Bhet hydrolase and use thereof

Abstract

The application discloses a BHET hydrolytic enzyme and application thereof, wherein the amino acid sequence of the BHET hydrolytic enzyme is shown as SEQ ID NO. 1, or has more than 80% homology with the amino acid sequence shown as SEQ ID NO. 1. The nucleotide sequence of the coding gene of the BHET hydrolytic enzyme is shown as SEQ ID NO. 2. The application artificially designs and screens a protein, and obtains a new protein with the function of the BHET hydrolytic enzyme, which can selectively catalyze the conversion of BHET to generate MHET, and is beneficial to realize the directional accumulation of the target intermediate MHET and improve the controllability of the degradation path.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and enzyme engineering, specifically to a dihydroxyethyl terephthalate (BHET) hydrolase and its applications. Background Technology

[0002] Polyethylene terephthalate (PET) is an important synthetic polyester widely used in packaging materials, fiber products, and engineering plastics. Due to its excellent mechanical properties and chemical stability, PET degrades extremely slowly in the natural environment, leading to the long-term accumulation of large amounts of waste PET in soil and water bodies, which has become a global environmental pollution problem. In recent years, the use of biocatalytic technology to degrade PET has been considered a green recycling pathway. Studies have shown that under enzymatic catalysis, PET typically first undergoes ester bond cleavage to generate oligomer intermediates such as dihydroxyethyl terephthalate (BHET) and monohydroxyethyl terephthalate (MHET), which are then further hydrolyzed to produce terephthalic acid (TPA) and ethylene glycol (EG).

[0003] In the aforementioned degradation process, the conversion of BHET to MHET is a key intermediate step. MHET, a low-molecular-weight monohydroxyethyl ester formed during the partial depolymerization of PET, contains both free carboxyl and hydroxyethyl ester groups. Based on these structural characteristics, MHET can serve as a dedicated intermediate for PET degradation pathway research, enzyme activity detection, reaction process analysis, and functionalization derivatization. Compared to bulk basic monomers such as TPA and EG, the commercial supply of MHET is relatively limited and its acquisition cost is high. Existing research indicates that the conversion of BHET to MHET typically relies on the catalysis of natural esterases or lipases derived from microorganisms. However, some natural enzymes suffer from insufficient substrate specificity or complex product compositions, potentially generating multiple products such as MHET and TPA simultaneously during catalysis, making it difficult to obtain a product system dominated by MHET. Developing hydrolases capable of directionally converting BHET to MHET would be beneficial for obtaining PET-derived intermediates with MHET as the main product and expanding its resource utilization methods.

[0004] Furthermore, existing BHET hydrolases are mostly derived from nature, and their sequence composition and catalytic characteristics are limited by natural evolutionary processes, leaving limited room for further rational design to achieve specific substrate and product selectivity. Therefore, developing artificially designed enzymes with novel structures capable of selectively catalyzing BHET conversion is of great significance for improving the controllability of PET degradation pathways and the resource utilization of intermediates. Summary of the Invention

[0005] To address the technical problem of low substrate selectivity in the existing BHET hydrolysis process, which makes it difficult to achieve targeted accumulation of MHET, the present invention aims to provide a BHET hydrolase and its application. By artificially designing the SEQ25 protein, it can selectively catalyze the conversion of BHET into MHET, with MHET as the main reaction product. This facilitates the targeted accumulation of the target intermediate and improves the controllability of the degradation pathway.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: A BHET hydrolase, wherein the amino acid sequence of SEQ 25 is shown in SEQ ID NO.1.

[0007] The BHET hydrolase has more than 80% sequence homology with the amino acid sequence shown in SEQ ID NO.1.

[0008] A gene encoding a BHET hydrolase.

[0009] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0010] A recombinant expression vector comprising the coding gene.

[0011] The construction process of the recombinant expression vector includes: using NdeI and XhoI restriction endonucleases to double digest the nucleotide sequence of the encoding gene and the pET-22b(+) vector, and then ligating them to obtain the recombinant expression vector, denoted as SEQ25-pET22b.

[0012] A recombinant engineered bacterium, wherein the recombinant engineered bacterium contains the recombinant expression vector.

[0013] The construction process of the recombinant engineered bacteria includes: transforming the recombinant expression vector SEQ25-pET22b into Escherichia coli BL21(DE3) competent cells and screening to obtain recombinant engineered bacteria.

[0014] Application of a BHET hydrolase as described above in catalyzing the hydrolysis of dihydroxyethyl terephthalate (BHET) to monohydroxyethyl terephthalate (MHET).

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The BHET hydrolase provided by the present invention can selectively catalyze the conversion of BHET into MHET. The reaction product is mainly MHET, which is conducive to the directional accumulation of the target intermediate and improves the controllability of the degradation pathway.

