BhrPETase mutant and application thereof

Abstract

The invention belongs to the technical field of enzyme engineering, and discloses a BhrPETase mutant and application thereof. The invention provides a BhrPETase mutant, the amino acid sequence of the BhrPETase mutant is shown as SEQ ID No.2, and the BhrPETase mutant is marked as a mutant F208I. Compared with mutant wild type BhrPETase, the PET degradation activity of the BhrPETase mutant is improved by 1.39 times. On the basis of the mutant F208I, the amino acid of the mutant F208I is further subjected to rational mutation design, and 21 BhrPETase mutants are provided. According to the BhrPETase mutant provided by the invention, the PET (Polyethylene Terephthalate) degradation activity is improved by 1.39 to 4.24 times under the condition that the thermal stability is basically equivalent to that of wild type BhrPETase. The BhrPETase mutant provided by the invention can be applied to the fields of PET degradation, PET degradation product recovery or PET degradation agent preparation and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of enzyme engineering, and relates to a PET degrading enzyme, in particular to a BhrPETase mutant and application thereof. BACKGROUND

[0002] Plastics are indispensable materials in modern life, among which polyethylene terephthalate (PET) is widely used in packaging and textile industries due to its light weight, corrosion resistance and strong plasticity. However, the high crystallinity and stable chemical structure of PET make it difficult to degrade naturally, leading to a large accumulation of plastic waste, which poses a serious threat to the ecological environment and human health.

[0003] Enzymatic degradation of PET shows great potential. Compared with traditional chemical recycling methods, enzymatic degradation has the advantages of mild reaction conditions, no pollution, and recyclable products. In recent years, scientists have found that many microorganisms can secrete PET hydrolases that can specifically break the ester bonds in PET molecules, providing inspiration for the application of biological enzymes in PET waste management and sustainable recycling of PET. Among them, through the strategy of artificial intelligence assisted protein engineering, researchers developed a BhrPETase mutant (named TurboPETase) which pushed the PET depolymerization efficiency to a new height. Under the load of 200g / kg, a high substrate concentration, it only takes 8 hours to achieve nearly 99% conversion rate, which is significantly better than the previous optimal LCC-ICCG. The maximum production rate of TurboPETase reaches 61.3g hydrolyzed PET L -1 h -1 , and the reaction can be carried out in aqueous solution without the need for expensive buffer systems. This breakthrough provides an innovative solution for sustainable recycling of plastics and promotes the development of green chemistry and circular economy.

[0004] In high temperature environment, the crystallinity of PET increases significantly, and the molecular structure becomes more compact. This characteristic makes it difficult for existing PET hydrolases to efficiently act on the substrate, thereby restricting the industrialization of PET biodegradation technology. BhrPETase, as a thermophilic PET degrading enzyme, although has certain advantages in thermal stability (Tm≈97℃) and PET catalytic activity, but its catalytic efficiency still has a lot of room for improvement. Therefore, exploring the BhrPETase mutant with enhanced PET degradation activity by molecular modification without significantly reducing or not reducing the thermal stability of BhrPETase, so as to further improve the catalytic efficiency, has important significance to meet the practical application requirements of industrial scale production. SUMMARY

[0005] In view of the above problems existing in the prior art, the present application provides a BhrPETase mutant with significantly improved PET degradation activity and application thereof on the basis of wild-type BhrPETase.

[0006] To achieve the above-mentioned purposes, the embodiments of the present application adopt the following technical solutions: In a first aspect, the present application provides a BhrPETase mutant, which is a BhrPETase mutant obtained by site-directed mutagenesis of phenylalanine at position 208 in the amino acid sequence of wild-type BhrPETase to isoleucine, and the amino acid residues at other positions are unchanged, and the obtained amino acid sequence is shown as SEQ ID No. 2, and the obtained BhrPETase mutant is denoted as mutant F208I.

[0007] The present application provides a BhrPETase mutant, i.e., mutant F208I, which is obtained by site-directed mutagenesis of phenylalanine at position 208 in the amino acid sequence of wild-type BhrPETase to isoleucine. The PET degradation activity of mutant F208I is increased by 1.39 times compared with that of wild-type BhrPETase.

[0008] The amino acid sequence of wild-type BhrPETase is shown as SEQ ID No. 1, and the amino acid sequence of mutant F208I is shown as SEQ ID No. 2.

