Recombinant ferulic acid esterase and its mutants and applications

Abstract

The application discloses a recombinant feruloyl esterase and a mutant and application thereof, and belongs to the technical field of enzyme engineering. Clostridium butyricum, The wild-type enzyme amino acid sequence is shown as SEQ ID NO:1, and the coding gene sequence is shown as SEQ ID NO:2; the target enzyme is obtained by constructing a recombinant expression vector containing the CbFAE gene, inducing expression in Escherichia coli BL21 (DE3) and purifying; substrate spectrum analysis shows that the enzyme not only has catalytic decomposition activity of model substrates such as methyl ferulate, but also has catalytic activity of decomposing mono (2-hydroxyethyl) terephthalate (MHET) into terephthalic acid (TPA) and ethylene glycol (EG); the application also constructs the CbFAE mutant L168R based on sequence and structure analysis, the amino acid sequence of the mutant enzyme is shown as SEQ ID NO:3, and the catalytic MHET decomposition activity of the mutant enzyme is increased by nearly 4 times compared with the wild type under the same reaction condition. The recombinant feruloyl esterase and the mutant provided by the application expand the types of MHET hydrolytic enzymes and provide a new way for the biodegradation of MHET.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of enzyme engineering, and particularly relates to a recombinant feruloyl esterase and a mutant and application thereof. BACKGROUND

[0002] Feruloyl esterase (Feruloyl esterase, FAE) is a kind of carboxylic acid esterase capable of catalyzing the hydrolysis reaction of phenolic acid ester bond, belongs to the carbon-oxygen ester hydrolase family, and is widely distributed in bacteria, fungi and plants. FAE was first discovered in fungi, and with its ability to hydrolyze aromatic ester bonds, FAE shows important application value in bio-refining, feed processing, functional food development and natural product modification. Existing researches mainly focus on the hydrolysis of FAE on plant-derived phenolic acid ester substrates, and the degradation activity of FAE on artificial synthetic polyester compounds is still lack of reports.

[0003] Polyethylene terephthalate (PET) is a thermoplastic polyester with excellent performance, and is widely used in beverage packaging bottles, synthetic fibers, films and other engineering plastics. Due to its difficulty in natural degradation, the overuse and accumulation of PET waste have caused serious environmental pollution, becoming a global concern of the "white pollution" problem. In recent years, PET biodegradation technology has developed rapidly, among which PET hydrolase (PETase) can catalyze the degradation of PET into oligomeric esters and monomers such as bis(2-hydroxyethyl) terephthalate (BHET) and mono(2-hydroxyethyl) terephthalate (MHET), and MHET is further hydrolyzed by MHET hydrolase (MHETase) to TPA and EG, realizing the complete depolymerization of PET.

[0004] However, the current PETase mainly has high catalytic activity on PET and BHET, and limited degradation ability on MHET. The accumulation of MHET not only reduces the overall degradation efficiency, but also affects the recovery purity of TPA and EG, becoming the rate-limiting step of PET degradation. At the same time, the reported MHETase is extremely limited, mainly derived from Ideonella sakaiensis , and the enzymatic properties, stability and industrial applicability still have deficiencies. Therefore, developing new MHET hydrolase, especially enzymes with diverse sources and high MHET activity, has become a technical bottleneck that needs to be broken through in the field of PET biodegradation. SUMMARY

[0005] In view of the problems that the MHET hydrolase in the prior art is single in source and limited in types, and the MHET degradation efficiency in the existing PET decomposition system is low, the purpose of the present application is to enrich the types of MHET hydrolase, improve the efficiency of PET waste biodegradation and resource utilization, and provide a recombinant feruloyl esterase CbFAE and a mutant L186R with decomposition MHET activity. Clostridium butyricumA feruloyl esterase CbFAE was obtained by medium cloning, and the recombinant enzyme was obtained by heterologous expression, and the substrate spectrum characterization was carried out, it was found that CbFAE not only can hydrolyze common phenolic acid ester substrates, but also has the ability to catalyze the hydrolysis of MHET to generate TPA and EG, further, the activity of CbFAE mutant L168R constructed based on sequence and structure analysis is nearly 4 times higher than that of wild type under the same reaction conditions; the recombinant CbFAE and its mutant enrich the types of MHET hydrolytic enzymes, and provide a new technical scheme for efficient biodegradation and resource recycling of PET waste, which has important environmental protection and industrial application prospect.

