CN115287274B - Cutinase ICCG mutant and application thereof - Google Patents
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Abstract
The invention discloses a cutinase ICCG mutant and application thereof. According to the invention, single-site and multi-site mutation of cutinase ICCG is reasonably designed, and finally, a single-site mutant obtained by performing single-site mutation of any one amino acid of S32L, D18T, S98R, T157P, E173Q or N213P on cutinase ICCG and a multi-site mutant obtained by performing multi-site mutation of S32L/D18T/S98R/T157P/E173Q/N213P are discovered, so that the optimal reaction temperature and the catalytic activity of the mutant are obviously improved compared with those of wild cutinase ICCG. The invention further provides application of the mutant in the aspects of efficiently degrading ethylene terephthalate plastic products and the like.
Description
Technical Field
The invention relates to a cutinase mutant, in particular to a single-site or multi-site mutant of cutinase ICCG, and also relates to application of the mutant in degradation of polyethylene terephthalate (PET) plastics, belonging to the cutinase ICCG mutant and the application field thereof.
Background
Plastic products are widely used in the fields of food packaging, chemical industry, agriculture and the like due to the characteristics of durability, strong plasticity, good safety and the like, and meanwhile, due to the reasons of strong hydrophobicity, high crystallinity and the like, the plastic products are difficult to degrade and utilize by microorganisms or enzymes, so that plastic waste is continuously accumulated, the global ecological environment is seriously threatened, and meanwhile, urgent social and environmental problems are brought. Polyethylene terephthalate (PET) plastic is a polycondensate of terephthalic acid and ethylene glycol, and is widely used for producing packaging film sheets of foods, medicines and the like, packaging bottles, automobile parts and the like. In recent years, biodegradation studies on such plastics have been extensive.
In 2019, A, marty et al excavated cutinase LCC from a leaf and branch compost metagenome (the amino acid sequence of cutinase LCC is published in Protein Data Bank, PDB; https:// www.rcsb.org, ID 4EB 0), further mutant ICCG (A, marty et, al. An engineered PET polymerized to broken down and recycle plastic bottles. Nature. Vol.580.9 April 2020) was obtained by mutating cutinase LCC, and the depolymerization efficiency of 10 h could reach 90% with pretreated bottle-grade PET powder (crystallinity 14.6%) as a substrate under the condition of 72 ℃. However, the inventors have found through experiments that the optimal reaction temperature or catalytic activity of ICCG is not satisfactory and needs to be improved.
Therefore, the cutinase ICCG is mutated by means of rational design and the like to improve the enzymatic properties such as catalytic activity and the like, and the industrial application value of the cutinase ICCG is further improved.
Disclosure of Invention
One of the objects of the present invention would be to provide a mutant of the cutinase ICCG;
the second purpose of the invention is to apply the cutinase ICCG mutant and the coding gene thereof to degrading plastic products such as ethylene terephthalate and the like.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
as a specific embodiment of the present invention, there is provided a single-site mutant of cutinase ICCG obtained by single-site mutation of an amino acid sequence of the cutinase ICCG into any one of S32L, D18T, S98R, T157P, E173Q or N213P; preferably, the single-site mutant is obtained by performing single-site mutation on the amino acid sequence of cutinase ICCG by using any one amino acid of S32L, D18T, S98R or T157; more preferably, the single-site mutant is obtained by subjecting the amino acid sequence of the cutinase ICCG to S32L or D18T amino acid single-site mutation.
The amino acid single-site mutation 'S32L' of the invention means that the 32 nd amino acid of cutinase ICCG is mutated from serine (S) to leucine (L); the remaining single site mutations are described in analogy.
As another specific embodiment of the present invention, the present invention provides a multi-site mutant of cutinase ICCG, which is obtained by subjecting the amino acid sequence of cutinase ICCG to S32L/D18T multi-site mutation; preferably, the multi-site mutant is obtained by carrying out multi-site mutation on the amino acid sequence of cutinase ICCG, namely S32L/D18T/S98R/T157P; more preferably, the cutinase ICCG amino acid sequence is subjected to S32L/D18T/S98R/T157P/E173Q/N213P multi-site mutation to obtain the multi-site mutant.
The amino acid multi-site mutation "S32LL/D18T" of the invention means that the 32 nd amino acid of cutinase ICCG is mutated from serine (S) to leucine (L) and the 18 th amino acid is simultaneously mutated from aspartic acid (D) to threonine (T); the rest of the multi-site mutations are expressed and so on.