[0009] In a second aspect, the present application further provides 11 kinds of BhrPETase mutants obtained by single-point mutation based on mutant F208I, and the amino acid sequences of the mutants are any one of (1)~(11) as follows: (1) the serine at position 27 in the amino acid sequence shown as SEQ ID No. 2 is site-directed mutated to valine, and the amino acid residues at other positions are unchanged, and the obtained mutant is denoted as mutant I-S27V; (2) the valine at position 28 in the amino acid sequence shown as SEQ ID No. 2 is site-directed mutated to isoleucine, and the amino acid residues at other positions are unchanged, and the obtained mutant is denoted as mutant I-V28I; (3) the serine at position 113 in the amino acid sequence shown as SEQ ID No. 2 is site-directed mutated to proline, and the amino acid residues at other positions are unchanged, and the obtained mutant is denoted as mutant I-S113P; (4) the glutamine at position 142 in the amino acid sequence shown as SEQ ID No. 2 is site-directed mutated to leucine, and the amino acid residues at other positions are unchanged, and the obtained mutant is denoted as mutant I-Q142L; (5) the glutamine at position 142 of the amino acid sequence shown in SEQ ID No. 2 is mutated to tryptophan, and the amino acid residues at other positions are unchanged, and the obtained mutant is recorded as mutant I-Q142W; (6) the threonine at position 157 of the amino acid sequence shown in SEQ ID No. 2 is mutated to proline, and the amino acid residues at other positions are unchanged, and the obtained mutant is recorded as mutant I-T157P; (7) the threonine at position 160 of the amino acid sequence shown in SEQ ID No. 2 is mutated to tyrosine, and the amino acid residues at other positions are unchanged, and the obtained mutant is recorded as mutant I-T160Y; (8) the glutamine at position 167 of the amino acid sequence shown in SEQ ID No. 2 is mutated to methionine, and the amino acid residues at other positions are unchanged, and the obtained mutant is recorded as mutant I-Q167M; (9) the histidine at position 183 of the amino acid sequence shown in SEQ ID No. 2 is mutated to tyrosine, and the amino acid residues at other positions are unchanged, and the obtained mutant is recorded as mutant I-H183Y; (10) the asparagine at position 211 of the amino acid sequence shown in SEQ ID No. 2 is mutated to methionine, and the amino acid residues at other positions are unchanged, and the obtained mutant is recorded as mutant I-N211M; (11) the serine at position 212 of the amino acid sequence shown in SEQ ID No. 2 is mutated to methionine, and the amino acid residues at other positions are unchanged, and the obtained mutant is recorded as mutant I-S212M.

[0010] The present application found through experiments that, compared with the wild-type BhrPETase, the PET degradation activity of the above-mentioned 11 mutants is increased by 1.48-2.78 times. Compared with mutant F208I, the PET degradation activity of the above-mentioned 11 mutants is increased by 4%-58%. Compared with mutant F208I (Tm value is 91.5℃), the thermal stability of the above-mentioned 11 mutants is basically the same, and the Tm value is 88.26-96.61℃, among which the Tm value of mutant I-Q142W is 96.43℃, and the Tm value of mutant I-N211M is 96.61℃. The Tm value of mutant I-H183Y is 92.36℃, and its PET degradation activity is 3.78 times that of wild-type BhrPETase.

[0011] In a third aspect, the present application further provides 10 BhrPETase mutants based on mutant F208I, and the amino acid sequence of the mutants is any one of the following (12)-(21), specifically: (12) the histidine at position 183 of the amino acid sequence shown in SEQ ID No. 2 is mutated to tyrosine, the valine at position 28 is mutated to isoleucine, and the amino acid residues at other positions are unchanged, and the obtained mutant is recorded as mutant IY-V28I; (13) the histidine at position 183 of the amino acid sequence shown in SEQ ID No. 2 is mutated to tyrosine, the serine at position 113 is mutated to proline, and the amino acid residues at other positions are unchanged, and the obtained mutant is recorded as mutant IY-S113P; (14) the histidine at position 183 of the amino acid sequence shown in SEQ ID No. 2 is mutated to tyrosine, the glutamine at position 142 is mutated to leucine, and the amino acid residues at other positions are unchanged, and the obtained mutant is recorded as mutant IY-Q142L; (15) the histidine at position 183 of the amino acid sequence shown in SEQ ID No. 2 is mutated to tyrosine, the glutamine at position 142 is mutated to tryptophan, and the amino acid residues at other positions are unchanged, and the obtained mutant is recorded as mutant IY-Q142W; (16) the histidine at position 183 of the amino acid sequence shown in SEQ ID No. 2 is mutated to tyrosine, the threonine at position 157 is mutated to proline, and the amino acid residues at other positions are unchanged, and the obtained mutant is recorded as mutant IY-T157P; (17) the histidine at position 183 of the amino acid sequence shown in SEQ ID No. 2 is mutated to tyrosine, the threonine at position 160 is mutated to tyrosine, and the amino acid residues at other positions are unchanged, and the obtained mutant is recorded as mutant IY-T160Y; (18) the histidine at position 183 of the amino acid sequence shown in SEQ ID No. 2 is mutated to tyrosine, the glutamine at position 167 is mutated to methionine, and the amino acid residues at other positions are unchanged, and the obtained mutant is recorded as mutant IY-Q167M; (19) the histidine at position 183 of the amino acid sequence shown in SEQ ID No. 2 is mutated to tyrosine, the glutamic acid at position 201 is mutated to leucine, and the amino acid residues at other positions are unchanged, and the obtained mutant is recorded as mutant IY-E201L; (20) the histidine at position 183 of the amino acid sequence shown in SEQ ID No. 2 is mutated to tyrosine, the asparagine at position 211 is mutated to methionine, and the amino acid residues at other positions are unchanged, and the obtained mutant is recorded as mutant IY-N211M; (21) the histidine at position 183 of the amino acid sequence shown in SEQ ID No. 2 is site-mutated into tyrosine, the serine at position 212 is site-mutated into methionine, and the amino acid residues at other positions are unchanged, and the obtained mutant is denoted as mutant IY-S212M.

