGAGAATGATAAAATCGCACCAGTGAACTCATCAGCCTTGCCAATTTACGACTCGATGTCGCGTAACGCTAAACAGTTTCTTGAGATTTGTGGGGGCTCTCACTCTTGTGCCAACAGTGGGAATTCCAATCAAGCCCTTATCGGAAAAAAGGGCGTTGCTTGGATGAAGCGCTTCATGGACAATGACACTCGTTATTCAACTTTCGCTTGCGAAAACCCAAACTCAACCATAGTATGTGATTTTCGCACTGCTAACTGCAGC。
in some embodiments, the expression vector may comprise an expression control sequence, preferably an AOX1 promoter. In some embodiments, the polynucleotide according to the third aspect is operably linked to an expression control sequence, preferably an AOX1 promoter. In some embodiments, the polynucleotide according to the third aspect may be located downstream of the AOX1 promoter.
In a fifth aspect, the present invention provides a host cell comprising the expression vector of the fourth aspect, obtained by introducing the expression vector into a cell.
In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the prokaryotic cell may be e. In some embodiments, the eukaryotic cell may be a yeast cell. In some embodiments, the yeast cell may be selected from one or more of a pichia cell, a brewer's yeast cell, or a polytype of a yeast cell.
In some embodiments, the eukaryotic cell is a pichia cell. In some embodiments, the pichia cell may be selected from one or more of X33, GS115, KM71H, or SMD 116. In some embodiments, the pichia cell is GS115.
In a sixth aspect the present invention provides the use of a mutant according to the first aspect, a polynucleotide according to the third aspect, an expression vector according to the fourth aspect and/or a host cell according to the fifth aspect for degrading plastics.
In some embodiments, the plastic is PET and/or derivatives thereof, such as APET, RPET, PETG, and the like.
The high temperature resistant plastic degrading enzyme mutant HotPETase is obtained by performing site-directed mutagenesis and combined mutagenesis on the amino acid sequence of the PETase by rational design and directed evolution strategy on the basis of the wild plastic degrading enzyme PETase shown as SEQ ID NO. 1. The mutant has better stability, particularly better thermal stability and optimal pH value of 9.0. The optimum temperature is 60 ℃, the melting temperature is up to 86.5 ℃, and the enzyme activity of more than 90 percent can be maintained after the treatment for 2 hours at 75 ℃; the degradation efficiency is high, and the degradation efficiency of the mutant obtained according to rational design is improved by 3.5 times compared with that of a wild type. The high temperature resistant characteristic of the mutant can meet the requirement of the high temperature environment of industrial production, and the mutant has wide application prospect.
Drawings
FIG. 1 shows a bar graph of enzyme activity of high temperature resistant plastic degrading enzyme mutants measured at different pH conditions.
FIG. 2 shows a bar graph of enzyme activity of high temperature resistant plastic degrading enzyme mutants measured at different temperatures.
FIG. 3 shows the enzyme activity retention of high temperature resistant plastic degrading enzyme mutants under high temperature conditions.
Detailed Description
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.
The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.
The PETase in the invention is derived from Idenonella sakaiensis201-F6 strain, can grow and reproduce by taking plastic as the only carbon source and energy source, and can be adsorbed on the surface of the plastic to decompose PET into monomers (ethylene glycol phthalate) in the presence of water. The term "plastic" in the present invention may include, but is not limited to, PET and derivatives thereof, such as APET, RPET, PETG and the like. Wherein the "derivative" is a compound produced by modification of a parent organic compound by one or more chemical reactions.
The term "mutation" in the present invention includes, but is not limited to, site-directed mutagenesis, error-prone PCR mutagenesis or saturation mutagenesis. Preferably, the invention adopts site-directed mutagenesis to carry out genetic information modification on PETase so as to obtain mutants with more excellent performance. Site-directed mutagenesis is the introduction of desired changes or modifications, including addition, deletion, point mutation, etc., of bases into a DNA fragment of interest by Polymerase Chain Reaction (PCR) or the like. The site-directed mutagenesis can rapidly and efficiently improve the properties and characterization of target proteins expressed by DNA.
The term "amino acid substitution" or "amino acid substitution" in the present invention refers to the replacement of an amino acid at a particular position in a polypeptide sequence with a different amino acid, and non-naturally occurring analogs can also be used, provided that the modified sequence substantially retains the desired activity or ability.
