Hydrophilic PET (Polyethylene Terephthalate) depolymerizing enzyme mutant and application thereof

Abstract

The invention relates to the field of PET plastic depolymerization through genetic engineering and a biological enzyme method, in particular to a hydrophilic PET depolymerization enzyme mutant and application thereof. According to the present invention, the 68 , or the 137 , or the 243 amino acid residue of the PETase protein amino acid sequence is mutated into R, such that the hydrophilic PET depolymerizing enzyme mutant V68R, or A137R or I243R is obtained; compared with a wild type PETase protein, on the premise that the depolymerization efficiency of PET / BHET is not reduced and PET / BHET is effectively depolymerized, in an escherichia coli, bacillus subtilis or pichia pastoris expression system, the expression level of soluble protein is improved, formation of inclusion bodies is reduced, economic feasibility and PETase high-yield universality are achieved, and the application prospect is wide.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzymatic depolymerization of PET plastics, specifically to a hydrophilic PET depolymerization enzyme mutant and its applications. Background Technology

[0002] Polyethylene terephthalate (PET) is one of the most widely used synthetic polyesters globally. Due to its excellent mechanical properties, chemical stability, and plasticity, it is widely used in beverage packaging, textile fibers, food containers, and other fields. However, this chemical stability also allows PET to resist natural degradation in the environment, leading to a serious problem of "white pollution."

[0003] PET depolymerases (PETases) are a class of hydrolytic enzymes that can break down the ester bonds in PET, primarily derived from microorganisms such as bacteria and fungi. Through specific catalytic action, these enzymes decompose PET polymers into terephthalic acid (TPA), ethylene glycol (EG), and intermediate products. These monomers can be reused to produce new PET products, achieving a closed-loop "plastic-to-plastic" cycle and representing a potential biological approach to solving PET pollution problems. Currently, relatively well-studied PETases include IsPETase derived from Idobacterium ossaka 201-F6, leaf compost keratinase (LCC), and various engineered PETases. These enzymes share a common catalytic mechanism: they possess a classic α / β hydrolase fold structure, and their active center typically consists of a catalytic triplet (Ser-His-Asp), which cleaves the ester bonds in the PET polymer chain through a nucleophilic attack mechanism.

[0004] Large-scale enzyme production platforms are one of the most ideal technologies for real-world application of enzyme-based PET decoy localization. Several microbial strains have been used to produce PETase, including secretory expression in Escherichia coli. However, in the industrial preparation of PETase, its heterologous expression still faces the following prominent problems: (1) Low expression efficiency: PETase has low expression efficiency in microbial (such as Escherichia coli) systems, resulting in limited recombinant protein production, which is difficult to meet the requirements of large-scale industrial applications for enzyme dosage; (2) Easy formation of inclusion bodies: PETase is prone to misfolding and aggregation to form inclusion bodies during heterologous expression, resulting in a very low proportion of soluble and active enzymes, which not only increases the difficulty of purification but also seriously affects the enzyme yield and catalytic availability; (3) Insufficient industrial economics: The expression level of PETase is not high, and it still faces the bottleneck of economics and scale in industrial applications, making it difficult to meet the strict requirements of large-scale biorecycling processes for enzyme preparation cost and supply. This seriously hinders the progress of industrial application of PETase.

[0005] Therefore, improving the soluble expression level of PETase, reducing inclusion body formation, and increasing the yield of bioactive proteins are important factors and key links in promoting its industrial application. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to address the shortcomings of the existing technology, such as low PETase expression efficiency, easy formation of inclusion bodies, and production bottleneck in industrialization, and to provide a hydrophilic PET depolymerase mutant.

[0007] Another technical problem to be solved by the present invention is to provide the encoding gene of the hydrophilic PET depolymerase mutant.

[0008] Another technical problem that this invention aims to solve is to provide a recombinant expression vector and a recombinant bacterial strain.

[0009] Another technical problem to be solved by the present invention is to provide the application of the hydrophilic PET depolymerase mutant in the expression of PET depolymerase protein, and a method for expressing PET depolymerase protein.

[0010] The final technical problem to be solved by the present invention is to provide the application of the hydrophilic PET depolymerase mutant in the depolymerization of PET and / or BHET, and a method for depolymerizing PET and / or BHET.