[0012] It is found through experiments of the present application that, compared with the wild-type BhrPETase, the PET degradation activity of the above-mentioned 10 mutants is increased by 2.03-4.24 times. Compared with the mutant F208I, the PET degradation activity of the above-mentioned 10 mutants is increased by 27.0%-120%, and compared with the mutant F208I (Tm value of 91.5℃), the thermal stability of the above-mentioned 10 mutants is basically equivalent, and the Tm value is 85.1-97.2℃. Among them, the Tm value of the mutant IY-Q142L is 92.99℃, and the PET degradation activity is increased by 4.24 times compared with the wild-type BhrPETase.

[0013] In a fourth aspect, the present application provides a nucleic acid which can encode the BhrPETase mutant of the first to third aspects.

[0014] In a fifth aspect, the present application provides a recombinant vector comprising the nucleic acid of the fourth aspect.

[0015] In a sixth aspect, the present application provides a recombinant strain comprising the recombinant vector of the fifth aspect.

[0016] Illustratively, the host cell of the recombinant strain is Escherichia coli.

[0017] In a seventh aspect, the present application provides a method for producing a PET hydrolytic enzyme, which specifically comprises: culturing the recombinant strain of the sixth aspect in large scale, and inducing expression to obtain the PET hydrolytic enzyme.

[0018] Preferably, the culture medium used for inducing expression is ZYM auto-induction culture medium.

[0019] Illustratively, the ZYM auto-induction culture medium comprises the following components: 8-12 g of tryptone, 4-6 g of yeast powder, 20-30 mM of Na2HPO4·12H2O, 20-30 mM of KH2PO4, 45-5 mM of NH4Cl, 4-6 mM of Na2SO4, 1.5-2.5 mM of MgSO4·7H2O, 4-6 g of glycerol, 0.3-0.6 g of anhydrous glucose, and 1.5-2.5 g of lactose monohydrate per 1 L.

[0020] In an eighth aspect, the present application provides use of the nucleic acid provided in the fourth aspect, the recombinant vector provided in the fifth aspect, or the recombinant strain provided in the sixth aspect in the preparation of a PET hydrolytic enzyme.

[0021] In a ninth aspect, the present application provides use of the BhrPETase mutant provided in the first aspect to the third aspect in the degradation of PET, the recovery of a PET degradation product, or the preparation of a PET degradation agent.