[0006] In order to achieve the above purpose, the technical scheme provided by the present application is as follows:

[0007] In the first aspect, the present application provides a feruloyl esterase CbFAE or its mutant L186R, the amino acid sequence of the feruloyl esterase CbFAE is shown as SEQ ID NO: 1; the amino acid sequence of the feruloyl esterase CbFAE mutant L186R is shown as SEQ ID NO: 3.

[0008] Based on the above technical scheme, further, the feruloyl esterase CbFAE is derived from Clostridium butyricum .

[0009] Based on the above technical scheme, further, the feruloyl esterase CbFAE or its mutant L186R has the activity of catalyzing the decomposition of MHET.

[0010] Based on the above technical scheme, further, the mutant L186R is obtained by mutating the leucine at the 186th position of the feruloyl esterase CbFAE to arginine, and its yield in catalyzing the hydrolysis of MHET under the same reaction conditions is nearly 4 times higher than that of the wild type.

[0011] In the second aspect, the present application provides the coding gene of the above-mentioned feruloyl esterase CbFAE or its mutant L186R.

[0012] Based on the above technical scheme, further, the nucleotide sequence of the coding gene of the feruloyl esterase CbFAE is shown as SEQ ID NO: 2; the nucleotide sequence of the coding gene of the feruloyl esterase CbFAE mutant L186R is shown as SEQ ID NO: 4.

[0013] In the third aspect, the present application provides a recombinant expression vector inserted with the coding gene of the above-mentioned feruloyl esterase CbFAE or its mutant L186R.

[0014] Based on the above technical scheme, further, the recombinant expression vector comprises pET28a plasmid.

[0015] In a fourth aspect, the present application provides a recombinant engineering bacterium carrying the recombinant expression vector.

[0016] Based on the above technical solution, further, the recombinant engineering bacterium comprises Escherichia coli BL 21 (DE3).

[0017] In a fifth aspect, the present application provides application of the feruloyl esterase CbFAE or the mutant L186R thereof, the recombinant expression vector or the recombinant engineering bacterium in hydrolysis of monohydroxyethyl terephthalate (MHET) into terephthalic acid and ethylene glycol.

[0018] Based on the above technical solution, further, the hydrolysis reaction is carried out in a buffer solution with pH 3.0-10.0 at a temperature of 20-70℃, and the preferred conditions are pH 7.0 and 50℃.

[0019] Based on the above technical solution, further, the buffer solution comprises a phosphate buffer.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] 1. Rich MHET hydrolysis enzyme species: the recombinant enzyme sequence is derived from a novel feruloyl esterase of Clostridium butyricum , which is first found to have the activity of decomposing MHET, and expands the substrate adaptation range of feruloyl esterase.

[0022] 2. Improved PET depolymerization efficiency: the enzyme L186R mutant can efficiently catalyze MHET to generate TPA and EG, and the enzyme activity is nearly four times higher than that of the wild type, thereby improving the decomposition efficiency of the PET depolymerization system, providing a new technical approach for green recycling of PET waste, and having important environmental protection significance and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present application, the drawings involved in the embodiments will be briefly introduced below.

[0024] Figure 1 It is a SDS-PAGE analysis diagram of the recombinant CbFAE protein.

[0025] Figure 2 It is a HPLC analysis diagram of the MHET hydrolysis product catalyzed by CbFAE.

[0026] Figure 3 It is a CbFAE sequence conservation analysis diagram, wherein A is a WebLogo diagram, and B is a visualization analysis diagram of the active center (Ser-His-Asp catalytic triad) and the lid domain (Loop region) covering the same (deep red represents high conservation).

[0027] Figure 4 A is the optimum temperature of CbFAE, B is the thermal stability of CbFAE, C is the optimum pH of CbFAE, and D is the pH tolerance of CbFAE.

[0028] Figure 5 Relative activity of CbFAE and mutants in catalyzing the MHET reaction. DETAILED DESCRIPTION

[0029] The application will be described in detail below with reference to the embodiments, but the embodiments of the application are not limited thereto. Obviously, the embodiments described below are only part of the embodiments of the application, and other similar embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0030] Example 1 Clostridium Cloning of CbFAE gene and construction of vector

