Hydrophilic pet depolymerase mutant and use thereof

Abstract

The present application relates to the field of genetic engineering and biological enzyme depolymerization of PET plastics, and particularly relates to a hydrophilic PET depolymerase mutant and application thereof. The present application obtains a hydrophilic PET depolymerase mutant V68R, or A137R or I243R by mutating the 68th, or 137th, or 243th amino acid residue of the PETase protein amino acid sequence into R. Compared with the wild-type PETase protein, under the premise of ensuring the depolymerization efficiency of PET / BHET and effectively depolymerizing PET / BHET, the expression level of soluble protein is improved, and the formation of inclusion bodies is reduced in the expression system of Escherichia coli, Bacillus subtilis or Pichia pastoris, which has economic feasibility, PETase high-yield universality, and wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering and biological enzyme depolymerization of PET plastics, in particular to a hydrophilic PET depolymerase mutant and its application. BACKGROUND

[0002] Polyethylene terephthalate (PET) is one of the most widely used synthetic polyesters in the world. 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 makes PET resistant to natural degradation in the environment, leading to a serious "white pollution" problem.

[0003] PET depolymerase (PETase) is a class of hydrolases that can hydrolyze the ester bond in PET, mainly 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 "plastic-to-plastic" closed-loop cycle, which is a potential biological means to solve the problem of PET pollution. Currently, the relatively in-depth PETases include IsPETase derived from Idesia osakensis 201-F6, leaf compost keratinase (LCC) and various engineered PETases. These enzymes have a common catalytic mechanism: they have a classic α / β hydrolase folding structure, and their active centers are usually composed of a catalytic triad (Ser-His-Asp), which cleaves the ester bond in the PET polymer chain through a nucleophilic attack mechanism.

[0004] Large-scale enzyme production platform is one of the most ideal technologies for PET bait positioning based on enzymes to be applied in the real world. Several microbial strains have been used to produce PETase, including secretory expression in Escherichia coli. However, in the industrial production process of PETase, its heterologous expression still faces the following outstanding problems: (1) low expression efficiency: PETase has low expression efficiency in microbial (such as Escherichia coli) systems, and the yield of recombinant protein is limited, which is difficult to meet the demand for enzyme preparation amount in industrial large-scale application; (2) easy to form inclusion bodies: PETase is prone to misfolding and aggregation to form inclusion bodies when expressed heterologously, resulting in a very low proportion of soluble and active enzymes, which not only increases the difficulty of purification, but also seriously affects the yield and catalytic availability of the enzyme; (3) lack of industrial economy: PETase has low expression level, which still faces the economic and scale bottlenecks of industrial application, making it difficult to meet the strict requirements of enzyme preparation cost and supply in large-scale biological recycling process. This seriously hinders the industrial application progress of PETase.

[0005] Therefore, how to improve the soluble expression level of PETase, reduce the formation of inclusion bodies, and improve the yield of active protein is an important factor and key link to promote its industrial application. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a hydrophilic PET depolymerase mutant to solve the problems of low expression efficiency, easy formation of inclusion bodies and yield bottleneck in industrialization of PETase in the prior art.

[0007] The technical problem to be solved by the present application is to provide a hydrophilic PET depolymerase mutant to solve the problems of low expression efficiency, easy formation of inclusion bodies and yield bottleneck in industrialization of PETase in the prior art.

[0008] The technical problem to be solved by the present application is to provide a hydrophilic PET depolymerase mutant to solve the problems of low expression efficiency, easy formation of inclusion bodies and yield bottleneck in industrialization of PETase in the prior art.

[0009] The technical problem to be solved by the present application is to provide a hydrophilic PET depolymerase mutant to solve the problems of low expression efficiency, easy formation of inclusion bodies and yield bottleneck in industrialization of PETase in the prior art.

[0010] The technical problem to be solved by the present application is to provide a hydrophilic PET depolymerase mutant to solve the problems of low expression efficiency, easy formation of inclusion bodies and yield bottleneck in industrialization of PETase in the prior art.

[0011] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0012] In a first aspect, the present application provides a hydrophilic PET depolymerase mutant, wherein the hydrophilic PET depolymerase mutant is a mutant in which the amino acid at any of the following positions is mutated to R:

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

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

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

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

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

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

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

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

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

[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 the amino acid at the mutation site is mutated to R:

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

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

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

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

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

[0029] In a third aspect, the present application provides a recombinant expression vector containing the coding gene.

