Carboxylesterase est30 mutants and uses thereof

Abstract

The application belongs to the technical field of enzyme engineering, and specifically discloses a carboxylic esterase Est30 mutant and application thereof. The application carries out site-directed mutagenesis on the basis of the mutant Est30-KL, and provides a series of carboxylic esterases Est30 mutants with excellent thermal stability and BHET degradation activity. Compared with the mutant Est30-KL, the BHET degradation activity of the carboxylic esterase Est30 mutant provided by the application is increased by 156.2% to 598.7% at 72 DEG C, and the Tm value is increased by 11.34 to 23.97 DEG C, which is of great significance for industrial efficient degradation of PET. In view of this, the carboxylic esterase Est30 mutant provided by the application can be used to construct a PET degradation system in combination with a PETase enzyme, and can also be used as a BHET or MHET degradation agent to efficiently degrade PET intermediates, thereby laying a foundation for industrial efficient biodegradation and closed-loop recycling of PET.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology and relates to a degradation enzyme, specifically a carboxylesterase Est30 mutant and its applications. Background Technology

[0002] Polyethylene terephthalate (PET) is one of the most widely used synthetic polyester materials, used extensively in beverage bottles, food packaging, textile fibers, films, and engineering plastics. Due to the highly stable aromatic ester bonds in its molecular chain, along with its high crystallinity and hydrophobicity, PET is difficult to degrade effectively by microorganisms or conventional biological processes in the natural environment. With the continuous expansion of PET product production and consumption, large amounts of PET waste have accumulated in the environment, becoming a significant problem in global plastic pollution control. Therefore, developing efficient, green, and sustainable PET recycling and degradation technologies has significant environmental and industrial value.

[0003] In recent years, enzymatic degradation of PET has become an important research direction in the field of PET resource recycling due to its mild reaction conditions, high selectivity, and environmental friendliness. PET biodegradation typically involves multiple enzymatic reactions, in which PETase enzymes first act on the PET polymer chain to generate hydrolysis products such as BHET, MHET, and a small amount of terephthalic acid. MHET, or mono(2-hydroxyethyl) terephthalate, is a key intermediate in the PET enzymatic hydrolysis process. If MHET cannot be further efficiently hydrolyzed in the system, its accumulation will affect the sustained catalytic efficiency of PETase and reduce the efficiency of complete depolymerization of PET into monomers. Therefore, further hydrolyzing MHET into terephthalic acid and ethylene glycol is a crucial step in achieving efficient PET biodegradation and closed-loop recycling.

[0004] Significant progress has been made in the molecular modification of PETase enzymes. Through strategies such as rational design, semi-rational design, directed evolution, and machine learning-assisted screening, researchers have obtained several performance-enhanced PET-degrading enzyme variants, such as LCCICCG, TurboPETase, and FASTPETase. These engineered PETases are generally well-suited for medium- and high-temperature degradation processes. By increasing the catalytic temperature, they can significantly accelerate the depolymerization rate of PET polymer chains, overcome the technical bottleneck of the difficult degradation of highly crystalline PET, and promote rapid depolymerization of PET under medium- and high-temperature conditions.

[0005] Currently, the optimal degradation temperature for high-performance PET hydrolases generally reaches the high-temperature range of 60 ℃–70 ℃. High-temperature reaction systems can effectively enhance the wetting effect of PET substrates and significantly improve overall degradation efficiency, making it the mainstream process direction for large-scale enzymatic PET degradation. However, the matching MHET and BHET hydrolases generally suffer from thermal instability. Most natural and existing engineered intermediate degradation enzymes have low temperature tolerance and cannot adapt to the reaction conditions of high-temperature PET degradation processes, making it difficult to achieve synchronous compounding and synergistic catalysis with high-temperature PET hydrolases. This system compatibility defect means that most existing processes can only adopt a stepwise low-temperature degradation mode, which not only significantly reduces the overall degradation efficiency of PET and prolongs the reaction cycle, but also easily leads to a large accumulation of MHET and BHET intermediates, producing a product inhibition effect, further limiting the complete depolymerization of PET, and severely restricting the industrial application of enzymatic PET degradation technology.

[0006] The carboxylesterase Est30 from *Bacillus thermophilus* has attracted widespread attention due to its excellent MHET hydrolysis specificity and superior thermal stability (wild-type Tm = 74.07 ℃). Based on this, researchers have developed a high-performance double-point mutant, Est30-G128L-I169K (referred to as mutant Est30-KL), using enzyme engineering. While its thermal stability Tm is slightly lower than that of wild-type Est30 (67.93 ℃), its catalytic efficiency is 36 times that of wild-type Est30. Although this mutant possesses high catalytic activity, its current thermal stability is still insufficient for high-temperature PET degradation systems at 70 ℃ and above. Therefore, further modification is needed to simultaneously improve its thermal stability and catalytic performance, solve the challenge of high-temperature complexation of multi-enzyme systems, and achieve efficient, integrated biodegradation and closed-loop recycling of PET. Summary of the Invention

[0007] In view of the above-mentioned problems in the prior art, the present invention modifies the Est30-KL mutant to provide a carboxylesterase Est30 mutant with significantly improved BHET degradation activity and its application.

[0008] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions: In a first aspect, the present invention provides a carboxylesterase Est30 mutant, the amino acid sequence of which is shown in SEQ ID No. 3.

[0009] This invention, based on the mutant Est30-KL (whose amino acid sequence is shown in SEQ ID No. 1), further mutates serine at position 219 to cysteine, aspartic acid at position 226 to cysteine, and glutamic acid at position 207 to arginine, while keeping other amino acid residues unchanged. The resulting mutant is denoted as mutant CCR. Compared to mutant Est30-KL, mutant CCR exhibits a 156.2% increase in BHET degradation activity at 72℃ and a 11.34℃ increase in Tm value (79.27℃).

