CN114891767B

CN114891767B - Cutinase-esterase fusion protein and application thereof

Info

Publication number: CN114891767B
Application number: CN202210557888.3A
Authority: CN
Inventors: 王永华; 庞亚星; 蓝东明; 王方华; 杨博
Original assignee: Guangdong Youjiang Biological Manufacturing Research Institute Co ltd; South China University of Technology SCUT
Current assignee: Guangdong Youjiang Biological Manufacturing Research Institute Co ltd; South China University of Technology SCUT
Priority date: 2022-05-19
Filing date: 2022-05-19
Publication date: 2023-04-28
Anticipated expiration: 2042-05-19
Also published as: CN114891767A

Abstract

The invention discloses a cutinase-esterase fusion protein and application thereof, wherein the amino acid sequence of the cutinase-esterase fusion protein is shown as SEQ ID NO.5, and when the cutinase-esterase fusion protein is used for degrading PET, compared with HRC single enzyme and HRC/PCEST double-free enzyme, the degradation efficiency and the yield of a final product terephthalic acid can be remarkably improved. The cutinase-esterase fusion protein has extremely high temperature, pH, metal ion, organic solvent and surfactant tolerance, and has better industrial application prospect.

Description

Cutinase-esterase fusion protein and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and protein expression, and particularly relates to a cutinase-esterase fusion protein (HRC-Linker-PCEST) and application thereof in efficient degradation of PET.

Background

Plastic products are widely used in various fields such as textile manufacturing and packaging materials due to their characteristics of good heat resistance, electrical insulation, biosafety and the like. It is counted by researchers that about 1 million tons of plastic products are produced every year worldwide and the trend is rising year by year. Plastic particles have been found in more than 240 species, and if not emphasized, would necessarily endanger the survival of humans themselves.

Polyesters are polymers obtained by polycondensation of polyols and polyacids, generally linear thermoplastic resins. PET (polyethylene terephthalate) is a common plastic from petroleum, and is polymerized from TPA (terephthalic acid) and EG (ethylene glycol) via an ester linkage. PET is widely used in packaging, construction, electrical and other industries, such as beverage bottles, films and engineering plastics. PET is widely used and, at the same time, it causes serious environmental pollution due to the difficulty in natural degradation of its wastes. Most plastics become garbage finally, so that serious environmental pollution is caused, and great social attention is paid.

In general, methods for degrading PET are mainly classified into physical methods including high Wen Chongsu and the like, chemical methods including alcoholysis ammonolysis and the like, and biological methods. These methods are based on high temperature or extreme chemical agents, and have high degradation costs and are prone to additional environmental pollutants.

With the development of biotechnology, the biodegradable PET has great application prospect. Compared with chemical degradation, the biological method not only greatly reduces degradation cost, but also does not produce additional pollutants. Biological methods degrade PET macromolecular polymers mainly by microbial action. Since the PET macromolecules cannot enter the microorganism, the microorganism needs to degrade the PET polymers into water-soluble small molecules with small molecular weight by secreting some extracellular degrading enzymes, and the water-soluble small molecules are absorbed into the microorganism again, and finally hydrolyzed into substances such as water, carbon dioxide and the like by further digestion of in-vivo enzymes. From these microorganisms, researchers isolated and identified functional enzymes that exert degradation in vitro, and found that these degrading enzymes can hydrolyze PET into raw material components such as MHET (ethylene terephthalate), TPA, EG, etc. The effect of the degrading enzymes not only solves the pollution problem of PET plastics, but also can further recycle raw material components, and is more environment-friendly, so that the microbial enzyme degradation method has a great application prospect.

Researchers use PET or a mimic thereof as a substrate, perform strain screening in compost, isolate a plurality of microorganisms with degradation activity, and in order to further study the biodegradation of PET, the researchers try to isolate microbial enzymes with degradation activity from the degradation strains. In these microbial enzymes, the cutinase and its homologs exhibit higher degradability than other enzymes because the catalytic center of the cutinase does not have a "cap" structure that aids the enzyme in recognizing the PET polymer substrate. Japanese scientists in 2016 isolated and identified a bacterium from PET that had developed natural attack in the landfill (Ideonella sakaiensis) and recombinantly expressed a key hydrolase for degrading PET, designated PETase, the first PET hydrolase named PETase (previous studies have generally used hydrolases such as esterases, lipases, cutinases, etc. to degrade PET). However, the enzyme can only degrade PET slowly at normal temperature, and the degradation efficiency is not high.

PET is a typical polymer, which has a glass transition temperature (T _g ) T of PET in aqueous solution at about 70-75deg.C _g The values will be reduced to 60-65 c (because water molecules enter between the polymer chains, weakening intermolecular hydrogen bonds). The polymer chains are bonded at temperatures above the glass transition temperature (T _g ) The transition to the high-elastic state occurs at a temperature at which the mobility of the polymer chains increases, thereby increasing the accessibility of the enzyme to the intramolecular ester bonds. Thus, higher reaction temperatures will significantly increase the degradation rate of PET. Therefore, in the enzymatic degradation of PET, the temperature resistance to PET hydrolase is particularly high. ACS Sustainable Chemistry in 2020&EnginIn a review by eering, enzymes with PET hydrolysis ability are classified into PET surface modifying enzymes (only capable of hydrolyzing terminal ester bonds on the PET surface) and PETase (significantly eroding the PET film as a whole) based on the efficiency of the enzyme degradation on PET. And it is believed that PETase should have two main characteristics: (1) The temperature tolerance is more than 65 ℃ (preferably more than 70 ℃); (2) The catalytic pocket is capable of containing at least one PET monomer (e.g., MHET). According to this standard, enzymes capable of achieving efficient degradation of PET are very rare.

After enzymatic hydrolysis of PET, the resulting intermediates (BHET and MHET) competitively bind to the enzyme's substrate binding site, thereby inhibiting further degradation of PET. The degradation efficiency can be effectively improved by structurally modifying the existing cutinase, and the enzymolysis of PET is also facilitated by adopting a double-enzyme system which generally comprises polyester degrading enzyme and intermediate product removing enzyme. In 2016, degradation of PET was achieved by PETase-MHETase found by Japanese scientist in Ideonella sakaiensis bacteria. However, this double enzyme can only degrade PET at 37℃and the end product can only reach around 1.5 mM.

Therefore, it is very interesting to further explore more enzymes that can efficiently degrade PET.

Disclosure of Invention

Based on this, it is an object of the present invention to provide a cutinase-esterase fusion protein which can degrade PET efficiently.

The specific technical scheme for realizing the aim of the invention comprises the following steps:

a cutinase-esterase fusion protein has the amino acid sequence shown in SEQ ID No. 5.

The invention also provides application of the cutinase-esterase fusion protein in degrading PET.

The invention also provides a gene for encoding the cutinase-esterase fusion protein, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 6.

The invention also provides application of the gene for encoding the cutinase-esterase fusion protein in degrading PET.

The invention also provides a recombinant expression vector inserted with the gene for encoding the cutinase-esterase fusion protein.

The invention also provides application of the recombinant expression vector inserted with the gene for encoding the cutinase-esterase fusion protein in degrading PET.

The invention also provides a recombinant engineering strain transferred with the recombinant expression vector.

In some embodiments, the host strain of the recombinant engineering strain is escherichia coli.

In some of these embodiments, the E.coli is E.coli BL21 (DE 3).