[0031] According to Clostridium butyricum The coding gene sequence (SEQ ID NO: 2, and the amino acid sequence is SEQ ID NO: 1) of ferulic acid esterase CbFAE was obtained by searching the genome database, 5'-end Nde I (CATATG) and 3'-end Xho I (CTCGAG) restriction enzyme cutting sites were introduced to ensure the integrity of the ORF and meet the requirements of the multiple cloning site reading frame of pET28a(+). The primers used are as follows: Clostridium butyricum Genomic DNA was used as a template for PCR amplification using high-fidelity Phusion DNA Polymerase. The PCR product was detected by 1.0% (w / v) agarose gel electrophoresis to confirm a single target band. Subsequently, the PCR product and pET28a(+) vector were subjected to Nde I / Xho I double digestion (37°C, 2 h), and the enzyme digestion products were purified by gel recovery. T4 DNA ligase (Thermo Fisher Scientific) was used for overnight ligation at 16°C (molar ratio of insert to vector was 3:1). The ligation product was purified by ethanol precipitation, and the competent cells of DH5α were transformed by heat shock method, and then spread on LB plates containing 50 μg / mL kanamycin. Single colonies were picked for colony PCR screening, and positive clones were sent to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing. Finally, the recombinant expression plasmid pET28a-CbFAE with correct sequence was obtained. Escherichia coli -1 DH5α competent cells, and then spread on LB plates containing 50 μg / mL kanamycin. Single colonies were picked for colony PCR screening, and positive clones were sent to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing. Finally, the recombinant expression plasmid pET28a-CbFAE with correct sequence was obtained.

[0032] Example 2 Expression and purification of CbFAE protein

[0033] ​The pET28a-CbFAE plasmid was transformed into E. coli BL21(DE3) competent cells. Single colonies were picked and inoculated into LB medium containing kanamycin and cultured at 37°C and 220 rpm until OD600. 600 ≈0.6, add 0.5 mM IPTG to induce induction, and continue culturing at 16℃ for 16 h. After induction, centrifuge the bacterial culture at 4500×g for 15 min, discard the supernatant, resuspend the precipitate in lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0), sonicate, centrifuge at 12000×g for 30 min, and load the supernatant onto Ni 2+ The NTA affinity column was washed and eluted sequentially with buffers containing 20 mM and 350 mM imidazole. SDS-PAGE analysis revealed a recombinant CbFAE protein with a molecular weight of approximately 28 kDa. (Electrophoresis image shown below.) Figure 1 As shown. The collected eluent was concentrated to 5 mL by ultrafiltration and then purified using an AKTA pure protein purification system combined with a Superdex 200 gel filtration column. The protein collected by the gel chromatography column was concentrated to a certain volume and stored at -80°C.

[0034] Example 3: High-performance liquid chromatography (HPLC) analysis of MHET substrate and TPA product.

[0035] Analysis was performed using an Agilent 1260 Infinity II high-performance liquid chromatography system. The column was a Z0RBAXSB-C18 column (4.6 × 150 mm, 5 μm), the detection wavelength was 240 nm, the injection volume was 10 μL, the flow rate was 0.8 mL / min, and the column temperature was 30℃. Separate standard solutions of MHET and TPA (0.05–20 mM) were prepared. Isocratic elution was performed for 8 min in a mobile phase of a mixture of 0.1% trifluoroacetic acid and acetonitrile (v:v = 4:1) to obtain standard curves for MHET and TPA. The separated MHET and TPA were then quantitatively analyzed and their yields calculated based on the standard curves.

[0036] Example 4: Determination of CbFAE activity against MHET

[0037] The reaction system (500 μL) consisted of 50 mM Tris-HCl (pH 8.0), 5 mM MHET and 22.5 μg CbFAE. The mixture was incubated at 50°C for 15 min. After the reaction was terminated, the product was quantitatively analyzed by HPLC.

[0038] Chromatographic results as follows Figure 2As shown, CbFAE can catalyze the hydrolysis of MHET to terephthalic acid, with an enzyme activity of 0.0607 U (1 U = μmol / min). -1 per mg of enzyme).