[0010] Secondly, the present invention provides a carboxylesterase Est30 mutant, the amino acid sequence of which is obtained by site-directed mutation of valine at position 96 of the amino acid sequence shown in SEQ ID No. 3 to leucine, while the amino acid residues at other positions remain unchanged, and is denoted as mutant CCR-V96L.

[0011] Compared to the mutant Est30-KL, the mutant CCR-V96L provided by this invention has a 27.4% higher BHET degradation activity at 50 ℃, a 4.89% higher BHET degradation activity at 60 ℃, and a 13.28℃ higher Tm value (81.21 ℃).

[0012] Thirdly, the present invention provides a carboxylesterase Est30 mutant, whose amino acid sequence is based on the amino acid sequence shown in SEQ ID No. 3, with proline at position 182 mutated to cysteine ​​and valine at position 210 mutated to cysteine, while the amino acid residues at other positions remain unchanged, and is denoted as mutant CCRCC.

[0013] Compared to the mutant Est30-KL, the mutant CCRCC provided by this invention showed a 407.4% increase in BHET degradation activity at 72 °C and a 14.48 °C increase in Tm value (82.41 °C).

[0014] Fourthly, the present invention provides a carboxylesterase Est30 mutant, the amino acid sequence of which is obtained by replacing five or six amino acid residues in the amino acid sequence of the mutant CCRCC, and the amino acid sequence is any one of the following (1) to (6): (1) The amino acid sequence of the mutant CCRCC is mutated at position 28 (alanine) to asparagine, position 54 (valine) to lysine, position 96 (valine) to leucine, and position 114 (cysteine) to asparagine. At the same time, position 119 (isoleucine) is mutated to glutamic acid. The amino acid residues at other positions remain unchanged. This mutant is called CCRCCNKLNE. (2) The amino acid sequence of the mutant CCRCC is mutated at position 28 (alanine) to asparagine, position 54 (valine) to arginine, position 96 (valine) to leucine, and position 114 (cysteine) to asparagine. At the same time, position 119 (isoleucine) is mutated to glutamic acid. The amino acid residues at other positions remain unchanged. This mutant is called CCRCCNRLNE. (3) The amino acid sequence of the mutant CCRCC is mutated at position 28 (alanine) to asparagine, position 54 (valine) to lysine, position 98 (serine) to alanine, and position 114 (cysteine) to asparagine. At the same time, position 119 (isoleucine) is mutated to glutamic acid. The amino acid residues at other positions remain unchanged. This mutant is called CCRCCNKANE. (4) The amino acid sequence of the mutant CCRCC is mutated at position 28 (alanine) to asparagine, position 54 (valine) to arginine, position 98 (serine) to alanine, position 114 (cysteine) to asparagine, and position 119 (isoleucine) to glutamic acid. The amino acid residues at other positions remain unchanged. This mutant is called CCRCCNRANE. (5) The amino acid sequence of the mutant CCRCC is mutated at position 28 (alanine) to asparagine, position 54 (valine) to lysine, position 96 (valine) to leucine, position 98 (serine) to alanine, and position 114 (cysteine) to asparagine. At the same time, position 119 (isoleucine) is mutated to glutamic acid. The amino acid residues at other positions remain unchanged. This mutant is called CCRCCNKLANE. (6) The amino acid sequence of the mutant CCRCC is mutated at position 28 (alanine) to asparagine, position 54 (valine) to arginine, position 96 (valine) to leucine, position 98 (serine) to alanine, and position 114 (cysteine) to asparagine. At the same time, position 119 (isoleucine) is mutated to glutamic acid. The amino acid residues at other positions remain unchanged. This mutant is called CCRCCNRLANE.

[0015] Compared to the mutant Est30-KL, the mutants CCRCCNKLNE, CCRCCNRLNE, CCRCCNKANE, CCRCCNRANE, CCRCCNKLANE, and CCRCCNRLANE provided by this invention exhibited Tm values ​​increased by 22.86 ℃ to 23.97 ℃, and their BHET degradation activity at 72 ℃ increased by 383.9% to 598.7%. Among them, the mutant CCRCCNRLNE had the highest Tm value at 91.91 ℃, and its BHET degradation activity was 598.7% higher than that of the mutant Est30-KL.

[0016] Fifthly, the present invention provides the encoding gene of the carboxylesterase Est30 mutant as described in any one of the first to fourth aspects.

[0017] In a sixth aspect, the present invention provides a recombinant vector comprising the coding gene described in the fifth aspect.

[0018] In a seventh aspect, the present invention provides a recombinant strain comprising the recombinant vector described in the sixth aspect.

[0019] For example, the host cell of the recombinant strain is Escherichia coli.

[0020] Eighthly, the present invention provides the use of the carboxylesterase Est30 mutant described in any one of the first to fourth aspects, the encoding gene described in the fifth aspect, the recombinant vector described in the sixth aspect, or the recombinant strain described in the seventh aspect in the degradation of MHET or BHET.

[0021] In a ninth aspect, the present invention provides the use of the carboxylesterase Est30 mutant described in any one of the first to fourth aspects, the encoding gene described in the fifth aspect, the recombinant vector described in the sixth aspect, or the recombinant strain described in the seventh aspect in the preparation of MHET degrading agents or BHET degrading agents.

[0022] In a tenth aspect, the present invention provides a PET degrading agent comprising a PET hydrolase and a carboxylesterase Est30 mutant as described in any one of the first to fourth aspects.

[0023] For example, the PET hydrolase includes FASTPETase.