[0016] (2) The BHET hydrolase provided by the present invention is an artificially designed de novo protein with low sequence similarity to natural enzymes, providing a new molecular template for enzyme engineering optimization and functional regulation.

[0017] (3) The BHET hydrolase provided by the present invention can realize the directional conversion of BHET to MHET, which helps to improve the pathway regulation ability of PET biodegradation process and has potential resource utilization value. Attached Figure Description

[0018] Figure 1 This is a plasmid map of the SEQ25-pET22b recombinant expression vector.

[0019] Figure 2 This is an electrophoresis image of the SEQ25 protein.

[0020] Figure 3 The results are from the liquid chromatography of SEQ25 protein after hydrolysis with BHET for 30 min.

[0021] Figure 4 These are the liquid chromatography results for the blank control group.

[0022] Figure 5 These are the liquid chromatography results of MHET standards.

[0023] Figure 6 These are the liquid chromatography results of BHET standards.

[0024] Figure 7 The results show the concentration of MHET, the product of protein hydrolysis BHET (SEQ25), compared to the blank control group. Detailed Implementation

[0025] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. All materials and reagents used in the following embodiments are commercially available unless otherwise specified. The raw materials and equipment used in the specific embodiments of this invention are all known products and were obtained by purchasing commercially available products.

[0026] Example 1: Expression and preparation of SEQ25 protein The novel BHET hydrolase designed in this invention is a protein with an amino acid sequence as shown in SEQ ID NO.1, denoted as SEQ25; the nucleotide sequence of the gene encoded by this protein SEQ25 is shown in SEQ ID NO.2.

[0027] The present invention also provides a BHET hydrolase, which is a protein composed of an amino acid sequence having more than 80% homology with the one shown in SEQ ID NO.1, and has BHET hydrolase activity.

[0028] SEQ ID NO.1 is as follows: MTHQIVTTQYGKVKGTTENGVHKWKGIPYAKPPVGELRFKAPVPPEPWEGVRDATEFGNICPQTIPNTDILGEEDCLYLNVYTPELPEDKKLPVMVWIHGGGFVSGSGSDYDGSKLAANGVIVVTINYRLGALGFLAHPALTAESEHHASGNYGLLDQQAALQWVQRNIAAFGGDPDNVTIFGESAGGLSVHSQLASPLAKGLFHKAISESGAYMRSQQSLAEAEAIGEKFAAAAGCSSQTDAAACLRALPAEKILELVDYAGFTPDIDGKVLTQSPEAALAAGEFNKVPVIAGSNADEGRALTAFLFPLSGDQYLANVKQAFGELADEALELYPAGSDEEALASQLVTDRDFGGGAAFVAEAVTAAHPGAKVYLYQFSRRSPANPFRKFLGAAHASEI SEQ ID NO.2 is as follows: The nucleotide sequence (SEQ ID NO.2) containing the SEQ25 gene was inserted between the NdeI and XhoI restriction sites of the pET-22b(+) vector to construct a recombinant expression vector, denoted as SEQ25-pET22b. Figure 1 As shown, the vector expresses the protein SEQ ID NO.1 (SEQ 25); the target gene for BHET hydrolase was synthesized by Beijing Liuhe BGI Genomics Co., Ltd.

[0029] The recombinant expression vector SEQ25-pET22b was transformed into Escherichia coli BL21(DE3) competent cells to obtain recombinant engineered bacteria. Positive clones were screened using LB agar plates containing ampicillin sodium resistance (100 μg / mL) and cultured overnight at 37°C.

[0030] Positive clones were picked and cultured in 3 mL of liquid LB medium at 37°C and 200 rpm for 14 h. Then, they were inoculated into 150 mL of liquid LB medium for expansion culture, and cultured at 37°C and 200 rpm until the absorbance OD of the bacterial culture reached a certain level. 600 The concentration was approximately 0.8. Isopropyl-β-D-thiogalactoside (IPTG, final concentration 0.5 mM) was added to induce the expression of recombinant engineered bacteria at 16°C and 200 rpm for 20 hours. Cells were collected by centrifugation at 8000 rpm for 10 min, resuspended in buffer (100 mM K₂HPO₄-HCl, 100 mM NaCl, pH 7.0), and hyperbarically lysed at 1500 bar. The supernatant of protein SEQ25 was collected by centrifugation at 12000 rpm for 45 min at 4°C.

