CN115029332A - Mutant protein of thermophilic PET hydrolase and application thereof - Google Patents

Mutant protein of thermophilic PET hydrolase and application thereof Download PDF

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CN115029332A
CN115029332A CN202210771873.7A CN202210771873A CN115029332A CN 115029332 A CN115029332 A CN 115029332A CN 202210771873 A CN202210771873 A CN 202210771873A CN 115029332 A CN115029332 A CN 115029332A
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陈必强
张子璇
陈长京
邵超锋
谭天伟
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Beijing University of Chemical Technology
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Abstract

The invention belongs to the technical field of protein engineering and biocatalysis application, and relates to a mutant protein of thermophilic polyethylene terephthalate hydrolase, which is obtained by modification through rational design and site-specific mutation, is suitable for various PET hydrolases, has high enzyme activity and thermal stability at 70 ℃, and can realize high-efficiency degradation of polyester materials.

Description

Mutant protein of thermophilic PET hydrolase and application thereof
Technical Field
The invention belongs to the technical field of protein engineering and biocatalysis application, and relates to a mutant protein of thermophilic PET hydrolase and application thereof.
Background
Plastics are one of indispensable materials in our daily life due to the characteristics of stability, lightness, low price and the like, however, most of the post-consumer plastics are not effectively treated. The cumulative plastic waste amount in 2050 is reported to reach 330 hundred million tons. The common treatment method of the waste plastics mainly comprises the following steps: heat treatment, landfill, chemical treatment and mechanical recovery. However, the treatment methods have obvious limitations, such as serious environmental pollution caused by incineration and pyrolysis; the landfill treatment has great waste and serious pollution to land resources; chemical methods are inefficient and more harmful to the environment; the mechanical recovery cost is high and the output value is low. Biodegradation is an effective method for controlling plastic pollution, is green and economical in process, and can be used for treating environmental waste management problems taking plastic as a main component.
Polyester plastics are one of the most produced and widely applied plastics in the world, and the common polyester plastics mainly comprise polyethylene terephthalate (PET), polybutylene succinate (PBS), Polycaprolactone (PCL), Polyhydroxybutyrate (PHB), polylactic acid (PLA), Polyurethane (PU) and the like. Biodegradation is typically catalyzed by enzymes that catalyze the hydrolysis of the ester bonds of the polymer, degrading it into monomers that are readily processed further. In the case of PET, PET is difficult to biopolymehze due to the limited accessibility of ester linkages in PET. A variety of enzymes that catalyze hydrolysis of PET have been reported in the past few years, and these enzymes are biocatalysts that are promising replacements for traditional chemical recovery means. It has been reported that these enzymes are mostly present in saprophytic or phytopathogenic organisms (including fungi and bacteria) or in an environment rich in vegetable organic substances or plastic debris, and in extreme cases can utilize PET as the main carbon source for cell growth.
Bulk of PET at glass transition temperature (T) g ) The following are extremely difficult to degrade by enzymes. Many enzymes (e.g., lipases, cutinases, esterases, carboxylesterases, and papain) hydrolyze only the surface and do not degrade the bulk, and these enzymes can be classified as PET surface modifying enzymes. In addition, the reaction temperature (A) can be optimized<At 65-70 ℃), enzymes which significantly degrade PET main bodies are classified as PET hydrolases, and all known PET hydrolases are cutinases at present and have high sequence similarity. Cutinases (EC 3.1.1.74) belong to the serine hydrolase superfamily, with a typical catalytic triad, Ser-His-Asp, and an oxygen anion pore. Cutinases are capable of catalyzing ester hydrolysis reactions (aqueous environment) and esterification and transesterification reactions of large and small molecules (anhydrous conditions) in vitro. In addition, cutinases have an open active pocket, without a lid, which is shallow and can accommodate bulky polyester chains and make them available to active serine in the catalytic triad.
The PET hydrolases which have been widely studied at present mainly include ThcCut1 and ThcCut2 derived from Thermobifida cellulolytica, TfCut2 derived from Thermobifida fusca, and a novel cutinase LCC isolated from branched leaf compost. In recent years, enzyme optimization by biotechnology has been successful to some extent, but no enzyme has been developed so far which can completely penetrate and degrade thick-layer high crystalline PET in an economically efficient and environmentally friendly manner. However, the hydrolysis efficiency of PET is always limited by low enzyme activity and poor thermal stability, so that the development of PET hydrolase mutants with high activity under high temperature conditions is very important for solving the problem of polyester plastic pollution.
Disclosure of Invention
The invention aims to solve the technical problem that the conventional polyethylene terephthalate (PET) hydrolase has low enzyme activity under a high-temperature condition, and provides a mutant protein of a thermophilic PET hydrolase, which is obtained by modification through rational design and site-specific mutation, is suitable for various PET hydrolases, has high enzyme activity at 70 ℃ after modification, and can realize high-efficiency degradation of polyester materials.
To this end, the present invention provides in a first aspect a mutant protein of a thermophilic PET hydrolase, which is a mutant of a wild thermophilic PET hydrolase and which has hydrolytic activity towards materials containing ester and/or amide linkages, wherein the wild thermophilic PET hydrolase is any one of the following proteins:
(a1) protein with an amino acid sequence of SEQ ID NO. 1;
(a2) a protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more with the amino acid sequence defined in (a1) and having a PET hydrolyzing activity;
(a3) a protein comprising the amino acid sequence defined in (a1) or (a2) in the sequence;
(a4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (a1) to (a 3);
preferably, the amino acid sequence of the protein having more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% homology with the amino acid sequence defined in (a1) and having PET hydrolytic activity is shown as SEQ ID NO.2 or SEQ ID NO. 3.
According to the present invention, the sequence of the mutant protein comprises one or more of mutants in which Thr at the 62 th position, Gly at the 63 rd position, Gly at the 93 rd position, Asp at the 205 th position, Cys, Phe at the 210 th position, Met, Phe at the 210 th position, Val at the 210 th position, Phe at the 210 th position, Trp at the 210 th position, Asn at the 213 th position, Asp at the 213 th position, Asn at the 213 th position, and Glu at the 254 th position are mutated into Cys from the N-terminus to the C-terminus;
preferably, the amino acid sequence of said wild thermophilic PET hydrolase is SEQ ID No.1 or SEQ ID No.2 or SEQ ID No. 3.
In some specific embodiments of the invention: the mutant protein is a No.1 thermophilic PET hydrolase mutant protein, and the sequence of the mutant protein comprises a mutation of Gly to Ala from N end to 63 th position of C end of an amino acid sequence shown in SEQ ID NO.1 of the wild thermophilic PET hydrolase; preferably, the sequence of the No.1 thermophilic PET hydrolase mutant protein is shown in SEQ ID NO. 4;
and/or the mutant protein is a No.2 thermophilic PET hydrolase mutant protein, the amino acid sequence of the wild thermophilic PET hydrolase shown as SEQ ID NO.1 is changed from 63 Gly to Ala from N end to C end, and 210 Phe is changed into Ile; preferably, the sequence of the No.2 thermophilic PET hydrolase mutant protein is shown in SEQ ID NO. 5;
and/or the mutant protein is a No.3 thermophilic PET hydrolase mutant protein, the sequence of the mutant protein comprises a mutation from the amino acid sequence shown as SEQ ID NO.1 of the wild thermophilic PET hydrolase from the N end to the C end, wherein the 63 rd Gly is changed into Ala, the 210 th Phe is changed into Ile, the 205 th Asp is changed into Cys, and the 254 th Glu is changed into Cys; preferably, the sequence of the No.3 thermophilic PET hydrolase mutant protein is shown in SEQ ID NO. 6;
and/or the mutant protein is a No.4 thermophilic PET hydrolase mutant protein, the sequence of the mutant protein comprises a mutation from the 63 rd Gly to the Ala, the 210 th Phe to the Ile, the 205 th Asp to the Cys, the 254 th Glu to the Cys and the 93 rd Gln to the Gly of the amino acid sequence shown in SEQ ID NO.1 of the wild thermophilic PET hydrolase; preferably, the sequence of the No.4 thermophilic PET hydrolase mutant protein is shown in SEQ ID NO. 7;
and/or, the mutant protein is a No.5 thermophilic PET hydrolase mutant protein, the sequence of the mutant protein comprises a mutation from the amino acid sequence shown as SEQ ID NO.2 of the wild thermophilic PET hydrolase from the N end to the C end, wherein the 63 rd Gly is changed into Ala, the 210 th Phe is changed into Ile, the 205 th Asp is changed into Cys, the 254 th Glu is changed into Cys, and the 93 th Gln is changed into Gly; preferably, the sequence of the No.5 thermophilic PET hydrolase mutant protein is shown in SEQ ID NO. 8;
and/or the mutant protein is a No.6 thermophilic PET hydrolase mutant protein, the sequence of the mutant protein comprises a mutation from the 63 rd Gly to the Ala, the 210 th Phe to the Ile, the 205 th Asp to the Cys, the 254 th Glu to the Cys and the 93 rd Gln to the Gly of the amino acid sequence shown in SEQ ID NO.3 of the wild thermophilic PET hydrolase; preferably, the sequence of the No.6 thermophilic PET hydrolase mutant protein is shown in SEQ ID NO. 9.
In a second aspect, the present invention provides a nucleotide molecule encoding a mutant protein according to the first aspect of the present invention.
According to the invention, the nucleotide sequence of the nucleotide molecule comprises a nucleotide sequence which is positioned in the direction from 5 'end to 3' end and contains a mutation from 185 th position C to T, a mutation from 186 th position C to G, a mutation from 188 th position G to C, a mutation from 189 th position T to G, a mutation from 277 th position C to G, a mutation from 278 th position A to G, a mutation from 279 th position G to A, a mutation from 279 th position G to T, a mutation from 613 th position G to T, a mutation from 614 th position A to G, a mutation from 615 th position T to C, a mutation from 628 th position T to A, a mutation from 628 th position T to G, a mutation from 629 position T to G, a mutation from 630 th position T to G, a mutation from 637 position A to G, a mutation from 638 th position A to T, a mutation from 639 th position T to C, a mutation from 639 th position T to G, a mutation from 761 th position G to T, a mutation from 761 position G to T to G, a mutation from 761 position to G, The 762 th A mutation is C, and the 762 th A mutation is one or more of nucleotide mutants of T;
preferably, the nucleotide sequence encoding the amino acid sequence of the wild thermophilic PET hydrolase is the nucleotide sequence shown as SEQ ID No.10 encoding the amino acid sequence shown as SEQ ID No.1 of the wild thermophilic PET hydrolase, or the nucleotide sequence shown as SEQ ID No.11 encoding the amino acid sequence shown as SEQ ID No.2 of the wild thermophilic PET hydrolase, or the nucleotide sequence shown as SEQ ID No.12 encoding the amino acid sequence shown as SEQ ID No.3 of the wild thermophilic PET hydrolase.