[0024] This invention utilizes site-directed mutagenesis on the Est30-KL mutant to provide a series of Est30 carboxylesterase mutants with significantly enhanced thermostability and BHET degradation activity. Compared to the Est30-KL mutant, at 72 °C, the BHET degradation activity of the Est30 carboxylesterase mutants provided by this invention is increased by 156.2%~598.7%, and the Tm value is increased by 11.34 °C~23.97 °C, laying the foundation for efficient and complete depolymerization conversion of PET. Among them, the Tm value of the mutant CCRCCNRLNE can reach 91.91 °C, and after reacting in a system at 72 °C for 1 h, the TPA content in the system can reach 0.229, and its BHET degradation activity is increased by 598.7% compared to Est30-KL.

[0025] The Est30 mutant carboxylesterase provided by this invention has both excellent thermal stability and BHET degradation activity. It can be combined with medium- and high-temperature PETase enzymes to construct PET degradation systems, and can also be used as a BHET or MHET degrading agent to efficiently degrade PET intermediates, laying the foundation for efficient, integrated biodegradation and closed-loop recycling of PET in the industry. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a schematic diagram of the plasmid map of the recombinant plasmid pET-22b-Est30-KL in Example 1 of the present invention; Figure 2 The results of BHET degradation activity and Tm determination of the mutants obtained by disulfide bond design in Example 1 of the present invention at 50 °C and 60 °C; Figure 3 The results of BHET degradation activity and Tm determination of mutants obtained by salt bridge design in Example 1 of the present invention at 50 °C and 60 °C; Figure 4 The results of BHET degradation activity and Tm determination of mutants obtained by rational design in Example 1 of the present invention at 50 °C and 60 °C; Figure 5 The results of BHET degradation activity and Tm value determination for different mutants in Example 2 of this invention; Figure 6 The results of measuring the BHET degradation activity and Tm value of different mutants at 50 °C and 60 °C in Example 3 of the present invention; Figure 7 This is a schematic diagram of the plasmid map of the recombinant plasmid pET-22b-CCRCCNRLNE in Example 4 of the present invention; Figure 8 The results of measuring the BHET degradation activity and Tm value of different mutants at 72 °C in Example 4 of this invention are shown. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0029] The mutants described in this invention are named according to the conventional naming methods of those skilled in the art. For example, mutant CCR indicates that the serine (S) at position 219 of the mutant Est30-KL amino acid sequence is mutated to cysteine ​​(C), the aspartic acid (D) at position 226 is mutated to cysteine ​​(C), and the glutamic acid (E) at position 207 is mutated to arginine (R), while the amino acid residues at other positions remain unchanged. The mutant CCR-V96L represents a site-directed mutation of valine (V) at position 96 of the amino acid sequence of the mutant CCR to leucine (L), while the amino acid residues at other positions remain unchanged.

[0030] Example 1 This embodiment provides a method for preparing, expressing, purifying, and detecting BHET degradation activity based on the Est30-KL single-point mutant. The method includes obtaining the mutated target gene via polymerase chain reaction (PCR), introducing it into an *E. coli* expression vector, preparing a recombinant plasmid using molecular biology methods such as DMT enzyme (TransGold, GD111) and seamless cloning, and transforming it into *E. coli* BL21(DE3) (TransGold, CD601) competent cells. After culturing, recombinant *E. coli* expressing the target protein heterologously is obtained. The specific details are as follows: I. Obtaining the Est30 mutant of carboxylesterase 1. Construction of disulfide bond-designed mutant recombinant plasmids and recombinant strains The amino acid sequence of the mutant Est30-KL is shown in SEQ ID No. 1. Its coding gene was obtained through codon optimization, and its coding gene sequence is shown in SEQ ID No. 2.

[0031] The recombinant plasmid pET-22b-Est30-KL was synthesized by Suzhou Genewiz Biotechnology Co., Ltd., and its plasmid map is shown in the figure below. Figure 1As shown.

[0032] Site-directed mutagenesis was used, with recombinant plasmid pET-22b-Est30-KL as a template. Primers were designed using the disulfide bonds shown in Table 1, and PCR was performed to obtain linearized plasmid fragments containing the corresponding mutation sites. The nucleotide sequences of the primers used are shown in SEQ ID No. 4~SEQ ID No. 27. The PCR reaction system was 20 μL, including 1 μL of template (plasmid), 1 μL of forward primer (F), 1 μL of reverse primer (R), 10 μL of high-fidelity amplification reagent, and the remainder being enzyme-free water. The PCR reaction conditions in this step were: pre-denaturation at 98℃ for 3 min; followed by 30 cycles, each cycle including: denaturation at 98℃ for 15 s, annealing at 54℃ for 15 s, extension at 72℃ for 3 min; and final extension at 72℃ for 5 min.

[0033] The PCR-obtained fragments were digested with DMT enzyme (TransGen, GD111), and further validated using molecular biology methods such as seamless cloning and Sanger sequencing. Six recombinant plasmids, including pET-22b-P5C, pET-22b-F10C, pET-22b-P56C, pET-22b-P182C, pET-22b-Q187C, and pET-22b-S219C, were obtained.

[0034] Table 1 Primers used for disulfide bond design Furthermore, using six recombinant plasmids—pET-22b-P5C, pET-22b-F10C, pET-22b-P56C, pET-22b-P182C, pET-22b-Q187C, and pET-22b-S219C—as templates, and with G53C-F / R, E38C-F / R, A133C-F / R, V210C-F / R, A199C-F / R, and D226C-F / R, respectively, as templates, Using primers, further PCR was performed to obtain recombinant plasmids pET-22b-P5C-G53C, pET-22b-F10C-E38C, pET-22b-P56C-A133C, pET-22b-P182C-V210C, pET-22b-Q187C-A199C, and pET-22b-S219C-D226C.