[0011] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0012] In a first aspect, the present invention provides a hydrophilic PET depolymerase mutant, wherein the hydrophilic PET depolymerase mutant is any one of the following mutants in which all amino acids at the mutation site are mutated to R:

[0013] (1) Mutate F to R at position 56 of the amino acid sequence of PETase;

[0014] (2) Mutate V to R at position 68 of the amino acid sequence of PETase;

[0015] (3) Mutate F to R at position 71 of the amino acid sequence of PETase;

[0016] (4) Mutate the amino acid sequence of PETase at position 137 from A to R;

[0017] (5) Mutate V to R at position 150 of the amino acid sequence of PETase;

[0018] (6) Mutate P to R at position 227 of the amino acid sequence of PETase;

[0019] (7) Mutate I to R at position 243 of the amino acid sequence of PETase;

[0020] (8) Mutate I to R at position 257 of the amino acid sequence of PETase;

[0021] (9) Mutate L to R at position 262 of the amino acid sequence of PETase;

[0022] The amino acid sequence of the PETase is shown in SEQ ID NO.1.

[0023] Preferably, the hydrophilic PET depolymerase mutant is any one of the following mutants in which all amino acids at the mutation site are mutated to R:

[0024] (1) Mutate V to R at position 68 of the amino acid sequence of PETase;

[0025] (2) Mutate the amino acid sequence of PETase at position 137 from A to R;

[0026] (3) Mutate I to R at position 243 of the amino acid sequence of PETase.

[0027] The mutation site of the hydrophilic PET depolymerase mutant is more than 5 Å away from the active center and the mutation energy is less than 0.

[0028] In a second aspect, the present invention provides a coding gene encoding the hydrophilic PET depolymerase mutant.

[0029] Thirdly, the present invention provides a recombinant expression vector containing the aforementioned coding gene.

[0030] Fourthly, the present invention provides a recombinant strain, which is obtained by introducing the recombinant expression vector into a host.

[0031] The host is any one of Escherichia coli, Bacillus subtilis, or Pichia pastoris.

[0032] In some embodiments of the present invention, the Escherichia coli is BL21, the Bacillus subtilis is WB600, and the Pichia pastoris is GS115.

[0033] Fifthly, the present invention provides the use of the hydrophilic PET depolymerase mutant, the recombinant expression vector, or the recombinant strain in expressing PET depolymerase protein.

[0034] In a sixth aspect, the present invention provides a method for expressing a PET depolymerase mutant protein, wherein a plasmid containing the hydrophilic PET depolymerase mutant is transformed into competent cells of a host to obtain a recombinant strain; and the seed culture of the recombinant strain is then inoculated into a fermentation medium to express the PET depolymerase mutant protein.

[0035] In some embodiments of the present invention, when the host is Escherichia coli, the culture conditions in the fermenter are: fermenter temperature 16~37℃, stirring speed 200~800 rpm, tank pressure 0.03~0.08 MPa, pH 5.0~6.0, maximum aeration ratio 0.3~2vvm, and dissolved oxygen value controlled at 30±5%.

[0036] In some embodiments of the present invention, when the host is Bacillus subtilis, the culture conditions in the fermenter are as follows: fermenter temperature 35~37℃, stirring speed 200~800 rpm, tank pressure 0.03~0.06 MPa, pH 5.0~6.0, maximum aeration ratio of 0.3~2 vvm, and dissolved oxygen value controlled at 30~50%.

[0037] In some embodiments of the present invention, when the host is Pichia pastoris, the culture conditions in the fermenter are: fermenter temperature 25~30℃, stirring speed 200~800 rpm, pH 5.0~6.0, maximum aeration ratio of 0.3~2 vvm, and dissolved oxygen value controlled at 25~35%.

[0038] In a seventh aspect, the present invention provides the use of the hydrophilic PET depolymerase mutant, or the recombinant expression vector, or the recombinant strain in depolymerizing PET and / or BHET.

[0039] The depolymerization is carried out using PET and / or BHET as substrates and an enzyme solution of a hydrophilic PET depolymerase mutant as a catalyst to depolymerize PET and / or BHET.

[0040] Eighthly, the present invention provides a method for depolymerizing PET and / or BHET, wherein at the start of the reaction, an enzyme solution of the hydrophilic PET depolymerase mutant is added to a buffer solution containing the substrate PET and / or BHET to carry out the depolymerization reaction.

[0041] In some embodiments of the present invention, the PET and / or BHET are added in powder form.

[0042] The buffer solution is a 40-60 mM phosphate buffer with a pH of 6.0-8.0.

[0043] In some embodiments of the present invention, the buffer solution is a pH 8.0 50 mM phosphate buffer.

[0044] The final concentration of the hydrophilic PET depolymerase mutant enzyme solution in the depolymerization reaction system is 2-6 mg / L.

[0045] In some embodiments of the present invention, the final concentration of the enzyme solution of the hydrophilic PET depolymerase mutant in the depolymerization reaction system is 2 mg / L, 4 mg / L, or 6 mg / L.

[0046] The depolymerization reaction is carried out under the following conditions: temperature 40~85℃ and time 3~20 h.

