Recombinant feruloyl esterase as well as mutant and application thereof

Abstract

The invention discloses recombinant feruloyl esterase as well as a mutant and application thereof, and belongs to the technical field of enzyme engineering. The feruloyl esterase CbFAE is derived from Clostridium butyricum, the amino acid sequence of a wild enzyme is as shown in SEQ ID NO: 1, the sequence of an encoding gene of the feruloyl esterase CbFAE is as shown in SEQ ID NO: 2, and a target enzyme is obtained by constructing a recombinant expression vector containing a CbFAE gene and performing induced expression and purification in escherichia coli BL21 (DE3). Substrate spectrum analysis shows that the enzyme not only has the activity of catalyzing decomposition of mode substrates such as methyl ferulate, but also has the catalytic activity of decomposing mono (2-hydroxyethyl) terephthalate (MHET) into terephthalic acid (TPA) and ethylene glycol (EG); the invention also constructs a CbFAE mutant L168R based on sequence and structure analysis, the amino acid sequence of the mutant enzyme is as shown in SEQ ID NO: 3, and the activity of catalyzing MHET decomposition under the same reaction condition is nearly 4 times higher than that of a wild type. According to the recombinant feruloyl esterase and the mutant thereof provided by the invention, the variety of MHET hydrolase is expanded, and a new way is provided for 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 that can catalyze 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 has not been reported.

[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 its 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 In view of the problems in the prior art that the source of MHET hydrolase is single, the types are limited, and the degradation efficiency of MHET 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.

[0005] In order to achieve the above purpose, the technical scheme provided by the present application is as follows: In a 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.

[0006] Based on the above technical scheme, further, the feruloyl esterase CbFAE is derived from Clostridium butyricum .

[0007] Based on the above technical scheme, further, the feruloyl esterase CbFAE or its mutant L186R has the activity of catalyzing the decomposition of MHET.

[0008] 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 is nearly 4 times higher than that of the wild type under the same reaction conditions.

[0009] In a second aspect, the present application provides the coding gene of the above-mentioned feruloyl esterase CbFAE or its mutant L186R.

[0010] 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.

[0011] In a 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.

[0012] Based on the above technical scheme, further, the recombinant expression vector comprises a pET28a plasmid.

[0013] In a fourth aspect, the present application provides a recombinant engineering bacterium carrying the recombinant expression vector.

[0014] Based on the above technical solution, further, the recombinant engineering bacterium comprises Escherichia coli BL 21 (DE3).

[0015] 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.

[0016] 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 condition is pH 7.0 and 50℃.

[0017] Based on the above technical solution, further, the buffer solution comprises a phosphate buffer.

[0018] Compared with the prior art, the present application has the following beneficial effects: 1. Rich MHET hydrolytic 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. 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

[0019] In order to more clearly illustrate the embodiments of the present application, the drawings involved in the embodiments will be briefly introduced below.

[0020] Figure 1 It is a SDS-PAGE analysis diagram of the recombinant CbFAE protein.

[0021] Figure 2 It is a HPLC analysis diagram of the MHET hydrolysis product catalyzed by CbFAE.

[0022] 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).

[0023] Figure 4A, B, C and D are the comprehensive effects of pH and temperature on the catalytic activity and stability of CbFAE, wherein 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.

[0024] Figure 5 Relative activity of CbFAE and mutants in catalyzing MHET reaction. DETAILED DESCRIPTION

[0025] The application will be described in detail below with reference to the embodiments, but the embodiments of the application are not limited thereto. It is obvious that 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.

[0026] Embodiment 1 Clostridium Cloning of CbFAE gene and construction of vector 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 were designed by Primer Premier 5.0 software. Clostridium butyricum The genomic DNA was used as a template for PCR amplification by 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, then the PCR product and pET28a(+) vector were subjected to Nde I / Xho I double enzyme digestion (37°C, 2 h), the enzyme digestion products were purified by gel recovery, and then T4 DNA ligase (Thermo Fisher Scientific) was used for ligation at 16°C overnight (the molar ratio of the insert and the vector was 3:1), the ligation product was purified by ethanol precipitation, and the heat shock method was used to transform into Escherichia coli DH5α competent cells, and then coated on LB plates containing 50 μg / mL kanamycin. Single colonies were picked for colony PCR primary screening, and positive clones were sent to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing, and finally the recombinant expression plasmid pET28a-CbFAE with correct sequence was obtained. -1