[0035] 2. Construction of recombinant plasmids for salt-bridged mutants Using the recombinant plasmid pET-22b-Est30-KL as a template, and with the primers designed using the salt bridge shown in Table 2, linearized plasmid fragments containing the corresponding mutation sites were obtained by PCR. The nucleotide sequences of the primers used in this step are shown in SEQ ID No. 28~SEQ ID No. 43; the PCR reaction system and conditions are the same as those described in "1. Construction of disulfide bond-designed mutant recombinant plasmids and recombinant strains".

[0036] The obtained fragments were digested with DMT enzyme (TransGen, GD111), and further validated by molecular biology methods such as seamless cloning and Sanger sequencing. Eight recombinant plasmids were constructed, including pET-22b-F10R, pET-22b-V54K, pET-22b-V54R, pET-22b-M117D, pET-22b-I119E, pET-22b-K137D, pET-22b-I201D, and pET-22b-E207R.

[0037] Table 2 Primers used in the design of the salt bridge 3. Construction of rationally designed mutant recombinant plasmids Using the recombinant plasmid pET-22b-Est30-KL as a template, and employing the rationally designed primers shown in Table 3, linearized plasmid fragments containing the corresponding mutation sites were obtained by PCR. The nucleotide sequences of the primers used in this step are shown in SEQ ID No. 44~SEQ ID No. 63. The PCR reaction system and conditions were identical to those described in "1. Construction of disulfide bond-designed mutant recombinant plasmids and recombinant strains".

[0038] The obtained fragments were digested with DMT enzyme (TransGen, GD111), and further validated by molecular biology methods such as seamless cloning and Sanger sequencing. Ten recombinant plasmids, including pET-22b-A28N, pET-22b-H45I, pET-22b-V96L, pET-22b-S98A, pET-22b-S98I, pET-22b-S98L, pET-22b-C114N, pET-22b-C114Q, pET-22b-K137L, and pET-22b-K137M, were successfully obtained.

[0039] Table 3 Primers used in rational design II. Construction of recombinant strains and expression and purification of recombinant proteins The recombinant plasmids were introduced into *E. coli* BL21(DE3) (CellGold, CD601) competent cells via heat shock (42 ℃ water bath for 45 s). After heat shock, the cells were rapidly transferred to an ice bath for 2 min, and 500 μL of sterile LB medium (antibiotic-free) was added. The cells were mixed and incubated at 37 ℃ and 200 rpm for 1 h to allow the bacteria to recover. After recovery, the cells were centrifuged at 6000 rpm for 90 s, and 450 μL of supernatant was discarded. The remaining bacterial culture was resuspended and added to LB agar medium containing ampicillin. The cells were spread evenly until the liquid was absorbed, and the plates were inverted and incubated at 37 ℃ for 12 h.

[0040] Then, positive monoclonal strains were selected and transferred to test tubes containing 5 mL LB medium (containing ampicillin). After incubation at 37 ℃ and 220 rpm for 8 h, they were inoculated into 80 mL LB medium shake flasks at a 1% inoculum size for fermentation. The culturing was then carried out at 37 ℃ and 220 rpm for 2.5 h to allow the bacterial concentration to reach OD500. 600 The concentration was brought to between 0.8 and 1, and then isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM. The expression was induced at 16 ℃ and 160 rpm for 14 h to obtain bacterial cultures rich in the mutant Est30-KL and the mutants obtained by further site-directed mutagenesis.

[0041] The different fermentation broths were treated using a high-speed refrigerated centrifuge (8000 g, 5 min), and the cells were collected. Each cell was resuspended in 10 mL of lysis buffer (each 1 L of lysis buffer contains 50 mM Tris-HCl, 150 mM NaCl, and 10 mM imidazole, pH=7.5), and then the cells were lysed using an autoclave. After lysis, the cells were centrifuged at 10000 rpm for 1 h to remove cell debris. The resulting supernatant was the total protein solution containing Est30-KL and its mutants.

[0042] The total protein solution was filtered through a 0.45 μm filter to remove impurities, followed by gradient elution purification using a Ni-NTA packed column to obtain the target protein. The specific purification steps included: equilibration with lysis buffer for 2 min, then repeated column loading of the filtered total protein solution three times, followed by washing three times with washing buffer (each 1L of washing buffer contains 50mM Tris-HCl, 150mM NaCl, and 20mM imidazole, pH=7.5) to remove impurities; finally, elution with elution buffer (each 1L of elution buffer contains 50mM Tris-HCl, 300mM NaCl, and 300mM imidazole, pH=7.5) to obtain the protein eluent; finally, the eluent was changed three times with replacement buffer (each 1L of replacement buffer contains 20mM Tris-HCl and 300mM NaCl, pH=7.5) to dilute the imidazole in the protein eluent to 1‰ of its original concentration, and then concentrated to obtain the concentrated protein solution.

[0043] III. Performance Characterization Methods 1. Determination of BHET degradation activity BHET substrate (purchased from Maclean's, catalog number B796598) was dissolved in dimethyl sulfoxide to obtain a 500 mM BHET stock solution. For each enzymatic digestion, the stock solution was diluted to 5 mM before use.

[0044] The reaction system in this invention is as follows: The concentrated protein solution was placed in 300 μL of reaction solution (100 mM potassium phosphate buffer, pH=8) at the corresponding concentration (250 nM), and 5 mM BHET was added. The reaction was carried out in a water bath at 50 ℃, 60 ℃, or 72 ℃ for 1 h. After the reaction was completed, an equal volume of acetonitrile was added to terminate the reaction, and the TPA, MHET, and BHET produced in the reaction were analyzed by high performance liquid chromatography (HPLC). In this invention, the proportion of TPA in the total content of the above three substances is used to characterize the degradation activity of different mutants on BHET.