[0047] In some embodiments of the present invention, the depolymerization reaction is carried out under the following conditions: temperature of 68-80°C and time of 6 hours.

[0048] Beneficial effects:

[0049] (1) The present invention obtains a hydrophilic PET depolymerase mutant V68R, A137R, or I243R by mutating any one of the amino acid residues at positions 56, 68, 71, 137, 150, 227, 243, 257 or 262 of the amino acid sequence of the PETase protein to R.

[0050] (2) Compared with wild-type PETase, the hydrophilic PET depolymerase mutant obtained by increasing the hydrophilicity of PET depolymerization in this invention improves the expression level of soluble protein and reduces the formation of inclusion bodies while ensuring that the depolymerization efficiency of PET / BHET is not reduced and effectively depolymerizing PET / BHET. This achieves a balance between expression level and enzyme activity, improves the overall economic efficiency and environmental sustainability of the process, and realizes a balance between cost-effectiveness and catalytic efficiency.

[0051] (3) The PETase mutant of the present invention can not only effectively reduce the formation of protein inclusion bodies during enzyme production in Escherichia coli, significantly improve the protein expression level, and reduce the protein production cost, but also improve the expression level of the PETase mutant of the present invention in Bacillus subtilis and Pichia pastoris, and has universal applicability to achieve high production of PETase in different engineered strains. Attached Figure Description

[0052] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0053] Figure 1 This is a pET29a-PETase plasmid map.

[0054] Figure 2 Protein gel electrophoresis image of broken bacterial sludge from a hydrophilic PET depolymerase mutant.

[0055] Figure 3 The results show the heat resistance of hydrophilic PET depolymerase mutant protein at different reaction temperatures.

[0056] Figure 4 This image shows the depolymerization effect of the hydrophilic PET depolymerase mutant protein BHET.

[0057] Figure 5 This image shows the depolymerization effect of the hydrophilic PET depolymerase mutant protein PET.

[0058] Figure 6 This is a pP43NMK-PETase plasmid map.

[0059] Figure 7 This is a pPICZαA-PETase plasmid map. Detailed Implementation

[0060] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0061] The culture media involved in the following examples are as follows:

[0062] (1) LB medium: 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract.

[0063] (2) YPD medium: 1% yeast extract, 2% peptone, 2% glucose.

[0064] (3) Escherichia coli fermentation medium: 24 g / L yeast extract, 12 g / L peptone, 5 g / L glycerol, 12.54 g / L K2HPO4, 2.31 g / L KH2PO4, with an initial pH of 7.

[0065] (4) Bacillus subtilis fermentation medium: 24 g / L yeast extract, 12 g / L soybean peptone, 5 g / L glycerol, 12.54 g / L K2HPO4, 2.31 g / L KH2PO4, with an initial pH of 6-7.

[0066] (5) Pichia pastoris fermentation medium: 26.7 g / L H3PO4, 0.93 g / L CaSO4·2H2O, 18.2 g / L K2SO4, 14.9 g / L MgSO4·2H2O, 4.13 g / L KOH, 4% glycerol, 0.4% PMT1, with an initial pH of 5~6.

[0067] The detection methods involved in the following embodiments are as follows:

[0068] (1) Biomass determination method

[0069] The wet cell weight (WCW) of the cells during fermentation was determined using a specific gravity method. A 2 mL capped centrifuge tube was dried to constant weight, weighed, and then 1 mL of fermentation broth was added. The tube was centrifuged at 12000 rpm for 5 min. After removing the supernatant, the cell pellet was collected and washed twice with 1 mL of physiological saline. After centrifugation, the supernatant was discarded, and the cells were weighed. The wet cell weight was calculated as follows: Wet cell weight (g / L) = Weight of centrifuge tube containing wet cells (g) - Net weight of centrifuge tube (g) / Sampling volume of fermentation broth (L).

[0070] The turbidimetric method was used to determine the cellular optical density (OD) during fermentation. 1 mL of fermentation broth was added to a capped centrifuge tube and centrifuged at 12000 rpm for 5 min. The supernatant was discarded, and 1 mL of physiological saline was added and mixed thoroughly. After appropriate dilution, the absorbance of physiological saline was used as a blank control. The appropriate dilution factor (n) was considered when the absorbance at 600 nm was between 0.2 and 0.8. Cellular optical density (OD) = n × OD 600 .

[0071] (2) Enzyme activity detection method

[0072] Add 10 μL of 10 mM p-nitrophenol octanoate (pNPO), 10 μL of enzyme solution, and 980 μL of PBS buffer (50 mM, pH 8.0) to a 5 mL centrifuge tube, bringing the final volume to 1 mL. Heat the assay system at 37°C for 10 min, then immediately cool on ice. Measure the absorbance at 410 nm using a microplate reader. The absorbance is determined by p-nitrophenol and OD... 410 The standard curve was plotted to calculate the molar amount of p-nitrophenol. Enzyme activity is defined as the amount of enzyme required to produce 1 μmol of p-nitrophenol per minute at 37°C.