The invention also provides a preparation method of the cutinase-esterase fusion protein, which comprises the following steps: and (3) carrying out expression and purification on the recombinant engineering strain transferred into the recombinant expression vector to obtain the recombinant engineering strain.

In some of these embodiments, the expression temperature is 18±3 ℃, preferably 18±2 ℃, and the expression time is 20 hours to 22 hours; and/or the purification pH is 8.0 to 9.5, preferably 8.0 to 8.5.

The invention also provides application of the recombinant engineering strain transferred into the recombinant expression vector in degrading PET.

The invention also provides a method for degrading PET, which is to react the cutinase-esterase fusion protein with PET.

Compared with the prior art, the invention has the following beneficial effects:

the cutinase-esterase fusion protein of the invention is obtained by the inventors by skillfully and reasonably connecting fragments of HRC (cutinase) and PCEST (esterase) together according to a rich experience. Compared with HRC single enzyme and HRC/PCEST double free enzyme, the cutinase-esterase fusion protein can obviously improve the degradation efficiency and the yield of the final product terephthalic acid when degrading PET. The cutinase-esterase fusion protein has extremely high temperature, pH, metal ion, organic solvent and surfactant tolerance, and has better industrial application prospect.

Drawings

FIG. 1 shows a pET-30a (+) -HRC positive plasmid map of example 1 of the present invention.

FIG. 2 is a map of pET-23a (+) -PCEST positive plasmid in example 1 of the present invention.

FIG. 3 is a map of pET-23a (+) -HRC-GS6Linker-PCEST positive plasmid constructed in example 1 of the present invention.

FIG. 4 is a graph showing comparison of PET degradation efficiency catalyzed by fusion proteins of different Linker lengths according to example 2 of the present invention.

FIG. 5 shows how HRC-Linker-PCEST fusion proteins expressed by different hosts were examined by SDS-PAGE in example 3 of the present invention, wherein A is E.coli BL21 (DE 3), B is E.coli Sheffer T7, C is E.coli Arcticexpress (DE 3), D is E.coli BL21 Star (DE 3) pLyss, line5 is the pure enzyme of HRC-Linker-PCEST, and the theoretical molecular weight is 64.01kDa.

FIG. 6 is a graph showing the results of an optimum temperature and temperature tolerance test for the HRC-Linker-PCEST fusion protein of example 4 of the present invention; wherein A is an optimal temperature test result; b is the temperature tolerance test result.

FIG. 7 shows the results of the pH optimum and pH tolerance tests for the HRC-Linker-PCEST fusion protein of example 4 of the present invention; wherein A is the optimal pH test result; b is the pH tolerance test result.

FIG. 8 shows the effect of metal ions on HRC-Linker-PCEST fusion protease activity in example 4 of the present invention.

FIG. 9 shows the effect of organic solvents on HRC-Linker-PCEST fusion protease activity in example 4 of the present invention.

FIG. 10 results of the effect of surfactant on HRC-Linker-PCEST fusion protease activity in example 4 of the present invention.

FIG. 11 is a comparison of the amount of TPA produced by degrading PET with HRC-Linker-PCEST fusion protein, HRC single enzyme, PCEST single enzyme, HRC+PCEST double free enzyme in example 5 of the present invention.

FIG. 12 is a comparison result of the yield of the total product MHET+TPA in example 5 of the present invention, wherein the PET is degraded by HRC-Linker-PCEST fusion protein, HRC single enzyme, PCEST single enzyme, and HRC+PCEST double-free enzyme.

Detailed Description

The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

In the invention, the inventor obtains cutinase-esterase fusion protein (in the following examples, the fusion protein is uniformly named as HRC-Linker-PCEST) engineering bacteria through a large number of experimental screening according to abundant experiences; then expressing HRC-Linker-PCEST recombinant protein engineering bacteria, separating and purifying to obtain HRC-Linker-PCEST fusion protein, and exploring relevant factors to obtain optimal fusion protein; finally, the obtained HRC-Linker-PCEST fusion protein is applied to degradation of PET to improve the conversion efficiency of the degradation of PET into the final product TPA. Because the HRC-Linker-PCEST fusion protein has extremely high temperature tolerance (the enzyme activity is still more than 90% after incubation for 6 hours at the optimal reaction temperature of 75 ℃), the HRC-Linker-PCEST fusion protein is more suitable for the glass transition temperature of PET in temperature, is favorable for the damage of PET crystallinity, and has higher degradation efficiency. And the double enzyme coupling further reduces the product inhibition, thereby achieving the effect of degrading PET more efficiently. The invention provides support for reducing the degradation cost of PET and improving the conversion efficiency, aims to enrich the research on the enzymatic degradation application of PET, and also provides theoretical experiment basis for the construction of fusion expression fusion proteins in cells.

The experimental procedure of the present invention, in which no specific conditions are noted in the following examples, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor LaboratoryPress, 1989) or as recommended by the manufacturer.

Materials and reagents used in the following examples: both the cutinase plasmid pET-30a (+) -HRC and the esterase plasmid pET-23a (+) -PCEST are stored in the applicant's laboratory (and can be constructed by conventional methods according to the following gene sequences); coli TOP10, E.coli Shuffle T7, E.coli BL21 (DE 3), E.coli Arcticexpress (DE 3) and E.coli BL21 Star (DE 3) pLySs competent cells, purchased from Biotech only Co., ltd; seamless cloning kit, purchased from zhongmeitai and biotechnology (beijing) limited; plasmid extraction kit, purchased from the division of bioengineering (Shanghai); amorphous PET, available from Goodfelt. Other reagents which are not described are all common chemical reagents and all commercial products.

The amino acid sequence of cutinase HRC (SEQ ID No. 1):

SNPYQRGPNPTRSALTTDGPFSVATYSVSRLSVSGFGGGVIYYPTGTTLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVIVINTNSRLDFPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGATLRISEQIPTLKAGVPLTPWHTDKTFNTPVPQLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELDNATHFAPNSPNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALSDFRSNNRHCQ。

HRC encoding nucleic acid sequence (SEQ ID No. 2):

ATGTCTAACCCGTACCAGCGTGGCCCGAACCCGACCCGTAGCGCGCTGACCACCGACGGCCCGTTCTCTGTTGCGACCTACTCTGTTAGCCGTCTGAGCGTGAGCGGCTTCGGCGGCGGTGTTATCTACTACCCGACCGGCACCACCCTGACCTTCGGCGGCATCGCGATGTCTCCGGGCTACACCGCGGATGCGTCCAGCCTGGCGTGGCTGGGTCGTCGTCTGGCGTCCCACGGCTTCGTTGTTATCGTGATCAACACCAACAGCCGTCTGGATTTCCCGGATAGCCGCGCGAGCCAGCTGTCTGCGGCGCTGAACTACCTGCGTACCTCTTCCCCGAGCGCGGTTCGTGCGCGTCTGGATGCGAACCGTCTGGCGGTGGCAGGCCACTCTATGGGCGGTGGCGCGACCCTGCGTATCAGCGAACAGATCCCGACCCTGAAAGCGGGCGTTCCGCTGACCCCGTGGCACACCGACAAAACCTTCAACACCCCGGTTCCGCAGCTGATCGTTGGTGCGGAAGCGGATACCGTTGCGCCGGTTAGCCAGCACGCGATCCCGTTCTACCAGAACCTGCCGAGCACCACCCCGAAAGTTTACGTTGAACTGGATAACGCGACCCACTTCGCGCCGAACTCTCCGAACGCGGCGATCAGCGTTTACACCATCTCTTGGATGAAACTGTGGGTTGATAACGATACCCGTTACCGTCAGTTCCTGTGCAACGTTAACGATCCGGCGCTGAGCGATTTCCGTAGCAACAACCGTCACTGCCAG

amino acid sequence of esterase PCEST (SEQ ID No. 3):