[0045] Under the above reaction system and reaction conditions, a group without added enzyme was set up as a blank control group (Control group).

[0046] 2. Methods for determining Tm Protein melting temperature (Tm) was determined using differential scanning fluorometry (DSF). Protein samples were added to eight-cell arrays, with each well containing 25 μL of the following: 15 μL of enzyme stock solution (containing 20 mM Tris-HCl and 300 mM NaCl per liter), 9 μL of protein solution (0.4 mg / mL), and 1 μL of SYPRO Orange dilution solution. DSF experiments were performed using a real-time quantitative PCR system with 465 nm excitation and a 580 nm emission filter. Samples were heated from 25 °C to 100 °C at a rate of 0.05 °C / s, with fluorescence measured every 2 s. Tm was determined using first derivative curves.

[0047] IV. Experimental Results 1. Results of mutants obtained from disulfide bond design To improve the thermal stability of the mutant Est30-KL, disulfide bond design was first performed. The BHET degradation activity and Tm of the mutant obtained through disulfide bond design were measured at 50 °C and 60 °C, as shown below. Figure 2 As shown.

[0048] Depend on Figure 2 It can be seen that among the six pairs of disulfide bonds provided by this invention, except for the mutants P56C-A133C and Q187C-A199C, whose Tm values ​​are slightly lower than those of the mutant Est30-KL, the Tm values ​​of the other four pairs of disulfide bond mutants are 3.30℃ to 6.86℃ higher than those of the mutant Est30-KL (Tm = 67.93℃). Among them, the mutant S219C-D226C has the best thermal stability, with a Tm of 74.79℃, which is 6.86℃ higher than that of the mutant Est30-KL. The thermal stability of the mutant P182C-V210C is also significantly improved, with a Tm value 4.27℃ higher than that of the mutant Est30-KL.

[0049] Regarding BHET degradation activity, different mutants exhibited varying effects. The mutant P182C-V210C showed a 4.27 °C increase in Tm compared to the Est30-KL mutant, and its BHET degradation activity at 50 °C and 60 °C was increased by 46.8% and 56.7%, respectively, compared to the Est30-KL mutant. The remaining mutants showed decreased BHET degradation activity at both 50 °C and 60 °C compared to the Est30-KL mutant. However, considering that the mutant S219C-D226C exhibited the best thermal stability and could degrade BHET at both 50 °C and 60 °C, further combination mutations were proposed based on this mutant.

[0050] 2. Results of mutants obtained from salt bridge design This invention incorporates salt bridge design based on the Est30-KL mutant. The BHET degradation activity and Tm determination results of the salt bridge-designed mutant at 50 °C and 60 °C are as follows: Figure 3 As shown.

[0051] Depend on Figure 3 As shown, among the eight mutants F10R, V54K, V54R, M117D, I119E, K137D, I201D, and E207R, the Tm values ​​of mutants V54K, V54R, I119E, and E207R were 2.48–5.90 °C higher than those of mutant Est30-KL. Among them, mutant E207R exhibited the best thermal stability, with a Tm value of 73.83 °C, which was 5.90 °C higher than that of mutant Est30-KL. The thermal stability of mutants V54R and I119E also showed significant improvements, with Tm values ​​increasing by 3.42 °C and 2.58 °C, respectively, compared to mutant Est30-KL.

[0052] Regarding BHET degradation activity, the mutant F10R exhibited 1.69 times the BHET degradation activity of the mutant Est30-KL at 50 °C, but its BHET degradation activity decreased slightly at 60 °C. Notably, the mutant E207R not only had the highest Tm value (73.83 °C), but its BHET degradation activity at both 50 °C and 60 °C was comparable to that of the mutant Est30-KL, and it will be considered for future combined mutations.

[0053] 3. Results of mutants obtained through rational design This invention utilizes computer-aided rational design to further modify the mutant Est30-KL. Specifically, the BHET degradation activity and Tm determination results of the mutant obtained through rational design under reaction conditions of 50 ℃ and 60 ℃ are as follows: Figure 4 As shown.

[0054] Depend on Figure 4It can be seen that among the 10 mutants obtained through rational design, the Tm values ​​of mutants A28N, V96L, S98A, and C114N were 2.54–3.10 °C higher than those of mutant Est30-KL. Among them, mutant V96L exhibited the best thermal stability, with a Tm value of 71.03 °C. Mutant C114N had a Tm value of 70.90 °C, which was 2.97 °C higher than that of mutant Est30-KL. Although the improvement in thermal stability of these mutants was not significant, they performed well in terms of BHET degradation activity. Mutant A28N showed the best BHET degradation activity, with BHET degradation activities at 50 °C and 60 °C being 137.1% and 92.1% higher than those of mutant Est30-KL, respectively, and its Tm value was 2.86 °C higher than that of mutant Est30-KL. The BHET degradation activity of mutant V96L was increased by 79.2% and 75.9% compared with mutant Est30-KL under reaction conditions of 50 ℃ and 60 ℃, respectively.

[0055] The BHET degradation activities of mutants H45I, K137L, and K137M at 50 °C were increased by 31.9%, 76.4%, and 27.9% respectively compared to mutant Est30-KL, but their BHET degradation activities at 60 °C were all reduced to varying degrees compared to mutant Est30-KL.