The coding gene of the single-site mutant or the multi-site mutant of the cutinase ICCG also belongs to the protection scope of the invention.
The invention also discloses a recombinant expression vector or a recombinant host cell containing the coding gene of the cutinase ICCG mutant; wherein, the recombinant expression vector can be a recombinant prokaryotic expression vector or a recombinant eukaryotic vector.
The present invention further provides a process for the preparation of a single-site or multi-site mutant of the cutinase ICCG according to any one of the preceding claims comprising:
(1) Connecting the coding gene of the single-site mutant or the multi-site mutant of the cutinase ICCG with an expression regulation element in an operable way to construct a recombinant expression vector;
(2) Transforming the recombinant expression vector into a host cell, culturing the host cell, inducing and expressing the recombinant protein, and purifying to obtain the recombinant protein.
The invention also discloses the single-site mutant or multi-site mutant of cutinase ICCG, a coding gene thereof and application of a recombinant expression vector containing the coding gene in degradation of plastic products such as polyethylene terephthalate (PET).
The single-point mutant and the multi-point mutant with the optimal reaction temperature and the obviously improved catalytic activity are successfully screened by rationally designing the single-point mutant and the multi-point mutant of the cutinase ICCG, and the mutants have application prospects in the aspects of efficiently degrading plastic products such as polyethylene terephthalate (PET) and the like.
Detailed description of the overall solution of the invention
ICCG single-point mutant screening and experimental verification
The invention utilizes a machine learning tool Preoptem combined with evolutionary analysis to provide two strategies for reasonably designing ICCG: the first method is that Preoptem combines hidden Markov model to obtain 18 single point mutants; the second is Preoptem combined coevolution analysis, and 18 single point mutants are obtained. These 36 single-point mutants were further experimentally verified to determine the amount of accumulated product of all variants degrading PET powder (crystallinity 39.07%) at 55 ℃, 65 ℃ and 75 ℃, respectively. The results show that 4 of the 18 single-point mutants screened by the first strategy showed higher activity than the wild-type cutinase ICCG at 75 ℃, while 2 of the 18 single-point mutants screened by the second strategy showed higher activity than the wild-type cutinase ICCG at 75 ℃. Overall, the 6 mutation sites identified by both strategies, i.e., D18T, T157P, E173Q, N213P, S32L and S98R, produced higher amounts of degradation products than the wild-type cutinase ICCG at 75 ℃, and the enzymatic activities of these mutants were higher.
ICCG multipoint mutant construction and experimental verification
In order to determine whether the combination mutation can further increase the optimal reaction temperature, variants containing 2, 4 or 6 single point mutations, namely, ICCG _ I2M (S32L/D18T), ICCG _ I4M (S32L/D18T/S98R/T157P) and ICCG _ I6M (S32L/D18T/S98R/T157P/E173Q/N213P) were constructed separately and subjected to heterologous expression, purification and activity detection. The activity detection result shows that the hydrolysis efficiency of PET at high temperature is gradually increased along with the increase of the number of mutation sites, particularly ICCG _ I6M shows the highest activity to PET powder (crystallinity is 39.07%) at 80 ℃, and the mutant is intensively researched subsequently.
In order to further compare the analyses of the wild-type cutinases ICCG, ICCG _ RIP and ICCG _ I6M, the present invention first compared their optimum reaction temperature for hydrolyzing PET powder and the experimental results show that on commercial high crystalline PET material (39.07% crystallinity), the optimum temperature of ICCG _ I6M is increased from 65 deg.C (ICCG) to 80 deg.C, which is also higher than ICCG _ RIP (70-75 deg.C). Furthermore, the yields of ICCG-I6M (4.355. + -. 0.153 mM) at 80 ℃ were 3.05-fold and 1.30-fold respectively for ICCG (1.426. + -. 0.207 mM) and ICCG-RIP (3.351. + -. 0.080 mM). The results of ICCG, ICCG _ RIP and ICCG _ I6M PET degradation of PET powder in 24 hours at 80 ℃ showed that ICCG _ I6M accumulated more MHET and TPA than ICCG or ICCG _ RIP (FIGS. 4B, 4C, 4D), and that ICCG-I6M produced 10 times and 1.5 times higher amounts of product than ICCG and ICCG _ RIP, respectively, and that the final product reached a plateau of 8.69. + -. 0.56 mM after 24 hours of reaction, 9.38 times and 1.56 times higher than ICCG (0.93. + -. 0.07 mM) and ICCG _ RIP (5.56. + -. 0.45 mM), respectively.