[0073] (3) Protein content detection methods

[0074] Protein concentrations were determined using a BSA protein assay kit, and the purity of each protein was analyzed by SDS-PAGE. First, a 1 mg / mL bovine serum albumin (BSA) stock solution was prepared. Then, PBS (50 mM, pH 8.0) was added to the stock solution to prepare a series of BSA stock solutions with concentrations ranging from 0.2 to 1 mg / L. These solutions were then diluted 10-fold to obtain a series of diluted BSA solutions with concentrations ranging from 0.02 to 0.1 mg / L. 20 μL of each concentration gradient of BSA standard solution was added to a 96-well plate, followed by 200 μL of Coomassie Brilliant Blue solution. After thorough mixing, the plates were allowed to stand for 3 min. The absorbance of the different concentrations of BSA standard solutions at 595 nm was measured using a microplate reader, and a standard curve was plotted with protein concentration on the x-axis and absorbance on the y-axis. The absorbance of the samples could be measured using the same method, and the protein concentration could be calculated from the standard curve.

[0075] In this invention, the protein expression level refers to the concentration of active protein in the protein solution, and the calculation formula is: protein expression level (mg / L) = protein solution enzyme activity (U / mL) / protein specific enzyme activity (U / mg).

[0076] (4) Liquid chromatography method for detecting the depolymerization rate of PET and BHET

[0077] PET degradation enzymatic hydrolysates were filtered using a 0.22 μm filter, diluted appropriately according to product concentration, and then analyzed using an Agilent 1260 Infinity II high-performance liquid chromatography system via a C18 column (Thermo Fisher Scientific, 150 × 4.6 mm, 5 μm). A mobile phase of 18% acetonitrile, 1% formic acid, and 81% water (pH 2.5) was used. The detection time was 25 min, the flow rate was 0.8 mL / min, and the detection wavelength was 254 nm. Standard curves were constructed using TPA and BHET gradient standard solutions at concentrations of 1 mM, 0.1 mM, 0.01 mM, 0.001 mM, and 0.0001 mM. The product concentration of the enzymatic hydrolysate could be calculated from the standard curves.

[0078] Example 1: Selection of mutation sites for hydrophilic PET depolymerase (PETase) and construction of recombinant expression vector

[0079] The three-dimensional structure of PET depolymerase (PDB:8QRJ) was observed in pymol. Hydrophilic mutation sites on the surface of the PETase protein were selected. To ensure that the protein activity and stability were not affected while increasing hydrophilicity, mutation sites (F56R, V68R, F71R, A137R, V150R, P227R, I243R, I257R, or L262R) that were more than 5 Å away from the active site and had a mutation energy of less than 0 were selected. The mutation energy was calculated as shown in Table 1.

[0080] Table 1 Mutation energy of hydrophilic PET depolymerase mutants

[0081]

[0082] Using whole-plasmid PCR, site-directed mutagenesis was performed at positions 56, 68, 71, 137, 150, 227, 243, 257, or 262. Based on the PETase gene sequence (as shown in SEQ ID NO. 2), primers introducing mutations of F56R, V68R, F71R, A137R, V150R, P227R, I243R, I257R, or L262R were designed and synthesized, as shown in Table 2. The PETase gene fragment was synthesized into the pET29a vector by GenScript Technology Co., Ltd., resulting in the pET29a-PETase plasmid. Figure 1 Using this plasmid as a template, PCR amplification was performed with the corresponding primers. The amplified PCR product was digested and transformed with enzymes, and verified by sequencing. The plasmid was extracted using a plasmid extraction kit to obtain the recombinant expression vectors containing the PETase mutant gene: pET29a-PETase-F56R, pET29a-PETase-V68R, pET29a-PETase-F71R, pET29a-PETase-A137R, pET29a-PETase-V150R, pET29a-PETase-P227R, pET29a-PETase-I243R, pET29a-PETase-I257R, and pET29a-PETase-L262R. The amino acid sequences of the hydrophilic PET depolymerase mutants F56R, V68R, F71R, A137R, V150R, P227R, I243R, I257R, and L262R obtained by mutation are shown in SEQ ID NO. 3~11.