[0056] Example 2 As shown in Example 1, compared to the mutant Est30-KL, the mutant S219C-D226C (hereinafter referred to as mutant CC) exhibits the greatest improvement in thermal stability. Since one of the main objectives of this invention is to improve the thermal stability of the mutant Est30-KL, the mutation points that improve its thermal stability compared to Est30-KL are superimposed onto the mutant CC, which has the best thermal stability. This results in mutants S219C-D226C-P5C-G53C (hereinafter referred to as mutant CC-P5C-G53C), S219C-D226C-F10C-E38C (hereinafter referred to as mutant CC-F10C-E38C), S219C-D226C-P182C-V210C (hereinafter referred to as mutant CC-P182C-V210C), and S219C-D226C-V54K (hereinafter referred to as mutant CC). The following mutant strains are listed: CC-V54K, S219C-D226C-V54R (abbreviated as mutant CC-V54R), S219C-D226C-I119E (abbreviated as mutant CC-I119E), S219C-D226C-E207R (abbreviated as mutant CC-E207R), S219C-D226C-A28N (abbreviated as mutant CC-A28N), S219C-D226C-V96L (abbreviated as mutant CC-V96L), S219C-D226C-S98A (abbreviated as mutant CC-S98A), and S219C-D226C-C114N (abbreviated as mutant CC-C114N).

[0057] The method for constructing recombinant plasmids for the above-mentioned three- or four-point combination mutants includes: using the recombinant plasmid pET-22b-S219C-D226C constructed in Example 1 as a template, and using the corresponding primers in Tables 1 to 3, performing PCR according to the PCR conditions described in Example 1. After Sanger sequencing, the following recombinant plasmids were obtained: pET-22b-S219C-D226C-P5C, pET-22b-S219C-D226C-F10C, pET-22b-S219C-D226C-P182C, pET-22b-S219C-D226C-V54K, pET-22b-S219C-D226C-V54R, and pET-2... 2b-S219C-D226C-I119E, recombinant plasmid pET-22b-S219C-D226C-E207R, recombinant plasmid pET-22b-S219C-D226C-A28N, recombinant plasmid pET-22b-S219C-D226C-V96L, recombinant plasmid pET-22b-S219C-D226C-S98A, and recombinant plasmid pET-22b-S219C-D226C-C114N.

[0058] Then, using recombinant plasmids pET-22b-S219C-D226C-P5C, pET-22b-S219C-D226C-F10C, and pET-22b-S219C-D226C-P182C as templates, and primer pairs G53C-F / R, E38C-F / R, and V210C-F / R as primers, PCR was performed to obtain recombinant plasmids pET-22b-S219C-D226C-P5C-G53C, pET-22b-S219C-D226C-F10C-E38C, and pET-22b-S219C-D226C-P182C-V210C.

[0059] Based on the 11 recombinant plasmids constructed above, corresponding mutant recombinant strains were further constructed using the method described in Example 1. Eleven Est30 carboxylesterase mutants, including CC-P5C-G53C, CC-F10C-E38C, CC-P182C-V210C, CC-V54K, CC-V54R, CC-I119E, CC-E207R, CC-A28N, CC-V96L, CC-S98A, and CC-C114N, were prepared and purified. The BHET degradation activity and Tm of different mutants were further determined. The results of the BHET degradation activity and Tm values ​​of different mutants at 50 ℃ and 60 ℃ are shown below. Figure 5 As shown.

[0060] Depend on Figure 5 It was found that, compared to the mutant Est30-KL (Tm = 67.93 ℃), the Tm values ​​of the 11 carboxylesterase Est30 mutants constructed in this embodiment were increased by 8.13 ℃ to 11.34 ℃. Among them, the mutant CC-E207R (referred to as mutant CCR) exhibited the best thermostability, with a Tm value of 79.27 ℃. Although most mutants showed reduced BHET degradation activity at 50 ℃ and 60 ℃ compared to mutant Est30-KL, they were still able to degrade BHET substrates into TPA monomers, and their activity can be further enhanced through other methods.

[0061] Example 3 This embodiment further superimposes other mutation sites on the mutant CCR (whose amino acid sequence is shown in SEQ ID No. 3) provided in Example 2, resulting in mutants S219C-D226C-E207R-P5C-G53C (referred to as mutant CCR-P5C-G53C), S219C-D226C-E207R-F10C-E38C (referred to as mutant CCR-F10C-E38C), S219C-D226C-E207R-P182C-V210C (referred to as mutant CCR-P182C-V210C), S219C-D226CE207R-V54K (referred to as mutant CCR-V54K), and S219C-D226C-E207R-P5C-G53C (referred to as mutant CCR-P5C-G53C), respectively. C-E207R-V54R (abbreviated as mutant CCR-V54R), S219C-D226C-E207R-I119E (abbreviated as mutant CCR-I119E), S219C-D226C-E207R-A28N (abbreviated as mutant CCR-A28N), S219C-D226C-E207R-V96L (abbreviated as mutant CCR-V96L), S219C-D226C-E207R-S98A (abbreviated as mutant CCR-S98A), and S219C-D226C-E207R-C114N (abbreviated as mutant CCR-C114N).

[0062] Using site-directed mutagenesis, with the recombinant plasmid pET-22b-S219C-D226C-E207R constructed in Example 2 as a template, and with the corresponding primers shown in Tables 1-3, PCR was performed according to the method provided in Example 1 to construct 10 recombinant plasmids, including pET-22b-S219C-D226C-E207R-P5C-G53C.

[0063] Further, after constructing the corresponding recombinant strains using the method described in Example 1, corresponding mutants were prepared, and the BHET degradation activity and Tm of different carboxylesterase Est30 mutants were measured. The results of the BHET degradation activity and Tm values ​​of different mutants at 50 °C and 60 °C are as follows: Figure 6 As shown.