[0022] The present application provides a series of BhrPETase mutants having a PET degradation activity 1.39-4.24 times that of the wild-type BhrPETase and a thermal stability substantially equivalent to that of the wild-type BhrPETase, by performing single-point mutation or combined mutation on the wild-type BhrPETase. Among them, the Tm value of the mutant IY-Q142L is 92.99℃, and the PET degradation activity thereof is 4.24 times that of the wild-type BhrPETase. The BhrPETase mutants provided by the present application can be applied in the fields of degrading PET, recovering a PET degradation product, or preparing a PET degradation agent. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0024] Figure 1 FIG. 1 is a schematic diagram of the plasmid map of the recombinant plasmid pET-22b-BhrPETase in the embodiment 1 of the present application; Figure 2 FIG. 2 is a schematic diagram of the plasmid map of the recombinant plasmid pET-22b-F208I in the embodiment 1 of the present application; Figure 3 FIG. 3 is the PET degradation activity determination result of the wild-type BhrPETase and the mutants thereof in the embodiment 1 of the present application; Figure 4 FIG. 4 is the Tm value determination result of the wild-type BhrPETase and the mutants thereof in the embodiment 1 of the present application; T m Figure 5 FIG. 5 is a schematic diagram of the plasmid map of the recombinant plasmid pET-22b-F208I-H183Y in the embodiment 2 of the present application; Figure 6 FIG. 6 is the PET degradation activity determination result of the wild-type BhrPETase and the mutants thereof in the embodiment 2 of the present application; Figure 7 ​The determination results of the PET degradation activity of the wild-type BhrPETase and the mutants thereof in Example 2 of the present application are shown in Table 2. T m The determination results of the PET degradation activity of the wild-type BhrPETase and the mutants thereof in Example 2 of the present application are shown in Table 2. Figure 8 The plasmid map of the recombinant plasmid pET-22b-F208I-H183Y-Q142L in Example 3 of the present application is shown in Figure 3. Figure 9 The determination results of the PET degradation activity of the wild-type BhrPETase and the mutants thereof in Example 3 of the present application are shown in Table 3. Figure 10 The determination results of the PET degradation activity of the wild-type BhrPETase and the mutants thereof in Example 3 of the present application are shown in Table 3. T m The determination results of the PET degradation activity of the wild-type BhrPETase and the mutants thereof in Example 2 of the present application are shown in Table 2. DETAILED DESCRIPTION

[0025] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the examples. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application.

[0026] The mutants described in the present application are named according to the conventional naming method of those skilled in the art, for example: the mutant F208I indicates that the phenylalanine (F) at the 208th position in the amino acid sequence of the wild-type BhrPETase is mutated to isoleucine (I), and the amino acid residues at other positions remain unchanged; The mutant I-H183Y indicates that the histidine (H) at the 183rd position in the amino acid sequence of the mutant F208I is mutated to tyrosine (Y), and the amino acid residues at other positions remain unchanged.

[0027] Example 1 The present example provides a preparation method, expression and purification method and activity detection method of the single-point mutant based on the wild-type BhrPETase, which comprises obtaining the mutated target gene by polymerase chain reaction (PCR), introducing the target gene into an E. coli expression vector, preparing a recombinant plasmid by using DMTase (GenScript, GD111), seamless cloning and other molecular biology methods, transforming the recombinant plasmid into competent cells of E. coli BL21 (DE3) (GenScript, CD601), and culturing to obtain recombinant E. coli for heterologous expression of the target protein. The specific content is as follows: I. Obtaining of the BhrPETase mutant 1. Construction of the single-point mutant recombinant plasmid and recombinant strain The amino acid sequence of the wild-type BhrPETase is shown as SEQ ID No. 1, and its encoding gene is obtained by codon optimization, and the sequence of the encoding gene is shown as SEQ ID No. 3.

[0028] The recombinant plasmid pET-22b-BhrPETase was synthesized by Suzhou Juyizhi Biotechnology Co., Ltd. in the following specific process: after amplifying the gene with the nucleotide sequence shown as SEQ ID No. 3, the gene was cleaved by Nco I and Xho I restriction endonuclease and then connected to the pET-22b vector to obtain the recombinant plasmid pET-22b-BhrPETase, and the schematic diagram of the plasmid map is shown as Figure 1 .

[0029] The recombinant plasmid pET-22b-BhrPETase was synthesized by Suzhou Juyizhi Biotechnology Co., Ltd. in the following specific process: after amplifying the gene with the nucleotide sequence shown as SEQ ID No. 3, the gene was cleaved by Nco I and Xho I restriction endonuclease and then connected to the pET-22b vector to obtain the recombinant plasmid pET-22b-BhrPETase, and the schematic diagram of the plasmid map is shown as

[0030] The PCR products obtained were digested with DMTase (full-size gold company, GD111) to digest the template, and 18 kinds of single-point mutant recombinant plasmids containing pET-22b-S27V, pET-22b-V28I, pET-22b-S29F and pET-22b-F208I were obtained by molecular biology methods such as seamless cloning. The schematic diagram of the plasmid map of the recombinant plasmid pET-22b-F208I is shown as Figure 2 .

