Bhr petase mutants and uses thereof

Abstract

The application belongs to the technical field of enzyme engineering, and discloses a BhrPETase mutant and application thereof. The application provides a BhrPETase mutant, wherein the amino acid sequence is that a serine at the 27th position in the amino acid sequence shown in SEQ ID No. 2 is mutated to valine, and the amino acid residues at other positions are unchanged. The BhrPETase mutant provided by the application has a PET degradation activity significantly higher than that of the wild-type BhrPETase and the mutant F208I under the condition that the thermal stability of the BhrPETase mutant is basically equivalent to that of the wild-type BhrPETase. The BhrPETase mutant provided by the application can be applied to the fields of degrading PET, recycling PET degradation products or preparing a PET degradation agent.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology and relates to a PET-degrading enzyme, specifically a BhrPETase mutant and its applications. Background Technology

[0002] Plastics are indispensable materials in modern life. Among them, polyethylene terephthalate (PET) is widely used in the packaging and textile industries due to its advantages such as being lightweight, corrosion-resistant, and highly malleable. However, PET's high crystallinity and stable chemical structure make it difficult to degrade naturally, leading to the accumulation of large amounts of plastic waste, which poses a serious threat to the ecological environment and human health.

[0003] Enzymatic degradation of PET has shown great potential. Compared with traditional chemical recycling methods, enzymatic degradation has advantages such as mild reaction conditions, no pollution, and recyclable products. In recent years, scientists have discovered that various microorganisms can secrete PET hydrolases that can specifically break ester bonds in PET molecules, providing inspiration for the application of bio-enzymes in PET waste management and sustainable PET recycling. Among them, researchers developed a BhrPETase mutant (named TurboPETase) using an artificial intelligence-assisted protein engineering strategy, pushing PET depolymerization efficiency to a new level. At a high substrate concentration loading of 200 g / kg, it achieved a conversion rate of nearly 99% in just 8 hours, significantly outperforming the previous best LCC-ICCG. The maximum product generation rate of TurboPETase reached 61.3 g of hydrolyzed PET L... -1 h -1 Furthermore, the reaction can be carried out in aqueous solution without the need for expensive buffer systems. This breakthrough provides an innovative solution for the sustainable recycling of plastics, driving the development of green chemistry and the circular economy.

[0004] At high temperatures, PET exhibits significantly increased crystallinity and a more compact molecular structure. This characteristic hinders the efficient action of existing PET hydrolases on substrates, thus restricting the industrial-scale advancement of PET biodegradation technology. While BhrPETase, a thermophilic PET degrading enzyme, demonstrates advantages in thermal stability (Tm≈97℃) and catalytic activity, its catalytic efficiency still has considerable room for improvement. Therefore, exploring molecular modification to obtain BhrPETase mutants with enhanced PET degradation activity, while moderately reducing or maintaining the thermal stability of BhrPETase, is crucial for meeting the practical application needs of large-scale industrial production. Summary of the Invention

[0005] In view of the above-mentioned problems in the prior art, the present invention modifies the wild-type BhrPETase to provide a BhrPETase mutant with significantly improved PET degradation activity and its application.

[0006] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions: In a first aspect, the present invention provides a BhrPETase mutant, the amino acid sequence of which is obtained by site-directed mutation of serine at position 27 in the amino acid sequence shown in SEQ ID No. 2 to valine, while the amino acid residues at other positions remain unchanged.

[0007] Based on the mutant F208I (whose amino acid sequence is shown in SEQ ID No. 2), this invention further involves a site-directed mutation of serine at position 27 to valine, resulting in a mutant denoted as mutant I-S27V. This mutant exhibits significantly enhanced PET degradation activity compared to both wild-type BhrPETase and mutant F208I, while maintaining thermal stability comparable to wild-type BhrPETase.

[0008] In a second aspect, the present invention provides a nucleic acid that can encode the BhrPETase mutant described in the first aspect.

[0009] Thirdly, the present invention provides a recombinant vector comprising the nucleic acid described in the second aspect.

[0010] Fourthly, the present invention provides a recombinant strain comprising the recombinant vector described in the third aspect.

[0011] For example, the host cell of the recombinant strain is Escherichia coli.