[0030] In a fourth aspect, the present application provides a recombinant strain obtained by introducing the recombinant expression vector into a host.

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

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

[0033] In a fifth aspect, the present application provides the use of the hydrophilic PET depolymerase mutant, or the recombinant expression vector, or the recombinant strain in expressing a PET depolymerase protein.

[0034] In a sixth aspect, the present application provides a method for expressing a PET depolymerase mutant protein. The method comprises the following steps: transforming a plasmid containing the hydrophilic PET depolymerase mutant into a competent cell of a host to obtain a recombinant strain; and inoculating seed liquid of the recombinant strain into a fermentation medium to express the PET depolymerase mutant protein.

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

[0036] In some embodiments of the present application, 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 0.3-2 vvm, and controlled dissolved oxygen value 30-50%.

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

[0038] In a seventh aspect, the present application 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] In the depolymerization, PET and / or BHET is used as the substrate, and the enzyme solution of the hydrophilic PET depolymerase mutant is used as the catalyst.

[0040] In an eighth aspect, the present application provides a method for depolymerizing PET and / or BHET, in which the enzyme solution of the hydrophilic PET depolymerase mutant is added into a buffer solution containing the substrate PET and / or BHET at the beginning of the reaction to carry out the depolymerization reaction.

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

[0042] In the buffer solution, the pH is 6.0-8.0, and the phosphate buffer concentration is 40-60 mM.

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

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

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

[0046] The depolymerization reaction is carried out at a temperature of 40-85°C for 3-20 hours.

[0047] In some embodiments of the present application, the depolymerization reaction is carried out at a temperature of 68-80°C for 6 hours.

[0048] Advantages:

[0049] (1) The present application preferably obtains a hydrophilic PET depolymerase mutant V68R, or 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 PETase protein amino acid sequence to R.

[0050] (2) Compared with the wild-type PETase, the hydrophilic PET depolymerase mutant obtained by increasing the hydrophilicity of PET depolymerization in the present application improves the expression level of soluble protein and reduces the formation of inclusion bodies while ensuring the depolymerization efficiency of PET / BHET and effectively depolymerizing PET / BHET, thereby balancing the expression level and enzyme activity, improving the economic efficiency and environmental sustainability of the overall process, and achieving a balance between cost efficiency and catalytic efficiency.

[0051] (3) The PETase mutant of the present application not only effectively reduces the formation of protein inclusion bodies during enzyme production in Escherichia coli, significantly improves the protein expression level, and reduces the production cost of protein. At the same time, the expression amount of the PETase mutant of the present application in Bacillus subtilis and Pichia pastoris is also improved to a certain extent, which is universal for achieving high yield of PETase in different engineering strains. BRIEF DESCRIPTION OF DRAWINGS

[0052] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

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

[0054] Figure 2 Figure 4 is a protein gel electrophoresis map of the hydrophilic PET depolymerase mutant broken bacterial slurry.

[0055] Figure 3 Figure 6 is the heat resistance detection result of the hydrophilic PET depolymerase mutant protein at different reaction temperatures.

[0056] Figure 4 Figure for PET depolymerization effect of hydrophilic PET depolymerase mutant protein BHET.

[0057] Figure 5 Figure for PET depolymerization effect of hydrophilic PET depolymerase mutant protein BHET.

[0058] Figure 6 Figure for pP43NMK-PETase plasmid map.

[0059] Figure 7 Figure for pPICZαA-PETase plasmid map. DETAILED DESCRIPTION

[0060] In the following examples, the experimental methods are described, and if no special instructions, they are all conventional methods; the reagents and materials, if no special instructions, can be obtained from commercial channels.

[0061] In the following examples, the culture medium involved is as follows:

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

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

[0064] (3) Escherichia coli fermentation medium: 24 g / L yeast powder, 12 g / L peptone, 5 g / L glycerol, 12.54 g / L K2HPO4, 2.31 g / L KH2PO4, initial pH 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, initial pH 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, initial pH 5~6.