[0027] Embodiment 2 Expression and purification of CbFAE protein ​The pET28a-CbFAE plasmid was transformed into E. coli BL21 (DE3) competent cells, a single colony was inoculated in LB medium containing kanamycin, and cultured at 37°C, 220 rpm until OD 600 ≈0.6, and 0.5 mM IPTG was added to induce, and the culture was continued at 16°C for 16 h. After induction, the bacterial solution was centrifuged at 4500 x g for 15 min, the supernatant was discarded, and the precipitate was resuspended with lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0). After ultrasonic disruption, the solution was centrifuged at 12000 x g for 30 min, and the supernatant was loaded onto a Ni 2+ -NTA affinity column, and then rinsed and eluted with buffers containing 20 mM and 350 mM imidazole, respectively. SDS-PAGE was used to detect the purified product, and a recombinant CbFAE protein with a molecular weight of about 28 kDa was obtained, as shown in Figure 1 After the collected eluate was ultrafiltrated and concentrated to 5 mL, it was purified using an AKTA pure protein purification system combined with a Superdex 200 gel filtration column. The protein collected from the gel filtration column was concentrated to a certain volume and stored in a -80°C refrigerator.

[0028] Example 3 HPLC analysis of MHET substrate and TPA product An Agilent 1260 Infinity II high-performance liquid chromatography system was used for analysis, the chromatographic column was a Z0RBAX SB-C18 column (4.6 x 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°C; MHET and TPA standard solutions (0.05-20 mM) were prepared. Under the isocratic elution of a mobile phase of 0.1% trifluoroacetic acid and acetonitrile (v:v=4:1) for 8 min, the standard curves of MHET and TPA were obtained, respectively, and the separated MHET and TPA were quantitatively analyzed and the yield was calculated according to the standard curves.

[0029] Example 4 Determination of CbFAE enzyme activity on MHET The reaction system (500 μL) consisted of 50 mM Tris-HCl (pH 8.0), 5 mM MHET, and 22.5 μg CbFAE, which was incubated at 50°C for 15 min. After the reaction was terminated, the product was quantitatively analyzed by HPLC.

[0030] The chromatography results are shown in 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).

[0031] Example 5: Screening, Cloning, and Vector Construction of CbFAE Mutants (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%).

[0032] (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. -1Lytic buffer resuspension, 30°C static lysis for 20 min, the resulting enzyme and wild type CbFAE were tested under the same conditions, process steps refer to Example 4: CbFAE activity determination of MHET, immediately quantitatively determined by HPLC (Example 3) enzyme activity.

[0033] (3) Positive mutant verification: the mutant strain with activity increased by ≥2 times compared with the wild type obtained by preliminary screening was streaked on LB plates containing 50 μg mL -1 kanamycin, 37 °C incubated overnight. Single colonies were picked for sequencing verification, and after confirming the mutation, the corresponding plasmid was re-transformed into E. coli BL21(DE3) competent cells. Protein expression and purification process refer to Example 2: Expression and purification of CbFAE. The resulting pure enzyme was compared with wild type CbFAE under the same conditions, process steps refer to Example 4: CbFAE activity determination of MHET.

[0034] The high-activity mutant obtained by screening was sequenced, and after sequence analysis, CbFAE mutant L186R was obtained, the amino acid sequence is shown in SEQ ID NO: 3 (the sequence of the encoding gene is shown in SEQ ID NO: 4). Example 6 Effect of temperature and pH on enzyme activity To characterize the temperature and pH adaptability of recombinant CbFAE, HPLC was used to detect the generation rate of MHET hydrolysis products. The optimal temperature experiment was carried out at 20~70°C with 10°C as the gradient; all reactions were carried out in 50 mM PBS (pH 7.0), enzyme amount was 0.1 mg, total reaction system volume was 500 μL, substrate final concentration was 10 mM, reaction time was 15 min. The results are shown in Figure 4 A, the results show that the optimal reaction temperature of CbFAE is 50°C; when the temperature rises to 70°C, the residual activity decreases, showing that the enzyme is irreversibly inactivated at high temperature.

[0035] The pH adaptability experiment was carried out at 30°C and substrate concentration of 10 mM, the buffer system was as follows: 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 system volume was 500 μL, enzyme amount was 0.1 mg, reaction time was 15 min.

[0036] The results are shown in Figure 4As 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.

[0037] Example 7: Enzyme thermal stability and pH tolerance 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.

[0038] 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.

[0039] Example 8: Comparison of MHET enzyme activities of CbFAE and its mutant with PET 46 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.

[0040] 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.

[0041] 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)

[0042] 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 includes 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 mentioned include 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 is carried out in a buffer solution with a pH of 3.0 to 10.0 at a temperature of 20°C to 70°C, with the preferred conditions being pH 7.0 and 50°C.

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

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