In some specific embodiments of the invention, the nucleotide molecule is a nucleotide molecule encoding a mutant protein of thermophilic PET hydrolase No.1, and the nucleotide molecule comprises a nucleotide sequence which is located in the direction from 5 'end to 3' end of the nucleotide sequence shown in SEQ ID No.10 of the amino acid sequence shown in SEQ ID No.1 encoding wild thermophilic PET hydrolase, wherein G at position 188 is mutated into C, and T at position 189 is mutated into C; preferably, the nucleotide sequence of the nucleotide molecule for coding the No.1 thermophilic PET hydrolase mutant protein is shown as SEQ ID NO. 13;
and/or the nucleotide molecule is a nucleotide molecule for coding No.2 thermophilic PET hydrolase mutant protein, the sequence of the nucleotide molecule comprises a 188 th G mutation which is positioned in the direction from the 5 'end to the 3' end of the nucleotide sequence shown in SEQ ID NO.10 of the amino acid sequence shown in SEQ ID NO.1 for coding wild thermophilic PET hydrolase, the 189 th T mutation is C, 628 th T mutation is A, and 630 th T mutation is A; preferably, the nucleotide sequence of the nucleotide molecule for coding the No.2 thermophilic PET hydrolase mutant protein is shown as SEQ ID NO. 14;
and/or the nucleotide molecule is a nucleotide molecule for coding No.3 thermophilic PET hydrolase mutant protein, the sequence of the nucleotide molecule comprises a nucleotide sequence which is positioned in an amino acid sequence shown as SEQ ID NO.1 and used for coding wild thermophilic PET hydrolase and is provided with a mutation from the 5 'end to the 3' end, wherein the 188 th G position is mutated into C, the 189 th T position is mutated into C, the 613 th G position is mutated into T, the 614 th A position is mutated into G, the 615 th T position is mutated into C, the 628 th position is mutated into A, the 630 th T position is mutated into A, the 760 th G position is mutated into T, the 761A position is mutated into G, and the 762 th A position is mutated into C; preferably, the nucleotide sequence of the nucleotide molecule for coding the No.3 thermophilic PET hydrolase mutant protein is shown as SEQ ID NO. 15;
and/or the nucleotide molecule is a nucleotide molecule for coding a No.4 thermophilic PET hydrolase mutant protein, the sequence of the nucleotide molecule comprises a nucleotide sequence which is positioned in an amino acid sequence shown as SEQ ID NO.1 and used for coding a wild thermophilic PET hydrolase and is characterized in that the 188 th G position in the direction from the 5 'end to the 3' end is mutated into C, the 189 th T position is mutated into C, the 277 th C position is mutated into G, the 278 th A position is mutated into G, the 279 th G position is mutated into A, the 613 th G position is mutated into T, the 614 th A position is mutated into G, the 615 th T position is mutated into C, the 628 th T position is mutated into A, the 630 th T position is mutated into A, the 760 th G position is mutated into T, the 761A position is mutated into G, and the 762 th A position is mutated into C; preferably, the nucleotide sequence of the nucleotide molecule for coding the No.4 thermophilic PET hydrolase mutant protein is shown as SEQ ID NO. 16;
and/or the nucleotide molecule is a nucleotide molecule for coding a No.5 thermophilic PET hydrolase mutant protein, wherein the nucleotide molecule comprises a nucleotide sequence which is positioned in an amino acid sequence shown as SEQ ID NO.2 for coding a wild thermophilic PET hydrolase and is provided with a mutation from a 5 'end to a 3' end, wherein a 188 th G position is mutated into C, a 189 th T position is mutated into C, a 277 th C position is mutated into G, a 278 th A position is mutated into G, a 279 th G position is mutated into T, a 613 th G position is mutated into T, a 614 th A position is mutated into G, a 628 th T position is mutated into A, a 761 th A position is mutated into G, and a 762 th A position is mutated into T; preferably, the nucleotide sequence of the nucleotide molecule for coding the No.5 thermophilic PET hydrolase mutant protein is shown in SEQ ID NO. 17;
and/or the nucleotide molecule is a nucleotide molecule for coding a No.6 thermophilic PET hydrolase mutant protein, the nucleotide sequence comprises a nucleotide sequence which is positioned in an amino acid sequence shown as SEQ ID NO.3 for coding a wild thermophilic PET hydrolase and is provided with a mutation from 5 'end to 3' end of the nucleotide sequence shown as SEQ ID NO.12, wherein the 188 th G position is mutated into C, the 189 th T position is mutated into G, the 277 th C position is mutated into G, the 278 th A position is mutated into G, the 279 th G position is mutated into T, the 613 th G position is mutated into T, the 614 th A position is mutated into G, the 628 th T position is mutated into A, the 761 th A position is mutated into T, and the 762 th A position is mutated into T; preferably, the nucleotide sequence of the nucleotide molecule for coding the No.6 thermophilic PET hydrolase mutant protein is shown as SEQ ID NO. 18.
In some further specific embodiments of the invention, the nucleotide molecule is a DNA molecule as follows:
(b1) the coding region comprises DNA molecules of nucleotide sequences shown as SEQ ID NO.13, SEQ ID NO14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO. 18;
(b2) DNA molecule with nucleotide sequence shown in SEQ ID NO.13, SEQ ID NO14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO. 18;
(b3) a DNA molecule which has 75% or more identity to the nucleotide sequence of (b1) or (b2) and which encodes a protein of the first aspect of the invention;
(b4) a DNA molecule which hybridizes under stringent conditions to the nucleotide sequence of (b1) or (b2) and which encodes a protein according to the first aspect of the present invention.
In a third aspect, the invention provides an expression cassette, recombinant vector or recombinant microorganism comprising a nucleotide molecule according to the second aspect of the invention.
In a fourth aspect, the present invention provides the use of a mutant protein according to the first aspect of the invention or a mutant protein encoded by a nucleotide molecule according to the second aspect of the invention or a mutant protein produced from an expression cassette, a recombinant vector or a recombinant microorganism according to the third aspect of the invention for the hydrolysis of a material comprising an ester linkage and an amide linkage.
In some embodiments of the present invention, the material containing ester and amide linkages comprises one or more of a polyethylene terephthalate (PET) material, a polylactic acid (PLA) material, and a Polyurethane (PU) material.
The thermophilic polyethylene terephthalate hydrolase mutant protein provided by the invention comprises at least three PET hydrolases, and is modified through rational design and site-directed mutagenesis to obtain the corresponding mutant protein, so that the thermal stability and PET hydrolytic activity of the PET hydrolases can be greatly improved, the modified mutant has higher enzyme activity at 70 ℃, and the high-efficiency degradation of PET bottles after consumption can be realized.
Drawings
The invention is described in further detail below with reference to the attached drawing figures:
FIG. 1 is a ThcCut1-pET-22b plasmid map.
FIG. 2 shows the HPLC detection results of the wild type and the mutant of ThcCut 1; wherein, (A) is a wild-type liquid phase detection map of ThcCut1, (B) is a liquid phase detection map of a ThcCut1-G63A mutant, (C) is a liquid phase detection map of a ThcCut1-G63A/F210I mutant, (D) is a liquid phase detection map of a ThcCut1-G63A/F210I/D205C/E254C mutant, and (E) is a liquid phase detection map of a ThcCut1-G63A/F210I/D205C/E254C/Q93G mutant.
FIG. 3 is a liquid phase detection chart of TPA standard.
FIG. 4 is a liquid phase detection image of MHET standard.
FIG. 5 is a schematic diagram of the hydrolysis reaction process of PET.
FIG. 6 is a time course chart of PET bottle granules after hydrolysis consumption by ThcCut1 wild type and G63A/F210I/D205C/E254C/Q93G mutant.
FIG. 7 shows the electron microscopy results after degradation of PLA material by the G63A/F210I/D205C/E254C/Q93G variant; wherein, (A) is PLA straw, (B) is PLA film.
FIG. 8 shows the HPLC assay of the wild type and mutant of ThcCut 2; wherein, (A) is a wild type liquid phase detection map of ThcCut2, and (B) is a liquid phase detection map of ThcCut2-G63A/F210I/D205C/E254C/Q93G mutant.
Fig. 9 is a liquid phase detection chart of the BHET standard.
FIG. 10 shows the HPLC detection results of Tfcut2 wild type and mutant; wherein, (A) is a TfCut2 wild type liquid phase detection map, and (B) is a TfCut2-G63A/F210I/D205C/E254C/Q93G mutant liquid phase detection map.
Detailed Description
In order that the invention may be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. However, before the present invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
Term (I)
As used herein, the term "PET" refers to polyethylene terephthalate, "PLA" refers to polylactic acid, "TPA" refers to terephthalic acid, "MHET" refers to mono (hydroxyethyl) terephthalate, "BHET" refers to bis (hydroxyethyl) terephthalate, "HPLC" refers to high performance liquid chromatography, "T m "means the melting temperature.
The term "nucleotide mutant" as used herein refers to the smallest unit in the nucleotide series of a gene in which a mutation can occur.
Similarly, the term "amino acid mutant" as used herein refers to the smallest unit in the amino acid sequence of a protein in which a mutation can occur.
The terms "wild type" and "wild type" as used herein may be used interchangeably.
The terms "specific enzyme activity" and "specific activity" as used herein refer to the enzyme activity per unit mass of enzyme protein.
The terms "hydrolysis" and "degradation" as used herein may be used interchangeably.
The terms "thermophilic" and "high temperature resistant", "high temperature" and "thermophilic" as used herein are used interchangeably.
The terms "protein" and "protein" as used herein may be used interchangeably.
The terms "variant" and "mutant" as used herein may be used interchangeably.
Embodiments II
As described above, currently, the PET hydrolysis efficiency of PET hydrolase is always limited by low enzymatic activity and poor thermal stability, and the enzymatic activity of the existing polyethylene terephthalate hydrolase is low under high temperature conditions.
The present inventors have studied and found that thermal stability and PET hydrolytic activity of PET hydrolase can be greatly improved by rational design and site-directed mutagenesis, and a mutant protein of high-activity thermophilic PET hydrolase, which comprises one or more of mutants in which the amino acid sequence of wild thermophilic PET hydrolase is mutated from N-terminus to C-terminus from Thr at position 62 to Met, Gly at position 63 to Ala, Gln at position 93 to Gly, Asp at position 205 to Cys, Phe at position 210 to Ile, Phe at position 210 to Met, Phe at position 210 to Val, Phe at position 210 to Trp, Asn at position 213 to Asp, Asn at position 213 to Met, and Glu at position 254 to Cys, is obtained.
It can be understood that the invention provides a modification method of thermophilic polyethylene terephthalate hydrolase, and the modified mutant protein of thermophilic PET hydrolase is applied to hydrolysis of polyester materials.
The wild thermophilic PET enzyme modified by the invention is any one of the following (a1) - (a 4):
(a1) a protein (marked as thermophilic PET hydrolase ThcCut1) with an amino acid sequence of SEQ ID NO. 1;
(a2) a protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more with the amino acid sequence defined in (a1) and having a PET hydrolyzing activity;
(a3) a protein comprising the amino acid sequence defined in (a1) or (a2) in the sequence;
(a4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (a1) to (a 3);
preferably, the amino acid sequence of the protein having more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% homology with the amino acid sequence defined in (a1) and having PET hydrolytic activity is shown as SEQ ID NO.2 or SEQ ID NO. 3.
The protein with the PET hydrolytic activity and the amino acid sequence shown as SEQ ID NO.2 is marked as thermophilic PET hydrolytic enzyme ThcCut2 in the invention.
The protein with the PET hydrolytic activity and the amino acid sequence shown as SEQ ID NO.3 is marked as thermophilic PET hydrolytic enzyme TfCut2 in the invention.
The mutant protein is obtained by site mutation on the basis of wild thermophilic PET hydrolase, and the sequence of the mutant protein of the thermophilic PET hydrolase contains one or more of mutants of mutation from the amino acid sequence of the wild thermophilic PET hydrolase from N end to C end, wherein the mutation from the 62 th position Thr to the Met, the 63 th position Gly to the Ala, the 93 rd position Gln to the Gly, the 205 th position Asp to the Cys, the 210 th position Phe to the Ile, the 210 th position Phe to the Met, the 210 th position Phe to the Val, the 210 th position Phe to the Trp, the 213 th position Asn to the Asp, the 213 th position Asn to the Met and the 254 th position Glu to the Cys.
Preferably, the wild thermophilic PET hydrolase has the amino acid sequence of SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO. 3.
In some embodiments of the invention, the mutant protein is a number 1 thermophilic PET hydrolase mutant protein (designated as thcut 1 mutant G63A) comprising a Gly to Ala mutation from N-terminus to C-terminus of the amino acid sequence shown in SEQ ID No.1 of the wild thermophilic PET hydrolase.
In some particularly preferred embodiments of the invention, the sequence of the No.1 thermophilic PET hydrolase mutant protein is shown in SEQ ID NO. 4.
In some embodiments of the invention, the mutant protein is a number 2 thermophilic PET hydrolase mutant protein (designated as thcut 1 mutant G63A/F210I) comprising a mutation from Gly to Ala at position 63 and a mutation from Phe to Ile at position 210 from N-terminus to C-terminus of the amino acid sequence shown in SEQ ID No.1 of the wild thermophilic PET hydrolase.
In some particularly preferred embodiments of the invention, the sequence of the mutant protein No.2 thermophilic PET hydrolase is shown in SEQ ID No. 5.
In some embodiments of the invention, the mutant protein is a mutant protein of thermophilic PET hydrolase No.3 (designated as the thcut 1 mutant G63A/F210I/D205C/E254C), which comprises a mutation from Gly to Ala at position 63, a mutation from Phe to Ile at position 210, a mutation from Asp to Cys at position 205, and a mutation from Glu to Cys at position 254, from the N-terminus to C-terminus of the amino acid sequence shown in SEQ ID No.1 of the wild thermophilic PET hydrolase.
In some particularly preferred embodiments of the invention, the sequence of the No.3 thermophilic PET hydrolase mutant protein is shown in SEQ ID NO. 6.
In some embodiments of the invention, the mutant protein is a number 4 thermophilic PET hydrolase mutant protein (designated as thcut 1 mutant G63A/F210I/D205C/E254C/Q93G) comprising a mutation from Gly to Ala at position 63, a mutation from Phe to Ile at position 210, a mutation from Asp to Cys at position 205, a mutation from Glu to Cys at position 254, and a mutation from gin to Gly at position 93 in the amino acid sequence shown in SEQ ID No.1 of the wild thermophilic PET hydrolase.
In some particularly preferred embodiments of the invention, the sequence of the No.4 thermophilic PET hydrolase mutant protein is shown in SEQ ID No. 7.
In some embodiments of the invention, the mutant protein is a mutant protein of thermophilic PET hydrolase No.5 (designated as the thcut 2 mutant G63A/F210I/D205C/E254C/Q93G), which comprises a mutation from Gly to Ala at position 63, a mutation from Phe to Ile at position 210, a mutation from Asp to Cys at position 205, a mutation from Glu to Cys at position 254, and a mutation from gin to Gly in the amino acid sequence shown in SEQ ID No.2 of the wild thermophilic PET hydrolase.
In some particularly preferred embodiments of the invention, the sequence of the mutant protein No.5 thermophilic PET hydrolase is shown in SEQ ID No. 8.