[0031] Table 1

[0032] Subsequently, the 18 single-point mutant recombinant plasmids and the recombinant plasmid pET-22b-BhrPETase were introduced into the competent cells of Escherichia coli BL21 (DE3) (full type gold, CD601) by heat shock (42°C water bath heat shock for 45s), and after heat shock, they were quickly transferred to an ice bath for 2 minutes, 500 μL of sterile LB medium (without antibiotics) was added, mixed, and then placed in a 37°C, 200 rpm incubator for 1 hour to recover the bacteria. After the end, centrifugation was performed at 6000 rpm for 90 seconds, 450 μL of supernatant was removed, and the remaining was added to LB agar medium, the cells were evenly spread to the liquid was absorbed, and the plate was inverted and incubated at 37°C for 12h. Then, positive monoclonal strains were selected, transferred to 5mL small test tube LB medium (containing 100mg / L ampicillin), incubated at 37°C for 12h, and the correctness of the mutant construction was ensured by Sanger sequencing, and 18 single-point mutant recombinant strains including recombinant strains S27V, V28I, S29F, S113P, Q142L, Q142W, T153A, T157P, T160Y, N162S, H183Y, etc. and the recombinant strain BhrPETase were constructed.

[0033] 2. Preparation of wild-type BhrPETase and its single-point mutant The 18 single-point mutant recombinant strains and the recombinant strain BhrPETase were inoculated in 5mL of LB medium, incubated at 37°C, 220 rpm for 12h, then inoculated in a shake flask containing 80mL ZYM auto-induction medium at a 1% inoculation amount for fermentation, induced to express at 21°C, 160 rpm for 20h, and the fermentation liquid rich in the corresponding BhrPETase mutant was obtained.

[0034] Each 1L of ZYM auto-induction medium contains: 10g tryptone, 5g yeast powder, 25mM Na2HPO4·12H2O, 25mM KH2PO4, 50mM NH4Cl, 5mM Na2SO4, 2mM MgSO4·7H2O, 5g glycerol, 0.5g anhydrous glucose, 2g lactose monohydrate.

[0035] The different fermentation broths were treated with a high-speed refrigerated centrifuge (8000g, 5 min) to collect the bacterial cells, and 10 mL of a cell disruption buffer (containing 50 mM Tris-HCl, 150 mM NaCl and 10 mM imidazole per 1 L of the cell disruption buffer, pH = 7.5) was added to resuspend the bacterial cells, and then a high-pressure disrupter was used to disrupt the cells. After the disruption was completed, the cells were centrifuged at a speed of 10000 rpm for 1 h to remove the cell debris, and the obtained supernatant was the whole protein liquid containing BhrPETase and the mutants thereof. The whole protein liquid was filtered through a 0.45 μm filter membrane to remove impurities, and then a gradient elution was performed on a Ni-NTA packed column to obtain the target protein. The specific steps of the purification included: the column was equilibrated with the cell disruption buffer for 2 min, and then the whole protein liquid after the membrane filtration was repeatedly hung on the column for 3 times, and the column was washed with a washing buffer (containing 50 mM Tris-HCl, 150 mM NaCl and 40 mM imidazole per 1 L of the washing buffer, pH = 7.5) for 3 times to remove impure proteins; finally, an eluent (containing 50 mM Tris-HCl, 300 mM NaCl and 300 mM imidazole per 1 L of the eluent, pH = 7.5) was used for elution to obtain a protein eluent; and then the protein was further concentrated and the high-concentration imidazole was removed, so as to obtain a concentrated wild-type BhrPETase and mutant enzyme liquid.

[0036] II. Performance characterization method 1. Determination of PET degradation activity In the present application, amorphous PET film (purchased from Goodfellow, with a crystallinity of about 8%) was used as a PET substrate, and the mode substrate was sequentially washed with 1% SDS, anhydrous ethanol and double-distilled water, and then a puncher was used to punch it into a disc with a diameter of 6 mm (weighting 8 mg), and 3 repeated determinations were performed each time.

[0037] The concentrated wild-type BhrPETase and mutant enzyme liquid was placed in 300 μL of a reaction liquid (100 mM potassium phosphate buffer, pH = 8) according to the corresponding concentration (500 nM), one piece of PET disc was added to the above system, and the reaction was performed in a 70℃ water bath for 5 h. After the reaction was completed, acetonitrile was added to terminate the reaction, and the TPA, MHET and BHET produced in the reaction were analyzed by high performance liquid chromatography. The sum of the concentrations of TPA, MHET and BHET was used as an index for evaluating the PET degradation activity.

[0038] 2. Determination method of Tm The differential scanning fluorimetry was used to determine the protein melting temperature. The DSF experiment used a real-time fluorescent quantitative PCR system, and 465 nm excitation and 580 nm emission filters were used. The sample was heated from 25℃ to 100℃ at a rate of 0.3℃ / s, and the fluorescence was measured every 0.03 s. Tm It is determined by the first derivative curve.

[0039] III. Experimental Results The PET degradation activity of wild-type BhrPETase and its mutants was determined in this embodiment. T m Values. Results of PET degradation activity assays for wild-type BhrPETase and its mutants are as follows: Figure 3 As shown, wild-type BhrPETase and its mutants T m Value measurement results are as follows Figure 4 As shown in the figure. Bhr represents wild-type BhrPETase.