[0067] In the following examples, the detection methods involved are as follows:

[0068] (1) Biomass determination method

[0069] The wet cell weight (WCW) in the fermentation process was determined by specific gravity method. 2 mL centrifuge tube with cover was dried to constant weight, weighed, then 1 mL fermentation broth was added, and centrifuged at 12000 rpm for 5 min. After removing the supernatant, the bacterial pellet was collected and washed twice with 1 mL physiological saline. After centrifugation, the supernatant was removed, and the bacterial pellet was weighed. The wet cell weight was: wet cell weight (g / L) = weight of centrifuge tube containing wet bacteria (g) - net weight of centrifuge tube (g) / fermentation broth sampling volume (L).

[0070] The cell optical density (OD) in the fermentation process was determined by turbidimetry. 1 mL of fermentation broth was added to a centrifuge tube with cover, and centrifuged at 12000 rpm for 5 min. The supernatant was discarded, and 1 mL of physiological saline was added and mixed well. After appropriate dilution of the sample, the absorbance of physiological saline was used as a blank control. When the absorbance at 600 nm was between 0.2 and 0.8, the dilution factor (n) at this time was the appropriate dilution condition. Cell optical density (OD) = n x OD 600 .

[0071] (2) Enzyme activity detection method

[0072] In a 5 mL centrifuge tube, 10 μL of 10 mM p-nitrophenyl octanoate (pNPO), 10 μL of enzyme solution and 980 μL of PBS buffer solution (50 mM, pH 8.0) were added to a final volume of 1 mL. The enzyme activity system was heated at 37°C for 10 min, then immediately cooled in ice, and the absorbance at 410 nm was detected using an enzyme marker. The standard curve of p-nitrophenol and OD 410 was drawn to calculate the molar amount of p-nitrophenol. Enzyme activity definition: the amount of enzyme required to generate 1 μmol of p-nitrophenol per minute at 37°C is defined as one enzyme activity unit.

[0073] (3) Protein amount detection method

[0074] The protein concentration was detected by a protein assay kit (BSA protein assay kit), and the purity of each protein was detected by SDS-PAGE analysis. First, a 1 mg / mL bovine serum albumin (BSA) mother liquor was prepared, and then PBS (50 mM, pH 8.0) was added to the mother liquor to prepare a group of BSA mother liquor with a concentration range of 0.2-1 mg / L. Then, the group of solutions was diluted 10 times to obtain a group of BSA dilution solutions with a concentration of 0.02-0.1 mg / L. First, 20 μL of BSA standard solution with different concentration gradients was added to a 96-well plate, then 200 μL of coomassie brilliant blue solution was added, and after mixing thoroughly, it was left to react for 3 min. The absorbance value of the BSA standard solution with different concentrations at 595 nm was detected by an enzyme-labeled instrument, and the standard curve was drawn with the protein concentration as the abscissa and the absorbance as the ordinate. The absorbance value of the sample can be measured by the same method, and the protein concentration can be calculated by the standard curve.

[0075] In the present application, the protein expression amount is the concentration of active protein in the protein solution, and the calculation formula is: protein expression amount (mg / L) = protein enzyme activity (U / mL) / protein specific enzyme activity (U / mg).

[0076] (4) Liquid chromatography detection method of PET and BHET depolymerization rate

[0077] The PET degradation enzyme product was filtered using a 0.22 μm filter, and then diluted according to the product concentration. Then, the product was analyzed by a C18 chromatographic column (Thermo Fisher Scientific, 150×4.6 mm, 5 μm) using an Agilent 1260 Infinity II high-performance liquid chromatography system. The solvent used was 18% acetonitrile, 1% formic acid and 81% water (pH 2.5), the detection time was 25 min, the flow rate was 0.8 mL / min, and the detection wavelength was 254 nm. The detection results of 1 mM, 0.1 mM, 0.01 mM, 0.001 mM and 0.0001 mM TPA and BHET gradient standard solutions were used to make a standard curve. The product concentration of the enzyme hydrolysis sample can be calculated by the standard curve.

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

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

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

[0081]

[0082] The full plasmid PCR method was used to perform site-directed mutagenesis at the 56th, 68th, 71st, 137th, 150th, 227th, 243rd, 257th or 262nd sites. According to the gene sequence of PETase (as shown in SEQ ID NO. 2), primers introducing F56R, V68R, F71R, A137R, V150R, P227R, I243R, I257R or L262R mutations were designed and synthesized, as shown in Table 2. The PETase gene fragment was synthesized by Jinersi Technology Co., Ltd. into pET29a vector to obtain pET29a-PETase plasmid (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). Figure 1 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, respectively.