In some embodiments of the invention, the mutant protein is a No.6 thermophilic PET hydrolase mutant protein (denoted as Tfcut2 mutant G63A/F210I/D205C/E254C/Q93G) comprising a mutation from Gly to Ala at position 63, a mutation from Phe to Ile at position 210, a mutation from Asp to Cys at position 205, a mutation from Glu to Cys at position 254, and a mutation from Gln to Gly at position 93 of the amino acid sequence shown in SEQ ID No.3 of the wild thermophilic PET hydrolase.
In some particularly preferred embodiments of the invention, the sequence of the No.6 thermophilic PET hydrolase mutant protein is shown as SEQ ID NO. 9.
All nucleotide molecules encoding the above proteins are also within the scope of the present invention. Accordingly, in a second aspect, the present invention provides a nucleotide molecule encoding a mutant thermophilic PET hydrolase protein according to the first aspect of the invention.
According to the invention, the nucleotide sequence of the nucleotide molecule comprises a nucleotide sequence which is positioned in the direction from 5 'end to 3' end and contains a mutation from 185 th position C to T, a mutation from 186 th position C to G, a mutation from 188 th position G to C, a mutation from 189 th position T to G, a mutation from 277 th position C to G, a mutation from 278 th position A to G, a mutation from 279 th position G to A, a mutation from 279 th position G to T, a mutation from 613 th position G to T, a mutation from 614 th position A to G, a mutation from 615 th position T to C, a mutation from 628 th position T to A, a mutation from 628 th position T to G, a mutation from 629 position T to G, a mutation from 630 th position T to G, a mutation from 637 position A to G, a mutation from 638 th position A to T, a mutation from 639 th position T to C, a mutation from 639 th position T to G, a mutation from 761 th position G to T, a mutation from 761 position G to T to G, a mutation from 761 position to G, The 762 th A mutation is C, and the 762 th A mutation is one or more of nucleotide mutants of T;
preferably, the nucleotide sequence encoding the amino acid sequence of the wild thermophilic PET hydrolase is the nucleotide sequence shown as SEQ ID No.10 encoding the amino acid sequence shown as SEQ ID No.1 of the wild thermophilic PET hydrolase, or the nucleotide sequence shown as SEQ ID No.11 encoding the amino acid sequence shown as SEQ ID No.2 of the wild thermophilic PET hydrolase, or the nucleotide sequence shown as SEQ ID No.12 encoding the amino acid sequence shown as SEQ ID No.3 of the wild thermophilic PET hydrolase.
The nucleotide molecule with the nucleotide sequence shown in SEQ ID NO.10, which encodes the wild thermophilic PET hydrolase with the amino acid sequence shown in SEQ ID NO.1, is marked as the nucleotide molecule encoding the thermophilic PET hydrolase ThcCut1 in the invention.
The nucleotide molecule with the nucleotide sequence shown in SEQ ID NO.11, which encodes the wild thermophilic PET hydrolase with the amino acid sequence shown in SEQ ID NO.2, is marked as the nucleotide molecule encoding the thermophilic PET hydrolase ThcCut2 in the invention.
The nucleotide molecule with the nucleotide sequence shown in SEQ ID NO.12 of the wild thermophilic PET hydrolase with the amino acid sequence shown in SEQ ID NO.3 is recorded as the nucleotide molecule coding the thermophilic PET hydrolase TfCut2 in the invention.
In some embodiments of the invention, the nucleotide molecule is a nucleotide molecule encoding a mutant protein of thermophilic PET hydrolase No.1 (denoted as a nucleotide molecule encoding a thcut 1 mutant G63A), and the sequence of the nucleotide molecule comprises a mutation from the 188 th position G to the 3 th position G of the nucleotide sequence shown in SEQ ID No.10 located in the direction from the 5 'end to the 3' end of the amino acid sequence shown in SEQ ID No.1 encoding wild thermophilic PET hydrolase to C, and a mutation from the 189 th position T to C.
In some particularly preferred embodiments of the invention, the nucleotide sequence of the nucleotide molecule encoding the mutant protein No.1 thermophilic PET hydrolase is shown in SEQ ID NO. 13;
in some embodiments of the invention, the nucleotide molecule is a nucleotide molecule encoding a mutant protein of thermophilic PET hydrolase No.2 (denoted as a nucleotide molecule encoding a thcut 1 mutant G63A/F210I), and the sequence of the nucleotide molecule comprises a mutation from the 188 th position G to the C, the 189 th position T to the C, the 628 th position T to the a and the 630 th position T to the a in the direction from the 5 'end to the 3' end of the nucleotide sequence of SEQ ID No.10 located in the amino acid sequence shown in SEQ ID No.1 encoding wild thermophilic PET hydrolase.
In some particularly preferred embodiments of the invention, the nucleotide sequence of the nucleotide molecule encoding the mutant protein No.2 thermophilic PET hydrolase is shown in SEQ ID NO. 14;
in some embodiments of the invention, the nucleotide molecule is a nucleotide molecule encoding a mutant protein of thermophilic PET hydrolase No.3 (denoted as a nucleotide molecule encoding a thcut 1 mutant G63A/F210I/D205C/E254C), and the sequence of the nucleotide molecule comprises a mutation from the 188 th position G to the 189 th position C, a mutation from the 613 th position G to the T, a mutation from the 614 th position a to the G, a mutation from the 615 th position T to the C, a mutation from the 628 th position T to the a, a mutation from the 630 th position T to the a, a mutation from the 760 th position G to the T, an mutation from the 761th position a to the G, and a mutation from the 762 th position a to the C, which are located in the direction from the 5 'end to the 3' end of the nucleotide sequence of SEQ ID No.10 encoding the amino acid sequence of the wild thermophilic PET hydrolase, as shown in SEQ ID No. 1.
In some particularly preferred embodiments of the present invention, the nucleotide sequence of the nucleotide molecule encoding the mutant protein No.3 thermophilic PET hydrolase is shown as SEQ ID NO. 15;
in some embodiments of the invention, the nucleotide molecule is a nucleotide molecule encoding a mutant protein of thermophilic PET hydrolase No.4 (denoted as a nucleotide molecule encoding a thcut 1 mutant G63A/F210I/D205C/E254C/Q93G), and the sequence of the nucleotide molecule comprises a nucleotide sequence of SEQ ID No.10 which is located in the amino acid sequence shown in SEQ ID No.1 encoding the wild thermophilic PET hydrolase, wherein the mutation from the 188 th position G to the 3' end of the nucleotide sequence is C, the mutation from the 189 th position T to C, the mutation from the 277 th position C to G, the mutation from the 278 th position a to G, the mutation from the 279 th position G to a, the 613 th position G to T, the 614 th position a to G, the 615 th position T to C, the 628 th position T to a, the mutation from the 630 th position T to a, the 760 th position G to T, the 761 to G, and the 762 th position a to C.
In some particularly preferred embodiments of the present invention, the nucleotide sequence of the nucleotide molecule encoding the mutant protein No.4 thermophilic PET hydrolase is shown as SEQ ID NO. 16;
in some embodiments of the invention, the nucleotide molecule is a nucleotide molecule encoding a mutant protein of thermophilic PET hydrolase No.5 (denoted as a nucleotide molecule encoding a thcut 2 mutant G63A/F210I/D205C/E254C/Q93G), and the nucleotide sequence includes a nucleotide molecule encoding a wild thermophilic PET hydrolase such as SEQ ID No.2, in which the 188 th position G from the 5 'end to the 3' end of the nucleotide sequence described in SEQ ID No.11 is mutated into C, the 189 th position T into C, the 277 th position C into G, the 278 th position a into G, the 279 th position G into T, the 613 th position G into T, the 614 th position a into G, the 628 th position T into a, the 760 th position G into T, the 761 th position a into G and the 762 th position a into T.
In some particularly preferred embodiments of the present invention, the nucleotide sequence of the nucleotide molecule encoding the mutant protein No.5 thermophilic PET hydrolase is shown as SEQ ID NO. 17;
in some embodiments of the invention, the nucleotide molecule is a nucleotide molecule encoding a mutant protein of thermophilic PET hydrolase No.6 (denoted as a nucleotide molecule encoding a TfCut2 mutant G63A/F210I/D205C/E254C/Q93G), and the sequence of the nucleotide molecule comprises a nucleotide molecule which is located in the direction from 5 'end to 3' end of the nucleotide sequence shown in SEQ ID No.12 of the amino acid sequence shown in SEQ ID No.3 encoding wild thermophilic PET hydrolase, wherein the mutation at position 188G is C, the mutation at position 189 is G, the mutation at position 277C is G, the mutation at position 278 a is G, the mutation at position 279 is T, the mutation at position 613 is G, the mutation at position 614 is G, the mutation at position 628 is a, the mutation at position 760 is G, the mutation at position 761 is G, and the mutation at position 762 is T.
In some particularly preferred embodiments of the present invention, the nucleotide sequence of the nucleotide molecule encoding the mutant protein No.6 thermophilic PET hydrolase is shown as SEQ ID NO. 18.
In some further specific embodiments of the invention, the nucleotide molecule is a DNA molecule as follows:
(b1) the coding region comprises DNA molecules of nucleotide sequences shown as SEQ ID NO.13, SEQ ID NO14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO. 18;
(b2) DNA molecule with nucleotide sequence shown in SEQ ID NO.13, SEQ ID NO14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO. 18;
(b3) a DNA molecule which has 75% or more identity to the nucleotide sequence of (b1) or (b2) and which encodes a protein of the first aspect of the invention;
(b4) a DNA molecule which hybridizes under stringent conditions to the nucleotide sequence of (b1) or (b2) and which encodes a protein according to the first aspect of the present invention.
In a third aspect, the invention provides an expression cassette, recombinant vector or recombinant microorganism comprising a nucleotide molecule according to the second aspect of the invention, it being understood that in the third aspect of the invention there is provided an expression cassette, recombinant vector or recombinant microorganism comprising a nucleotide molecule corresponding to a protein according to the first aspect of the invention.
In a fourth aspect, the present invention provides the use of a mutant protein according to the first aspect of the invention or a mutant protein encoded by a nucleotide molecule according to the second aspect of the invention or a mutant protein produced from an expression cassette, a recombinant vector or a recombinant microorganism according to the third aspect of the invention for the hydrolysis of a material comprising an ester linkage and an amide linkage.
In some embodiments of the present invention, the material containing ester and amide linkages comprises one or more of a polyethylene terephthalate (PET) material, a polylactic acid (PLA) material, and a Polyurethane (PU) material.
In some particularly preferred embodiments of the present invention, the present invention provides the use of a mutant protein according to the present invention or a mutant protein produced from a nucleotide molecule according to the present invention or an expression cassette, a recombinant vector or a recombinant microorganism according to the present invention for catalyzing the hydrolysis of polyethylene terephthalate and other esters.
In some further particularly preferred embodiments of the invention, the application comprises catalyzing hydrolysis of polyethylene terephthalate, increasing terephthalic acid production, and increasing efficiency of thermophilic PET hydrolase catalysis.
Example III
The experimental procedures described below are, unless otherwise specified, conventional laboratory procedures. The experimental materials described below are commercially available without specific reference. The invention will be further described with reference to specific examples in order to better understand the invention, but the scope of the invention is not limited to the following description.
In some embodiments of the invention, the expression of the thermophilic PET hydrolase mutant protein is carried out using E.coli BL21(DE3) (available from Beijing Quanyu Biotech Co., Ltd.) as host cell, and the following media formulations are involved in the following examples:
LB liquid medium: peptone 1%, yeast extract 0.5%, NaCl 1%;
LB solid medium: 1.5% of agar, 1% of peptone, 0.5% of yeast extract and 1% of NaCl;
the unit in the medium is% (W/V).
Example 1: construction of thermophilic PET hydrolase expression vector
(1) Acquisition of thermophilic PET hydrolase mutant Gene
The thermophilic PET hydrolase used in the embodiment is ThcCut1 (GenBank: ADV92526.1), the source of the coding gene is Thermobifida cellulolysitica, the amino acid sequence of ThcCut1 is shown as SEQ ID NO.1, and one of the nucleotide sequences of the coding gene of ThcCut1 is shown as SEQ ID NO. 10.
Single-point mutation or multi-point mutation was performed on thcut 1, respectively, to give thcut 1 mutants shown in table 1.
The mutant ThcCut1-G63A is an amino acid sequence (SEQ ID NO.4) obtained by mutating Gly at the 63 th site of the ThcCut1 amino acid sequence (SEQ ID NO.1) into Ala and keeping other amino acid residues unchanged, wherein one of the corresponding nucleotide sequences is shown as SEQ ID NO. 13.
The ThcCut1-G63A/F210I mutant is a sequence (SEQ ID NO.5) obtained by mutating Gly at position 63 of an amino acid sequence (SEQ ID NO.1) of ThcCut1 to Ala, mutating Phe at position 210 to Ile and keeping other amino acid residues unchanged, wherein one of corresponding nucleotide sequences is shown as SEQ ID NO. 14.
The mutant ThcCut1-G63A/F210I/D205C/E254C is a sequence (SEQ ID NO.6) obtained by mutating Gly at position 63 of an amino acid sequence (SEQ ID NO.1) of ThcCut1 into Ala, mutating Phe at position 210 into Ile, mutating Asp at position 205 into Cys, mutating Glu at position 254 into Cys and keeping other amino acid residues unchanged, wherein one of corresponding nucleotide sequences is shown as SEQ ID NO. 15.