[0083] Table 2 Primer information used in Example 1

[0084]

[0085] Example 2: Construction of recombinant Escherichia coli with hydrophilic PET depolymerase mutant

[0086] (1) Preparation of Escherichia coli BL21 competent cells

[0087] In a clean bench, pick a single colony from a freshly activated E. coli BL21 agar plate and inoculate it into 3-5 mL LB broth. Incubate at 37°C with shaking for approximately 12 h until the logarithmic growth phase. Transfer the bacterial suspension at a ratio of 1:50-100 to 20 mL LB broth and incubate at 37°C with shaking to expand the culture. Once the culture medium begins to become turbid, measure the OD (octane rating) every 20-30 min. 600 , to OD 600 Stop culturing when the concentration is ≤0.5. Transfer 1.5 mL of culture medium to a microcentrifuge tube, cool on ice for 10 min, and centrifuge at 5000 rpm for 10 min at 4°C (from this step onwards, all operations should be performed on ice, and the speed should be as fast and steady as possible). Discard the supernatant culture medium, gently resuspend the cells in 1 mL of ice-cold 0.1 mol / L CaCl2 solution, incubate on ice, and centrifuge at 5000 rpm for 10 min at 0–4°C. Discard the supernatant, add 500 µL of ice-cold 0.1 mol / L CaCl2 solution, and carefully resuspend the cells. Centrifuge at 5000 rpm for 10 min at 0–4°C. Discard the supernatant, add 100 µL of ice-cold 0.1 mol / L CaCl2 solution, carefully resuspend the cells, and place on ice for a short time to prepare the E. coli BL21 competent cell suspension. The prepared E. coli BL21 competent cell suspension can be used directly for transformation experiments, or approximately 15% of autoclaved glycerol can be added, mixed well, and then dispensed into Eppendorf microcentrifuge tubes in a clean bench, flash-frozen in liquid nitrogen, and placed at -80°C.

[0088] (2) Construction of recombinant Escherichia coli

[0089] Remove the E. coli BL21 competent cell suspension prepared in step (1) from the -80℃ freezer, thaw at room temperature, and immediately place on ice. Add the recombinant expression vector DNA solution from Example 1, with a concentration not exceeding 50 ng and a volume not exceeding 10 μL, and gently shake well. Then place on ice for 30 min. Heat shock the reaction mixture in a 42℃ water bath for 90 s, and then quickly place on ice to cool for 3-5 min. Add 1 mL of LB liquid medium to the reaction tube, mix well, and incubate at 37℃ with shaking for 1 h. After shaking the above bacterial suspension well, take 100 μL and spread it on a selection plate containing 100 μg / mL kana, place it face up for 30 min, and after the bacterial suspension has been completely absorbed by the medium, invert the culture dish and incubate at 37℃ for 16-24 hours. h, thus obtaining recombinant Escherichia coli: pET29a-PETase-F56R / E.coil, pET29a-PETase-V68R / E.coil, pET29a-PETase-F71R / E.coil, pET29a-PETase-A137R / E.coil, pET29a-PETase-V150R / E.coil, pET29a-PETase-P227R / E.coil, pET29a-PETase-I243R / E.coil, pET29a-PETase-I257R / E.coil, and pET29a-PETase-L262R / E.coil. Recombinant Escherichia coli prepared using wild-type PETase was used as a control.

[0090] Example 3: Detection of protein expression and fermentation level in recombinant Escherichia coli

[0091] (1) Preparation of seed liquid

[0092] The recombinant Escherichia coli obtained in Example 2 was activated and inoculated into LB medium. After being cultured at 37°C and 220 rpm for 12-14 h, the corresponding seed culture was obtained.

[0093] (2) Expression of recombinant Escherichia coli proteins

[0094] The seed culture from step (1) was inoculated into Escherichia coli fermentation medium at an inoculation rate of 5% v / v. The medium was then cultured at 37°C, stirring speed of 200-800 rpm, pressure of 0.03-0.08 MPa, pH of 5.0-6.0, maximum aeration ratio of 0.3-2 vvm, and dissolved oxygen level of 30±5% until OD. 600The temperature was raised to 25℃, then lowered to 25℃, and IPTG was added to a final concentration of 0.1 mM for induction expression for 12–18 h. After fermentation, 10 mL of fermentation broth was centrifuged at 4℃ and 12000 r / min for 10 min to obtain fermented bacterial sludge. The sludge was resuspended in 10 mL of phosphate buffer (pH 8.0) and then sonicated. After disruption, the sludge was centrifuged at 4℃ and 12000 r / min for 10 min to obtain the supernatant. Enzyme activity and protein content of the supernatant were measured, and the results are shown in Table 3. The disrupted bacterial sludge with increased enzyme activity and mutant protein was subjected to SDS-PAGE to detect intracellular inclusion body production. The results are shown in Table 3. Figure 2 As shown.

[0095] Combining the results of enzyme activity, soluble protein expression level measurement and protein gel electrophoresis, it was found that during the expression of V68R, A137R and I243R, the soluble protein expression level was much higher than that of wild-type PETase, and the amount of inclusion body formation was significantly reduced.