MPLSPILRQILQQLAAQLQFRPDMDVKTVREQFEKSSLILVKMANEPIHRVEDITIPGRGGPIRARVYRPRDGERLPAVVYYHGGGFVLGSVETHDHVCRRLANLSGAVVVSVDYRLAPEHKFPAAVEDAYDAAKWVADNYDKLGVDNGKIAVAGDSAGGNLAAVTAIMARDRGESFVKYQVLIYPAVNLTGSPTVSRVEYSGPEYVILTADLMAWFGRQYFSKPQDALSPYASPIFADLSNLPPALVITAEYDPLRDEGELYAHLLKTRGVRAVAVRYNGVIHGFVNFYPILEEGREAVSQIAASIKSMAVA

nucleic acid sequence encoding PCEST (SEQ ID No. 4):

ATGCCTCTGTCACCAATCCTGAGACAAATCCTGCAACAACTGGCTGCACAATTGCAGTTTAGACCAGATATGGATGTAAAGACGGTGAGAGAGCAGTTCGAGAAGTCAAGTCTTATCCTGGTGAAGATGGCAAACGAGCCTATCCATAGAGTGGAAGACATCACAATTCCAGGAAGAGGTGGACCTATTAGAGCAAGAGTGTACAGACCAAGAGACGGAGAAAGATTGCCTGCAGTTGTATACTACCATGGAGGTGGATTCGTACTTGGTTCTGTGGAAACTCATGACCACGTTTGTAGACGACTTGCTAACTTGTCCGGAGCTGTTGTTGTATCTGTTGACTACAGGCTAGCACCAGAACACAAATTCCCAGCTGCTGTTGAAGATGCATACGATGCTGCCAAATGGGTAGCTGATAATTACGACAAATTGGGTGTTGACAACGGTAAAATTGCCGTCGCAGGTGACTCAGCAGGTGGTAACTTAGCAGCTGTTACAGCTATTATGGCTCGTGATCGTGGAGAATCATTTGTCAAGTACCAGGTGCTAATATATCCCGCTGTTAACTTGACCGGTTCTCCAACTGTTTCCCGTGTTGAATATTCCGGACCTGAATACGTTATTCTTACTGCCGATCTAATGGCCTGGTTTGGTAGGCAGTATTTCTCCAAACCTCAAGATGCTTTGTCTCCCTATGCCAGTCCTATATTTGCTGACTTGTCTAATCTTCCCCCTGCCTTGGTCATTACCGCTGAGTATGATCCATTAAGGGATGAGGGCGAGTTATATGCCCACTTGTTAAAGACTAGGGGCGTTCGAGCTGTCGCTGTTCGTTATAATGGGGTCATTCATGGCTTTGTCAATTTCTATCCAATATTAGAGGAAGGGCGAGAAGCCGTCAGTCAAATTGCTGCTAGTATTAAGTCTATGGCCGTCGCC

amino acid sequence of HRC-Linker-PCEST fusion protein (SEQ ID No. 5):

MSNPYQRGPNPTRSALTTDGPFSVATYSVSRLSVSGFGGGVIYYPTGTTLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVIVINTNSRLDFPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGATLRISEQIPTLKAGVPLTPWHTDKTFNTPVPQLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELDNATHFAPNSPNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALSDFRSNNRHCQGGSGGSMPLSPILRQILQQLAAQLQFRPDMDVKTVREQFEKSSLILVKMANEPIHRVEDITIPGRGGPIRARVYRPRDGERLPAVVYYHGGGFVLGSVETHDHVCRRLANLSGAVVVSVDYRLAPEHKFPAAVEDAYDAAKWVADNYDKLGVDNGKIAVAGDSAGGNLAAVTAIMARDRGESFVKYQVLIYPAVNLTGSPTVSRVEYSGPEYVILTADLMAWFGRQYFSKPQDALSPYASPIFADLSNLPPALVITAEYDPLRDEGELYAHLLKTRGVRAVAVRYNGVIHGFVNFYPILEEGREAVSQIAASIKSMAVA

nucleic acid sequence encoding HRC-Linker-PCEST fusion protein (SEQ ID No. 6):

ATGTCTAACCCGTACCAGCGTGGCCCGAACCCGACCCGTAGCGCGCTGACCACCGACGGCCCGTTCTCTGTTGCGACCTACTCTGTTAGCCGTCTGAGCGTGAGCGGCTTCGGCGGCGGTGTTATCTACTACCCGACCGGCACCACCCTGACCTTCGGCGGCATCGCGATGTCTCCGGGCTACACCGCGGATGCGTCCAGCCTGGCGTGGCTGGGTCGTCGTCTGGCGTCCCACGGCTTCGTTGTTATCGTGATCAACACCAACAGCCGTCTGGATTTCCCGGATAGCCGCGCGAGCCAGCTGTCTGCGGCGCTGAACTACCTGCGTACCTCTTCCCCGAGCGCGGTTCGTGCGCGTCTGGATGCGAACCGTCTGGCGGTGGCAGGCCACTCTATGGGCGGTGGCGCGACCCTGCGTATCAGCGAACAGATCCCGACCCTGAAAGCGGGCGTTCCGCTGACCCCGTGGCACACCGACAAAACCTTCAACACCCCGGTTCCGCAGCTGATCGTTGGTGCGGAAGCGGATACCGTTGCGCCGGTTAGCCAGCACGCGATCCCGTTCTACCAGAACCTGCCGAGCACCACCCCGAAAGTTTACGTTGAACTGGATAACGCGACCCACTTCGCGCCGAACTCTCCGAACGCGGCGATCAGCGTTTACACCATCTCTTGGATGAAACTGTGGGTTGATAACGATACCCGTTACCGTCAGTTCCTGTGCAACGTTAACGATCCGGCGCTGAGCGATTTCCGTAGCAACAACCGTCACTGCCAGGGCGGATCGGGAGGTTCAATGCCTCTGTCACCAATCCTGAGACAAATCCTGCAACAACTGGCTGCACAATTGCAGTTTAGACCAGATATGGATGTAAAGACGGTGAGAGAGCAGTTCGAGAAGTCAAGTCTTATCCTGGTGAAGATGGCAAACGAGCCTATCCATAGAGTGGAAGACATCACAATTCCAGGAAGAGGTGGACCTATTAGAGCAAGAGTGTACAGACCAAGAGACGGAGAAAGATTGCCTGCAGTTGTATACTACCATGGAGGTGGATTCGTACTTGGTTCTGTGGAAACTCATGACCACGTTTGTAGACGACTTGCTAACTTGTCCGGAGCTGTTGTTGTATCTGTTGACTACAGGCTAGCACCAGAACACAAATTCCCAGCTGCTGTTGAAGATGCATACGATGCTGCCAAATGGGTAGCTGATAATTACGACAAATTGGGTGTTGACAACGGTAAAATTGCCGTCGCAGGTGACTCAGCAGGTGGTAACTTAGCAGCTGTTACAGCTATTATGGCTCGTGATCGTGGAGAATCATTTGTCAAGTACCAGGTGCTAATATATCCCGCTGTTAACTTGACCGGTTCTCCAACTGTTTCCCGTGTTGAATATTCCGGACCTGAATACGTTATTCTTACTGCCGATCTAATGGCCTGGTTTGGTAGGCAGTATTTCTCCAAACCTCAAGATGCTTTGTCTCCCTATGCCAGTCCTATATTTGCTGACTTGTCTAATCTTCCCCCTGCCTTGGTCATTACCGCTGAGTATGATCCATTAAGGGATGAGGGCGAGTTATATGCCCACTTGTTAAAGACTAGGGGCGTTCGAGCTGTCGCTGTTCGTTATAATGGGGTCATTCATGGCTTTGTCAATTTCTATCCAATATTAGAGGAAGGGCGAGAAGCCGTCAGTCAAATTGCTGCTAGTATTAAGTCTATGGCCGTCGCC

the invention is described in detail below with reference to the drawings and the specific embodiments.