[0064] Depend on Figure 6It can be seen that, compared with the mutant Est30-KL, the thermostability of the 10 carboxylesterase Est30 mutants provided in this embodiment is significantly improved, with their Tm values ​​increasing by 11.34 ℃ to 14.48 ℃. Among them, the Tm value of mutant CCR-P182C-V210C is 82.41 ℃, but the BHET degradation activity of this mutant at 50 ℃ and 60 ℃ is reduced to some extent compared with mutant Est30-KL. However, compared with mutant CCR, mutant CCR-P182C-V210C not only has a Tm value increased by 3.13 ℃, but also has BHET degradation activity increased by 8.86% and 32.7% respectively.

[0065] Example 4 Given the highly reducing environment of the E. coli cytoplasm, recombinant protein expression is generally suitable for proteins containing at most two pairs of disulfide bonds. Furthermore, based on the experimental results above, the proteins exhibit good additive thermal stability. To obtain mutants with significantly enhanced thermal stability more quickly, some or all sites of V54K, V54R, I119E, A28N, V96L, S98A, and C114N were subsequently added to the mutant CCR-P182C-V210C (referred to as mutant CCRCC), resulting in a total of six mutants: S219C-D226C-E207R-P18. 2C-V210C-A28N-V54K-V96L-C114N-I119E (abbreviated as mutant CCRCCNKLNE), S219C-D226C-E207R-P182C-V210C-A28N-V54R-V96L-C114N-I119E (abbreviated as mutant CCRCCNRLNE), S219C-D226C-E207R- P182C-V210C-A28N-V54K-S98A-C114N-I119E (abbreviated as mutant CCRCCNKANE), S219C-D226C-E207R-P182C-V210C-A28N-V54R-S98A-C114N-I119E (abbreviated as mutant CCRCCNRANE), S219C-D226C-E20 7R-P182C-V210C-A28N-V54K-V96L-S98A-C114N-I119E (referred to as mutant CCRCCNKLANE) and S219C-D226C-E207R-P182C-V210C-A28N-V54R-V96L-S98A-C114N-I119E (referred to as mutant CCRCCNRLANE).

[0066] Using site-directed mutagenesis, and with the recombinant plasmid pET-22b-S219C-D226C-E207R-P5C-G53C constructed in Example 3 as a template, PCR was performed using the corresponding primers shown in Tables 1-3, following the method provided in Example 1, to construct six recombinant plasmids. A schematic diagram of the plasmid map for recombinant plasmid pET-22b-CCRCCNRLNE is shown below. Figure 7 As shown.

[0067] Furthermore, after constructing the corresponding recombinant strains using the method described in Example 1, the corresponding mutants were prepared. Given that the thermal stability of the mutants has been significantly improved, to fully evaluate the degradation performance of different mutants under high-temperature conditions, this invention evaluated the BHET degradation activity and Tm of the mutants Est30-KL, CC, CCR, and CCRCC prepared in Examples 1-3, as well as the six mutants prepared in this example, at 72 °C. The results of the BHET degradation activity and Tm values ​​of different mutants at 72 °C are as follows: Figure 8 As shown.

[0068] Depend on Figure 8 It can be seen that, compared with the mutant Est30-KL, the mutants CCRCCNKLNE, CCRCCNRLNE, CCRCCNKANE, CCRCCNRANE, CCRCCNKLANE and CCRCCNRLANE provided in this embodiment have increased Tm values ​​by 22.86 ℃ to 23.97 ℃, and their BHET degradation activity at 72 ℃ has increased by 383.9% to 598.7%.

[0069] Specifically, compared to the mutant Est30-KL, the Tm value (90.80 ℃) of the mutant CCRCCNKLNE increased by 22.86 ℃, and the BHET degradation activity (TPA content in the mutant Est30-KL system was 0.033 vs. TPA content in the mutant CCRCCNKLNE system was 0.216) increased by 556.9%.

[0070] Compared to the mutant Est30-KL, the mutant CCRCCNRLNE showed a 23.97 ℃ increase in Tm value (91.91 ℃) and a 598.7% increase in BHET degradation activity (TPA content in the mutant Est30-KL system was 0.033 vs. TPA content in the mutant CCRCCNRLNE system was 0.229).

[0071] Compared to the mutant Est30-KL, the mutant CCRCCNKANE showed a 23.08 ℃ increase in Tm value (91.01 ℃) and a 383.9% increase in BHET degradation activity (TPA content in the mutant Est30-KL system was 0.033 vs. TPA content in the mutant CCRCCNKANE system was 0.159).

[0072] Compared to the mutant Est30-KL, the mutant CCRCCNRANE had a Tm value (90.92 ℃) that increased by 22.99 ℃ and a BHET degradation activity (TPA content in the mutant Est30-KL system was 0.033 vs. TPA content in the mutant CCRCCNRANE system was 0.166) that increased by 407.1%.

[0073] Compared to the mutant Est30-KL, the mutant CCRCCNKLANE had a Tm value (91.51 ℃) that increased by 23.58 ℃ and a BHET degradation activity (TPA content in the mutant Est30-KL system was 0.033 vs. TPA content in the mutant CCRCCNKLNE system was 0.173) that increased by 428.2%.

[0074] Compared to the mutant Est30-KL, the Tm value (91.72 ℃) of the mutant CCRCCNRLANE increased by 23.78 ℃, and the BHET degradation activity (TPA content in the mutant Est30-KL system was 0.033 vs. TPA content in the mutant CRCCNRLANE system was 0.170) increased by 417.0%.

[0075] Depend on Figure 8 It can also be seen that, compared with the mutant Est30-KL, the Tm values ​​of the mutants CCR and CCRCC provided in this embodiment are increased by 11.34 ℃~14.48 ℃, and the BHET degradation activity at 72 ℃ is increased by 156.2%~407.4%.