Definitions of terms related to the invention
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 term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, and the like). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues.
The terms "mutation" and "mutant" have their usual meanings herein, and refer to a genetic, naturally occurring or introduced change in a nucleic acid or polypeptide sequence, which has the same meaning as is commonly known to those of skill in the art.
The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used for insertion to produce the recombinant host cell, e.g., direct uptake, transduction, f-pairing or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome.
The term "transformation" refers to the process by which eukaryotic cells acquire a new genetic marker due to the incorporation of foreign DNA.
Drawings
FIG. 1 is a temperature optimum test for enzymatic hydrolysis of PET substrates with different degrees of crystallinity and glass transition temperatures by the wild-type cutinase ICCG; FIG. A is the detection of the optimum reaction temperature of ICCG hydrolyzed low-crystallinity PET film (crystallinity 6.04%); graph B optimum reaction temperature detection for high crystallinity PET powder (crystallinity 39.07%); panel C is a low crystallinity PET film glass transition temperature Tg test; graph D shows the glass transition temperature Tg measurements of high crystallinity PET powders.
FIG. 2 shows the results of the detection of total soluble products released by hydrolysis of PET powder with a single-site mutant of the wild-type cutinase ICCG: panel a is the total soluble product assay results released from each single point mutant hydrolyzed PET powder screened by strategy 1, and panel B is the total soluble product assay released from each single point mutant hydrolyzed PET powder screened by strategy 2.
FIG. 3 shows the result of the optimum temperature measurement of hydrolyzed PET powder of a multi-site mutant of wild-type cutinase ICCG.
FIG. 4 shows the optimum temperature detection of wild-type cutinase ICCG and its mutants and the time course product analysis of hydrolyzed PET powder; FIG. A shows the optimal temperature detection of ICCG and its mutants; panel B is a time course product analysis of ICCG hydrolyzed PET powder; panel C is a time course product analysis of ICCG _ RIP (C) hydrolyzed PET powder; panel D is a time course product analysis of ICCG I6M hydrolyzed PET powder.
Detailed Description
The invention is further described below in conjunction with specific embodiments, and the advantages and features of the invention will become more apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1 optimal temperature detection of ICCG degradation of PET plastics of different crystallinity
In order to increase the PET degrading ability of the PET degrading enzyme ICCG, it was first measured that it degrades PET film (crystallinity 6.04%) and PET powder (crystallinity 39.07%) while releasing BHET, MHET and TPA at different temperatures for 5 hours.
The results show that the optimum reaction temperatures for the formation of TPA and MHET are both 70 ℃ when the substrate is a low crystallinity PET film, wherein the proportion of TPA build-up is higher (FIG. 1A). Whereas when hydrolyzing high crystallinity PET powder, the optimum reaction temperature for the formation of TPA is about 65 ℃ and the optimum reaction temperature for the formation of MHET is close to 55 ℃ (FIG. 1B). Differential Scanning Calorimetry (DSC) measurements showed that the glass transition temperature Tg of the PET film was about 73-76 deg.C (FIG. 1C), whereas the glass transition temperature of the high crystallinity PET powder could not be determined (FIG. 1D). The difference in crystallinity may be responsible for the difference in optimal reaction temperature for ICCG. In addition, the catalytic activity of ICCG decreased significantly beyond 70 ℃ for low crystallinity films and high crystallinity powders. Because the high temperature is more favorable for PET degradation, the optimal reaction temperature and the catalytic activity of ICCG are to be improved.
Example 2 ICCG Single Point mutant screening and Experimental validation
The invention provides two strategies for reasonably designing ICCG by combining a machine learning tool Preoptem with evolution analysis: the first method is that Preoptem combines hidden Markov model to obtain 18 single point mutants (Table 1); the second was a Preoptem binding co-evolution assay, which also yielded 18 single point mutants (Table 1).