The ThcCut1-G63A/F210I/D205C/E254C/Q93G mutant is a sequence (SEQ ID NO.7) obtained by mutating Gly at the 63 th position of a ThcCut1 amino acid sequence (SEQ ID NO.1) into Ala, Phe at the 210 th position into Ile, Asp at the 205 th position into Cys, Glu at the 254 th position into Cys, Gln at the 93 th position into Gly and keeping other amino acid residues unchanged, wherein one of corresponding nucleotide sequences is shown as SEQ ID NO. 16.
(2) Construction of expression vectors
The vector for expressing the wild type ThcCut1 is a vector (shown in figure 1) obtained by inserting a nucleotide sequence (SEQ ID NO.10) of a ThcCut1 coding gene between NdeI and XhoI enzyme cutting sites of a pET-22b vector, and is named as ThcCut1-pET-22 b.
Each vector for expressing the ThcCut1 mutant was obtained by inserting the gene encoding each ThcCut1 mutant into pET-22b vector between the NdeI and XhoI cleavage sites.
Example 2: preparation of thermophilic PET hydrolase mutant protein
(1) Expression of wild-type and mutant ThcCut1
In order to test the enzyme activity and thermostability of the wild type and mutant of ThcCut1, it was expressed and purified in E.coli.
The vector plasmids of ThcCut1 and the mutant thereof constructed in example 1 are respectively transferred into competent cells of escherichia coli BL21(DE3) to obtain recombinant bacteria. Ampicillin-resistant plates (containing 100. mu.g/mL Ampicillin) were used for screening of positive clones and cultured overnight at 37 ℃.
Selecting a single clone to be cultured in 4mL of sterile LB liquid culture medium (containing 100 mu g/mL Ampicill) at 37 ℃ (180rpm) for 12-14 h; inoculating 500 μ L of the bacterial liquid into 30mL of sterile LB liquid medium (containing 100 μ g/mL Ampicill), and culturing at 37 deg.C (180rpm) to OD 600 0.8 to 1.0; adding IPTG (isopropyl-beta-D-thiogalactoside) until the final concentration is 0.2mM, inducing for 16-22 h at 16 ℃ (180rpm), and centrifuging to collect thalli; 6mL of lysis buffer (100mM pH 8.0K) was added 2 HPO 4 -KH 2 PO 4 300mM NaCl), resuspending the cells; carrying out ultrasonic crushing for 15min in ice bath; and centrifuging and collecting the supernatant to obtain a corresponding crude enzyme solution.
(2) Purification of proteins
The wild-type and mutant of thcut 1 were purified using a nickel column:
(1) column assembling: adding 2mL of nickel column filler into each 12cm gravity column;
(2) 5mL of phosphate buffer (100mM pH 8.0K) was added 2 HPO 4 -KH 2 PO 4 )；
(3) 5mL of eluent (100mM pH 8.0K) was added 2 HPO 4 -KH 2 PO 4 300mM NaCl, 250mM imidazole);
(4) 5mL of a washing solution (100mM pH 8.0K) was added 2 HPO 4 -KH 2 PO 4 300mM NaCl, 20mM imidazole);
(5) adding a thermophilic PET hydrolase crude enzyme solution, and repeatedly penetrating for 3 times;
(6) adding 5mL of cleaning solution, and repeating for 5 times;
(7) adding 5mL of eluent, repeating for 4 times, and collecting effluent after 3 times;
(8) 5mL of eluent and cleaning solution are used for washing for 5 times respectively;
(9) imidazole was washed off with 5mL of phosphate buffer;
(10) pre-sealing the column with 5mL of 20% ethanol;
(11) blocking the lower plug, adding 5mL of 20% ethanol, sealing the column, and storing at 4 ℃.
(3) Concentration and concentration determination of proteins
Adding the effluent collected in the nickel column purification process into a 15mL ultrafiltration tube (10kDa), centrifuging at 4 ℃ and 4500rpm for 15min, and removing the lower solution; add 10mL lysis buffer to the ultrafiltration tube to further remove imidazole; centrifuging at 4 deg.C and 4500rpm for 15min, and collecting upper layer enzyme solution.
Protein concentration was measured at 280nm using a NanoPhotometer-N50 ultramicro spectrophotometer.
Example 3: evaluation of Properties of thermophilic PET hydrolase mutant protein
(1) Enzyme activity assay of ThcCut1 wild type and mutant
The enzyme activity of the wild type and the mutant of ThcCut1 is measured by taking PET nano-particles as a substrate, and the measuring method is as follows:
purified thermophilic PET hydrolase was added to the reaction buffer (100mM K) 2 HPO 4 -KH 2 PO 4 pH 8.0) to 50. mu.g/mL. Then, 300 μ L of the protein solution and 300 μ L of PET nanoparticles (0.25mg/mL) were added to a 2mL centrifuge tube, incubated at 60 ℃ for 2 hours on a thermal shaker (400rpm), the reaction was stopped by adding an equal volume of 160mM phosphate buffer (pH 2.5) containing 20% (v/v) DMSO, centrifuged (12000rpm, 3min) to collect the supernatant, and the product was analyzed by high performance liquid chromatography (selmefei, UltiMate 3000) on an Acclaim 120-C18(4.6 × 250mM), phase a: phosphate buffer (20mM, pH 2.5), phase B: chromatographically pure methanol, flow rate: 1mL/min, detection wavelength: 240nm, elution procedure: 0 to 15 minutes, 25% methanol; 15 to 25 minutes, 25% -100% methanol, 25 to 26 minutes, 100% methanol; 26 to 30 minutes, 25% -100% methanol. TPA and MHET were quantified.
The High Performance Liquid Chromatography (HPLC) detection results of the wild type ThcCut1 and the mutant thereof are shown in FIG. 2, and peaks at retention times of 18.6min and 21.5min, respectively.
The peak appearance at retention time 18.6min was consistent with that of the standard TPA (TCI, CAS: 100-21-0) (FIG. 3), so that the substance with retention time 18.6min was TPA.
The peak at retention time 21.5min was consistent with that of the standard MHET (MACKLIN, CAS: 1137-99-1) (FIG. 4), so that the material at retention time 21.5min was MHET.
The above results show that the hydrolysates of wild-type and mutant ThcCut1 of the present invention have TPA and MHET, while MHET is only an intermediate product in the PET hydrolysis reaction (FIG. 5), and TPA is a real reaction end product, so that the amount of TPA produced is used as a definition standard of the activity of PET hydrolase, and one unit of enzyme activity (U) is defined as the amount of enzyme required to produce 1. mu. mol of TPA per hour at 60 ℃.
The results of enzyme activity determination are shown in Table 1, the hydrolytic activity of the 4 mutants on PET is higher than that of a wild type ThcCut1, wherein the specific enzyme activity of the G63A/F210I/D205C/E254C/Q93G variant is improved by 2.2 times than that of the wild type, and the specific enzyme activity of the G63A, G63A/F210I, G63A/F210I/D205C/E254C variant is improved by more than 1.6 times than that of the wild type. (2) Thermostability assay for ThcCut1 wild-type and mutant
The PET main body is difficult to hydrolyze below the glass transition temperature, so that the thermal stability of the enzyme is improved, and the hydrolysis at higher temperature is important for improving the hydrolysis effect of PET. Therefore, the thermostability of wild-type and mutant ThcCut1 was determined using NanoDSF (NanoTemper, Prometheus NT.48). The excitation wavelength of the device is 280nm, and the emission wavelengths are 330nm and 350 nm. Wild-type thcut 1 and variants thereof were diluted to 0.1mg/mL using lysis buffer. The sample was heated from 25 ℃ to 100 ℃ at a rate of 1 ℃/min at 100% excitation power. Melting temperature (T) m ) Reported by Prometheus software (I) 330nm /I 350nm The temperature corresponding to the maximum slope value of the ratio).
The measurement results of the melting temperature are shown in table 1. The G63A/F210I/D205C/E254C variant increases the melting temperature of ThcCut1 by 15.7 ℃, and the G63A/F210I/D205C/E254C/Q93G variant increases the melting temperature by 18.3 ℃, so that the thermal stability of ThcCut1 is greatly improved.
TABLE 1 evaluation of Properties of thermophilic PET hydrolase mutant proteins
Figure BDA0003724488030000151
Example 4: application of thermophilic PET hydrolase mutant protein
(1) Hydrolysis of post-consumer PET bottles
Post-consumer PET bottles (Yibao) were cut into 1cm by 1cm pieces, washed and dried for use. The post-consumer PET bottle flakes were crushed using a high-speed rotary grinder (ant science and technology, AM500S) equipped with 1.0mm trapezoidal hole sieve rings to obtain post-consumer PET bottle particles having a diameter of about 1 mm. About 20mg of post-consumer PET bottle particles were weighed into a 5mL reaction flask and 3mL of reaction buffer containing 30ppm dodecyltrimethylammonium bromide and 75. mu.g of purified protein (ThcCut1 wild-type and G63A/F210I/D205C/E254C/Q93G variant) were added. The reaction was stopped by adding an equal volume of 160mM phosphate buffer (pH 2.5) containing 20% (v/v) DMSO after 96h reaction at 70 ℃ (400rpm) with sampling at 24h intervals, diluted by a suitable multiple, centrifuged (12000rpm, 3min) to collect the supernatant, and the product was analyzed by high performance liquid chromatography. The post-consumer PET bottle pellets after reaction were washed, dried and weighed to calculate the weight loss ratio, and the process hydrolysis ratio was calculated as the product variation, with the results shown in fig. 6. G63A/F210I/D205C/E254C/Q93G variant reacts for 96 hours, namely 96.2% of post-consumer PET bottle particles can be hydrolyzed, and the post-consumer PET bottle particles are 86.1 times of wild ThcCut 1.
(2) Hydrolysis of polylactic acid
Cutting plastic PLA straws and PLA films into square sheets with sides not larger than 1cm, weighing about 30mg of PLA straw sheets and PLA sheets into 5mL glass bottles, adding 3mL of reaction buffer containing 30ppm dodecyltrimethylammonium bromide and 75 μ G of purified protein (ThcCut1 wild-type and G63A/F210I/D205C/E254C/Q93G variant), reacting at 70 ℃ (400rpm) for 96h, adding an equal volume of 160mM phosphate buffer (pH 2.5) containing 20% (v/v) DMSO to terminate the reaction, centrifuging (12000rpm, 3min) to collect PLA straw sheets and PLA sheets, washing and drying. And adhering conductive adhesive on the reacted PLA straw slice and the PLA film, sputtering gold on the surface of the sample, and then using a scanning electron microscope to adjust proper focal length and multiplying power under the condition of 15kv to perform characterization. The electron microscope characterization result is shown in FIG. 7, and the G63A/F210I/D205C/E254C/Q93G variant has a certain depolymerization effect on the PLA straw and the PLA film, and a large crack gap appears.
Example 5: modification of thermophilic PET hydrolase ThcCut2
The thermophilic PET hydrolase used in the embodiment is ThcCut2 (GenBank: ADV92527.1), the source of the coding gene is Thermobifida cellulolysitica, the amino acid sequence of ThcCut2 is shown as SEQ ID NO.2, and one of the nucleotide sequences of the coding gene of ThcCut2 is shown as SEQ ID NO. 11.
The ThcCut2-G63A/F210I/D205C/E254C/Q93G mutant is a sequence (SEQ ID NO.8) obtained by mutating Gly at the 63 th position of a ThcCut2 amino acid sequence (SEQ ID NO.2) into Ala, Phe at the 210 th position into Ile, Asp at the 205 th position into Cys, Glu at the 254 th position into Cys, Gln at the 93 th position into Gly and keeping other amino acid residues unchanged, wherein one of corresponding nucleotide sequences is shown as SEQ ID NO. 17.
The vector for expressing the wild type ThcCut2 is a vector obtained by inserting the nucleotide sequence (SEQ ID NO.11) of a ThcCut2 coding gene between NdeI and XhoI enzyme cutting sites of a pET-26b vector. The vector for expressing the ThcCut2-G63A/F210I/D205C/E254C/Q93G mutant is obtained by inserting a coding gene (SEQ ID NO.17) for coding the mutant between NdeI and XhoI enzyme cutting sites of a pET-26b vector.
ThcCut2 and mutants thereof were screened using kanamycin sulfate, and expression and purification procedures were as shown in example 2. The enzyme activities of the wild type and the mutant of ThcCut2 are measured by using PET nano particles as substrates, the measuring method is the same as that in example 3, and the detection result of the wild type ThcCut2 by high performance liquid chromatography is shown in figure 8(A), and peaks appear at retention time of 18.0min, 21.4min and 22.1min respectively. The high performance liquid chromatography detection result of the ThcCut2-G63A/F210I/D205C/E254C/Q93G mutant is shown in FIG. 8(B), and peaks appear at retention time of 18.0min and 21.4min respectively. The peak at retention time of 18.0min was consistent with that of the standard TPA (fig. 3), so the substance at retention time of 18.0min was TPA. The peak-out time at retention time 21.4min was consistent with that of the standard MHET (fig. 4), so the material at retention time 21.4min was MHET. The peak at retention time of 22.1min was consistent with that of the standard BHET (TCI, CAS: 959-26-2) (FIG. 9), so that the material at retention time of 22.1min was BHET. The wild-type hydrolysate of ThcCut2 mainly comprises TPA, MHET and BHET, while the hydrolysate of the mutant ThcCut2-G63A/F210I/D205C/E254C/Q93G is TPA and MHET, and the reaction is more complete. The PET hydrolase activity is calculated by taking the generation amount of TPA as a standard, and the enzyme activity determination result is shown in Table 2, wherein the specific enzyme activity of the ThcCut2-G63A/F210I/D205C/E254C/Q93G mutant is improved by 5.6 times compared with that of a wild type.