[0083] Table 2 Primer information used in Example 1

[0084]

[0085] Example 2: Construction of recombinant E. coli of hydrophilic PET depolymerase mutant

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

[0087] In the super-clean bench, a single colony was picked from a newly activated E. coli BL21 bacterial plate and inoculated into 3-5 mL of LB liquid culture. The culture was incubated at 37°C for about 12 h with shaking until the logarithmic growth phase. The bacterial suspension was transferred into 20 mL of LB liquid medium at a ratio of 1:50-100, and the culture was expanded at 37°C with shaking. When the culture solution began to appear turbid, the OD 600 was measured every 20-30 min until the OD 600 was ≤0.5. Then 1.5 mL of the culture solution was transferred into a microcentrifuge tube and cooled on ice for 10 min. The solution was centrifuged at 4°C and 5000 rpm for 10 min. From this step, all operations were performed on ice as quickly and stably as possible. The supernatant was discarded, and the cells were gently suspended with 1 mL of ice-cold 0.1 mol / L CaCl2 solution. The solution was centrifuged at 0-4°C and 5000 rpm for 10 min. The supernatant was discarded, and 500 μL of ice-cold 0.1 mol / L CaCl2 solution was added to the cells. The cells were carefully suspended and centrifuged at 0-4°C and 5000 rpm for 10 min. The supernatant was discarded, and 100 μL of ice-cold 0.1 mol / L CaCl2 solution was added to the cells. The cells were carefully suspended and placed on ice for a while to prepare the E. coli BL21 competent cell suspension. The prepared E. coli BL21 competent cell suspension can be directly used for transformation experiments, or it can be mixed with about 15% of autoclaved glycerol based on the total volume. After mixing, the solution was aliquoted into Eppendorf microcentrifuge tubes in a super-clean bench, frozen in liquid nitrogen, and stored at -80°C.

[0088] (2) Construction of recombinant E. coli

[0089] The E. coli BL21 competent cell suspension prepared in step (1) was taken out from the -80 °C refrigerator, and immediately placed on ice after thawing at room temperature. The recombinant expression vector DNA solution in Example 1 was added in an amount of not more than 50 ng and a volume of not more than 10 μL, and shaken gently. Then it was placed on ice for 30 min. After heat shock at 42 °C for 90 s, the reaction mixture was quickly placed on ice for 3-5 min. 1 mL of LB liquid medium was added to the reaction tube, and after mixing, it was placed in a 37 °C shaker for 1 h. After shaking the above bacterial solution, 100 μL was spread on a screening plate containing 100 μg / mL kana, and placed upside down for 30 min. After the bacterial solution was completely absorbed by the culture medium, the culture dish was inverted and incubated at 37 °C for 16-24 h to obtain recombinant E. coli: pET29a-PETase-F56R / E. coli, pET29a-PETase-V68R / E. coli, pET29a-PETase-F71R / E. coli, pET29a-PETase-A137R / E. coli, pET29a-PETase-V150R / E. coli, pET29a-PETase-P227R / E. coli, pET29a-PETase-I243R / E. coli, pET29a-PETase-I257R / E. coli, pET29a-PETase-L262R / E. coli. The recombinant E. coli prepared with wild-type PETase was used as a control.

[0090] Example 3: Detection of recombinant E. coli protein expression and fermentation level

[0091] (1) Preparation of seed solution

[0092] The recombinant E. coli obtained in Example 2 was activated and inoculated into LB medium, and incubated at 37 °C, 220 rpm for 12-14 h to obtain the corresponding seed solution.