[0039] Example 5: Screening, Cloning, and Vector Construction of CbFAE Mutants

[0040] (1) Using the CbFAE amino acid sequence as a template, the WebLogo diagram was drawn by sequence conservation analysis using Clustal Omega multiple sequence alignment. Figure 3 A), and sequence annotation was performed using ESPript 3.0; based on this, the three-dimensional structure of CbFAE was predicted using AlphaFold2, and PyMOL was used to visualize and analyze the active center (Ser-His-Asp catalytic triplet) and the lid domain (Loop region) covering it. Figure 3 B). Based on the structure-sequence information, some sites adjacent to the lid region and substrate channel entrance (G179, T185, L186, Y188, T193) were selected as saturation mutation targets. Subsequently, a degenerate NNK primer library was constructed using a whole plasmid PCR strategy. Forward and reverse primers containing the degenerate codons NNK (N=A / T / C / G; K=G / T) were designed. Whole plasmid PCR amplification was performed using pET28a-CbFAE plasmid as a template. After digestion with DpnI (37℃, 1 h), gene cloning and pET-28a(+) vector construction were performed according to the procedure described in "Example 1". The cells were then heat-shocked to... E. coli BL21(DE3) competent cells were spread on a medium containing 100 μg / mL -1 After culturing kanamycin on LB solid medium, 96 independent single clones were randomly selected for initial screening by colony PCR. Positive clones were verified by Sanger bidirectional sequencing. Sequencing ensured that the mutation sites were correct, there were no additional mutations, and the results met the statistical requirements for saturation mutations (library coverage ≥75%).

[0041] (2) Recombinant expression and initial screening: Single clones were picked and inoculated into 96-well deep-well plates, each well containing 700 μL of LB medium (containing 50 μg mL of LB broth). -1 Kanamycin), cultured at 37°C with shaking at 220 rpm until OD. 600 ≈0.6, add 0.5 mM IPTG to induce induction, and bring the final volume to 900 μL. Continue culturing at 16 °C and 220 rpm for 16 h. After induction, collect the cells by centrifugation at 4 °C and 4500 × g for 15 min, discard the supernatant, and use a solution containing 1 mg / mL of IPTG. -1Lysozyme was resuspended in lysis buffer and lysed at 30°C for 20 min. The resulting enzyme and wild-type CbFAE were tested under the same conditions, following the procedure described in Example 4: Determination of CbFAE activity against MHET. Enzyme activity was immediately quantified by HPLC (Example 3).

[0042] (3) Validation of positive mutants: Mutants with ≥2 times higher activity than wild type obtained from the initial screening were streaked with glycerol in a solution containing 50 μg / mL -1 Kanamycin was incubated overnight at 37 °C on LB agar plates. Single clones were picked for sequencing verification. After confirming the mutation, the corresponding plasmid was retransformed into... E. coli BL21(DE3) competent cells. The protein expression and purification procedure was as described in Example 2: Expression and purification of CbFAE. The obtained purified enzyme was compared with wild-type CbFAE under the same conditions, and the procedure was as described in Example 4: Determination of CbFAE activity against MHET.

[0043] The highly active mutants obtained through screening were sequenced, and the CbFAE mutant L186R was obtained after sequence analysis. The amino acid sequence is shown in SEQ ID NO:3 (the sequence of the encoding gene is shown in SEQ ID NO:4).

[0044] Example 6: Effects of Temperature and pH on Enzyme Activity

[0045] To characterize the temperature and pH adaptability of recombinant CbFAE, the formation rate of MHET hydrolysis products was detected by HPLC. Optimal temperature experiments were conducted in 10°C increments within the range of 20–70°C. All reactions were performed in 50 mM PBS (pH 7.0), with an enzyme dosage of 0.1 mg, a total reaction volume of 500 μL, a final substrate concentration of 10 mM, and a reaction time of 15 min. Results are as follows: Figure 4 As shown in Figure A, the results indicate that the optimal reaction temperature for CbFAE is 50°C; when the temperature rises to 70°C, the residual activity decreases, indicating that the enzyme undergoes irreversible inactivation at high temperatures.

[0046] pH adaptability experiments were conducted at 30°C and a substrate concentration of 10 mM, using the following buffer system: 0.2 mol L... -1 Citric acid – sodium citrate (pH 3.0–5.0), 0.2 mol L -1 Phosphate (PBS, pH 6.0–8.0) and 0.2 mol L -1 Glycine–NaOH (pH 9.0–10.0); total reaction volume 500 μL, enzyme amount 0.1 mg, reaction time 15 min.

[0047] The results are as followsFigure 4 As shown in Figure C, the optimal pH for CbFAE is 7.0; under pH ≤ 4.0 or pH ≥ 9.0 conditions, the relative activity is less than 40% and 20%, respectively, indicating that the enzyme has the best catalytic efficiency in a neutral to slightly alkaline environment.