[0012] Fifthly, the present invention provides a method for producing PET hydrolase, the specific method comprising: expanding the culture of the recombinant strain as described in the fourth aspect, inducing expression, and obtaining PET hydrolase.

[0013] Preferably, the culture medium used for the induced expression is ZYM self-inducing medium.

[0014] For example, the ZYM self-inducing medium comprises the following components per 1L: 8g-12g tryptone, 4g-6g yeast extract, 20mM-30mM Na2HPO4·12H2O, 20mM-30mM KH2PO4, 45mM-50mM NH4Cl, 4mM-6mM Na2SO4, 1.5mM-2.5mM MgSO4·7H2O, 4g-6g glycerol, 0.3g-0.6g anhydrous glucose, and 1.5g-2.5g lactose monohydrate.

[0015] In a sixth aspect, the present invention provides the application of the nucleic acid provided in the second aspect, the recombinant vector provided in the third aspect, and the recombinant strain provided in the fourth aspect in the preparation of PET hydrolase.

[0016] In a seventh aspect, the present invention provides the application of the BhrPETase mutant described in the first aspect in the degradation of PET, the recovery of PET degradation products, or the preparation of PET degradation agents.

[0017] This invention involves a two-point mutation of wild-type BhrPETase to provide a mutant, I-S27V, which exhibits thermal stability comparable to wild-type BhrPETase but significantly enhanced PET degradation activity compared to both wild-type BhrPETase and mutant F208I. The BhrPETase mutant provided by this invention can be applied to PET degradation, PET degradation product recovery, and the preparation of PET degradation agents. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the plasmid map of the recombinant plasmid pET-22b-BhrPETase in Example 1 of the present invention; Figure 2 This is a schematic diagram of the plasmid map of the recombinant plasmid pET-22b-F208I in Example 1 of the present invention; Figure 3 The results of PET degradation activity determination of wild-type BhrPETase and its mutants in Example 1 of this invention; Figure 4 Wild-type BhrPETase and its mutants in Example 1 of this invention T m Value measurement results; Figure 5 This is a schematic diagram of the plasmid map of the recombinant plasmid pET-22b-F208I-H183Y in Example 2 of the present invention; Figure 6 The results of PET degradation activity determination of wild-type BhrPETase and its mutants in Example 2 of this invention; Figure 7 Wild-type BhrPETase and its mutants in Example 2 of this invention T m Value measurement results; Figure 8 This is a schematic diagram of the plasmid map of the recombinant plasmid pET-22b-F208I-H183Y-Q142L in Example 3 of the present invention; Figure 9 The results of PET degradation activity determination of wild-type BhrPETase and its mutants in Example 3 of this invention; Figure 10 Wild-type BhrPETase and its mutants in Example 3 of this invention T m Value measurement results. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0021] The mutants described in this invention are named in accordance with the conventional naming methods of those skilled in the art. For example, mutant F208I indicates that the phenylalanine (F) at position 208 in the amino acid sequence of wild-type BhrPETase is mutated to isoleucine (I), while the amino acid residues at other positions remain unchanged. The mutant I-H183Y represents a site-directed mutation of histidine (H) at position 183 of the amino acid sequence of mutant F208I to tyrosine (Y), while the amino acid residues at other positions remain unchanged.

[0022] Example 1 This embodiment provides a method for preparing, expressing, purifying, and detecting the activity of a wild-type BhrPETase single-point mutant. The method includes obtaining the mutated target gene via polymerase chain reaction (PCR), introducing it into an *E. coli* expression vector, preparing a recombinant plasmid using molecular biology methods such as DMT enzyme (TransGold, GD111) and seamless cloning, and transforming it into *E. coli* BL21(DE3) (TransGold, CD601) competent cells. After culturing, recombinant *E. coli* expressing the target protein heterologously is obtained. The specific details are as follows: I. Obtaining the BhrPETase mutant 1. Construction of single-point mutant recombinant plasmids and recombinant strains The amino acid sequence of wild-type BhrPETase is shown in SEQ ID No. 1. Its coding gene was obtained through codon optimization, and its coding gene sequence is shown in SEQ ID No. 3.