The thermal stability of the wild type and the mutant of ThcCut2 was measured by using NanoDSF in the same manner as in example 3, and the results of the measurement of the melting temperature are shown in Table 2. the melting temperature of ThcCut2-G63A/F210I/D205C/E254C/Q93G mutant was increased by 16.1 ℃ to greatly improve the thermal stability of ThcCut 2.
TABLE 2 evaluation of the Properties of ThcCut2 and its mutant proteins
Figure BDA0003724488030000171
Example 6: modification of thermophilic PET hydrolase Tfcut2
The thermophilic PET hydrolase used in the embodiment is TfCut2 (GenBank: CBY05530.1), the source of the coding gene is Thermobifida fusca, the amino acid sequence of TfCut2 is shown in SEQ ID NO.3, and one of the nucleotide sequences of TfCut2 coding gene is shown in SEQ ID NO. 12.
The TfCut2-G63A/F210I/D205C/E254C/Q93G mutant is a sequence (SEQ ID NO.9) obtained by mutating Gly at the 63 th position of a TfCut2 amino acid sequence (SEQ ID NO.3) into Ala, Phe at the 210 th position into Ile, Asp at the 205 th position into Cys, Glu at the 254 th position into Cys, Gln at the 93 th position into Gly and keeping other amino acid residues unchanged, wherein one of corresponding nucleotide sequences is shown as SEQ ID NO. 18.
The carrier for expressing the wild type TfCut2 is a carrier obtained by inserting the nucleotide sequence (SEQ ID NO.12) of a TfCut2 coding gene between NdeI and XhoI enzyme cutting sites of a pET-22b carrier. The vector for expressing the Tfcut2-G63A/F210I/D205C/E254C/Q93G mutant is obtained by inserting a coding gene (SEQ ID NO.18) for coding the mutant between NdeI and XhoI enzyme cutting sites of a pET-22b vector.
The expression and purification method of Tfcut2 and its mutants are shown in example 2. The enzyme activity determination of the wild type TfCut2 and the mutant is carried out by taking PET nano particles as a substrate, the determination method is the same as that in the embodiment 3, and the high performance liquid chromatography detection results of the wild type TfCut2 and the mutant are shown in figure 10, and peaks appear at retention time of 18.7min and 21.5min respectively. The peak at retention time of 18.7min was consistent with that of the standard TPA (fig. 3), so the substance at retention time of 18.7min was TPA. The peak-out time at retention time 21.8min was consistent with that of the standard MHET (fig. 4), so the material at retention time 21.8min was MHET. Therefore, hydrolysis products of wild type and mutant of the TfCut2 are TPA and MHET, the PET hydrolase activity is calculated by taking the generation amount of TPA as a standard, the enzyme activity determination result is shown in Table 3, and the specific enzyme activity of the TfCut2-G63A/F210I/D205C/E254C/Q93G mutant is improved by 3.4 times compared with that of the wild type.
The thermal stability of wild type and mutant of TfCut2 is measured by using NanoDSF, the measuring method is the same as that of example 3, the measuring result of the melting temperature is shown in Table 3, and the TfCut2-G63A/F210I/D205C/E254C/Q93G mutant improves the melting temperature of TfCut2 by 17.9 ℃ and greatly improves the thermal stability of TfCut 2.
TABLE 3 evaluation of the Properties of Tfcut2 and its mutant proteins
Figure BDA0003724488030000181
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are used for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.
Sequence listing
<110> Beijing university of chemical industry
<120> mutant protein of thermophilic PET hydrolase and application thereof
<130> RB2202851-FF
<141> 2022-06-30
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 262
<212> PRT
<213> (thermophilic PET hydrolase ThcCut1)
<400> 1
Met Ala Asn Pro Tyr Glu Arg Gly Pro Asn Pro Thr Asp Ala Leu Leu
1 5 10 15
Glu Ala Ser Ser Gly Pro Phe Ser Val Ser Glu Glu Asn Val Ser Arg
20 25 30
Leu Ser Ala Ser Gly Phe Gly Gly Gly Thr Ile Tyr Tyr Pro Arg Glu
35 40 45
Asn Asn Thr Tyr Gly Ala Val Ala Ile Ser Pro Gly Tyr Thr Gly Thr
50 55 60
Glu Ala Ser Ile Ala Trp Leu Gly Glu Arg Ile Ala Ser His Gly Phe
65 70 75 80
Val Val Ile Thr Ile Asp Thr Ile Thr Thr Leu Asp Gln Pro Asp Ser
85 90 95
Arg Ala Glu Gln Leu Asn Ala Ala Leu Asn His Met Ile Asn Arg Ala
100 105 110
Ser Ser Thr Val Arg Ser Arg Ile Asp Ser Ser Arg Leu Ala Val Met
115 120 125
Gly His Ser Met Gly Gly Gly Gly Thr Leu Arg Leu Ala Ser Gln Arg
130 135 140
Pro Asp Leu Lys Ala Ala Ile Pro Leu Thr Pro Trp His Leu Asn Lys
145 150 155 160
Asn Trp Ser Ser Val Thr Val Pro Thr Leu Ile Ile Gly Ala Asp Leu
165 170 175
Asp Thr Ile Ala Pro Val Ala Thr His Ala Lys Pro Phe Tyr Asn Ser
180 185 190
Leu Pro Ser Ser Ile Ser Lys Ala Tyr Leu Glu Leu Asp Gly Ala Thr
195 200 205
His Phe Ala Pro Asn Ile Pro Asn Lys Ile Ile Gly Lys Tyr Ser Val
210 215 220
Ala Trp Leu Lys Arg Phe Val Asp Asn Asp Thr Arg Tyr Thr Gln Phe
225 230 235 240
Leu Cys Pro Gly Pro Arg Asp Gly Leu Phe Gly Glu Val Glu Glu Tyr
245 250 255
Arg Ser Thr Cys Pro Phe
260
<210> 2
<211> 262
<212> PRT
<213> (thermophilic PET hydrolase ThcCut 2)
<400> 2
Met Ala Asn Pro Tyr Glu Arg Gly Pro Asn Pro Thr Asp Ala Leu Leu
1 5 10 15
Glu Ala Arg Ser Gly Pro Phe Ser Val Ser Glu Glu Arg Ala Ser Arg
20 25 30
Phe Gly Ala Asp Gly Phe Gly Gly Gly Thr Ile Tyr Tyr Pro Arg Glu
35 40 45
Asn Asn Thr Tyr Gly Ala Val Ala Ile Ser Pro Gly Tyr Thr Gly Thr
50 55 60
Gln Ala Ser Val Ala Trp Leu Gly Glu Arg Ile Ala Ser His Gly Phe
65 70 75 80
Val Val Ile Thr Ile Asp Thr Asn Thr Thr Leu Asp Gln Pro Asp Ser
85 90 95
Arg Ala Arg Gln Leu Asn Ala Ala Leu Asp Tyr Met Ile Asn Asp Ala
100 105 110
Ser Ser Ala Val Arg Ser Arg Ile Asp Ser Ser Arg Leu Ala Val Met
115 120 125
Gly His Ser Met Gly Gly Gly Gly Thr Leu Arg Leu Ala Ser Gln Arg
130 135 140
Pro Asp Leu Lys Ala Ala Ile Pro Leu Thr Pro Trp His Leu Asn Lys
145 150 155 160
Asn Trp Ser Ser Val Arg Val Pro Thr Leu Ile Ile Gly Ala Asp Leu
165 170 175
Asp Thr Ile Ala Pro Val Leu Thr His Ala Arg Pro Phe Tyr Asn Ser
180 185 190
Leu Pro Thr Ser Ile Ser Lys Ala Tyr Leu Glu Leu Asp Gly Ala Thr
195 200 205
His Phe Ala Pro Asn Ile Pro Asn Lys Ile Ile Gly Lys Tyr Ser Val
210 215 220
Ala Trp Leu Lys Arg Phe Val Asp Asn Asp Thr Arg Tyr Thr Gln Phe
225 230 235 240
Leu Cys Pro Gly Pro Arg Asp Gly Leu Phe Gly Glu Val Glu Glu Tyr
245 250 255
Arg Ser Thr Cys Pro Phe
260
<210> 3
<211> 262
<212> PRT
<213> (thermophilic PET hydrolase Tfcut 2)
<400> 3
Met Ala Asn Pro Tyr Glu Arg Gly Pro Asn Pro Thr Asp Ala Leu Leu
1 5 10 15
Glu Ala Arg Ser Gly Pro Phe Ser Val Ser Glu Glu Asn Val Ser Arg
20 25 30
Leu Ser Ala Ser Gly Phe Gly Gly Gly Thr Ile Tyr Tyr Pro Arg Glu
35 40 45
Asn Asn Thr Tyr Gly Ala Val Ala Ile Ser Pro Gly Tyr Thr Gly Thr
50 55 60
Glu Ala Ser Ile Ala Trp Leu Gly Glu Arg Ile Ala Ser His Gly Phe
65 70 75 80
Val Val Ile Thr Ile Asp Thr Ile Thr Thr Leu Asp Gln Pro Asp Ser
85 90 95
Arg Ala Glu Gln Leu Asn Ala Ala Leu Asn His Met Ile Asn Arg Ala
100 105 110
Ser Ser Thr Val Arg Ser Arg Ile Asp Ser Ser Arg Leu Ala Val Met
115 120 125
Gly His Ser Met Gly Gly Gly Gly Ser Leu Arg Leu Ala Ser Gln Arg
130 135 140
Pro Asp Leu Lys Ala Ala Ile Pro Leu Thr Pro Trp His Leu Asn Lys
145 150 155 160
Asn Trp Ser Ser Val Thr Val Pro Thr Leu Ile Ile Gly Ala Asp Leu
165 170 175
Asp Thr Ile Ala Pro Val Ala Thr His Ala Lys Pro Phe Tyr Asn Ser
180 185 190
Leu Pro Ser Ser Ile Ser Lys Ala Tyr Leu Glu Leu Asp Gly Ala Thr
195 200 205
His Phe Ala Pro Asn Ile Pro Asn Lys Ile Ile Gly Lys Tyr Ser Val
210 215 220
Ala Trp Leu Lys Arg Phe Val Asp Asn Asp Thr Arg Tyr Thr Gln Phe
225 230 235 240
Leu Cys Pro Gly Pro Arg Asp Gly Leu Phe Gly Glu Val Glu Glu Tyr
245 250 255
Arg Ser Thr Cys Pro Phe
260
<210> 4
<211> 262
<212> PRT
<213> (ThcCut1 mutant G63A)
<400> 4
Met Ala Asn Pro Tyr Glu Arg Gly Pro Asn Pro Thr Asp Ala Leu Leu
1 5 10 15
Glu Ala Ser Ser Gly Pro Phe Ser Val Ser Glu Glu Asn Val Ser Arg
20 25 30
Leu Ser Ala Ser Gly Phe Gly Gly Gly Thr Ile Tyr Tyr Pro Arg Glu
35 40 45
Asn Asn Thr Tyr Gly Ala Val Ala Ile Ser Pro Gly Tyr Thr Ala Thr
50 55 60
Glu Ala Ser Ile Ala Trp Leu Gly Glu Arg Ile Ala Ser His Gly Phe
65 70 75 80
Val Val Ile Thr Ile Asp Thr Ile Thr Thr Leu Asp Gln Pro Asp Ser
85 90 95
Arg Ala Glu Gln Leu Asn Ala Ala Leu Asn His Met Ile Asn Arg Ala
100 105 110
Ser Ser Thr Val Arg Ser Arg Ile Asp Ser Ser Arg Leu Ala Val Met
115 120 125
Gly His Ser Met Gly Gly Gly Gly Thr Leu Arg Leu Ala Ser Gln Arg
130 135 140
Pro Asp Leu Lys Ala Ala Ile Pro Leu Thr Pro Trp His Leu Asn Lys
145 150 155 160
Asn Trp Ser Ser Val Thr Val Pro Thr Leu Ile Ile Gly Ala Asp Leu
165 170 175
Asp Thr Ile Ala Pro Val Ala Thr His Ala Lys Pro Phe Tyr Asn Ser