Example 1 obtaining of HRC-Linker-PCEST fusion proteins

The embodiment firstly constructs an HRC-Linker gene fragment, then performs seamless cloning with PCEST to obtain an HRC-Linker-PCEST engineering bacterium, and specifically comprises the following steps:

1. construction of HRC-Linker Gene fragments

The HRC-Linker gene fragment is obtained by PCR amplification and PCR product purification by taking pET-30a (+) -HRC (structure shown in figure 1) as a template and taking SEQ ID NO.7 and SEQ ID NO.8 as primers.

The specific primer sequences are as follows:

N-HRC-F(SEQ ID NO.7)：ATGTCCCCGTACCAGTAACGT

N-HRC-R(SEQ ID NO.8)：

TGAACCTCCCGATCCGCCCTGGCAGTGACGGTTGTTGCT

note that: the underlined part is GS6Linker nucleic acid sequence

PCR amplification system: 2 XPrime STARMAX 25. Mu. L, prime-F/R1. Mu.L each, template DNA 1. Mu. L, ddH ₂ O makes up 50. Mu.L.

PCR amplification procedure: pre-denaturation: 98 ℃ for 3min;30 Xcycle (denaturation at 98℃for 30s; annealing at 60℃for 30s; extension at 72 ℃ (1 kb/min)); extending at 72deg.C for 5min.

2. Engineering bacteria for constructing HRC-Linker-PCEST

And (3) using pET-23a (+) -PCEST (structure shown in figure 2) as a template, using SEQ ID NO.9 and SEQ ID NO.10 as primers, performing PCR amplification (a reaction system and a reaction procedure are the same as those in step 1) and purifying a PCR product, and performing seamless cloning with an HRC-Linker gene fragment to obtain the pET-23a (+) -HRC-GS6Linker-PCEST positive plasmid (structure shown in figure 3). And then transferring into TOP10 clone strain, performing colony PCR verification, performing sequencing verification on the correct bacterial liquid, extracting the plasmid (fusion protein recombinant plasmid) with accurate sequencing, and then transferring into escherichia coli BL21 (DE 3) for recombinant protein expression.

C-PCEST-F(SEQ ID NO.9)：

GCGGGATCGGGAGGTTCAATGCCTCTGTCACCAATCCTG

C-PCEST-R(SEQ ID NO.10)：

GGCCACGCTGGTACGGGTTAGACATGTGGTGGTGGTGGTGGTGCATAT

Note that: the underlined part is GS6Linker nucleic acid sequence

The system of seamless cloning is: 5. Mu.L of mix, 5. Mu.L of DNA in-Fusion enzyme, where mix is n (HRC-Linker gene fragment): n (linearized pET-23a (+) -PCEST vector) =3: 1.

the procedure for seamless cloning was: 50 ℃ for 1h.

The amplification system of colony PCR is: 2 XTaq mix 7.5 mu L, prime-F/R (SEQ ID NO.9 and SEQ ID NO. 10) each 0.6 mu L, ddH ₂ O makes up 10. Mu.L.

The amplification procedure for colony PCR was: pre-denaturation: 94 ℃ for 4min;30 Xcycle (denaturation 94℃for 30s; annealing 60℃for 30s; extension 72 ℃ (1 kb/min)); extending at 72deg.C for 5min.

3. Expression and purification of fusion proteins

(1) The obtained fusion protein recombinant plasmid pET-23a (+) -HRC-GS6Linker-PCEST is introduced into E.coli BL21 (DE 3) by a heat shock transformation method, and after the obtained positive clone is amplified and cultured, IPTG (final concentration is 0.2 mM) is added for induction expression, and the culture is carried out for 20 hours at 20 ℃. And after the fermentation process is finished, obtaining the HRC-Linker-PCEST fusion protein crude enzyme.

Purifying the HRC-Linker-PCEST fusion protein crude enzyme by using an affinity chromatography technology, expressing E.coli BL21 (DE 3) to obtain enzyme protein with purity higher than 95%, collecting eluted protein, concentrating and split charging, quick freezing by using liquid nitrogen, and preserving at-20 ℃ for later use;

(2) Detecting the molecular weight of the obtained protein by SDS-PAGE electrophoresis, wherein the electrophoresis result shows that the molecular weight of the target band is consistent with the theoretical molecular weight; the protein concentration obtained was determined using the Bradford kit, and the HRC-PCEST fusion protein pure enzyme concentration was 1.75mg/mL, containing about 10% of the protein of interest.

EXAMPLE 2 construction and screening of fusion proteins of different Linker lengths

1. Fusion proteins of Linker (GGSGGS) length 6, linker (GGGSGGSGSG) length 10 and Linker (GGGSGGSGGGSGGSGGGS) length 18 were constructed, respectively, following the procedure of example 1. For convenience, the different length Linker is hereinafter denoted as GS6, GS10 and GS18, respectively. The required primers are shown in Table 1.

TABLE 1 major primers for the design of fusion proteins

2. A100 mL reaction flask contained an appropriate amount (250 mg) of PET powder followed by 49mL of 0.1M PBS (K) pH 8.0 ⁺ ) Buffer and 0.5mL of GS6, GS10 or GS18 fusion protein pure enzyme solution HRC-Linker-PCEST (0.3 mg/mL), and the change of degradation efficiency of different fusion proteins is detected by timing sampling. After the reaction is finished, the reaction is stopped on ice, and the conversion rate of each product is analyzed and compared.

As shown in fig. 4, it is clear from fig. 4 that the conversion rate of the fusion protein HRC-GS6-PCEST with the length of 6 of the Linker to the soluble intermediate MHET is the fastest in the first 10 hours, and when the reaction is approaching equilibrium, the MHET conversion rate in the system is only 26.82%, while the fusion proteins HRC-GS10-PCEST and HRC-GS18-PCEST with the length of 10 of the Linker and the length of 18 of the Linker reach about 40% as the cumulative MHET conversion rate; the conversion rate of the final product TPA generated by degrading PET by HRC-GS6-PCEST reaches 56.07%, the conversion rate of the final product TPA generated by HRC-GS10-PCEST and HRC-GS18-PCEST is only between 30% and 40%, and the conversion rate of the final product TPA generated by degrading PET by HRC-GS6-PCEST is faster than that of the final product TPA generated by degrading PET by HRC-GS10-PCEST and HRC-GS18-PCEST in the reaction process.

The results of this example demonstrate that the effect of the constructed fusion protein HRC-Linker-PCEST on PET degradation is optimal when the Linker is GGSGGS.