[0023] The recombinant plasmid pET-22b-BhrPETase was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. The specific process is as follows: After amplifying the gene with the nucleotide sequence shown in SEQ ID No. 3, it was digested with NcoⅠ and XhoⅠ restriction endonucleases and ligated into the pET-22b vector to obtain the recombinant plasmid pET-22b-BhrPETase. A schematic diagram of its plasmid map is shown below. Figure 1 As shown.

[0024] Site-directed mutagenesis was used, with the recombinant plasmid pET-22b-BhrPETase as a template, and PCR was performed using the primers shown in Table 1 to obtain the corresponding linearized plasmid fragments. The PCR reaction volume was 20 μL, including 1 μL template (plasmid), 1 μL forward primer (F), 1 μL reverse primer (R), 10 μL high-fidelity amplification reagent, and the remainder being enzyme-free water. The PCR reaction conditions in this step were: pre-denaturation at 98℃ for 3 min; followed by 30 cycles, each cycle consisting of: denaturation at 98℃ for 15 s, annealing at 68℃ for 15 s, extension at 72℃ for 3 min; and final extension at 72℃ for 5 min.

[0025] The PCR product was digested with DMT enzyme (TransGen, GD111) to obtain 18 recombinant plasmids containing single-point mutations, including pET-22b-S27V, pET-22b-V28I, pET-22b-S29F, and pET-22b-F208I, using molecular biology methods such as seamless cloning. A schematic diagram of the pET-22b-F208I recombinant plasmid is shown below. Figure 2 As shown.

[0026] Table 1

[0027] Subsequently, the 18 single-point mutant recombinant plasmids and the recombinant plasmid pET-22b-BhrPETase were introduced into *E. coli* BL21(DE3) (Full Gold, CD601) competent cells via heat shock (42℃ water bath for 45s). After heat shock, the cells were rapidly transferred to an ice bath for 2 minutes, and 500 μL of sterile LB medium (antibiotic-free) was added. After mixing, the cells were incubated at 37℃ and 200 rpm for 1 hour to allow the bacteria to recover. After the incubation period, the cells were centrifuged at 6000 rpm for 90 seconds, and 450 μL of the supernatant was discarded. The remainder was added to LB agar medium, and the cells were spread evenly until the liquid was absorbed. The plates were then inverted and incubated at 37℃ for 12 hours. Then, positive single-clone strains were selected and transferred to 5 mL test tubes of LB medium (containing 100 mg / L ampicillin). The cultures were incubated at 37°C for 12 h and the correctness of the mutant construction was confirmed by Sanger sequencing. Eighteen recombinant strains with single-point mutations, including recombinant strains S27V, V28I, S29F, S113P, Q142L, Q142W, T153A, T157P, T160Y, N162S, and H183Y, as well as the recombinant strain BhrPETase, were constructed.

[0028] 2. Preparation of wild-type BhrPETase and its single-point mutants The 18 single-point mutation recombinant strains and the recombinant strain BhrPETase were inoculated into 5 mL of LB medium and cultured at 37 °C and 220 rpm for 12 h. Then, they were inoculated into shake flasks containing 80 mL of ZYM self-induction medium at a 1% inoculation rate for fermentation. The expression was induced at 21 °C and 160 rpm for 20 h to obtain fermentation broth rich in the corresponding BhrPETase mutants.

[0029] Each 1L of ZYM self-induction medium contains: 10g tryptone, 5g yeast extract, 25mM Na2HPO4·12H2O, 25mM KH2PO4, 50mM NH4Cl, 5mM Na2SO4, 2mM MgSO4·7H2O, 5g glycerol, 0.5g anhydrous glucose, and 2g lactose monohydrate.

[0030] The different fermentation broths were processed using a high-speed refrigerated centrifuge (8000g, 5 min), and the cells were collected. Each cell was resuspended in 10 mL of lysis buffer (each 1 L of lysis buffer contains 50 mM Tris-HCl, 150 mM NaCl, and 10 mM imidazole, pH=7.5), and then the cells were lysed using a high-pressure homogenizer. After lysis, the cells were centrifuged at 10000 rpm for 1 h to remove cell debris. The resulting supernatant was the total protein solution containing BhrPETase and its mutants. The total protein solution was filtered through a 0.45 μm filter to remove impurities, and then purified using a Ni-NTA packed column by gradient elution to obtain the target protein. The specific purification steps include: equilibrating with lysis buffer for 2 min, then repeatedly loading the whole protein solution onto the column 3 times, washing 3 times with washing buffer (each 1L of washing buffer contains 50mM Tris-HCl, 150mM NaCl and 40mM imidazole, pH=7.5) to remove impurities; finally eluting with elution buffer (each 1L of elution buffer contains 50mM Tris-HCl, 300mM NaCl and 300mM imidazole, pH=7.5) to obtain protein eluent; further concentrating the protein and removing high concentrations of imidazole to obtain concentrated wild-type BhrPETase and its mutant enzyme solution.