[0093] (2) Expression of recombinant E. coli protein

[0094] The seed solution of step (1) was inoculated into E. coli fermentation medium at a 5% v / v inoculation amount, and incubated at a temperature of 37 °C, a stirring speed of 200-800 rpm, a tank pressure of 0.03-0.08 Mpa, a pH of 5.0-6.0, a maximum aeration ratio of 0.3-2 vvm, and a dissolved oxygen value of 30±5% to an OD 600to 25℃, and 0.1 mM IPTG was added to induce 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 fermentation slurry, and the fermentation slurry was resuspended in 10 mL of phosphate buffer (pH 8.0) and then subjected to ultrasonic disruption. After disruption, the disrupted supernatant was centrifuged at 4℃ and 12000 r / min for 10 min to obtain the disrupted supernatant, and the disrupted supernatant was subjected to enzyme activity and protein content detection, and the detection results are shown in Table 3. The disrupted slurry of the mutant protein with increased enzyme activity in the lower tank was subjected to SDS protein gel electrophoresis to detect the production of intracellular inclusion bodies, and the detection results are shown in Table 4. Figure 2

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

[0096] Table 3 Enzyme activity and soluble protein expression level of hydrophilic PET depolymerase mutant (Escherichia coli)

[0097]

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

[0099] Three hydrophilic PET depolymerase mutants V68R, A137R and I243R with high soluble protein expression level in Example 3 were selected, and the enzyme solutions of V68R, A137R, I243R and wild-type PETase were incubated at 70℃, 80℃ and 90℃ for 2 h, respectively, and the residual enzyme activity of each was determined. The detection results are shown in Table 5. Figure 3 It was found that the heat resistance of V68R, A137R and I243R did not decrease relative to wild-type PETase, which indicated that V68R, A137R and I243R could still tolerate high temperature of 70-90℃ while maintaining high enzyme activity and protein expression level.

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

[0101] ​The reaction system was 50 mL: 50 mM pH 8.0 phosphate buffer, 2 g / L BHET powder (purity > 90%), and the crude enzyme solution of PET depolymerase was added at the beginning of the reaction to make the final concentration of PET depolymerase 2 mg / L, 4 mg / L and 6 mg / L, respectively, and the reaction was carried out at a reaction temperature of 80°C for 6 h. The BHET depolymerization effect is shown in Figure 4 As shown in the table, V68R, A137R, I243R and wild-type PETase have similar depolymerization effects on BHET, indicating that V68R, A137R and I243R do not decrease the depolymerization effect on BHET while improving enzyme activity and protein expression level.

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

[0103] The reaction system was 50 mL: 50 mM pH 8.0 phosphate buffer, 2 g / L PET powder (purity > 90%), and the crude enzyme solution of PET depolymerase was added at the beginning of the reaction to make the final concentration of PET depolymerase 2 mg / L, 4 mg / L and 6 mg / L, respectively, and the reaction was carried out at a reaction temperature of 68°C for 6 h. The PET depolymerization effect is shown in Figure 5 As shown in the table, V68R, A137R, I243R and wild-type PETase have similar depolymerization effects on PET, indicating that V68R, A137R and I243R do not decrease the depolymerization effect on PET while improving enzyme activity and protein expression level.

[0104] Example 7: Construction of recombinant B. subtilis of hydrophilic PET depolymerase mutants

[0105] (1) Construction of recombinant expression vector

[0106] The recombinant expression vectors pET29a-PETase-V68R, pET29a-PETase-A137R and pET29a-PETase-I243R obtained in Example 1 were used as templates, and the PETase-F and PETase-R primers (Table 4) were used to amplify the PETase-V68R, PETase-A137R and PETase-I243R fragments, respectively. The pP43NMK-F and pP43NMK-R (Table 4) were used to amplify the vector fragment from the pP43NMK vector. Then the PETase, V68R, A137R and I243R fragments were respectively connected to the pP43NMK vector by Infusio, and the connected products were transformed into E. coli JM109 competent cells to obtain the transformation products. The plasmids in the transformation products were extracted and sequenced to obtain the recombinant plasmids pP43NMK-PETase (pP43NMK-PETase-V68R, pP43NMK-PETase-A137R and pP43NMK-PETase-I243R. Figure 6

[0107] Table 4 Primer sequences used in Example 7

[0108]

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

[0110] A single colony of Bacillus subtilis WB600 was inoculated into 10 mL of LB liquid medium, and incubated at 37°C and 200 rpm for 8 h overnight. 2.5 mL of the culture was transferred into 40 mL of LB medium containing 0.5 M sorbitol, and incubated at 37°C and 200 rpm for 4-5 h. The bacterial solution was placed in an ice water bath for 10 min, and then centrifuged at 4°C and 5000 rpm for 5 min to collect the bacterial cells. The bacterial cells were resuspended in 50 mL of pre-cooled electrotransformation buffer, and centrifuged at 4°C and 5000 rpm for 5 min. The supernatant was removed, and the washing was repeated 4 times. The washed bacterial cells were resuspended in 1 mL of electrotransformation medium, and divided into 1.5 mL EP tubes, each containing 200 μL, to obtain Bacillus subtilis WB600 competent cells.