Example 3 expression of HRC-Linker-PCEST fusion proteins in different expression hosts

In order to compare the expression conditions of fusion proteins in different expression hosts, the fusion protein recombinant plasmids pET-23a (+) -HRC-GS6Linker-PCEST obtained in the step 2 of the embodiment 1 are respectively introduced into E.coli buffer T7, E.coli BL21 (DE 3), E.coli Arctricexpress (DE 3) and E.coli BL21 Star (DE 3) pLyss, induced expression and purification are carried out according to the method of the embodiment 1, and after sample preparation is carried out on partial samples left in the purification process, SDS-PAGE protein electrophoresis is carried out, the loading amounts of HRC-PCEST eluents expressed by four different expression hosts are controlled to be the same, and the optimal expression hosts are screened by comparing the soluble expression amounts and the purification conditions.

As a result, as shown in FIG. 5, it is understood from FIG. 5 that the fusion protein has soluble expression in the expression host E.coli BL21 (DE 3), and that a purer protein can be obtained after purification by His PrepTM FF16/10 chromatography column. E.coli BL21 (DE 3) is therefore preferred for the expression host of the fusion protein HRC-Linker-PCEST.

Example 4 characterization of the enzymatic Properties of the HRC-Linker-PCEST fusion protein

The enzymatic properties of the HRC-Linker-PCEST fusion proteins were analyzed in this example. The method specifically comprises the influence of temperature, pH, metal ions, organic solvents and surfactants on the activity of HRC-Linker-PCEST hydrolase.

The cutinase hydrolyzes p-nitrophenol ester (pNPB) substrate to produce p-nitrophenol (pNP) and acid hydrolysis products by reaction. pNP was detectable at Abs405 nm. Based on the principle, the enzyme activity of the HRC selected by the invention is detected by taking the p-nitrophenyl butyrate as a reference substrate. The hydrolase activity is defined as: under certain conditions, the amount of enzyme required to hydrolyze pNPB to produce 1. Mu. Mol of p-nitrophenol per unit time (min) is one activity unit (U).

The 100 mu L reaction system comprises 80 mu LPBS buffer solution, 10 mu L10 mM pNPB and 10 mu L enzyme solution, after other experiments react for 5min at 75 ℃ except for temperature influence experiments, absolute ethyl alcohol which is equal to the system in volume is added for stopping the reaction, after cooling treatment, an enzyme label instrument is used for measuring the OD value of pNP at Abs405 nm, 3 parallel group controls are arranged for each protein sample, and the blank group is 10 mu L of protein denaturing enzyme solution. The average value of each group of parallel samples was recorded.

Determination of a standard curve: 0,1,2,4,6,8 and 10 mu L of p-nitrophenol with the concentration of 10mM are respectively added into a 96-well ELISA plate, 3 parallel groups are arranged for each concentration, buffer solution is added to fill up each well of solution to 100 mu L, absolute ethyl alcohol with the same volume as the system is added to terminate the reaction after the reaction is finished, three technical parallels are adopted for each group of data, and the linear relation between the absorbance (Y axis) and the concentration (X axis) of pNP at 405nm is measured.

1. Influence of temperature on HRC-Linker-PCEST hydrolase Activity

The method comprises the steps of taking 5 ℃ as an interval, respectively selecting six temperature gradients of 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃ and 90 ℃ to measure the optimal reaction temperature, taking pNPB as a substrate in a measuring system, and measuring the enzyme activity by adopting the method. Three techniques were performed in parallel for each set of data, and the average of the results measured was the relative enzyme activity. The results are shown as a in fig. 6: the HRC-Linker-PCEST has an optimum temperature of 75℃and retains 90% of its activity when the temperature is raised to 80℃indicating that the HRC-Linker-PCEST is a high temperature enzyme.

Based on the optimum reaction temperature, the thermal stability of the HRC-Linker-PCEST is measured, the HRC-Linker-PCEST fusion protein sample is incubated at three temperature gradients (70 ℃, 75 ℃ and 80 ℃) and sampled at fixed intervals, the hydrolase activity of each sampling point is measured to reflect the temperature tolerance, and the measured result is the relative enzyme activity. The results are shown in fig. 6B: after incubation for 30 hours at 70 ℃ and 75 ℃, relative enzyme activities of 95% and 77% can be still maintained, and under the environment of high temperature of 80 ℃, the half life of HRC-GS6-PCEST can still reach more than 30 hours. The enzyme is in a solution environment with the glass transition temperature (Tg=60-65 ℃) higher than PET, so that intermolecular forces are greatly weakened, and the collision of the enzyme and PET ester bonds is facilitated. Therefore, the HRC-Linker-PCEST fusion protein capable of tolerating high temperature of 70 ℃ or higher for a long time has great advantages in the aspect of efficiently degrading PET.

2. Influence of pH on HRC-Linker-PCEST hydrolase Activity

Respectively selecting buffers with pH 6.0,7.0,7.5,8.0,8.5,9.0,9.5, taking pNPB as a substrate, and measuring the enzyme activity by adopting the method under the condition of the optimal reaction temperature of 75 ℃. Three techniques were performed in parallel for each set of data, and the average of the results measured was expressed as relative enzyme activity. The results are shown as a in fig. 7: the optimum reaction pH for HRC-PCEST was 8.5, and the pH was extended to 9.5, with more than 80% activity remaining. In contrast, the hydrolysis activity of HRC-Linker-PCEST at pH 6.0 was reduced to 18%, indicating that HRC-Linker-PCEST is an alkaline enzyme.

HRC-Linker-PCEST enzyme solutions were placed in buffers of different pH values (pH 6.0,7.0,7.5,8.0,8.5,9.0,9.5, 10), respectively, and after incubation at 4 ℃ for 24 hours, their hydrolase activities were determined. Three techniques were performed in parallel for each set of data, and the average of the results measured was the relative enzyme activity. The results are shown in fig. 7B: the HRC-Linker-PCEST is incubated for a long time within the pH range of 7.0-9.5, and still maintains more than 70% of residual activity, which indicates that the HRC-Linker-PCEST has very good pH tolerance.

3. Influence of Metal ions on HRC-Linker-PCEST hydrolase Activity

ZnCl, KCl, cuCl is added to HRC-Linker-PCEST protein samples respectively ₂ 、MgCl ₂ 、CaCl ₂ 、MnCl ₂ 、NiCl ₂ 、FeCl ₃ And EDTA (final concentration 10 mM), and after incubation at 4℃for 24 hours, the hydrolase activity was measured by the method described above. Three techniques were performed in parallel for each set of data, and the average of the results measured was the relative enzyme activity.

The results are shown in FIG. 8: zn (zinc) ²⁺ 、K ⁺ 、Cu ²⁺ The existence of the reaction system has a certain activating effect on the HRC-Linker-PCEST, and the maximum hydrolysis activity can reach 109%. Mg of ²⁺ 、Ca ²⁺ Has little effect on the enzymatic activity of HRC-Linker-PCEST. In contrast, HRC-Linker-PCEST is found in Mn ²⁺ 、Ni ²⁺ 、Fe ³⁺ In the presence, the minimum residual enzyme activity was 88%. The HRC-Linker-PCEST has better tolerance to most metal ions, and the hrC-Linker-PCEST can be further deduced that the enzyme does not need the participation of metal ions in the catalytic reaction. The fusion proteins therefore do not fall into the category of metal-dependent enzymes. The HRC-Linker-PCEST fusion protein has better tolerance to metal ions and better industrial application prospect.