[0031] Protein purification was performed using a 5 mL Ni-NTA gravity column. The column was equilibrated with 50 mL of buffer I (100 mM K₂HPO₄-HCl, 100 mM NaCl, 20 mM imidazole, pH 7.0). The supernatant of the SEQ25 protein collected in the previous step was used for loading. Washing was performed sequentially with 50 mL of buffer I and 10 mL of buffer II (100 mM K₂HPO₄-HCl, 100 mM NaCl, 75 mM imidazole, pH 7.0). Elution was then performed with 10 mL of buffer III (100 mM K₂HPO₄-HCl, 100 mM NaCl, 100 mM imidazole, pH 7.0) and 20 mL of buffer IV (100 mM K₂HPO₄-HCl, 100 mM NaCl, 250 mM imidazole, pH 7.0), collecting both SEQ25 protein eluates. The SEQ25 supernatant and the two eluates were then subjected to protein electrophoresis, as shown below. Figure 2As shown, the SEQ25 protein effluent eluted with buffer IV was concentrated by ultrafiltration using a 10 kDa ultrafiltration tube to remove imidazole, and the purified SEQ25 protein was obtained. The protein concentration was determined using a BCA kit, and the protein concentration was approximately 4.66 mg / mL.

[0032] Example 2: Evaluation of the hydrolytic performance of SEQ25 protein on BHET In this embodiment, the activity test of BHET hydrolase is specifically implemented through the following steps: BHET was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 20 mg / mL. 5 μL of this stock solution was added to a 2 mL centrifuge tube, followed by the addition of purified SEQ25 protein (final concentration 1 μM). The volume was then adjusted to 500 μL using 100 mM phosphate buffer (K₂HPO₄-KH₂PO₄, pH 7.0). The reaction was incubated at 37°C with shaking at 1200 rpm for 30 min. An equal volume (500 μL) of acetonitrile was added to terminate the reaction. The mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane for later use.

[0033] High-performance liquid chromatography (HPLC) was performed using a Shimadzu LC-20AD system with a C18 column (SilGreen, 4.6 × 250 mm, 5 μm). The mobile phase was 0.1% formic acid aqueous solution (A) and acetonitrile (B), with a flow rate of 0.8 mL / min and a detection wavelength of 254 nm. The elution program was completed within 36 min, with the volume fraction of acetonitrile (B) varying from 5% to 70%, and the main linear ascent phase controlled within 5–25 min.

[0034] The liquid chromatography results of SEQ25 protein degradation of BHET for 30 min and the blank control group (without SEQ25 protein) are as follows: Figure 3 and Figure 4 As shown, peaks were observed at 20.37 min and 20.89 min, respectively. Using this liquid chromatography method, the retention time of MHET standard (Biode Pharmaceuticals, CAS: 1137-99-1) was 20.32 min. Figure 5 The retention time of BHET standard (TCI, CAS: 959-26-2) was 20.84 min. Figure 6The elution time of the peak at 20.37 min is very close to that of the MHET standard, therefore the substance with a retention time of 20.37 min is MHET. The elution time of the peak at 20.89 min is very close to that of the BHET standard, therefore the substance with a retention time of 20.89 min is BHET. The above results indicate that the SEQ25 protein obtained in this invention hydrolyzes BHET to produce MHET, and can directionally catalyze the conversion of BHET to MHET.

[0035] The results of the concentration determination of MHET, the product of protein hydrolysis BHET in the blank control group and SEQ25, are as follows: Figure 7 As shown, the SEQ25 protein, designed de novo according to this invention, demonstrates its ability to hydrolyze BHET to generate MHET, thus representing a novel BHET hydrolase.

Claims

1. A BHET hydrolase, characterized in that: The amino acid sequence of the BHET hydrolase is shown in SEQ ID NO.

1.

2. The BHET hydrolase according to claim 1, characterized in that: The BHET hydrolase has more than 80% homology with the amino acid sequence shown in SEQ ID NO.

1.

3. A gene encoding a BHET hydrolase, characterized in that: The encoding gene encodes the BHET hydrolase as described in claim 1.

4. The encoding gene according to claim 3, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

5. A recombinant expression vector, characterized in that: The recombinant expression vector contains the coding gene as described in claim 2 or 3.

6. The recombinant expression vector according to claim 5, characterized in that, The construction process of the recombinant expression vector includes: using NdeI and XhoI restriction endonucleases to double digest the nucleotide sequence of the coding gene and the pET-22b(+) vector, and then ligating them to obtain the recombinant expression vector, denoted as SEQ25-pET22b.

7. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria comprises the recombinant expression vector of claim 4 or 5.

8. The recombinant engineered bacteria according to claim 7, characterized in that, The construction process of the recombinant engineered bacteria includes: transforming the recombinant expression vector into Escherichia coli BL21(DE3) competent cells and screening to obtain recombinant engineered bacteria.

9. The use of the BHET hydrolase as described in claim 1 or 2 in catalyzing the hydrolysis of dihydroxyethyl terephthalate to monohydroxyethyl terephthalate.

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