[0111] (3) Construction of recombinant Bacillus subtilis

[0112] ​The competent cells of Bacillus subtilis WB600 obtained in step (2) were added with each recombinant plasmid obtained in step (1), respectively, and then subjected to ice bath for 18 min, and then subjected to electric shock once (2.4 kv, 25 μF, 200 Ω) in a pre-cooled electric transformation cup (2 mm). After the electric shock, 1 mL of pre-cooled RM medium (10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of NaCl, 91 g / L of sorbitol, 69 g / L of mannitol) was immediately added, and then the mixture was recovered at 37°C and 200 rpm for 3 h, and then coated on a plate containing 100 μg / mL Kana, to obtain recombinant Bacillus subtilis: pP43NMK-PETase / Bacillus subtilis, pP43NMK-PETase-V68R / Bacillus subtilis, pP43NMK-PETase-A137R / Bacillus subtilis, pP43NMK-PETase-I243R / Bacillus subtilis.

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

[0114] (1) Preparation of seed liquid

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

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

[0117] The seed liquid of step (1) was inoculated into Bacillus subtilis fermentation medium at an inoculation amount of 5% v / v, and then cultured under the following conditions: temperature 35-37°C, stirring speed 200-800 rpm, tank pressure 0.03-0.06 Mpa, pH 5.0-6.0, maximum aeration ratio 0.3-2 vvm, and control of dissolved oxygen value 30-50%, for 24-30 h. After the fermentation was completed, 10 mL of fermentation broth was centrifuged at 4°C and 12000 r / min for 10 min to obtain fermentation supernatant, and then the enzyme activity and protein amount of the fermentation supernatant were detected, respectively.

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

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

[0120]

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

[0122] (1) Construction of recombinant expression vector

[0123] The recombinant expression vectors pET29a-PETase-V68R, pET29a-PETase-A137R and pET29a-PETase-I243R obtained in Example 1 were used as templates, and the PETase-V68R, PETase-A137R and PETase-I243R fragments were amplified using the PETase-F and PETase-R primers (Table 6). The pPICZαA vector was used as a template, and the vector fragment was amplified using the pPICZαA-F and pPICZαA-R primers (Table 6). Then the PETase, V68R, A137R and I243R fragments were respectively connected to the pPICZαA vector through Infusio, and the connected products were transformed into E. coli JM109 competent cells to obtain the transformed products. The plasmids in the transformed products were extracted and sequenced to obtain the recombinant plasmids pPICZαA-PETase (pPICZαA-PETase-V68R, pPICZαA-PETase-A137R and pPICZαA-PETase-I243R. Figure 7

[0124] Table 6 Primer sequences used in Example 9

[0125]

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

[0127] Fresh Pichia pastoris GS115 plates were inoculated in 5 mL of YPD medium and cultured at 30°C for 12 h to OD 600 = 1.0~1.5. 2.5 mL of the overnight culture was inoculated into a 1 L flask containing 250 mL of YPD medium, and the culture was incubated to OD 600 ​= 0.3~0.5, centrifuged at 4°C, 4000 rpm for 5 min to collect the bacterial slurry, and the cells were resuspended gently using 500 mL of pre-cooled sterilized water. The above step was repeated, and the collected bacterial slurry was mixed with 10 mL of freshly prepared Pichia pastoris GS115 competent mother liquor to resuspend the cells gently. The cells were allowed to stand at 4°C for 30 min, with gentle shaking every 10 min. The bacterial slurry was collected by centrifugation at 4°C, 4000 rpm for 5 min, and resuspended with 5 mL of pre-cooled 1 M sorbitol. This step was repeated 3 times. The cells were resuspended with 1 mL of pre-cooled 1 M sorbitol to a final volume of about 1.5 mL, and 80 μL per tube was aliquoted.