[0031] II. Performance Characterization Methods 1. Determination of PET degradation activity In this invention, an amorphous PET film (purchased from Goodfellow, with a crystallinity of approximately 8%) was used as the PET substrate. The substrate was washed sequentially with 1% SDS, anhydrous ethanol, and double-distilled water, and then punched into discs with a diameter of 6 mm (weighing 8 mg) using a puncher. Each measurement was repeated 3 times.

[0032] The concentrated wild-type BhrPETase and its mutant enzyme solutions were respectively placed in 300 μL of reaction solution (100 mM potassium phosphate buffer, pH=8) at the corresponding concentrations (500 nM). A PET disc was added to the system, and the reaction was carried out 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 indicator to evaluate the PET degradation activity.

[0033] 2. Methods for determining Tm Protein melting temperature was determined using differential scanning fluorometry (DSF). The DSF experiment employed a real-time quantitative PCR system with 465 nm excitation and a 580 nm emission filter. Samples were heated from 25 °C to 100 °C at a rate of 0.3 °C / s, and fluorescence was measured every 0.03 s. Tm It is determined by the first derivative curve.

[0034] 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.

[0035] Depend on Figure 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.

[0036] Example 2 Based on the mutant F208I, which exhibits significantly enhanced PET degradation activity compared to wild-type BhrPETase, this embodiment further mutated 15 two-point combination mutants based on wild-type BhrPETase, denoted as mutants I-S27V (F208I-S27V), I-V28I (F208I-V28I), I-S113P (F208I-S113P), I-Q142L (F208I-Q142L), I-Q142W (F208I-Q142W), and I-T157P (F208I-S27V). 8I-T157P), I-T160Y (F208I-T160Y), I-N162S (F208I-N162S), I-Q167M (F208I-Q167M), I-V170I (F208I-V170I), I-H18 3Y (F208I-H183Y), I-E201L (F208I-E201L), I-N211M (F208I-N211M), I-S212M (F208I-S212M) and I-T233L (F208I-T233L).

[0037] In this embodiment, the site-directed mutagenesis primers used to construct the recombinant plasmid of the above-mentioned two-point combination mutant are shown in Table 2.

[0038] Table 2

[0039] Using site-directed mutagenesis, and with the recombinant plasmid pET-22b-F208I constructed in Example 1 as a template, PCR was performed using the primers shown in Table 2 and the PCR conditions described in Example 1 to prepare 15 BhrPETase mutant recombinant plasmids, including pET-22b-F208I-H183Y. The methods for constructing the corresponding recombinant plasmids were the same as in Example 1. A schematic diagram of the plasmid pET-22b-F208I-H183Y is shown below. Figure 5 As shown.

[0040] Based on the 15 recombinant plasmids constructed above, corresponding mutant recombinant strains were further constructed using the method described in Example 1. 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 and purified. The PET degradation activity and Tm of different BhrPETase mutants were then determined. The results of the PET degradation activity determination of wild-type BhrPETase and its mutants are as follows: Figure 6 As shown, wild-type BhrPETase and its mutants T m Value measurement results are as follows Figure 7 As shown.

[0041] 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℃.

[0042] 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).

[0043] The site-directed mutagenesis primers used in constructing the corresponding recombinant plasmids are shown in Table 3 above.

[0044] Table 3

[0045] Using site-directed mutagenesis, and with the recombinant plasmid pET-22b-F208I-H183Y constructed in Example 2 as a template, PCR was performed using the corresponding primers shown in Table 3 to prepare the corresponding recombinant plasmids. The method for preparing the recombinant plasmids was the same as in Example 1. A schematic diagram of the plasmid map of recombinant plasmid pET-22b-F208I-H183Y-Q142L is shown below. Figure 8 As shown.