4. Influence of organic solvent on HRC-Linker-PCEST hydrolase Activity

Methanol, ethanol, isopropanol, acetone, DMF and DMSO (final concentration 10% (v/v)) were added to the HRC-Linker-PCEST protein sample, respectively, and incubated at 4℃for 24 hours, and then the hydrolase activity was measured by the above method. Three techniques were performed in parallel for each set of data, and the average of the results measured was the relative enzyme activity.

The results are shown in FIG. 9: after the HRC-Linker-PCEST is incubated for 24 hours at 4 ℃ in 10% (v/v) of organic solvent, all six organic reagents have influence on the enzyme activity, and DMF has the greatest weakening effect on the enzyme activity, but still retains at least 85% of the activity. The HRC-Linker-PCEST fusion protein has better tolerance to organic solvents and better industrial application prospect.

5. Effect of surfactant on HRC-Linker-PCEST hydrolase Activity

SDS, triton X-100, tween 20 and Tween 80 (final concentration is 1% (m/v)) are added to the HRC-Linker-PCEST protein sample, and the sample is incubated at 4 ℃ for 24 hours, and then the hydrolase activity of the sample is determined by the method. Three techniques were performed in parallel for each set of data, and the average of the results measured was the relative enzyme activity.

The results are shown in FIG. 10: after HRC-Linker-PCEST is incubated for 24 hours at 4 ℃ in 1% (w/v) common surfactant, triton X-100, tween 20 and Tween 80 are used as nonionic surfactants to have adverse effects on enzyme activity, wherein the reduction range of the Tween 20 on the enzyme activity is large, but the residual enzyme activity still reaches 83%. The HRC-Linker-PCEST fusion protein has better tolerance to the surfactant and better industrial application prospect.

Example 5 efficiency experiment of degradation of PET by the HRC-Linker-PCEST fusion protein of the invention

1. Concentrating the purified HRC-Linker-PCEST fusion protein, HRC single enzyme and PCEST single enzyme to 1mg/mL to obtain enzyme solution.

2. The reaction substrate was purchased from Goodfall as amorphous PET (Amorphous Polyethylene Terephthalate) (250 μm, product number ES 301445) (with a crystallinity of about 6%), cut into strips of length 5mm and width 2mm, snap frozen in liquid nitrogen, ground with an overspeed grinder equipped with a 500 μm screen, and screened to obtain amorphous PET powder with a particle size of less than 500 μm.

3. 250mg of amorphous PET powder was weighed into a 100mL Erlenmeyer flask, and 49mL of 50mM glycine-sodium hydroxide buffer (containing 300mM NaCl) at pH 8.0 and 1mL of enzyme solution (each 0.5mL of HRC+PCEST double-free enzyme) were added to make a total volume of 50mL. At 75℃the reaction was carried out at 600rpm, samples were taken at intervals, and A260 was measured after appropriate dilution.

4. The results of comparing the amount of TPA produced by the single enzyme, the double free enzyme and the HRC-Linker-PCEST fusion protein with respect to the degradation end product of amorphous PET are shown in FIG. 11: the ability of the double free enzyme to catalyze PET degradation is better than that of the single enzyme, while the ability of the HRC-Linker-PCEST fusion protein to catalyze PET degradation is obviously better than that of the single enzyme or the double free enzyme, the amount of the final product generated by catalyzing PET degradation is 21.55mM, the amount of the final product generated by the double free enzyme HRC/PCEST is 12.00mM, and the amount of the final product generated by the HRC single enzyme is only 7.84mM.

As shown in FIG. 12, after the HRC-GS6-PCEST catalyzes the degradation reaction of PET for 36 hours, HPLC detection shows that 728.90 mu mol of TPA and 348.65 mu mol of MHET product are generated after degradation, and the degradation rate of PET is calculated to be 82.9% by referring to the added substrate concentration (1 mg of PET contains about 5.2 mu mol of MHET unit and 250mg of PET contains about 1300 mu mol of MHET), which is significantly higher than the hydrolysis efficiency of HRC single enzyme and double free enzyme HRC/PCEST (the hydrolysis efficiency of HRC single enzyme is 30.2% and the hydrolysis efficiency of double free enzyme HRC/PCEST is 46.1%).

Knott et al constructed PETase-MHETase fusion protein in 2020, which is the fusion protein with the best effect reported at present and can degrade PET, and the fusion protein reacts with PET substrate for 96 hours at 30 ℃ to generate about 1.5mM final product TPA, compared with HRC-Linker-PCEST, the fusion protein has better degradation effect.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Sequence listing

<110> Guangdong Uyo enzyme Biomanufacturing Co Ltd at university of North China

<120> cutinase-esterase fusion protein and use thereof

<130> 1

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 258

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Ser Asn Pro Tyr Gln Arg Gly Pro Asn Pro Thr Arg Ser Ala Leu Thr

1 5 10 15

Thr Asp Gly Pro Phe Ser Val Ala Thr Tyr Ser Val Ser Arg Leu Ser

20 25 30

Val Ser Gly Phe Gly Gly Gly Val Ile Tyr Tyr Pro Thr Gly Thr Thr

35 40 45

Leu Thr Phe Gly Gly Ile Ala Met Ser Pro Gly Tyr Thr Ala Asp Ala

50 55 60

Ser Ser Leu Ala Trp Leu Gly Arg Arg Leu Ala Ser His Gly Phe Val

65 70 75 80

Val Ile Val Ile Asn Thr Asn Ser Arg Leu Asp Phe Pro Asp Ser Arg

85 90 95

Ala Ser Gln Leu Ser Ala Ala Leu Asn Tyr Leu Arg Thr Ser Ser Pro

100 105 110

Ser Ala Val Arg Ala Arg Leu Asp Ala Asn Arg Leu Ala Val Ala Gly

115 120 125

His Ser Met Gly Gly Gly Ala Thr Leu Arg Ile Ser Glu Gln Ile Pro

130 135 140

Thr Leu Lys Ala Gly Val Pro Leu Thr Pro Trp His Thr Asp Lys Thr

145 150 155 160

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

165 170 175

Val Ala Pro Val Ser Gln His Ala Ile Pro Phe Tyr Gln Asn Leu Pro

180 185 190

Ser Thr Thr Pro Lys Val Tyr Val Glu Leu Asp Asn Ala Thr His Phe

195 200 205

Ala Pro Asn Ser Pro Asn Ala Ala Ile Ser Val Tyr Thr Ile Ser Trp

210 215 220

Met Lys Leu Trp Val Asp Asn Asp Thr Arg Tyr Arg Gln Phe Leu Cys

225 230 235 240

Asn Val Asn Asp Pro Ala Leu Ser Asp Phe Arg Ser Asn Asn Arg His

245 250 255

Cys Gln

<210> 2

<211> 777

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atgtctaacc cgtaccagcg tggcccgaac ccgacccgta gcgcgctgac caccgacggc 60

ccgttctctg ttgcgaccta ctctgttagc cgtctgagcg tgagcggctt cggcggcggt 120

gttatctact acccgaccgg caccaccctg accttcggcg gcatcgcgat gtctccgggc 180

tacaccgcgg atgcgtccag cctggcgtgg ctgggtcgtc gtctggcgtc ccacggcttc 240

gttgttatcg tgatcaacac caacagccgt ctggatttcc cggatagccg cgcgagccag 300

ctgtctgcgg cgctgaacta cctgcgtacc tcttccccga gcgcggttcg tgcgcgtctg 360

gatgcgaacc gtctggcggt ggcaggccac tctatgggcg gtggcgcgac cctgcgtatc 420

agcgaacaga tcccgaccct gaaagcgggc gttccgctga ccccgtggca caccgacaaa 480

accttcaaca ccccggttcc gcagctgatc gttggtgcgg aagcggatac cgttgcgccg 540

gttagccagc acgcgatccc gttctaccag aacctgccga gcaccacccc gaaagtttac 600

gttgaactgg ataacgcgac ccacttcgcg ccgaactctc cgaacgcggc gatcagcgtt 660

tacaccatct cttggatgaa actgtgggtt gataacgata cccgttaccg tcagttcctg 720

tgcaacgtta acgatccggc gctgagcgat ttccgtagca acaaccgtca ctgccag 777

<210> 3

<211> 313

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Met Pro Leu Ser Pro Ile Leu Arg Gln Ile Leu Gln Gln Leu Ala Ala