180 185 190
Leu Pro Ser Ser Ile Ser Lys Ala Tyr Leu Glu Leu Asp Gly Ala Thr
195 200 205
His Phe Ala Pro Asn Ile Pro Asn Lys Ile Ile Gly Lys Tyr Ser Val
210 215 220
Ala Trp Leu Lys Arg Phe Val Asp Asn Asp Thr Arg Tyr Thr Gln Phe
225 230 235 240
Leu Cys Pro Gly Pro Arg Asp Gly Leu Phe Gly Glu Val Glu Glu Tyr
245 250 255
Arg Ser Thr Cys Pro Phe
260
<210> 5
<211> 262
<212> PRT
<213> (ThcCut1 mutant G63A/F210I)
<400> 5
Met Ala Asn Pro Tyr Glu Arg Gly Pro Asn Pro Thr Asp Ala Leu Leu
1 5 10 15
Glu Ala Ser Ser Gly Pro Phe Ser Val Ser Glu Glu Asn Val Ser Arg
20 25 30
Leu Ser Ala Ser Gly Phe Gly Gly Gly Thr Ile Tyr Tyr Pro Arg Glu
35 40 45
Asn Asn Thr Tyr Gly Ala Val Ala Ile Ser Pro Gly Tyr Thr Ala Thr
50 55 60
Glu Ala Ser Ile Ala Trp Leu Gly Glu Arg Ile Ala Ser His Gly Phe
65 70 75 80
Val Val Ile Thr Ile Asp Thr Ile Thr Thr Leu Asp Gln Pro Asp Ser
85 90 95
Arg Ala Glu Gln Leu Asn Ala Ala Leu Asn His Met Ile Asn Arg Ala
100 105 110
Ser Ser Thr Val Arg Ser Arg Ile Asp Ser Ser Arg Leu Ala Val Met
115 120 125
Gly His Ser Met Gly Gly Gly Gly Thr Leu Arg Leu Ala Ser Gln Arg
130 135 140
Pro Asp Leu Lys Ala Ala Ile Pro Leu Thr Pro Trp His Leu Asn Lys
145 150 155 160
Asn Trp Ser Ser Val Thr Val Pro Thr Leu Ile Ile Gly Ala Asp Leu
165 170 175
Asp Thr Ile Ala Pro Val Ala Thr His Ala Lys Pro Phe Tyr Asn Ser
180 185 190
Leu Pro Ser Ser Ile Ser Lys Ala Tyr Leu Glu Leu Asp Gly Ala Thr
195 200 205
His Ile Ala Pro Asn Ile Pro Asn Lys Ile Ile Gly Lys Tyr Ser Val
210 215 220
Ala Trp Leu Lys Arg Phe Val Asp Asn Asp Thr Arg Tyr Thr Gln Phe
225 230 235 240
Leu Cys Pro Gly Pro Arg Asp Gly Leu Phe Gly Glu Val Glu Glu Tyr
245 250 255
Arg Ser Thr Cys Pro Phe
260
<210> 6
<211> 262
<212> PRT
<213> (ThcCut1 mutant G63A/F210I/D205C/E254C)
<400> 6
Met Ala Asn Pro Tyr Glu Arg Gly Pro Asn Pro Thr Asp Ala Leu Leu
1 5 10 15
Glu Ala Ser Ser Gly Pro Phe Ser Val Ser Glu Glu Asn Val Ser Arg
20 25 30
Leu Ser Ala Ser Gly Phe Gly Gly Gly Thr Ile Tyr Tyr Pro Arg Glu
35 40 45
Asn Asn Thr Tyr Gly Ala Val Ala Ile Ser Pro Gly Tyr Thr Ala Thr
50 55 60
Glu Ala Ser Ile Ala Trp Leu Gly Glu Arg Ile Ala Ser His Gly Phe
65 70 75 80
Val Val Ile Thr Ile Asp Thr Ile Thr Thr Leu Asp Gln Pro Asp Ser
85 90 95
Arg Ala Glu Gln Leu Asn Ala Ala Leu Asn His Met Ile Asn Arg Ala
100 105 110
Ser Ser Thr Val Arg Ser Arg Ile Asp Ser Ser Arg Leu Ala Val Met
115 120 125
Gly His Ser Met Gly Gly Gly Gly Thr Leu Arg Leu Ala Ser Gln Arg
130 135 140
Pro Asp Leu Lys Ala Ala Ile Pro Leu Thr Pro Trp His Leu Asn Lys
145 150 155 160
Asn Trp Ser Ser Val Thr Val Pro Thr Leu Ile Ile Gly Ala Asp Leu
165 170 175
Asp Thr Ile Ala Pro Val Ala Thr His Ala Lys Pro Phe Tyr Asn Ser
180 185 190
Leu Pro Ser Ser Ile Ser Lys Ala Tyr Leu Glu Leu Cys Gly Ala Thr
195 200 205
His Ile Ala Pro Asn Ile Pro Asn Lys Ile Ile Gly Lys Tyr Ser Val
210 215 220
Ala Trp Leu Lys Arg Phe Val Asp Asn Asp Thr Arg Tyr Thr Gln Phe
225 230 235 240
Leu Cys Pro Gly Pro Arg Asp Gly Leu Phe Gly Glu Val Cys Glu Tyr
245 250 255
Arg Ser Thr Cys Pro Phe
260
<210> 7
<211> 262
<212> PRT
<213> (ThcCut1 mutant G63A/F210I/D205C/E254C/Q93G)
<400> 7
Met Ala Asn Pro Tyr Glu Arg Gly Pro Asn Pro Thr Asp Ala Leu Leu
1 5 10 15
Glu Ala Ser Ser Gly Pro Phe Ser Val Ser Glu Glu Asn Val Ser Arg
20 25 30
Leu Ser Ala Ser Gly Phe Gly Gly Gly Thr Ile Tyr Tyr Pro Arg Glu
35 40 45
Asn Asn Thr Tyr Gly Ala Val Ala Ile Ser Pro Gly Tyr Thr Ala Thr
50 55 60
Glu Ala Ser Ile Ala Trp Leu Gly Glu Arg Ile Ala Ser His Gly Phe
65 70 75 80
Val Val Ile Thr Ile Asp Thr Ile Thr Thr Leu Asp Gly Pro Asp Ser
85 90 95
Arg Ala Glu Gln Leu Asn Ala Ala Leu Asn His Met Ile Asn Arg Ala
100 105 110
Ser Ser Thr Val Arg Ser Arg Ile Asp Ser Ser Arg Leu Ala Val Met
115 120 125
Gly His Ser Met Gly Gly Gly Gly Thr Leu Arg Leu Ala Ser Gln Arg
130 135 140
Pro Asp Leu Lys Ala Ala Ile Pro Leu Thr Pro Trp His Leu Asn Lys
145 150 155 160
Asn Trp Ser Ser Val Thr Val Pro Thr Leu Ile Ile Gly Ala Asp Leu
165 170 175
Asp Thr Ile Ala Pro Val Ala Thr His Ala Lys Pro Phe Tyr Asn Ser
180 185 190
Leu Pro Ser Ser Ile Ser Lys Ala Tyr Leu Glu Leu Cys Gly Ala Thr
195 200 205
His Ile Ala Pro Asn Ile Pro Asn Lys Ile Ile Gly Lys Tyr Ser Val
210 215 220
Ala Trp Leu Lys Arg Phe Val Asp Asn Asp Thr Arg Tyr Thr Gln Phe
225 230 235 240
Leu Cys Pro Gly Pro Arg Asp Gly Leu Phe Gly Glu Val Cys Glu Tyr
245 250 255
Arg Ser Thr Cys Pro Phe
260
<210> 8
<211> 262
<212> PRT
<213> (ThcCut 2 mutant G63A/F210I/D205C/E254C/Q93G)
<400> 8
Met Ala Asn Pro Tyr Glu Arg Gly Pro Asn Pro Thr Asp Ala Leu Leu
1 5 10 15
Glu Ala Arg Ser Gly Pro Phe Ser Val Ser Glu Glu Arg Ala Ser Arg
20 25 30
Phe Gly Ala Asp Gly Phe Gly Gly Gly Thr Ile Tyr Tyr Pro Arg Glu
35 40 45
Asn Asn Thr Tyr Gly Ala Val Ala Ile Ser Pro Gly Tyr Thr Ala Thr
50 55 60
Gln Ala Ser Val Ala Trp Leu Gly Glu Arg Ile Ala Ser His Gly Phe
65 70 75 80
Val Val Ile Thr Ile Asp Thr Asn Thr Thr Leu Asp Gly Pro Asp Ser
85 90 95
Arg Ala Arg Gln Leu Asn Ala Ala Leu Asp Tyr Met Ile Asn Asp Ala
100 105 110
Ser Ser Ala Val Arg Ser Arg Ile Asp Ser Ser Arg Leu Ala Val Met
115 120 125
Gly His Ser Met Gly Gly Gly Gly Thr Leu Arg Leu Ala Ser Gln Arg
130 135 140
Pro Asp Leu Lys Ala Ala Ile Pro Leu Thr Pro Trp His Leu Asn Lys
145 150 155 160
Asn Trp Ser Ser Val Arg Val Pro Thr Leu Ile Ile Gly Ala Asp Leu
165 170 175
Asp Thr Ile Ala Pro Val Leu Thr His Ala Arg Pro Phe Tyr Asn Ser
180 185 190
Leu Pro Thr Ser Ile Ser Lys Ala Tyr Leu Glu Leu Cys Gly Ala Thr
195 200 205
His Ile Ala Pro Asn Ile Pro Asn Lys Ile Ile Gly Lys Tyr Ser Val
210 215 220
Ala Trp Leu Lys Arg Phe Val Asp Asn Asp Thr Arg Tyr Thr Gln Phe
225 230 235 240
Leu Cys Pro Gly Pro Arg Asp Gly Leu Phe Gly Glu Val Cys Glu Tyr
245 250 255
Arg Ser Thr Cys Pro Phe
260
<210> 9
<211> 262
<212> PRT
<213> (TfCut 2 mutant G63A/F210I/D205C/E254C/Q93G)
<400> 9
Met Ala Asn Pro Tyr Glu Arg Gly Pro Asn Pro Thr Asp Ala Leu Leu
1 5 10 15
Glu Ala Arg Ser Gly Pro Phe Ser Val Ser Glu Glu Asn Val Ser Arg
20 25 30
Leu Ser Ala Ser Gly Phe Gly Gly Gly Thr Ile Tyr Tyr Pro Arg Glu
35 40 45
Asn Asn Thr Tyr Gly Ala Val Ala Ile Ser Pro Gly Tyr Thr Ala Thr
50 55 60
Glu Ala Ser Ile Ala Trp Leu Gly Glu Arg Ile Ala Ser His Gly Phe
65 70 75 80
Val Val Ile Thr Ile Asp Thr Ile Thr Thr Leu Asp Gly Pro Asp Ser
85 90 95
Arg Ala Glu Gln Leu Asn Ala Ala Leu Asn His Met Ile Asn Arg Ala
100 105 110
Ser Ser Thr Val Arg Ser Arg Ile Asp Ser Ser Arg Leu Ala Val Met
115 120 125
Gly His Ser Met Gly Gly Gly Gly Ser Leu Arg Leu Ala Ser Gln Arg
130 135 140
Pro Asp Leu Lys Ala Ala Ile Pro Leu Thr Pro Trp His Leu Asn Lys
145 150 155 160
Asn Trp Ser Ser Val Thr Val Pro Thr Leu Ile Ile Gly Ala Asp Leu
165 170 175
Asp Thr Ile Ala Pro Val Ala Thr His Ala Lys Pro Phe Tyr Asn Ser
180 185 190
Leu Pro Ser Ser Ile Ser Lys Ala Tyr Leu Glu Leu Cys Gly Ala Thr
195 200 205
His Ile Ala Pro Asn Ile Pro Asn Lys Ile Ile Gly Lys Tyr Ser Val
210 215 220
Ala Trp Leu Lys Arg Phe Val Asp Asn Asp Thr Arg Tyr Thr Gln Phe
225 230 235 240
Leu Cys Pro Gly Pro Arg Asp Gly Leu Phe Gly Glu Val Cys Glu Tyr
245 250 255
Arg Ser Thr Cys Pro Phe
260
<210> 10
<211> 786
<212> DNA
<213> (nucleotide molecule encoding thermophilic PET hydrolase ThcCut1)
<400> 10
atggcgaatc cgtatgaacg cggcccgaat ccgaccgatg cgctgttaga agcgagcagc 60
ggtccattta gcgtgagcga agaaaatgtg agccgcctga gcgcgagcgg ctttggtggt 120
ggtaccattt attatccgcg cgaaaataat acctacggcg cggtggcgat tagcccgggt 180
tataccggta ccgaagcgag cattgcgtgg ctgggtgaac gcattgcgag ccatggtttt 240
gtggtgatta ccattgatac cattaccacc ctggatcagc cggatagccg cgcggaacaa 300
ttaaatgcgg cgttaaatca catgattaac cgcgcgagca gcaccgtgcg cagtagaatt 360
gatagcagcc gtctggcggt tatgggccat agcatgggcg gtggtggtac cttacgtttg 420
gcgagccaac gcccagattt aaaagcggcg attccgttga ccccatggca tctgaataaa 480
aattggagca gcgtgaccgt gccgaccctg attattggcg cggatctgga taccattgcg 540
ccggtggcga ctcatgcgaa accattttat aatagcctgc cgagcagcat tagcaaagcg 600
tatctggaac tggatggcgc gacccatttt gcgccgaata ttccgaataa aattatcggc 660
aaatacagcg tggcgtggct gaaacgcttt gtggataatg atacccgcta tacccagttt 720
ctgtgcccgg gcccacgtga tggcttattt ggtgaagttg aagaatatcg cagcacctgc 780
ccgttt 786
<210> 11
<211> 786
<212> DNA
<213> (nucleotide molecule encoding thermophilic PET hydrolase ThcCut 2)
<400> 11
atggcgaacc cgtatgaacg tggtccgaac cctaccgatg cgctgttaga agcgcgtagc 60
ggtcctttta gcgtgagcga agaacgcgcg agccgttttg gtgcggatgg ttttggtggc 120
ggcaccattt attatccgcg cgagaacaac acttatggcg cggttgcgat ttcaccgggt 180
tataccggta cccaagcgtc agttgcgtgg ctgggtgaac gtattgcgag ccatggcttt 240
gtggtgatta ccattgatac caacaccacc ctggatcagc ctgatagccg tgcgcgtcaa 300
ttaaatgcgg cgctggacta tatgattaac gatgcgagca gcgcagttcg tagccgcatt 360
gattcaagcc gcctggcggt tatgggtcat agcatgggtg gtggcggcac cttacgttta 420