[0096] Table 3. Enzyme activity and soluble protein expression levels of hydrophilic PET depolymerase mutants (E. coli)

[0097]

[0098] Example 4: Heat resistance test of hydrophilic PET depolymerase mutants V68R, A137R and I243R

[0099] Three hydrophilic PET depolymerase mutants with high soluble protein expression levels (V68R, A137R, and I243R) from Example 3 were selected. Enzyme solutions of V68R, A137R, I243R, and wild-type PETase were incubated at 70℃, 80℃, and 90℃ for 2 h, respectively, and their residual enzyme activities were measured. Results Figure 3 As shown, the heat resistance of V68R, A137R, and I243R did not decrease compared to wild-type PETase. This indicates that while the enzyme activity and protein expression levels of V68R, A137R, and I243R increased, their heat resistance did not decrease, and they can still withstand high temperatures of 70-90℃.

[0100] Example 5: Degradation of BHET by hydrophilic PET depolymerase mutants V68R, A137R, and I243R

[0101] The reaction system consisted of 50 mL of 50 mM pH 8.0 phosphate buffer and 2 g / L BHET powder (purity >90%). At the beginning of the reaction, crude PET depolymerase solution was added to achieve final concentrations of 2 mg / L, 4 mg / L, and 6 mg / L, respectively. The reaction was carried out at 80℃ for 6 h. The depolymerization effect of BHET was as follows... Figure 4 As shown, V68R, A137R, I243R and wild-type PETase have similar depolymerization effects on BHET, indicating that V68R, A137R and I243R do not decrease their depolymerization effect on BHET while increasing enzyme activity and protein expression levels.

[0102] Example 6: Degradation of PET by hydrophilic PET depolymerase mutants V68R, A137R and I243R

[0103] The reaction system consisted of 50 mL of 50 mM pH 8.0 phosphate buffer and 2 g / L PET powder (purity >90%). At the beginning of the reaction, crude PET depolymerase solution was added to achieve final concentrations of 2 mg / L, 4 mg / L, and 6 mg / L, respectively. The reaction was carried out at 68℃ for 6 h. The PET depolymerization effect was as follows: Figure 5 As shown, V68R, A137R, I243R and wild-type PETase have similar depolymerization effects on PET, indicating that V68R, A137R and I243R do not decrease their depolymerization effect on PET while increasing enzyme activity and protein expression levels.

[0104] Example 7: Construction of recombinant Bacillus subtilis with hydrophilic PET depolymerase mutant

[0105] (1) Construction of recombinant expression vector

[0106] Using the recombinant expression vectors pET29a-PETase-V68R, pET29a-PETase-A137R, and pET29a-PETase-I243R obtained in Example 1 as templates, the PETase-V68R, PETase-A137R, and PETase-I243R fragments were amplified using PETase-F and PETase-R primers (Table 4). Using the pP43NMK vector as a template, the vector fragments were amplified using pP43NMK-F and pP43NMK-R (Table 4). Then, the PETase, V68R, A137R, and I243R fragments were ligated to the pP43NMK vector using Infusio, and the ligation products were transformed into E. coli JM109 competent cells to obtain transformation products. The plasmid was extracted from the transformation products for sequencing verification, obtaining the recombinant plasmid pP43NMK-PETase (… Figure 6 ), pP43NMK-PETase-V68R, pP43NMK-PETase-A137R, pP43NMK-PETase-I243R.

[0107] Table 4 Primer sequences used in Example 7

[0108]

[0109] (2) Preparation of Bacillus subtilis WB600 competent cells

[0110] Single colonies of Bacillus subtilis WB600 were picked and inoculated into 10 mL of LB liquid medium and cultured overnight at 37°C and 200 rpm for 8 h. 2.5 mL of the culture was then transferred to 40 mL of LB medium containing 0.5 M sorbitol and cultured with shaking at 37°C and 200 rpm for 4–5 h. The bacterial culture was then incubated in an ice-water bath for 10 min, followed by centrifugation at 4°C and 5000 rpm for 5 min to collect the cells. The cells were resuspended in 50 mL of pre-chilled electroporation buffer, centrifuged at 4°C and 5000 rpm for 5 min, and the supernatant was discarded. This washing process was repeated four times. The washed cells were resuspended in 1 mL of electroporation medium and aliquoted into 1.5 mL EP tubes (200 μL per tube) to obtain competent Bacillus subtilis WB600 cells.