[0076] Specifically, compared to the mutant Est30-KL, the Tm value (79.27 ℃) of the mutant CCR increased by 11.34 ℃, and the BHET degradation activity (TPA content in the mutant Est30-KL system was 0.033 vs. TPA content in the mutant CCRCCNKLNE system was 0.084) increased by 156.2%.

[0077] Compared to the mutant Est30-KL, the Tm value (82.41 ℃) of the mutant CCRCC increased by 14.48 ℃, and the BHET degradation activity (TPA content in the mutant Est30-KL system was 0.033 vs. TPA content in the mutant CCRCCNKLNE system was 0.167) increased by 407.4%.

[0078] In summary, this invention provides a series of carboxylesterase Est30 mutants with significantly improved thermostability and BHET degradation activity by site-directed mutagenesis based on the Est30-KL mutant. Compared to the Est30-KL mutant, the BHET degradation activity of the carboxylesterase Est30 mutant provided by this invention is increased by 156.2%~598.7% at 72℃, and the Tm value is increased by 11.34℃~23.97℃, which is of great significance for the efficient industrial degradation of PET.

[0079] Given that the Est30 mutant carboxylesterase provided by this invention has both excellent thermal stability and BHET degradation activity, it can be compounded with PETase enzymes to prepare PET degradation agents to adapt to high-temperature PET degradation processes; it can also be used as an MHET degradation agent or BHET degradation agent to efficiently degrade PET enzymatic hydrolysis intermediates for TPA recovery and other applications.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A carboxylesterase Est30 mutant, characterized in that: Its amino acid sequence is shown in SEQ ID No.

3.

2. A carboxylesterase Est30 mutant, characterized in that: Its amino acid sequence is obtained by site-directed mutation of valine at position 96 of the amino acid sequence shown in SEQ ID No.3 to leucine, while the amino acid residues at other positions remain unchanged, and it is denoted as mutant CCR-V96L.

3. A carboxylesterase Est30 mutant, characterized in that: Its amino acid sequence is based on the amino acid sequence shown in SEQ ID No.3, with proline at position 182 mutated to cysteine ​​and valine at position 210 mutated to cysteine, while the amino acid residues at other positions remain unchanged. It is denoted as mutant CCRCC.

4. A carboxylesterase Est30 mutant, characterized in that: Its amino acid sequence is obtained by replacing five or six amino acid residues in the amino acid sequence of the mutant CCRCC described in claim 3, and its amino acid sequence is any one of (1) to (6) below: (1) The amino acid sequence of the mutant CCRCC is mutated at position 28 (alanine) to asparagine, position 54 (valine) to lysine, position 96 (valine) to leucine, and position 114 (cysteine) to asparagine. At the same time, position 119 (isoleucine) is mutated to glutamic acid. The amino acid residues at other positions remain unchanged. This mutant is called CCRCCNKLNE. (2) The amino acid sequence of the mutant CCRCC is mutated at position 28 (alanine) to asparagine, position 54 (valine) to arginine, position 96 (valine) to leucine, and position 114 (cysteine) to asparagine. At the same time, position 119 (isoleucine) is mutated to glutamic acid. The amino acid residues at other positions remain unchanged. This mutant is called CCRCCNRLNE. (3) The amino acid sequence of the mutant CCRCC is mutated at position 28 (alanine) to asparagine, position 54 (valine) to lysine, position 98 (serine) to alanine, and position 114 (cysteine) to asparagine. At the same time, position 119 (isoleucine) is mutated to glutamic acid. The amino acid residues at other positions remain unchanged. This mutant is called CCRCCNKANE. (4) The amino acid sequence of the mutant CCRCC is mutated at position 28 (alanine) to asparagine, position 54 (valine) to arginine, position 98 (serine) to alanine, position 114 (cysteine) to asparagine, and position 119 (isoleucine) to glutamic acid. The amino acid residues at other positions remain unchanged. This mutant is called CCRCCNRANE. (5) The amino acid sequence of the mutant CCRCC is mutated at position 28 (alanine) to asparagine, position 54 (valine) to lysine, position 96 (valine) to leucine, position 98 (serine) to alanine, and position 114 (cysteine) to asparagine. At the same time, position 119 (isoleucine) is mutated to glutamic acid. The amino acid residues at other positions remain unchanged. This mutant is called CCRCCNKLANE. (6) The amino acid sequence of the mutant CCRCC is mutated at position 28 (alanine) to asparagine, position 54 (valine) to arginine, position 96 (valine) to leucine, position 98 (serine) to alanine, and position 114 (cysteine) to asparagine. At the same time, position 119 (isoleucine) is mutated to glutamic acid. The amino acid residues at other positions remain unchanged. This mutant is called CCRCCNRLANE.

5. The encoding gene of the carboxylesterase Est30 mutant according to any one of claims 1 to 4.

6. A recombinant vector, characterized in that: It contains the coding gene as described in claim 5.

7. A recombinant bacterial strain, characterized in that: It comprises the recombinant vector as described in claim 6.

8. The use of the Est30 mutant of carboxylesterase according to any one of claims 1 to 4, the encoding gene according to claim 5, the recombinant vector according to claim 6, or the recombinant strain according to claim 7 in the degradation of MHET or BHET.

9. The use of the carboxylesterase Est30 mutant according to any one of claims 1 to 4, the encoding gene according to claim 5, the recombinant vector according to claim 6, or the recombinant strain according to claim 7 in the preparation of MHET degrading agents or BHET degrading agents.

10. A PET degradation agent, characterized in that: The PET degrading agent includes PET hydrolase and the Est30 mutant of carboxylesterase as described in any one of claims 1 to 4.

Images