This experiment was further experimentally validated against these 36 single-point mutants and the amount of accumulated product of all variants that degraded PET powder (crystallinity 39.07%) at 55 ℃, 65 ℃ and 75 ℃ was determined, respectively. The results showed that 4 of the 18 single-point mutants screened by the first strategy showed higher activity than wild-type ICCG at 75 ℃ (a 117D was inactive, data not shown) (fig. 2A), while 2 of the 18 single-point mutants screened by the second strategy showed higher activity than wild-type ICCG at 75 ℃ (fig. 2B). In general, the 6 mutation sites identified by the two strategies, namely D18T, T157P, E173Q, N213P, S32L and S98R, generate higher amounts of degradation products than ICCG at 75 ℃ and have higher activity.
TABLE 1 mutation sites of 36 Single-Point mutants
Example 3 ICCG multipoint mutant construction and Experimental validation
In order to determine whether the combination mutation can further increase the optimal reaction temperature, variants containing 2, 4 or 6 single point mutations, namely, ICCG _ I2M (S32L/D18T), ICCG _ I4M (S32L/D18T/S98R/T157P) and ICCG _ I6M (S32L/D18T/S98R/T157P/E173Q/N213P) were constructed separately and subjected to heterologous expression, purification and activity detection.
The activity test results are shown in fig. 3, the hydrolysis efficiency of PET at high temperature is gradually increased with the increase of the number of mutation sites, especially ICCG _ I6M shows the highest activity to PET powder (crystallinity 39.07%) at 80 ℃, and the mutant is subsequently intensively studied.
Previous researches show that ICCG has high thermal stability, and the depolymerization efficiency can reach 90% in 10 hours under the condition of 72 ℃ by using pretreated bottle-grade PET powder (with the crystallinity of 14.6%) as a substrate. A recently published study by Guo et al shows that the mutant ICCG _ RIP designed on the basis of ICCG is the most thermostable mutant to date (Substrate-binding mode of a Thermophilic PET Hydrolaser and Engineering the Enzyme to the hydrolysis effect).
To further compare the analysis of ICCG, ICCG _ RIP and ICCG _ I6M, the experiment first compared their optimum reaction temperatures for hydrolysis of PET powder.
The results of the experiment are shown in FIG. 4A, where the optimum temperature for ICCG I6M was increased from 65 deg.C (ICCG) to 80 deg.C and higher than ICCG RIP (70-75 deg.C) on a commercial high crystalline PET material (39.07% crystallinity). Furthermore, the yields of ICCG-I6M (4.355. + -. 0.153 mM) at 80 ℃ were 3.05-fold and 1.30-fold respectively for ICCG (1.426. + -. 0.207 mM) and ICCG-RIP (3.351. + -. 0.080 mM). The results of ICCG, ICCG _ RIP and ICCG _ I6M PET degradation of PET powder in 24 hours at 80 ℃ showed that ICCG _ I6M accumulated more MHET and TPA than ICCG or ICCG _ RIP (FIGS. 4B, 4C, 4D), and that ICCG-I6M produced 10 times and 1.5 times higher amounts of product than ICCG and ICCG _ RIP, respectively, and that the final product reached a plateau of 8.69. + -. 0.56 mM after 24 hours of reaction, 9.38 times and 1.56 times as much as ICCG (0.93. + -. 0.07 mM) and ICCG _ RIP (5.56. + -. 0.45 mM), respectively (FIGS. 4B, 4C, 4D).
Claims (8)
1. A mutant of cutinase ICCG, which is a single-site mutant obtained by subjecting cutinase ICCG to S32L single-site mutation.
2. A mutant of cutinase ICCG, which is characterized in that the mutant is a multi-site mutant obtained by carrying out S32L/D18T multi-site mutation on the cutinase ICCG.
3. A mutant of cutinase ICCG, which is characterized in that the mutant is a multi-site mutant obtained by carrying out multi-site mutation on cutinase ICCG, wherein the multi-site mutation is S32L/D18T/S98R/T157P.
4. A mutant of cutinase ICCG, which is characterized in that the mutant is a multi-site mutant obtained by carrying out S32L/D18T/S98R/T157P/E173Q/N213P multi-site mutation on the cutinase ICCG.
5. A gene encoding a mutant of the cutinase ICCG according to any one of claims 1 to 4.
6. A recombinant expression vector comprising the coding gene of claim 5.
7. Use of a mutant of the cutinase ICCG according to any one of claims 1 to 4 for degrading polyethylene terephthalate plastic.
8. Use of the coding gene of claim 5 or the recombinant expression vector of claim 6 for degrading polyethylene terephthalate plastic.
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