[0048] Example 7: Enzyme thermal stability and pH tolerance

[0049] The purified CbFAE (0.1 mg) was resuspended in 100 µL of 0.1 M phosphate-buffered saline (PBS, pH 7.0). Then, 200 µL of a buffer system with pH gradients of 0.2 M from 3.0 to 10.0 (1 pH unit) was added. After standing at room temperature for 30 min, 600 µL of PBS buffer (0.1 M pH 7.0) was added. Finally, 1 mM MHET was rapidly added as substrate, bringing the final volume to 1 mL. The enzymatic reaction was carried out at 50 °C. After 15 min, the generated TPA was immediately quantitatively determined by HPLC. The residual enzyme activity was characterized by the amount of product generated. The optimal reaction activity was taken as 100%. The results are as follows: Figure 4 As shown in D.

[0050] The purified CbFAE was diluted to 0.1 mg / mL with 0.2 M phosphate-buffered saline (PBS, pH 7.0). -1 The enzymes were incubated at 20–70 °C (gradient of 10 °C) for 30 min each, followed immediately by cooling in an ice-water bath to terminate the reaction. Using 1 mM MHET as the substrate, the reaction was carried out at 30 °C in a 500 µL system for 15 min. After the reaction was terminated, the generated TPA was quantitatively determined by HPLC. The residual enzyme activity was calculated based on the amount of product generated, and the optimal reaction activity was taken as 100%. The experimental results are as follows: Figure 4 As shown in B.

[0051] Example 8: Comparison of MHET enzyme activities of CbFAE and its mutant with PET 46

[0052] The total volume of the enzymatic reaction system was 500 μL, containing 50 mM Tris-HCl (pH 8.0), 5 mM MHET, and 22.5 μg of purified CbFAE or CbFAE mutant L186R or the plastic degrading enzyme PET46 reported in the literature (Pablo Perez-Garcia et al., Communications Chemistry, 2023). The system was reacted in a constant temperature water bath at 50℃ for 15 min, and then immediately placed in an ice bath to terminate the reaction. After filtration through a 0.22 μm filter membrane, the product was quantitatively analyzed by HPLC.

[0053] The experimental results are shown in Table 1. CbFAE can effectively catalyze the hydrolysis of MHET to produce terephthalic acid and ethylene glycol. Under the same reaction conditions, the catalytic activity of CbFAE is similar to that of PET46 reported in the literature. The catalytic activity of the CbFAE mutant L186R is significantly better than that of CbFAE, with the enzyme activity increasing by nearly four times. The relative activity data are as follows: Figure 5 As shown.

[0054] Table 1 Comparison of catalytic activities of CbFAE and mutant L186R with previously reported MHET degrading enzymes (1 U = μmol min) - 1 per mg of enzyme)

[0055]

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A ferulic acid esterase CbFAE or its mutant L186R, characterized in that, The amino acid sequence of the ferulic acid esterase CbFAE is shown in SEQ ID NO:1; the amino acid sequence of the ferulic acid esterase CbFAE mutant L186R is shown in SEQ ID NO:

3.

2. The gene encoding the ferulic acid esterase CbFAE or its mutant L186R as described in claim 1.

3. The gene encoding ferulic acid esterase CbFAE or its mutant L186R according to claim 2, characterized in that, The nucleotide sequence of the gene encoding ferulic acid esterase CbFAE is shown in SEQ ID NO:2; the nucleotide sequence of the gene encoding the ferulic acid esterase CbFAE mutant L186R is shown in SEQ ID NO:

4.

4. A recombinant expression vector that inserts the encoding gene of the ferulic acid esterase CbFAE or its mutant L186R as described in claim 2 or 3.

5. The recombinant expression vector according to claim 4, characterized in that, The recombinant expression vector is the pET28a plasmid.

6. A recombinant engineered bacterium carrying the recombinant expression vector of claim 4 or 5.

7. The recombinant engineered bacteria according to claim 6, characterized in that, The recombinant engineered bacteria is Escherichia coli BL21(DE3).

8. The use of the ferulic acid esterase CbFAE of claim 1 or its mutant L186R, the recombinant expression vector of claim 4 or 5, or the recombinant engineered bacteria of claim 6 or 7 in the hydrolysis of mono(2-hydroxyethyl) terephthalic acid to terephthalic acid and ethylene glycol.

9. The application according to claim 8, characterized in that, The hydrolysis reaction was carried out in a buffer solution with a pH of 3.0 to 10.0 at a temperature of 20°C to 70°C.

10. The application according to claim 9, characterized in that, The buffer solution is a phosphate buffer.

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