1 5 10 15

Gln Leu Gln Phe Arg Pro Asp Met Asp Val Lys Thr Val Arg Glu Gln

20 25 30

Phe Glu Lys Ser Ser Leu Ile Leu Val Lys Met Ala Asn Glu Pro Ile

35 40 45

His Arg Val Glu Asp Ile Thr Ile Pro Gly Arg Gly Gly Pro Ile Arg

50 55 60

Ala Arg Val Tyr Arg Pro Arg Asp Gly Glu Arg Leu Pro Ala Val Val

65 70 75 80

Tyr Tyr His Gly Gly Gly Phe Val Leu Gly Ser Val Glu Thr His Asp

85 90 95

His Val Cys Arg Arg Leu Ala Asn Leu Ser Gly Ala Val Val Val Ser

100 105 110

Val Asp Tyr Arg Leu Ala Pro Glu His Lys Phe Pro Ala Ala Val Glu

115 120 125

Asp Ala Tyr Asp Ala Ala Lys Trp Val Ala Asp Asn Tyr Asp Lys Leu

130 135 140

Gly Val Asp Asn Gly Lys Ile Ala Val Ala Gly Asp Ser Ala Gly Gly

145 150 155 160

Asn Leu Ala Ala Val Thr Ala Ile Met Ala Arg Asp Arg Gly Glu Ser

165 170 175

Phe Val Lys Tyr Gln Val Leu Ile Tyr Pro Ala Val Asn Leu Thr Gly

180 185 190

Ser Pro Thr Val Ser Arg Val Glu Tyr Ser Gly Pro Glu Tyr Val Ile

195 200 205

Leu Thr Ala Asp Leu Met Ala Trp Phe Gly Arg Gln Tyr Phe Ser Lys

210 215 220

Pro Gln Asp Ala Leu Ser Pro Tyr Ala Ser Pro Ile Phe Ala Asp Leu

225 230 235 240

Ser Asn Leu Pro Pro Ala Leu Val Ile Thr Ala Glu Tyr Asp Pro Leu

245 250 255

Arg Asp Glu Gly Glu Leu Tyr Ala His Leu Leu Lys Thr Arg Gly Val

260 265 270

Arg Ala Val Ala Val Arg Tyr Asn Gly Val Ile His Gly Phe Val Asn

275 280 285

Phe Tyr Pro Ile Leu Glu Glu Gly Arg Glu Ala Val Ser Gln Ile Ala

290 295 300

Ala Ser Ile Lys Ser Met Ala Val Ala

305 310

<210> 4

<211> 939

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

atgcctctgt caccaatcct gagacaaatc ctgcaacaac tggctgcaca attgcagttt 60

agaccagata tggatgtaaa gacggtgaga gagcagttcg agaagtcaag tcttatcctg 120

gtgaagatgg caaacgagcc tatccataga gtggaagaca tcacaattcc aggaagaggt 180

ggacctatta gagcaagagt gtacagacca agagacggag aaagattgcc tgcagttgta 240

tactaccatg gaggtggatt cgtacttggt tctgtggaaa ctcatgacca cgtttgtaga 300

cgacttgcta acttgtccgg agctgttgtt gtatctgttg actacaggct agcaccagaa 360

cacaaattcc cagctgctgt tgaagatgca tacgatgctg ccaaatgggt agctgataat 420

tacgacaaat tgggtgttga caacggtaaa attgccgtcg caggtgactc agcaggtggt 480

aacttagcag ctgttacagc tattatggct cgtgatcgtg gagaatcatt tgtcaagtac 540

caggtgctaa tatatcccgc tgttaacttg accggttctc caactgtttc ccgtgttgaa 600

tattccggac ctgaatacgt tattcttact gccgatctaa tggcctggtt tggtaggcag 660

tatttctcca aacctcaaga tgctttgtct ccctatgcca gtcctatatt tgctgacttg 720

tctaatcttc cccctgcctt ggtcattacc gctgagtatg atccattaag ggatgagggc 780

gagttatatg cccacttgtt aaagactagg ggcgttcgag ctgtcgctgt tcgttataat 840

ggggtcattc atggctttgt caatttctat ccaatattag aggaagggcg agaagccgtc 900

agtcaaattg ctgctagtat taagtctatg gccgtcgcc 939

<210> 5

<211> 578

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Met Ser Asn Pro Tyr Gln Arg Gly Pro Asn Pro Thr Arg Ser Ala Leu