[0128] (3) Construction of recombinant Pichia pastoris

[0129] The linearized recovered expression vector containing 1~3 ng was added to the Pichia pastoris GS115 competent cells prepared in step (2), and then mixed gently using a pipette and placed on ice for 5 min. Then, the cells were transferred to an electroporation cuvette and treated in an ice water bath for 5 min. The electroporation parameters of the electroporator were set as follows: 2.0 kV, 25 μF, 250 Ω, and 0.2 cm cuvette. After the completion of the electroporation, 1 mL of pre-cooled 1 M sorbitol was immediately added to the electroporation cuvette, and the bacterial solution was transferred to a sterile 1.5 mL centrifuge tube. The centrifuge tube was placed in a 30°C incubator and incubated for 1 h. Then, the bacterial solution was centrifuged at 4000 rpm for 5 min at room temperature, and the bacterial cells were collected and resuspended with 200 μL / mL of YPDS. 100 μL of the bacterial suspension was evenly spread on a YPD plate containing 100 μg / mL Zeocin, thereby obtaining the 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 recombinant Pichia pastoris protein expression and fermentation level

[0131] (1) Preparation of seed solution

[0132] The recombinant Pichia pastoris obtained in Example 9 was activated and inoculated into YPD medium, and then cultured at 30°C, 220 rpm for 20~24 h to obtain the corresponding seed solution.

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

[0134] The seed liquid of step (1) is inoculated into the Pichia fermentation medium at an inoculation amount of 5% v / v, and is cultured at a temperature of 30°C, a stirring speed of 200-800 rpm, a pH of 5.0-6.0, a maximum aeration ratio of 0.3-2 vvm, and a controlled dissolved oxygen value of 25-35%, until the wet weight reaches 250 g / mL. Then, the temperature is reduced to 28°C, methanol is added for induction culture for 150-200 h. After the fermentation is completed, 10 mL of the fermentation liquid is 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 are detected.

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

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

[0137]

[0138] The present application provides a kind of hydrophilic PET depolymerase mutant and its application idea and method, the method and approach for specifically realizing this technical scheme are many, above-mentioned only preferred embodiment of the present application, it should be pointed out, for the ordinary skilled person in the art, without departing from the principles of the present application, can make several improvements and refinements, these improvements and refinements also should be regarded as the protection scope of the present application. The components not explicitly described in the embodiment can be realized by existing technology.

Claims

1. A hydrophilic PET depolymerase mutant, characterized in that, 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: (1) Mutate V to R at position 68 of the amino acid sequence of PETase; (2) Mutate the amino acid sequence of PETase at position 137 from A to R; (3) Mutate I to R at position 243 of the amino acid sequence of PETase; The amino acid sequence of the PETase is shown in SEQ ID NO.

1.

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

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains the encoding gene as described in 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 the host.

5. The recombinant strain according to claim 4, characterized in that, The host is any one of Escherichia coli, Bacillus subtilis, or Pichia pastoris.

6. The use of the encoding gene of claim 2, the recombinant expression vector of claim 3, or the recombinant strain of claim 4 in the expression of PET depolymerase protein.

7. A method for expressing a mutant protein of PET depolymerase, characterized in that, After transforming a plasmid containing the hydrophilic PET depolymerase mutant of claim 1 into competent cells of a host, a recombinant strain was obtained; then the seed culture of the recombinant strain was inoculated into a fermentation medium to express the PET depolymerase mutant protein.

8. The application 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. The application according to claim 8, characterized in that, The depolymerization is carried out using PET and / or BHET as substrates and the enzyme solution of the hydrophilic PET depolymerase mutant as a catalyst to depolymerize PET and / or BHET.

10. A method for depolymerizing PET and / or BHET, characterized in that, At the start of the reaction, the enzyme solution of the hydrophilic PET depolymerase mutant of claim 1 is added to a buffer containing substrate PET and / or BHET to carry out the depolymerization reaction; wherein the buffer is a pH 6.0~8.0, 40~60 mM phosphate buffer; wherein the reaction temperature of the depolymerization reaction is 37~80℃.

11. The method according to claim 10, characterized in that, 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 according to claim 10, characterized in that, The reaction time for the depolymerization reaction is 3 to 20 hours.

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