[0111] (3) Construction of recombinant Bacillus subtilis

[0112] Add the recombinant plasmids obtained in step (1) to the Bacillus subtilis WB600 competent cells obtained in step (2), incubate on ice for 18 min, and then add them to a pre-cooled electroporation cup (2 mm) for one electroporation (2.4 kV, 25 μF, 200 Ω). Immediately after electroporation, 1 mL of pre-cooled RM medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 91 g / L sorbitol, and 69 g / L mannitol) was added. After recovery at 37°C and 200 rpm for 3 h, the medium was spread onto plates containing 100 μg / mL Kana to obtain recombinant Bacillus subtilis: pP43NMK-PETase / Bacillus subtilis, pP43NMK-PETase-V68R / Bacillus subtilis, pP43NMK-PETase-A137R / Bacillus subtilis, and pP43NMK-PETase-I243R / Bacillus subtilis.

[0113] Example 8: Detection of protein expression and fermentation level in recombinant Bacillus subtilis

[0114] (1) Preparation of seed liquid

[0115] The recombinant Bacillus subtilis obtained in Example 7 was activated and inoculated into LB medium. After being cultured at 37°C and 220 rpm for 12 h, the corresponding seed culture was obtained.

[0116] (2) Expression of recombinant Bacillus subtilis protein

[0117] The seed culture from step (1) was inoculated into Bacillus subtilis fermentation medium at an inoculation rate of 5% v / v. The medium was cultured for 24–30 h at a temperature of 35–37 °C, a stirring speed of 200–800 rpm, a tank pressure of 0.03–0.06 MPa, a pH of 5.0–6.0, a maximum aeration ratio of 0.3–2 vvm, and a dissolved oxygen level of 30–50%. After fermentation, 10 mL of the fermentation broth was centrifuged at 4 °C and 12000 r / min for 10 min to obtain the fermentation supernatant. The enzyme activity and protein content of the fermentation supernatant were then measured.

[0118] The results are shown in Table 5. The expression levels of soluble proteins V68R, A137R, and I243R were higher than those of wild-type PETase, indicating that increasing protein hydrophilicity can improve the expression level of PET depolymerase in Bacillus subtilis.

[0119] Table 5 Enzyme activity and soluble protein expression levels of hydrophilic PET depolymerase mutants (Bacillus subtilis)

[0120]

[0121] Example 9: Construction of recombinant Pichia pastoris with a hydrophilic PET depolymerase mutant

[0122] (1) Construction of recombinant expression vector

[0123] Using the recombinant expression vectors pET29a-PETase-V68R, pET29a-PETase-A137R, and pET29a-PETase-I243R obtained in Example 1 as templates, the PETase-V68R, PETase-A137R, and PETase-I243R fragments were amplified using PETase-F and PETase-R primers (Table 6). Using the pPICZαA vector as a template, the vector fragments were amplified using pPICZαA-F and pPICZαA-R (Table 6). Then, the PETase, V68R, A137R, and I243R fragments were ligated to the pPICZαA vector using Infusio, respectively. The ligated products were transformed into E. coli JM109 competent cells to obtain the transformation products. The plasmid was extracted from the transformation products for sequencing verification, yielding the recombinant plasmid pPICZαA-PETase (… Figure 7 ), pPICZαA-PETase-V68R, pPICZαA-PETase-A137R and pPICZαA-PETase-I243R.

[0124] Table 6 Primer sequences used in Example 9

[0125]

[0126] (2) Preparation of Pichia pastoris GS115 competent cells

[0127] Spots were picked from fresh Pichia pastoris GS115 plates and inoculated into 5 mL of YPD medium, and cultured at 30°C for 12 h until OD. 600 =1.0~1.5. Take 2.5 mL of the overnight culture and inoculate it into a 1 L shake flask containing 250 mL of YPD medium. Incubate until OD reaches 1.0~1.5. 600=0.3~0.5, centrifuge at 4℃, 4000 rpm for 5 min to collect the bacterial sludge, and gently resuspend the cells in 500 mL of pre-cooled sterile water. Repeat the above steps, mixing the collected bacterial sludge with 10 mL of freshly prepared Pichia pastoris GS115 competent cell stock solution and gently resuspending the cells. Incubate at 4℃ for 30 min, gently shaking every 10 min. Centrifuge at 4℃, 4000 rpm for 5 min, collect the bacterial sludge, and add 5 mL of pre-cooled 1 M sorbitol for resuspending. Repeat this step 3 times. Resuspend with 1 mL of pre-cooled 1 M sorbitol to a final volume of approximately 1.5 mL, and aliquot into 80 μL tubes.