[0040] Depend on Figures 3-4 It was found that among the 18 single-point mutants, except for mutants S29F and T153A, whose PET degradation activities were slightly lower than those of the wild type, the PET degradation activities of the remaining 16 mutants were increased by 15.8% to 138.6% compared with the wild type BhrPETase. Among them, mutant F208I showed the best degradation effect on PET substrates, with a PET degradation activity 2.39 times that of the wild type BhrPETase. In terms of thermal stability, compared with the wild type BhrPETase (Tm value of 95.05℃), mutant Q142W had a Tm increase of 4.95℃, and mutant N211M had a Tm increase of 2.53℃. Compared with the wild type BhrPETase, the thermal stability of mutant F208I (its amino acid sequence is shown in SEQ ID No. 2) was slightly decreased, with a Tm of 91.5℃, but it still met the general requirements for PET degradation.

[0041] Example 2 Based on the mutant F208I which has significantly improved PET degradation activity compared to the wild-type BhrPETase, 15 double-point combination mutants based on the wild-type BhrPETase were further mutated, and were sequentially recorded as mutants I-S27V (F208I-S27V), I-V28I (F208I-V28I), I-S113P (F208I-S113P), I-Q142L (F208I-Q142L), I-Q142W (F208I-Q142W), I-T157P (F208I-T157P), I-T160Y (F208I-T160Y), I-N162S (F208I-N162S), I-Q167M (F208I-Q167M), I-V170I (F208I-V170I), I-H183Y (F208I-H183Y), I-E201L (F208I-E201L), I-N211M (F208I-N211M), I-S212M (F208I-S212M), and I-T233L (F208I-T233L).

[0042] In this embodiment, the site-directed mutation primers used in the construction of the recombinant plasmids of the above double-point combination mutants are shown in Table 2.

[0043] Table 2

[0044] Using the site-directed mutation technology, the recombinant plasmid pET-22b-F208I constructed in Example 1 as a template, the primers shown in Table 2, and the PCR conditions described in Example 1, 15 BhrPETase mutant recombinant plasmids including the recombinant plasmid pET-22b-F208I-H183Y were prepared, and the method for constructing the corresponding recombinant plasmid was the same as that described in Example 1. The plasmid map of the recombinant plasmid pET-22b-F208I-H183Y is shown in Figure 5 .

[0045] Based on the above 15 recombinant plasmids constructed, the corresponding mutant recombinant strains were further constructed by the method described in Example 1, and the corresponding mutants I-S27V, I-V28I, I-S113P, I-Q142L, I-Q142W, I-T157P, I-T160Y, I-N162S, I-Q167M, I-V170I, I-H183Y, I-E201L, I-N211M, I-S212M, and I-T233L were prepared, purified, and determined for PET degradation activity and Tm. Among them, the results of the determination of the PET degradation activity of the wild-type BhrPETase and its mutants are shown in Table 3.Figure 6 As shown, wild-type BhrPETase and its mutants T m Value measurement results are as follows Figure 7 As shown.

[0046] Depend on Figure 6 It was found that, except for mutants I-N162S, I-V170I, and I-T233L, the PET degradation activity of the remaining 12 mutants was improved to varying degrees. Specifically, the PET degradation activity of the remaining 12 mutants was increased by 3.80% to 58.5% compared to mutant F208I, and by 1.48 to 2.78 times compared to wild-type BhrPETase. Among them, mutants I-T157P, I-H183Y, and I-E201L showed increased degradation activity of PET substrates of 39.4%, 58.5%, and 53.5% respectively compared to the F208I mutant, and increased by 2.32 times, 2.78 times, and 2.66 times respectively compared to wild-type BhrPETase. Figure 7 It can be seen that among the 12 mutants other than I-N162S, I-V170I, and I-T233L, except for mutant I-E201L, whose Tm value dropped to 78.87℃ and thermal stability was significantly reduced, the Tm values ​​of the other 11 mutants ranged from 88.26℃ to 96.61℃, which is basically equivalent to the thermal stability of wild-type BhrPETase. Among them, mutant I-H183Y showed the best degradation activity, with a Tm of 92.36℃.