The term "protein" or "protein" in the present invention refers to at least two covalently linked amino acids, including proteins, polypeptides, oligopeptides and peptides. The term also includes post-expression modifications of the protein, such as glycosylation, acetylation, phosphorylation, and the like. The term also includes modifications such as deletions, substitutions, and insertions of the amino acid sequence of the native protein or polypeptide.
The term "site" or "position" in the present invention refers to a position in a protein sequence. In most cases, unless otherwise indicated, the position number is relative to the first amino acid of the wild-type protein, mature protein, or enzyme (e.g., excluding signal peptide). The term "homology" or "identity" in the present invention refers to the degree of similarity in nucleotide or amino acid sequences of different polynucleotides or different proteins. Sequence homology, similarity or identity between any two given polynucleotides or polypeptides can be assessed using known computer algorithms. Homology or identity between two or more sequences may be expressed in percent (%).
The term "expression vector" in the present invention refers to a vector capable of autonomous replication in a host cell, which is preferably a multicopy vector. In addition, vectors often have markers such as antibiotic resistance genes for selection of transformants. In addition, the vector may have a promoter and/or terminator for expressing the introduced gene. The vector may be, for example, a viral vector, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a phage-derived vector, a cosmid, a phagemid, or the like. Genetically engineered vectors refer to vectors that enable expression of a gene of interest in a cell, and are typically linear or circular DNA molecules comprising a polynucleotide encoding a protein or polypeptide and operably linked to expression control sequences.
The term "polynucleotide" in the present invention generally refers to polymers of nucleotides (e.g., ribonucleotides or deoxyribonucleotides) and may include naturally occurring (adenine, guanine, cytosine, uracil, and thymine), non-naturally occurring, and modified nucleic acids.
The term "amino acid" in the present invention refers to one of 20 naturally occurring amino acids encoded by DNA and RNA, or one of artificially synthesized amino acids.
The invention adopts a rational design and directed evolution strategy, simulates the evolution track of natural protein with the help of a computer program, rapidly screens and predicts target mutants through computer virtual mutation, finally verifies the obtained mutants through a large number of experiments, and screens out the mutants with better stability and higher degradation efficiency.
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.
Test materials and reagents
1. Strains and vectors: pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitrogen.
2. Enzymes and other biochemical reagents: restriction endonucleases were purchased from TaKaRa, and ligases were purchased from Invitrogen.
3. Culture medium:
(1) Coli culture medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0).
(2) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB,0.00004% Biotin,1% glycerol (V/V).
(3) BMMY medium: except that 1% methanol was used instead of glycerin, the other components were the same as BMGY, pH 4.0.
Description: the molecular biology experimental methods not specifically described in the following examples were carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j.
EXAMPLE 1 Gene cloning of Plastic degrading enzyme mutant
The inventor predicts the three-dimensional structure of the wild type plastic degrading enzyme, and the prediction result shows that the wild type plastic degrading enzyme is formed by stacking a series of spirals, corners and random curls, and the Ser-His-Asp region of the wild type plastic degrading enzyme is closely related to the catalytic process of a substrate and participates in nucleophilic attack on a polymeric plastic ester bond. After the molecular conformation of the wild plastic degrading enzyme is evaluated by MODEER software, the subsequent energy optimization is carried out by using a model with the minimum lnPDF value, such as the work selection of adding, replacing, removing and the like of amino acids around the structural conservation region of the protein.
Molecular docking is carried out on the mutant subjected to model optimization by using Auto Dock software, a substrate is selected to be an amorphous PET film with the crystallinity of 6.7, a GaussView is adopted to draw a 3D structure of the mutant, and the mutant is subjected to dynamic optimization, so that the most stable conformation of p-nitropalmitate (pNPB) in space is finally obtained. And obtaining an optimal result after the butt joint simulation analysis. Mutants were finally obtained that included four point mutations of S121D, D, 186K, R, 280I, and S207K.
The wild type Ideonella sakaiensis-F6 is taken as a template, and four mutation sites are introduced into the wild type plastic degrading enzyme by an overlap PCR method to obtain the gene of the plastic degrading enzyme mutant. The primers used are shown in Table 1:
TABLE 1 primers for constructing Plastic degrading enzyme mutant genes
EXAMPLE 2 preparation of Plastic degrading enzyme mutant
Double digestion (EcoRI+NotI) is carried out on the expression vector pPIC9 and the gene obtained in the example 1, the gene fragment which is obtained after the digestion and codes for the mature plastic degrading enzyme mutant is connected with the expression vector pPIC9, the expression vector containing Ideonella sakaiensis-F6 plastic degrading enzyme mutant genes is obtained, and the pichia pastoris GS115 is transformed, so that the recombinant pichia pastoris strain is obtained.