[0128] (3) Construction of recombinant Pichia pastoris

[0129] Add 1–3 ng of linearized recovered expression vector to the Pichia pastoris GS115 competent cells prepared in step (2), then gently mix with a pipette and place on ice for 5 min. Transfer to an electroporation cuvette and continue treatment in an ice-water bath for 5 min. The electroporation parameters were set as follows: 2.0 kV, 25 μF, 250 Ω, and 0.2 cm cuvett. After electroporation, immediately add 1 mL of pre-chilled 1 M sorbitol to the cuvette and transfer the bacterial culture to a sterile 1.5 mL centrifuge tube. Place the centrifuge tube in a 30°C incubator and incubate statically for 1 h. Centrifuge at 4000 rpm for 5 min at room temperature, then collect the cells and resuspend in 200 μL / mL YPDS. 100 μL of bacterial suspension was evenly spread on a YPD plate containing 100 μg / mL Zeocin to obtain recombinant Pichia pastoris: pPICZαA-PETase / Pichia pastoris, pPICZαA-PETase-V68R / Pichia pastoris, pPICZαA-PETase-A137R / Pichia pastoris, and pPICZαA-PETase-I243R / Pichia pastoris.

[0130] Example 10: Detection of protein expression and fermentation level in recombinant Pichia pastoris

[0131] (1) Preparation of seed liquid

[0132] The recombinant Pichia pastoris obtained in Example 9 was activated and inoculated into YPD medium. After being cultured at 30°C and 220 rpm for 20-24 h, the corresponding seed culture was obtained.

[0133] (2) Expression of recombinant Pichia pastoris protein

[0134] The seed culture from step (1) was inoculated into Pichia pastoris fermentation medium at an inoculation rate of 5% v / v. The culture was maintained at 30℃, stirring speed of 200–800 rpm, pH 5.0–6.0, maximum aeration ratio of 0.3–2 vvm, and dissolved oxygen level of 25–35% until the wet weight reached 250 g / mL. The temperature was then lowered to 28℃, and methanol was added for induction culture for 150–200 h. After fermentation, 10 mL of the fermentation broth was centrifuged at 4℃ and 12000 r / min for 10 min to obtain the fermentation supernatant. Enzyme activity and protein content were measured in the fermentation supernatant.

[0135] The results are shown in Table 7. The protein expression levels of V68R, A137R, and I243R were higher than those of wild-type PETase. This result indicates that increasing protein hydrophilicity can enhance the expression level of PETase in Pichia pastoris.

[0136] Table 7 Enzyme activity and soluble protein expression levels of hydrophilic PET depolymerase mutants (Pichia pastoris)

[0137]

[0138] This invention provides a hydrophilic PET depolymerase mutant and its application, along with a method and approach. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A hydrophilic PET depolymerase mutant, characterized in that, The hydrophilic PET depolymerase mutant is any one of the following mutants in which the amino acid at the mutation site is mutated to R: (1) the V at position 68 of the amino acid sequence of PETase is mutated to R; (2) the A at position 137 of the amino acid sequence of PETase is mutated to R; (3) the I at position 243 of the amino acid sequence of PETase is mutated to R; wherein the amino acid sequence of the PETase is shown in SEQ ID NO.

1.

2. A coding gene encoding the hydrophilic PET depolymerase mutant of claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains the coding gene of claim 2.

4. A recombinant bacterial strain, characterized in that, The recombinant strain is obtained by introducing the recombinant expression vector of claim 3 into a host.

5. The recombinant strain of claim 4, wherein, The host is any one of Escherichia coli, Bacillus subtilis, and Pichia pastoris.

6. Use of the hydrophilic PET depolymerase mutant of claim 1, or the recombinant expression vector of claim 3, or the recombinant strain of claim 4 in expressing a PET depolymerase protein.

7. A method of expressing a PET depolymerase mutant protein, characterized by, After transforming the plasmid containing the hydrophilic PET depolymerase mutant of claim 1 into a competent cell of a host, a recombinant strain is obtained; then the seed liquid of the recombinant strain is inoculated into a fermentation medium to express the PET depolymerase mutant protein.

8. Use of the hydrophilic PET depolymerase mutant of claim 1, or the recombinant expression vector of claim 3, or the recombinant strain of claim 4 in depolymerizing PET and / or BHET.

9. Use according to claim 8, characterized in that, The depolymerization is performed with the hydrophilic PET depolymerase mutant as a catalyst to depolymerize PET and / or BHET.

10. A method of depolymerizing PET and / or BHET, characterized in that, At the beginning of the reaction, the enzyme solution of the hydrophilic PET depolymerase mutant of claim 1 is added to a buffer solution containing the substrate PET and / or BHET to perform the depolymerization reaction; wherein the buffer solution is a 40-60 mM phosphate buffer with a pH of 6.0-8.

0.

11. The method of claim 10, wherein, The enzyme solution of the hydrophilic PET depolymerase mutant has a final concentration of 2-6 mg / L in the depolymerization reaction system.

12. The method of claim 10, wherein, The depolymerization reaction is performed under the following conditions: temperature of 40-85℃, and time of 3-20 h.

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