[0046] Furthermore, after constructing the corresponding recombinant strains using the method described in Example 1, corresponding mutants were prepared, and the PET degradation activity and Tm of different BhrPETase mutants were measured. The results of the PET degradation activity measurements of wild-type BhrPETase and its mutants are as follows: Figure 9 As shown, wild-type BhrPETase and its mutants T m Value measurement results are as follows Figure 10 As shown.

[0047] Depend on Figure 9-10It can be seen that, compared with wild-type BhrPETase, the PET degradation activity of the 10 three-point combination mutants provided in this embodiment is increased by 2.03 times to 4.24 times. Compared with mutant F208I, the PET degradation activity of the 10 three-point combination mutants provided in this embodiment is increased by 27.0% to 120%, and the thermal stability is basically equivalent to that of mutant F208I (Tm value of 91.5℃), with Tm values ​​ranging from 85.1℃ to 97.2℃. Among them, mutant IY-Q142L has the best PET degradation activity, which is 4.24 times higher than wild-type BhrPETase, 1.20 times higher than mutant F208I, and 38.6% higher than mutant I-H183Y; and the Tm value of this mutant is 92.99℃, which is basically equivalent to the thermal stability of wild-type BhrPETase. Furthermore, the PET degradation activities of mutants IY-S113P, IY-T160Y, IY-T157P, IY-N211M, and IY-S212M were 3.13-3.9 times higher than those of wild-type BhrPETase. Among them, mutant IY-N211M showed a 3.13-fold increase in PET degradation activity and a 2.12°C increase in Tm value compared to wild-type BhrPETase. In summary, this invention provides a series of BhrPETase mutants that are substantially equivalent to wild-type BhrPETase in terms of thermal stability, while exhibiting 1.39-4.24 times higher PET degradation activity compared to wild-type BhrPETase through single-point or combined mutations. Among them, mutant IY-Q142L showed the best PET degradation activity. The PET degradation activity of this mutant was 4.24 times higher than that of wild-type BhrPETase, 1.20 times higher than that of mutant F208I, and 38.6% higher than that of mutant I-H183Y. Moreover, the thermal stability of this mutant was basically equivalent to that of wild-type BhrPETase, which is of great significance for the efficient industrial degradation of PET.

[0048] Given the significant advantages of the BhrPETase mutant provided by this invention in PET degradation activity and its good thermal stability, it can be applied to fields such as PET degradation, PET degradation product recovery, or PET degradation agent preparation.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A BhrPETase mutant, characterized in that: Its amino acid sequence is obtained by site-directed mutation of serine at position 27 in the amino acid sequence shown in SEQ ID No. 2 to valine, while the amino acid residues at other positions remain unchanged.

2. A nucleic acid, characterized in that: It encodes the BhrPETase mutant as described in claim 1.

3. A recombinant vector, characterized in that: It contains the nucleic acid as described in claim 2.

4. A recombinant bacterial strain, characterized in that: It comprises the recombinant vector as described in claim 3.

5. A method for producing PET hydrolytic enzyme, characterized in that: The recombinant strain as described in claim 5 was expanded and cultured, and expression was induced to obtain PET hydrolase.

6. The method for producing PET hydrolase as described in claim 5, characterized in that: The culture medium used for the induced expression was ZYM self-inducing medium; The ZYM self-inducing culture medium comprises the following components per 1L: 8g-12g tryptone, 4g-6g yeast extract, 20mM-30mM Na2HPO4·12H2O, 20mM-30mM KH2PO4, 45mM-50mM NH4Cl, 4mM-6mM Na2SO4, 1.5mM-2.5mM MgSO4·7H2O, 4g-6g glycerol, 0.3g-0.6g anhydrous glucose, and 1.5g-2.5g lactose monohydrate.

7. The use of the nucleic acid of claim 2, the recombinant vector of claim 3, or the recombinant strain of claim 4 in the preparation of PET hydrolase.

8. The use of the BhrPETase mutant according to claim 1 in the degradation of PET, the preparation of PET degradation agents, or the recycling of PET degradation products.

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