1 5 10 15

Thr Thr Asp Gly Pro Phe Ser Val Ala Thr Tyr Ser Val Ser Arg Leu

20 25 30

Ser Val Ser Gly Phe Gly Gly Gly Val Ile Tyr Tyr Pro Thr Gly Thr

35 40 45

Thr Leu Thr Phe Gly Gly Ile Ala Met Ser Pro Gly Tyr Thr Ala Asp

50 55 60

Ala Ser Ser Leu Ala Trp Leu Gly Arg Arg Leu Ala Ser His Gly Phe

65 70 75 80

Val Val Ile Val Ile Asn Thr Asn Ser Arg Leu Asp Phe Pro Asp Ser

85 90 95

Arg Ala Ser Gln Leu Ser Ala Ala Leu Asn Tyr Leu Arg Thr Ser Ser

100 105 110

Pro Ser Ala Val Arg Ala Arg Leu Asp Ala Asn Arg Leu Ala Val Ala

115 120 125

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

130 135 140

Pro Thr Leu Lys Ala Gly Val Pro Leu Thr Pro Trp His Thr Asp Lys

145 150 155 160

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

165 170 175

Thr Val Ala Pro Val Ser Gln His Ala Ile Pro Phe Tyr Gln Asn Leu

180 185 190

Pro Ser Thr Thr Pro Lys Val Tyr Val Glu Leu Asp Asn Ala Thr His

195 200 205

Phe Ala Pro Asn Ser Pro Asn Ala Ala Ile Ser Val Tyr Thr Ile Ser

210 215 220

Trp Met Lys Leu Trp Val Asp Asn Asp Thr Arg Tyr Arg Gln Phe Leu

225 230 235 240

Cys Asn Val Asn Asp Pro Ala Leu Ser Asp Phe Arg Ser Asn Asn Arg

245 250 255

His Cys Gln Gly Gly Ser Gly Gly Ser Met Pro Leu Ser Pro Ile Leu

260 265 270

Arg Gln Ile Leu Gln Gln Leu Ala Ala Gln Leu Gln Phe Arg Pro Asp

275 280 285

Met Asp Val Lys Thr Val Arg Glu Gln Phe Glu Lys Ser Ser Leu Ile

290 295 300

Leu Val Lys Met Ala Asn Glu Pro Ile His Arg Val Glu Asp Ile Thr

305 310 315 320

Ile Pro Gly Arg Gly Gly Pro Ile Arg Ala Arg Val Tyr Arg Pro Arg

325 330 335

Asp Gly Glu Arg Leu Pro Ala Val Val Tyr Tyr His Gly Gly Gly Phe

340 345 350

Val Leu Gly Ser Val Glu Thr His Asp His Val Cys Arg Arg Leu Ala

355 360 365

Asn Leu Ser Gly Ala Val Val Val Ser Val Asp Tyr Arg Leu Ala Pro

370 375 380

Glu His Lys Phe Pro Ala Ala Val Glu Asp Ala Tyr Asp Ala Ala Lys

385 390 395 400

Trp Val Ala Asp Asn Tyr Asp Lys Leu Gly Val Asp Asn Gly Lys Ile

405 410 415

Ala Val Ala Gly Asp Ser Ala Gly Gly Asn Leu Ala Ala Val Thr Ala

420 425 430

Ile Met Ala Arg Asp Arg Gly Glu Ser Phe Val Lys Tyr Gln Val Leu

435 440 445

Ile Tyr Pro Ala Val Asn Leu Thr Gly Ser Pro Thr Val Ser Arg Val

450 455 460

Glu Tyr Ser Gly Pro Glu Tyr Val Ile Leu Thr Ala Asp Leu Met Ala

465 470 475 480

Trp Phe Gly Arg Gln Tyr Phe Ser Lys Pro Gln Asp Ala Leu Ser Pro

485 490 495

Tyr Ala Ser Pro Ile Phe Ala Asp Leu Ser Asn Leu Pro Pro Ala Leu

500 505 510

Val Ile Thr Ala Glu Tyr Asp Pro Leu Arg Asp Glu Gly Glu Leu Tyr

515 520 525

Ala His Leu Leu Lys Thr Arg Gly Val Arg Ala Val Ala Val Arg Tyr

530 535 540

Asn Gly Val Ile His Gly Phe Val Asn Phe Tyr Pro Ile Leu Glu Glu

545 550 555 560

Gly Arg Glu Ala Val Ser Gln Ile Ala Ala Ser Ile Lys Ser Met Ala

565 570 575

Val Ala

<210> 6

<211> 1734

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

atgtctaacc cgtaccagcg tggcccgaac ccgacccgta gcgcgctgac caccgacggc 60

ccgttctctg ttgcgaccta ctctgttagc cgtctgagcg tgagcggctt cggcggcggt 120

gttatctact acccgaccgg caccaccctg accttcggcg gcatcgcgat gtctccgggc 180

tacaccgcgg atgcgtccag cctggcgtgg ctgggtcgtc gtctggcgtc ccacggcttc 240

gttgttatcg tgatcaacac caacagccgt ctggatttcc cggatagccg cgcgagccag 300

ctgtctgcgg cgctgaacta cctgcgtacc tcttccccga gcgcggttcg tgcgcgtctg 360

gatgcgaacc gtctggcggt ggcaggccac tctatgggcg gtggcgcgac cctgcgtatc 420

agcgaacaga tcccgaccct gaaagcgggc gttccgctga ccccgtggca caccgacaaa 480

accttcaaca ccccggttcc gcagctgatc gttggtgcgg aagcggatac cgttgcgccg 540

gttagccagc acgcgatccc gttctaccag aacctgccga gcaccacccc gaaagtttac 600

gttgaactgg ataacgcgac ccacttcgcg ccgaactctc cgaacgcggc gatcagcgtt 660

tacaccatct cttggatgaa actgtgggtt gataacgata cccgttaccg tcagttcctg 720

tgcaacgtta acgatccggc gctgagcgat ttccgtagca acaaccgtca ctgccagggc 780

ggatcgggag gttcaatgcc tctgtcacca atcctgagac aaatcctgca acaactggct 840

gcacaattgc agtttagacc agatatggat gtaaagacgg tgagagagca gttcgagaag 900

tcaagtctta tcctggtgaa gatggcaaac gagcctatcc atagagtgga agacatcaca 960

attccaggaa gaggtggacc tattagagca agagtgtaca gaccaagaga cggagaaaga 1020

ttgcctgcag ttgtatacta ccatggaggt ggattcgtac ttggttctgt ggaaactcat 1080

gaccacgttt gtagacgact tgctaacttg tccggagctg ttgttgtatc tgttgactac 1140

aggctagcac cagaacacaa attcccagct gctgttgaag atgcatacga tgctgccaaa 1200

tgggtagctg ataattacga caaattgggt gttgacaacg gtaaaattgc cgtcgcaggt 1260

gactcagcag gtggtaactt agcagctgtt acagctatta tggctcgtga tcgtggagaa 1320

tcatttgtca agtaccaggt gctaatatat cccgctgtta acttgaccgg ttctccaact 1380

gtttcccgtg ttgaatattc cggacctgaa tacgttattc ttactgccga tctaatggcc 1440

tggtttggta ggcagtattt ctccaaacct caagatgctt tgtctcccta tgccagtcct 1500

atatttgctg acttgtctaa tcttccccct gccttggtca ttaccgctga gtatgatcca 1560

ttaagggatg agggcgagtt atatgcccac ttgttaaaga ctaggggcgt tcgagctgtc 1620

gctgttcgtt ataatggggt cattcatggc tttgtcaatt tctatccaat attagaggaa 1680

gggcgagaag ccgtcagtca aattgctgct agtattaagt ctatggccgt cgcc 1734

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

atgtccccgt accagtaacg t 21

<210> 8

<211> 39

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

tgaacctccc gatccgccct ggcagtgacg gttgttgct 39

<210> 9

<211> 39

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gcgggatcgg gaggttcaat gcctctgtca ccaatcctg 39

<210> 10

<211> 48

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

ggccacgctg gtacgggtta gacatgtggt ggtggtggtg gtgcatat 48

<210> 11

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

atgtctaacc cgtaccagcg t 21

<210> 12

<211> 51

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gccactacct gaacctcccg atccgccacc ctggcagtga cggttgttgc t 51

<210> 13

<211> 45

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

agtggcggat caggtggtgg aagtatgcct ctgtcaccaa tcctg 45

<210> 14

<211> 45

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

catacttcca ccacctgatc cgccactacc gcctcctgaa cctcc 45

Claims

1. A cutinase-esterase fusion protein is characterized in that the amino acid sequence of the fusion protein is shown in SEQ ID NO. 5.

2. A coding gene of a cutinase-esterase fusion protein as defined in claim 1, characterized in that the nucleotide sequence of the coding gene is shown in SEQ ID No. 6.

3. Use of a cutinase-esterase fusion protein of claim 1 or of a gene encoding of claim 2 for degrading PET to TPA.

4. A recombinant expression vector into which the coding gene of claim 2 has been inserted.

5. A recombinant engineering strain transformed with the recombinant expression vector of claim 4, wherein the host strain of the recombinant engineering strain is escherichia coli BL21 (DE 3).

6. Use of the recombinant expression vector of claim 4, or the recombinant engineering strain of claim 5, for degrading PET to TPA.

7. A method for preparing a cutinase-esterase fusion protein, comprising the steps of: expressing and purifying the recombinant engineering strain of claim 5; the expression temperature is 18+/-3 ℃, and the expression time is 20-22 h.

8. A method of degrading PET comprising reacting the cutinase-esterase fusion protein of claim 1 with PET to produce TPA.