[0047] Example 3 This embodiment further combines the mutant I-H183Y provided in Example 2 with mutation sites to obtain 10 three-point combination mutants based on wild-type BhrPETase, namely mutants IY-V28I (F208I-H183Y-V28I), IY-S113P (F208I-H183Y-S113P), IY-Q142L (F208I-H183Y-Q142L), and IY-Q142W (F208I-H183Y-V28I). Q142W), IY-T157P (F208I-H183Y-T157P), IY-T160Y (F208I-H183Y-T160Y), IY-Q167M (F208I-H183Y-Q16 7M), IY-E201L (F208I-H183Y-E201L), IY-N211M (F208I-H183Y-N211M) and IY-S212M (F208I-H183Y-S212M).

[0048] The site-directed mutagenesis primers used in constructing the corresponding recombinant plasmids are shown in Table 3 above.

[0049] Table 3

[0050] Using site-directed mutagenesis technique, the recombinant plasmid pET-22b-F208I-H183Y constructed in Example 2 was used as a template, and the corresponding primers shown in Table 3 were used to perform PCR to prepare the corresponding recombinant plasmids. The method for preparing the recombinant plasmids was the same as that in Example 1. The schematic diagram of the plasmid map of the recombinant plasmid pET-22b-F208I-H183Y-Q142L is shown in Figure 8 .

[0051] Further, after the corresponding recombinant strains were constructed by the method described in Example 1, the corresponding mutants were prepared, and the PET degradation activity and Tm of different BhrPETase mutants were determined. The results of the determination of the PET degradation activity of the wild-type BhrPETase and its mutants are shown in Figure 9 , and the results of the determination of the Tm of the wild-type BhrPETase and its mutants are shown in T m . Figure 10

[0052] Figures 9-10 ​​It can be known that, compared with the wild type BhrPETase, the PET degradation activity of the 10 three-point combined mutants provided in the embodiment is increased by 2.03-4.24 times. Compared with the mutant F208I, the PET degradation activity of the 10 three-point combined mutants provided in the embodiment is increased by 27.0%-120%, and the thermal stability is basically equivalent to that of the mutant F208I (Tm value is 91.5°C), and the Tm value is 85.1-97.2°C. Among them, the PET degradation activity of the mutant IY-Q142L is the best, and the PET degradation activity of the mutant IY-Q142L is increased by 4.24 times compared with the wild type BhrPETase, 1.20 times compared with the mutant F208I, and 38.6% compared with the mutant I-H183Y; and the Tm value of the mutant IY-Q142L is 92.99°C, which is basically equivalent to the thermal stability of the wild type BhrPETase. In addition, the PET degradation activity of the mutants IY-S113P, IY-T160Y, IY-T157P, IY-N211M and IY-S212M is increased by 3.13-3.9 times compared with the wild type BhrPETase, and the PET degradation activity of the mutant IY-N211M is increased by 3.13 times compared with the wild type BhrPETase and the Tm value is increased by 2.12°C compared with the wild type BhrPETase. In summary, by single-point mutation or combined mutation on the basis of the wild type BhrPETase, the application provides a series of BhrPETase mutants whose thermal stability is basically equivalent to that of the wild type BhrPETase, and the PET degradation activity is increased by 1.39-4.24 times compared with the wild type BhrPETase. Among them, the PET degradation activity of the mutant IY-Q142L is the best, and the PET degradation activity of the mutant IY-Q142L is increased by 4.24 times compared with the wild type BhrPETase, 1.20 times compared with the mutant F208I, and 38.6% compared with the mutant I-H183Y; and the thermal stability of the mutant is basically equivalent to that of the wild type BhrPETase, which has important significance for industrial efficient degradation of PET.

[0053] In view of the significant advantages of the BhrPETase mutants provided in the application in terms of PET degradation activity, and good thermal stability, the BhrPETase mutants can be applied to the fields of degrading PET, recycling PET degradation products or preparing PET degradation agents.

[0054] The above only describes the preferred embodiments of the application and should not be used to limit the application. Any modification, equivalent replacement or improvement made within the spirit and principle of the application should be included in the protection scope of the application.

Claims

1. A BhrPETase mutant, characterized in that: It is that the phenylalanine at position 208 in the amino acid sequence of the wild-type BhrPETase is site-mutated into isoleucine, and the amino acid residues at other positions are not changed, and the obtained amino acid sequence is shown in SEQ ID No.

2.