The GS115 strain containing the recombinant plasmid was inoculated into 300mL of BMGY culture medium, and after 48 hours of shaking culture at 30℃and 250rpm, the cells were collected by centrifugation. Then resuspended in 150mL BMMY medium and incubated at 30℃with shaking at 250 rpm. After 72h of induction, the supernatant was collected by centrifugation.
Example 3 determination of enzyme Activity of Plastic degrading enzyme mutant
The enzyme solution obtained in example 2 was incubated with amorphous PET plastic, and the activity of the plastic degrading enzyme to degrade PET plastic was evaluated using the amount of MHET and TPA released in the high performance liquid chromatography system.
The results show that after amino acid substitutions S121D, D, 186, K, R and S207K are performed on the wild type plastic degrading enzyme, the degradation efficiency of the obtained mutant on plastic films with the crystallinity of 6.7% is improved by 3.5 times compared with the wild type.
Example 4 Plastic degrading enzyme mutant thermal stability optimization
The inventor further carries out rational design on the basis of the mutant of the embodiment 1, and unexpectedly discovers that disulfide bonds formed between the 233 st and 282 th positions after introducing cysteine respectively are analyzed by computer assistance, so that the heat stability of the enzyme is effectively improved, and the high-temperature-resistant plastic degrading enzyme mutant HotPETase is obtained.
The gene sequence of the high temperature resistant plastic degrading enzyme mutant of example 1 was obtained by overlapping PCR using the gene sequence of the plastic degrading enzyme mutant as a template, and the cleavage sites of EcoRI and NotI were introduced at both ends thereof, respectively. The primers used are shown in Table 2:
TABLE 2 primers for introducing disulfide bonds and cleavage sites in mutants
Example 5 determination of Properties of high temperature resistant Plastic degrading enzyme mutant HotPETase
High temperature resistant plastic degrading enzyme mutants were prepared according to the method of example 2.
(1) The method for determining the optimum pH of the HotPETase is as follows:
purified HotPETase was subjected to enzymatic reactions at different pH to determine its optimum pH. Semi-crystalline polyester powder with the crystallinity of 29.8% is used as a substrate, tris-HCL buffer solutions with different pH values are used for setting different pH gradients, the reaction is carried out for 24 hours at 60 ℃, and the total amount of soluble substrates (MHET and TPA) generated in a reaction system is detected by high performance liquid chromatography to carry out plastic degradation enzyme activity measurement. As shown in FIG. 1, the results show that HotPETase has higher enzyme activity in the pH range of 4-10, and the optimal pH is 9.0.
(2) The method for determining the optimum temperature of the HotPETase is as follows:
the purified HotPETase was subjected to enzymatic reactions at different temperatures to determine its optimum temperature. Semi-crystalline polyester powder with the crystallinity of 29.8% is used as a substrate, tris-HCL buffer with the pH of 9.0 is used for respectively reacting for 24 hours at 30 ℃,35 ℃,40 ℃,45 ℃,50 ℃,55 ℃,60 ℃,65 ℃,70 ℃ and 75 ℃, and the total amount of soluble substrates (MHET and TPA) generated in a reaction system is detected by high performance liquid chromatography to carry out plastic degradation enzyme activity measurement. As shown in FIG. 2, hotPETase has a comparable enzyme activity in the range of 30-75deg.C, with an optimum temperature of 65deg.C.
(3) The thermal stability of the HotPETase was determined as follows:
the purified HotPETase was incubated at different temperatures for 2h and then subjected to an enzymatic reaction to determine its thermostability. The purified enzyme solution is respectively incubated at 50 ℃,55 ℃,60 ℃,65 ℃,70 ℃,75 ℃,80 ℃ and 85 ℃ for 2 hours, semi-crystalline polyester powder with the crystallinity of 29.8% is taken as a substrate, tris-HCL buffer with the pH of 9.0 is used, incubation is carried out at 60 ℃ for 24 hours, and then the total amount of soluble substrates (MHET and TPA) generated in a reaction system is detected by high performance liquid chromatography to carry out plastic degradation enzyme activity measurement. As shown in FIG. 3, hotPETase can still retain more than 94% of enzyme activity after being treated for 2 hours at 75 ℃, and the retention rate of the enzyme activity after being treated at 85 ℃ is as high as 60%.
Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.