gcgagccagc gccctgatct gaaagcggcg attccgttaa ctccgtggca tctgaacaaa 480
aactggagca gcgttcgtgt tccgaccctg attattggcg cggatctgga tactattgcg 540
ccggtgttaa cccatgcgcg cccgttttat aatagcctgc cgaccagcat tagcaaagcg 600
tatctggaac tggatggcgc gactcatttt gcgccgaaca ttccgaacaa gatcatcggc 660
aaatatagcg tggcgtggct gaaacgcttt gtggataacg atacccgcta tacccagttt 720
ctgtgccctg gtccgcgcga tggtttattt ggcgaggtgg aagaatatcg cagcacctgc 780
ccgttt 786
<210> 12
<211> 786
<212> DNA
<213> (nucleotide molecule encoding the thermophilic PET hydrolase Tfcut 2)
<400> 12
atggcgaatc cgtatgaacg cggtccgaat ccgaccgatg cgctgttgga agcgagaagc 60
ggtccattta gcgttagcga agaaaatgtg agccgcctga gcgcgagcgg ctttggtggt 120
ggaaccattt attatccgcg cgaaaataat acctacggcg cggtggcgat tagcccgggt 180
tataccggta ccgaagcgag cattgcgtgg ctgggtgaac gcattgcgag ccatggtttt 240
gtggtgatta ccattgatac cattaccacc ctggatcagc cggatagccg cgcggaacaa 300
ttgaatgcgg cgttgaatca catgattaac cgcgcgagca gcaccgtgcg cagtcgtatt 360
gatagcagcc gcttagcggt tatgggtcat agcatgggcg gcggtggtag cttacgtttg 420
gcgagccaac gtccagattt aaaagcggcg attccgttaa ccccgtggca tctgaataaa 480
aattggagca gcgtgaccgt gccgaccctg attattggcg cggatctgga taccattgcg 540
ccggtggcga cccatgcgaa accattttat aatagcctgc cgagcagcat tagcaaagcg 600
tatctggaac tggatggcgc gacccatttt gcgccgaata ttccgaataa aattatcggc 660
aaatacagcg tggcgtggct gaaacgcttt gtggataatg atacccgcta tacccagttt 720
ctgtgcccgg gcccacgtga tggtttgttt ggtgaagttg aagaatatcg cagcacctgc 780
ccgttt 786
<210> 13
<211> 786
<212> DNA
<213> (nucleotide molecule encoding ThcCut1 mutant G63A)
<400> 13
atggcgaatc cgtatgaacg cggcccgaat ccgaccgatg cgctgttaga agcgagcagc 60
ggtccattta gcgtgagcga agaaaatgtg agccgcctga gcgcgagcgg ctttggtggt 120
ggtaccattt attatccgcg cgaaaataat acctacggcg cggtggcgat tagcccgggt 180
tataccgcca ccgaagcgag cattgcgtgg ctgggtgaac gcattgcgag ccatggtttt 240
gtggtgatta ccattgatac cattaccacc ctggatcagc cggatagccg cgcggaacaa 300
ttaaatgcgg cgttaaatca catgattaac cgcgcgagca gcaccgtgcg cagtagaatt 360
gatagcagcc gtctggcggt tatgggccat agcatgggcg gtggtggtac cttacgtttg 420
gcgagccaac gcccagattt aaaagcggcg attccgttga ccccatggca tctgaataaa 480
aattggagca gcgtgaccgt gccgaccctg attattggcg cggatctgga taccattgcg 540
ccggtggcga ctcatgcgaa accattttat aatagcctgc cgagcagcat tagcaaagcg 600
tatctggaac tggatggcgc gacccatttt gcgccgaata ttccgaataa aattatcggc 660
aaatacagcg tggcgtggct gaaacgcttt gtggataatg atacccgcta tacccagttt 720
ctgtgcccgg gcccacgtga tggcttattt ggtgaagttg aagaatatcg cagcacctgc 780
ccgttt 786
<210> 14
<211> 786
<212> DNA
<213> (nucleotide molecule encoding ThcCut1 mutant G63A/F210I)
<400> 14
atggcgaatc cgtatgaacg cggcccgaat ccgaccgatg cgctgttaga agcgagcagc 60
ggtccattta gcgtgagcga agaaaatgtg agccgcctga gcgcgagcgg ctttggtggt 120
ggtaccattt attatccgcg cgaaaataat acctacggcg cggtggcgat tagcccgggt 180
tataccgcca ccgaagcgag cattgcgtgg ctgggtgaac gcattgcgag ccatggtttt 240
gtggtgatta ccattgatac cattaccacc ctggatcagc cggatagccg cgcggaacaa 300
ttaaatgcgg cgttaaatca catgattaac cgcgcgagca gcaccgtgcg cagtagaatt 360
gatagcagcc gtctggcggt tatgggccat agcatgggcg gtggtggtac cttacgtttg 420
gcgagccaac gcccagattt aaaagcggcg attccgttga ccccatggca tctgaataaa 480
aattggagca gcgtgaccgt gccgaccctg attattggcg cggatctgga taccattgcg 540
ccggtggcga ctcatgcgaa accattttat aatagcctgc cgagcagcat tagcaaagcg 600
tatctggaac tggatggcgc gacccatata gcgccgaata ttccgaataa aattatcggc 660
aaatacagcg tggcgtggct gaaacgcttt gtggataatg atacccgcta tacccagttt 720
ctgtgcccgg gcccacgtga tggcttattt ggtgaagttg aagaatatcg cagcacctgc 780
ccgttt 786
<210> 15
<211> 786
<212> DNA
<213> (nucleotide molecules encoding mutant ThcCut 1G 63A/F210I/D205C/E254C)
<400> 15
atggcgaatc cgtatgaacg cggcccgaat ccgaccgatg cgctgttaga agcgagcagc 60
ggtccattta gcgtgagcga agaaaatgtg agccgcctga gcgcgagcgg ctttggtggt 120
ggtaccattt attatccgcg cgaaaataat acctacggcg cggtggcgat tagcccgggt 180
tataccgcca ccgaagcgag cattgcgtgg ctgggtgaac gcattgcgag ccatggtttt 240
gtggtgatta ccattgatac cattaccacc ctggatcagc cggatagccg cgcggaacaa 300
ttaaatgcgg cgttaaatca catgattaac cgcgcgagca gcaccgtgcg cagtagaatt 360
gatagcagcc gtctggcggt tatgggccat agcatgggcg gtggtggtac cttacgtttg 420
gcgagccaac gcccagattt aaaagcggcg attccgttga ccccatggca tctgaataaa 480
aattggagca gcgtgaccgt gccgaccctg attattggcg cggatctgga taccattgcg 540
ccggtggcga ctcatgcgaa accattttat aatagcctgc cgagcagcat tagcaaagcg 600
tatctggaac tgtgcggcgc gacccatata gcgccgaata ttccgaataa aattatcggc 660
aaatacagcg tggcgtggct gaaacgcttt gtggataatg atacccgcta tacccagttt 720
ctgtgcccgg gcccacgtga tggcttattt ggtgaagttt gcgaatatcg cagcacctgc 780
ccgttt 786
<210> 16
<211> 786
<212> DNA
<213> (nucleotide molecules encoding the mutant ThcCut 1G 63A/F210I/D205C/E254C/Q93G)
<400> 16
atggcgaatc cgtatgaacg cggcccgaat ccgaccgatg cgctgttaga agcgagcagc 60
ggtccattta gcgtgagcga agaaaatgtg agccgcctga gcgcgagcgg ctttggtggt 120
ggtaccattt attatccgcg cgaaaataat acctacggcg cggtggcgat tagcccgggt 180
tataccgcca ccgaagcgag cattgcgtgg ctgggtgaac gcattgcgag ccatggtttt 240
gtggtgatta ccattgatac cattaccacc ctggatggac cggatagccg cgcggaacaa 300
ttaaatgcgg cgttaaatca catgattaac cgcgcgagca gcaccgtgcg cagtagaatt 360
gatagcagcc gtctggcggt tatgggccat agcatgggcg gtggtggtac cttacgtttg 420
gcgagccaac gcccagattt aaaagcggcg attccgttga ccccatggca tctgaataaa 480
aattggagca gcgtgaccgt gccgaccctg attattggcg cggatctgga taccattgcg 540
ccggtggcga ctcatgcgaa accattttat aatagcctgc cgagcagcat tagcaaagcg 600
tatctggaac tgtgcggcgc gacccatata gcgccgaata ttccgaataa aattatcggc 660
aaatacagcg tggcgtggct gaaacgcttt gtggataatg atacccgcta tacccagttt 720
ctgtgcccgg gcccacgtga tggcttattt ggtgaagttt gcgaatatcg cagcacctgc 780
ccgttt 786
<210> 17
<211> 786
<212> DNA
<213> (nucleotide molecules encoding mutant ThcCut 2G 63A/F210I/D205C/E254C/Q93G)
<400> 17
atggcgaacc cgtatgaacg tggtccgaac cctaccgatg cgctgttaga agcgcgtagc 60
ggtcctttta gcgtgagcga agaacgcgcg agccgttttg gtgcggatgg ttttggtggc 120
ggcaccattt attatccgcg cgagaacaac acttatggcg cggttgcgat ttcaccgggt 180
tataccgcca cccaagcgtc agttgcgtgg ctgggtgaac gtattgcgag ccatggcttt 240
gtggtgatta ccattgatac caacaccacc ctggatggtc ctgatagccg tgcgcgtcaa 300
ttaaatgcgg cgctggacta tatgattaac gatgcgagca gcgcagttcg tagccgcatt 360
gattcaagcc gcctggcggt tatgggtcat agcatgggtg gtggcggcac cttacgttta 420
gcgagccagc gccctgatct gaaagcggcg attccgttaa ctccgtggca tctgaacaaa 480
aactggagca gcgttcgtgt tccgaccctg attattggcg cggatctgga tactattgcg 540
ccggtgttaa cccatgcgcg cccgttttat aatagcctgc cgaccagcat tagcaaagcg 600
tatctggaac tgtgtggcgc gactcatatt gcgccgaaca ttccgaacaa gatcatcggc 660
aaatatagcg tggcgtggct gaaacgcttt gtggataacg atacccgcta tacccagttt 720
ctgtgccctg gtccgcgcga tggtttattt ggcgaggtgt gtgaatatcg cagcacctgc 780
ccgttt 786
<210> 18
<211> 786
<212> DNA
<213> (nucleotide molecule encoding mutant TfCut 2G 63A/F210I/D205C/E254C/Q93G)
<400> 18
atggcgaatc cgtatgaacg cggtccgaat ccgaccgatg cgctgttgga agcgagaagc 60
ggtccattta gcgttagcga agaaaatgtg agccgcctga gcgcgagcgg ctttggtggt 120
ggaaccattt attatccgcg cgaaaataat acctacggcg cggtggcgat tagcccgggt 180
tataccgcga ccgaagcgag cattgcgtgg ctgggtgaac gcattgcgag ccatggtttt 240
gtggtgatta ccattgatac cattaccacc ctggatggtc cggatagccg cgcggaacaa 300
ttgaatgcgg cgttgaatca catgattaac cgcgcgagca gcaccgtgcg cagtcgtatt 360
gatagcagcc gcttagcggt tatgggtcat agcatgggcg gcggtggtag cttacgtttg 420
gcgagccaac gtccagattt aaaagcggcg attccgttaa ccccgtggca tctgaataaa 480
aattggagca gcgtgaccgt gccgaccctg attattggcg cggatctgga taccattgcg 540
ccggtggcga cccatgcgaa accattttat aatagcctgc cgagcagcat tagcaaagcg 600
tatctggaac tgtgtggcgc gacccatatt gcgccgaata ttccgaataa aattatcggc 660
aaatacagcg tggcgtggct gaaacgcttt gtggataatg atacccgcta tacccagttt 720
ctgtgcccgg gcccacgtga tggtttgttt ggtgaagttt gtgaatatcg cagcacctgc 780
ccgttt 786

Claims (10)

1. A mutant protein of a thermophilic PET hydrolase, which is a mutant of a wild thermophilic PET hydrolase and has hydrolytic activity towards a material containing ester and/or amide linkages, wherein the wild thermophilic PET hydrolase is any one of the following proteins:
(a1) protein with an amino acid sequence of SEQ ID NO. 1;
(a2) a protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more with the amino acid sequence defined in (a1) and having a PET hydrolyzing activity;
(a3) a protein comprising the amino acid sequence defined in (a1) or (a2) in the sequence;
(a4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (a1) to (a 3);
preferably, the amino acid sequence of the protein having more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% homology with the amino acid sequence defined in (a1) and having PET hydrolytic activity is shown as SEQ ID NO.2 or SEQ ID NO. 3.