2. A BhrPETase mutant, characterized in that: The amino acid sequence thereof is obtained by replacing the amino acid residue at one position in the amino acid sequence shown in SEQ ID No. 2, and the amino acid sequence is any one of the following (1)~(11): (1) the serine at position 27 in the amino acid sequence shown in SEQ ID No. 2 is site-mutated into valine, and the amino acid residues at other positions are not changed; (2) the valine at position 28 in the amino acid sequence shown in SEQ ID No. 2 is site-mutated into isoleucine, and the amino acid residues at other positions are not changed; (3) the serine at position 113 in the amino acid sequence shown in SEQ ID No. 2 is site-mutated into proline, and the amino acid residues at other positions are not changed; (4) the glutamine at position 142 in the amino acid sequence shown in SEQ ID No. 2 is site-mutated into leucine, and the amino acid residues at other positions are not changed; (5) the glutamine at position 142 in the amino acid sequence shown in SEQ ID No. 2 is site-mutated into tryptophan, and the amino acid residues at other positions are not changed; (6) the threonine at position 157 in the amino acid sequence shown in SEQ ID No. 2 is site-mutated into proline, and the amino acid residues at other positions are not changed; (7) the threonine at position 160 in the amino acid sequence shown in SEQ ID No. 2 is site-mutated into tyrosine, and the amino acid residues at other positions are not changed; (8) the glutamine at position 167 in the amino acid sequence shown in SEQ ID No. 2 is site-mutated into methionine, and the amino acid residues at other positions are not changed; (9) the histidine at position 183 in the amino acid sequence shown in SEQ ID No. 2 is site-mutated into tyrosine, and the amino acid residues at other positions are not changed; (10) the asparagine at position 211 in the amino acid sequence shown in SEQ ID No. 2 is site-mutated into methionine, and the amino acid residues at other positions are not changed; (11) the serine at position 212 in the amino acid sequence shown in SEQ ID No. 2 is site-mutated into methionine, and the amino acid residues at other positions are not changed.

3. A BhrPETase mutant, characterized in that: The amino acid sequence thereof is obtained by replacing the amino acid residues at two positions in the amino acid sequence shown in SEQ ID No. 2, and the amino acid sequence is any one of the following (12)~(21): (12) the histidine at position 183 in the amino acid sequence shown in SEQ ID No. 2 is site-mutated into tyrosine, and the valine at position 28 is site-mutated into isoleucine, and the amino acid residues at other positions are not changed; (13) the histidine at position 183 in the amino acid sequence shown in SEQ ID No. 2 is site-mutated into tyrosine, and the serine at position 113 is site-mutated into proline, and the amino acid residues at other positions are not changed; (14) the histidine at position 183 and the glutamine at position 142 of the amino acid sequence shown in SEQ ID No. 2 are mutated to tyrosine and leucine respectively, and the amino acid residues at other positions are unchanged; (15) the histidine at position 183 and the glutamine at position 142 of the amino acid sequence shown in SEQ ID No. 2 are mutated to tyrosine and tryptophan respectively, and the amino acid residues at other positions are unchanged; (16) the histidine at position 183 and the threonine at position 157 of the amino acid sequence shown in SEQ ID No. 2 are mutated to tyrosine and proline respectively, and the amino acid residues at other positions are unchanged; (17) the histidine at position 183 and the threonine at position 160 of the amino acid sequence shown in SEQ ID No. 2 are mutated to tyrosine and tyrosine respectively, and the amino acid residues at other positions are unchanged; (18) the histidine at position 183 and the glutamine at position 167 of the amino acid sequence shown in SEQ ID No. 2 are mutated to tyrosine and methionine respectively, and the amino acid residues at other positions are unchanged; (19) the histidine at position 183 and the glutamic acid at position 201 of the amino acid sequence shown in SEQ ID No. 2 are mutated to tyrosine and leucine respectively, and the amino acid residues at other positions are unchanged; (20) the histidine at position 183 and the asparagine at position 211 of the amino acid sequence shown in SEQ ID No. 2 are mutated to tyrosine and methionine respectively, and the amino acid residues at other positions are unchanged; (21) the histidine at position 183 and the serine at position 212 of the amino acid sequence shown in SEQ ID No. 2 are mutated to tyrosine and methionine respectively, and the amino acid residues at other positions are unchanged.

4. A nucleic acid, characterized in that: The BhrPETase mutant of any one of claims 1-3.

5. A recombinant vector, characterized by: The nucleic acid of claim 4.

6. A recombinant bacterial strain, characterized in that: The recombinant vector of claim 5.

7. A method of producing a PET hydrolytic enzyme, characterized by: The recombinant strain of claim 6 is expanded and induced to express a PET hydrolytic enzyme.

8. The method of producing a PET hydrolytic enzyme as claimed in claim 7, characterized by: The medium used for the induction of expression is ZYM auto-induction medium.

9. Use of the nucleic acid of claim 4, the recombinant vector of claim 5, or the recombinant strain of claim 6 in the preparation of a PET hydrolytic enzyme.

10. Use of the BhrPETase mutant of any one of claims 1-3 in the degradation of PET, the preparation of a PET degradation agent, or the recovery of a PET degradation product.

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