Claims (16)

1. A high temperature resistant plastics degrading enzyme mutant, characterized in that the mutant comprises amino acid substitutions compared to the amino acid sequence of wild type plastics degrading enzyme SEQ ID No. 1:
bit 233, 282, 122, 186, 280 and 207.
2. The mutant of claim 1, wherein the amino acid substitution sites of the mutant comprise N233C and S282C.
3. The mutant according to claim 1 or 2, wherein the amino acid substitution sites of the mutant comprise S121D, D186K, R280I, and S207K.
4. The mutant according to claim 1, wherein the amino acid sequence of the mutant is shown in SEQ ID NO. 2.
5. A mutant according to claim 1, wherein the amino acid sequence of the mutant has more than 85% homology, preferably more than 90% homology, more preferably more than 95% homology, most preferably more than 99% homology to SEQ ID No. 1.
6. A mutant according to claim 1, wherein the plastic is PET or a derivative thereof.
7. A method of degrading plastics using a mutant according to any one of claims 1 to 6, wherein the method comprises one or more of the following steps:
(1) Lysing a host cell expressing the mutant to obtain a fermentation broth comprising the mutant having enzymatic activity and/or a purified enzyme broth; wherein the host cell is a eukaryotic cell or a prokaryotic cell;
preferably, the prokaryotic cell is Escherichia coli;
preferably, the eukaryotic cell is a yeast cell, preferably, the yeast cell is selected from one or more of pichia pastoris cell, brewer's yeast cell or polymorpha cell;
more preferably, the eukaryotic cell is a pichia cell, preferably the pichia cell is selected from one or more of X33, GS115, KM71H or SMD116, more preferably the pichia cell is GS115;
(2) Incubating the fermentation broth comprising the mutants having enzymatic activity and/or the purified enzyme broth with a substrate plastic for 12-36h, preferably 24h, at 20-95 ℃, preferably 30-85 ℃, more preferably 65 ℃, pH 2.0-12.0, preferably pH 4.0-10.0, more preferably pH 9.0;
preferably, the fermentation broth is a fermentation broth supernatant; and/or
The plastic is PET and/or derivatives thereof.
8. A polynucleotide encoding the mutant according to claim 1.
9. The polynucleotide according to claim 8, wherein the sequence of the polynucleotide is shown in SEQ ID NO. 3.
10. An expression vector comprising the polynucleotide of claim 8.
11. The expression vector of claim 10, wherein the expression vector is a pichia vector selected from ppic3.5, pPIC9, ppiczα A, pPICZ αb or ppiczαc.
12. The expression vector of claim 10, wherein the expression vector is pPIC9.
13. The expression vector of claim 10, wherein the expression vector comprises a signal peptide having an amino acid sequence as set forth in SEQ ID No. 6.
14. A host cell comprising the expression vector of claim 10, obtained by introducing the expression vector into a cell.
15. The host cell of claim 14, wherein the host cell is a eukaryotic cell or a prokaryotic cell;
preferably, the prokaryotic cell is Escherichia coli,
preferably, the eukaryotic cell is a yeast cell, preferably, the yeast cell is selected from one or more of pichia pastoris cell, brewer's yeast cell or polymorpha cell;
more preferably, the eukaryotic cell is a pichia cell, preferably the pichia cell is selected from one or more of X33, GS115, KM71H or SMD116, more preferably the pichia cell is GS115.
16. Use of a mutant according to claim 1, a polynucleotide according to claim 8, an expression vector according to claim 10 and/or a host cell according to claim 14 in degrading plastics, preferably PET or a derivative thereof.
CN202310461082.9A 2023-04-26 2023-04-26 High-temperature-resistant plastic degrading enzyme mutant and encoding gene and application thereof Pending CN116478958A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117821489A (en) * 2023-12-14 2024-04-05 湖北大学 Method for degrading renewable plastic by using recombinant yeast whole cells
CN117844665A (en) * 2023-12-14 2024-04-09 湖北大学 Recombinant yeast engineering strain, preparation thereof and application thereof in degradation of PET plastic

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117821489A (en) * 2023-12-14 2024-04-05 湖北大学 Method for degrading renewable plastic by using recombinant yeast whole cells
CN117844665A (en) * 2023-12-14 2024-04-09 湖北大学 Recombinant yeast engineering strain, preparation thereof and application thereof in degradation of PET plastic
CN117844665B (en) * 2023-12-14 2024-08-30 湖北大学 Recombinant yeast engineering strain, preparation thereof and application thereof in degradation of PET plastic

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