2. The mutant protein of claim 1,
the sequence of the mutant protein comprises one or more of mutants of amino acid sequence from N-terminal to C-terminal, wherein the mutation is from 62 th Thr to Met, 63 th Gly to Ala, 93 th Gln to Gly, 205 th Asp to Cys, 210 th Phe to Ile, 210 th Phe to Met, 210 th Phe to Val, 210 th Phe to Trp, 213 th Asn to Asp, 213 th Asn to Met and 254 th Glu to Cys;
preferably, the amino acid sequence of said wild thermophilic PET hydrolase is SEQ ID No.1 or SEQ ID No.2 or SEQ ID No. 3.
3. The mutant protein of claim 2,
the mutant protein is a No.1 thermophilic PET hydrolase mutant protein, and the sequence of the mutant protein comprises a mutation of Gly to Ala from N end to 63 th position of C end of an amino acid sequence shown in SEQ ID NO.1 of the wild thermophilic PET hydrolase; preferably, the sequence of the No.1 thermophilic PET hydrolase mutant protein is shown in SEQ ID NO. 4;
and/or the mutant protein is a No.2 thermophilic PET hydrolase mutant protein, the sequence of the mutant protein comprises a mutation from the 63 rd Gly to the Ala and a mutation from the 210 th Phe to the Ile of an amino acid sequence shown in SEQ ID NO.1 of the wild thermophilic PET hydrolase; preferably, the sequence of the No.2 thermophilic PET hydrolase mutant protein is shown in SEQ ID NO. 5;
and/or the mutant protein is a No.3 thermophilic PET hydrolase mutant protein, the sequence of the mutant protein comprises a mutation from the amino acid sequence shown as SEQ ID NO.1 of the wild thermophilic PET hydrolase from the N end to the C end, wherein the 63 rd Gly is changed into Ala, the 210 th Phe is changed into Ile, the 205 th Asp is changed into Cys, and the 254 th Glu is changed into Cys; preferably, the sequence of the No.3 thermophilic PET hydrolase mutant protein is shown in SEQ ID NO. 6;
and/or the mutant protein is a No.4 thermophilic PET hydrolase mutant protein, the sequence of the mutant protein comprises a mutation from the 63 rd Gly to the Ala, the 210 th Phe to the Ile, the 205 th Asp to the Cys, the 254 th Glu to the Cys and the 93 rd Gln to the Gly of the amino acid sequence shown in SEQ ID NO.1 of the wild thermophilic PET hydrolase; preferably, the sequence of the No.4 thermophilic PET hydrolase mutant protein is shown in SEQ ID NO. 7;
and/or, the mutant protein is a No.5 thermophilic PET hydrolase mutant protein, the sequence of the mutant protein comprises a mutation from the amino acid sequence shown as SEQ ID NO.2 of the wild thermophilic PET hydrolase from the N end to the C end, wherein the 63 rd Gly is changed into Ala, the 210 th Phe is changed into Ile, the 205 th Asp is changed into Cys, the 254 th Glu is changed into Cys, and the 93 th Gln is changed into Gly; preferably, the sequence of the No.5 thermophilic PET hydrolase mutant protein is shown in SEQ ID NO. 8;
and/or the mutant protein is a No.6 thermophilic PET hydrolase mutant protein, and the amino acid sequence of the wild thermophilic PET hydrolase shown as SEQ ID NO.3 is changed from the 63 rd Gly to the Ala, the 210 th Phe to the Ile, the 205 th Asp to the Cys, the 254 th Glu to the Cys and the 93 th Gln to the Gly; preferably, the sequence of the No.6 thermophilic PET hydrolase mutant protein is shown in SEQ ID NO. 9.
4. A nucleotide molecule encoding the mutant protein of any one of claims 1-3.
5. The nucleotide molecule according to claim 4, characterized in that the nucleotide sequence comprises, in the nucleotide sequence encoding the amino acid sequence of wild thermophilic PET hydrolase, a mutation from C at position 185 to T, a mutation from C at position 186 to G, a mutation from G at position 188 to C, a mutation from T at position 189 to G, a mutation from C at position 277 to G, a mutation from A at position 278 to G, a mutation from G at position 279 to A, a mutation from G at position 279 to T, a mutation from G at position 613 to T, a mutation from A at position 614 to G, a mutation from T at position 615 to C, a mutation from T at position 628 to A, a mutation from T at position 628 to G, a mutation from T at position 629 to G, a mutation from T at position 630 to A, a mutation from T at position 630 to G, a mutation from A at position 637 to G, a mutation from A at position 638 to T, a mutation from T at position 639 to C, a mutation from T at position 639 to G, The 760 th G mutation is T, the 761 th A mutation is G, the 762 th A mutation is C, and the 762 th A mutation is T;
preferably, the nucleotide sequence encoding the amino acid sequence of the wild thermophilic PET hydrolase is the nucleotide sequence shown as SEQ ID No.10 encoding the amino acid sequence shown as SEQ ID No.1 of the wild thermophilic PET hydrolase, or the nucleotide sequence shown as SEQ ID No.11 encoding the amino acid sequence shown as SEQ ID No.2 of the wild thermophilic PET hydrolase, or the nucleotide sequence shown as SEQ ID No.12 encoding the amino acid sequence shown as SEQ ID No.3 of the wild thermophilic PET hydrolase.
6. The nucleotide molecule of claim 5,
the nucleotide molecule is a nucleotide molecule for coding a No.1 thermophilic PET hydrolase mutant protein, the sequence of the nucleotide molecule comprises a 188 th G mutation position from 5 'end to 3' end of a nucleotide sequence shown in SEQ ID NO.10 of an amino acid sequence shown in SEQ ID NO.1 for coding wild thermophilic PET hydrolase, and the 189 th T mutation position is C; preferably, the nucleotide sequence of the nucleotide molecule for coding the No.1 thermophilic PET hydrolase mutant protein is shown as SEQ ID NO. 13;
and/or the nucleotide molecule is a nucleotide molecule for encoding No.2 thermophilic PET hydrolase mutant protein, the sequence of the nucleotide molecule comprises a nucleotide sequence which is positioned in the direction from the 5 'end to the 3' end and is used for encoding the amino acid sequence shown in SEQ ID NO.1 of wild thermophilic PET hydrolase, wherein the 188 th G of the nucleotide sequence is mutated into C, the 189 th T is mutated into C, the 628 th T is mutated into A, and the 630 th T is mutated into A; preferably, the nucleotide sequence of the nucleotide molecule for coding the No.2 thermophilic PET hydrolase mutant protein is shown in SEQ ID NO. 14;
and/or the nucleotide molecule is a nucleotide molecule for coding No.3 thermophilic PET hydrolase mutant protein, the sequence of the nucleotide molecule comprises a nucleotide sequence which is positioned in an amino acid sequence shown as SEQ ID NO.1 and used for coding wild thermophilic PET hydrolase and is provided with a mutation from the 5 'end to the 3' end, wherein the 188 th G position is mutated into C, the 189 th T position is mutated into C, the 613 th G position is mutated into T, the 614 th A position is mutated into G, the 615 th T position is mutated into C, the 628 th position is mutated into A, the 630 th T position is mutated into A, the 760 th G position is mutated into T, the 761A position is mutated into G, and the 762 th A position is mutated into C; preferably, the nucleotide sequence of the nucleotide molecule for coding the No.3 thermophilic PET hydrolase mutant protein is shown as SEQ ID NO. 15;
and/or the nucleotide molecule is a nucleotide molecule for coding a No.4 thermophilic PET hydrolase mutant protein, the sequence of the nucleotide molecule comprises a nucleotide sequence which is positioned in an amino acid sequence shown as SEQ ID NO.1 and used for coding a wild thermophilic PET hydrolase and is characterized in that the 188 th G position in the direction from the 5 'end to the 3' end is mutated into C, the 189 th T position is mutated into C, the 277 th C position is mutated into G, the 278 th A position is mutated into G, the 279 th G position is mutated into A, the 613 th G position is mutated into T, the 614 th A position is mutated into G, the 615 th T position is mutated into C, the 628 th T position is mutated into A, the 630 th T position is mutated into A, the 760 th G position is mutated into T, the 761A position is mutated into G, and the 762 th A position is mutated into C; preferably, the nucleotide sequence of the nucleotide molecule for coding the No.4 thermophilic PET hydrolase mutant protein is shown as SEQ ID NO. 16;
and/or the nucleotide molecule is a nucleotide molecule for coding a No.5 thermophilic PET hydrolase mutant protein, the sequence of the nucleotide molecule comprises a nucleotide sequence which is positioned in an amino acid sequence shown as SEQ ID NO.2 for coding a wild thermophilic PET hydrolase and is provided with a mutation from the 5 'end to the 3' end, wherein the 188 th G position is mutated into C, the 189 th T position is mutated into C, the 277 th C position is mutated into G, the 278 th A position is mutated into G, the 279 th G position is mutated into T, the 613 th G position is mutated into T, the 614 th A position is mutated into G, the 628 th T position is mutated into A, the 760 th G position is mutated into T, the 761A position is mutated into G, and the 762 th A position is mutated into T; preferably, the nucleotide sequence of the nucleotide molecule for coding the No.5 thermophilic PET hydrolase mutant protein is shown as SEQ ID NO. 17;
and/or the nucleotide molecule is a nucleotide molecule for coding a No.6 thermophilic PET hydrolase mutant protein, the nucleotide sequence comprises a nucleotide sequence which is positioned in an amino acid sequence shown as SEQ ID NO.3 for coding a wild thermophilic PET hydrolase and is provided with a mutation from 5 'end to 3' end of the nucleotide sequence shown as SEQ ID NO.12, wherein the 188 th G position is mutated into C, the 189 th T position is mutated into G, the 277 th C position is mutated into G, the 278 th A position is mutated into G, the 279 th G position is mutated into T, the 613 th G position is mutated into T, the 614 th A position is mutated into G, the 628 th T position is mutated into A, the 761 th A position is mutated into T, and the 762 th A position is mutated into T; preferably, the nucleotide sequence of the nucleotide molecule for coding the No.6 thermophilic PET hydrolase mutant protein is shown as SEQ ID NO. 18.
7. The nucleotide molecule according to claim 6, wherein the nucleotide molecule is a DNA molecule comprising:
(b1) the coding region comprises DNA molecules of nucleotide sequences shown as SEQ ID NO.13, SEQ ID NO14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO. 18;
(b2) DNA molecule with nucleotide sequence shown in SEQ ID NO.13, SEQ ID NO14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO. 18;
(b3) a DNA molecule having 75% or more 75% or identity to the nucleotide sequence set forth in (b1) or (b2) and encoding the protein set forth in claim 3;
(b4) a DNA molecule which hybridizes under stringent conditions to the nucleotide sequence set forth in (b1) or (b2) and which encodes the protein set forth in claim 2 to 3.
8. An expression cassette, recombinant vector or recombinant microorganism comprising the nucleotide molecule of any one of claims 4-7.
9. Use of a mutant protein according to any one of claims 2-3 or encoded by a nucleotide molecule according to any one of claims 4-7 or produced by an expression cassette, recombinant vector or recombinant microorganism according to claim 8 for hydrolysis of materials containing ester and amide bonds.
10. The use according to claim 9, wherein the material containing ester and amide bonds comprises one or more of a polyethylene terephthalate material, a polylactic acid material and a polyurethane material.
CN202210771873.7A 2022-06-30 2022-06-30 Mutant protein of thermophilic PET hydrolase and application thereof Active CN115029332B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108588052A (en) * 2018-05-11 2018-09-28 天津大学 The mutant of PET degrading enzymes and its application
CN112301015A (en) * 2020-11-03 2021-02-02 江南大学 Method for promoting extracellular expression of protein in bacillus subtilis by using cutinase
CN113684196A (en) * 2021-08-28 2021-11-23 北京化工大学 Purification method of high-temperature-resistant polyethylene terephthalate hydrolase

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108588052A (en) * 2018-05-11 2018-09-28 天津大学 The mutant of PET degrading enzymes and its application
CN112301015A (en) * 2020-11-03 2021-02-02 江南大学 Method for promoting extracellular expression of protein in bacillus subtilis by using cutinase
CN113684196A (en) * 2021-08-28 2021-11-23 北京化工大学 Purification method of high-temperature-resistant polyethylene terephthalate hydrolase

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