Furandicarboxylic acid decarboxylase based on ancestor sequence reconstruction and application of furandicarboxylic acid decarboxylase

Abstract

The invention discloses furandicarboxylic acid decarboxylase based on ancestor sequence reconstruction and application of furandicarboxylic acid decarboxylase, and belongs to the technical field of enzyme engineering and genetic engineering. The furandicarboxylic acid decarboxylase is named as HmfF-N10, the furandicarboxylic acid decarboxylase is obtained by performing ancestor sequence reconstruction on a plurality of homologous enzyme sequences of PrFMN dependent furandicarboxylic acid decarboxylase HmfF extracted from a database, the amino acid sequence of the furandicarboxylic acid decarboxylase HmfF is shown as SEQ ID NO: 1, experiments show that the HmfF-N10 has excellent thermal stability, the melting temperature of the furandicarboxylic acid decarboxylase HmfF-N10 is 86.83 DEG C, and 60% of catalytic activity of the furandicarboxylic acid decarboxylase HmfF-N10 is still kept at 80 DEG C. In addition, the HmfF-N10 has the activity of catalyzing terephthalic acid decarboxylation to generate benzoic acid, and can be used for biodegradation of terephthalic acid plastic wastes.

Description

Technical Field

[0001] The invention belongs to the technical fields of enzyme engineering and genetic engineering, and particularly relates to a furandicarboxylate decarboxylase reconstructed based on an ancestral sequence and application thereof in the decarboxylation of terephthalic acid. Background Art

[0002] Terephthalic acid (TPA) is an important aromatic dicarboxylic acid widely used in the production of polyester materials such as polyethylene terephthalate (PET). However, PET degrades slowly in the natural environment, and its microplastic fragments can accumulate in ecosystems and be transmitted through the food chain, posing a potential threat to environmental safety and human health. To achieve the green recycling of plastic waste, increasing research is focusing on the biodegradation and conversion of TPA. Decarboxylases are enzymes that catalyze the decarboxylation of carboxyl compounds to produce corresponding products and have important applications in organic synthesis and green chemistry. However, the sources of enzymes with TPA decarboxylation activity are currently limited, and the development of efficient and stable TPA decarboxylases holds great potential. In recent years, ancestral sequence reconstruction (ASR) has been widely used in enzyme functional modification and novel enzyme design, enabling the discovery of ancestral protein sequences with high stability and excellent enzymatic activity.

[0003] Furandicarboxylic acid decarboxylase (HmfF) is an enzyme that catalyzes the decarboxylation of the biomass-derived platform compound 2,5-furandicarboxylic acid to 2-furancarboxylic acid. This enzyme belongs to the UbiD enzyme family and relies on prenylated flavin mononucleotide (prFMN) as a cofactor. It achieves efficient removal of the carboxyl group through a unique catalytic mechanism, and has significant application potential in the field of green catalysis. Studies have shown that HmfF has a broad substrate spectrum and can catalyze the decarboxylation of a variety of 2,5-furandicarboxylic acid analogs, exhibiting good substrate promiscuity. However, natural HmfF has not yet been found to catalyze the decarboxylation of terephthalic acid. Summary of the Invention

[0004] To expand the catalytic properties of furandicarboxylate decarboxylase and enhance its stability and substrate adaptability, such as for use with terephthalate substrates, the present invention provides a furandicarboxylate decarboxylase based on ancestral sequence reconstruction and its application in terephthalate decarboxylation. Using ancestral sequence reconstruction (ASR) technology, computational biology analysis, and protein engineering optimization, the present invention successfully constructed an ancestral furandicarboxylate decarboxylase sequence, HmfF-N10, with novel catalytic activity. HmfF-N10 not only exhibits excellent enzymatic stability and other properties but also achieves terephthalate decarboxylation. This overcomes the existing issues of HmfF's low stability and limited terephthalate decarboxylation activity, providing a novel enzymatic catalytic tool for the biodegradation of terephthalate.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a furandicarboxylate decarboxylase HmfF-N10, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] In a second aspect, the present invention provides a gene encoding the above-mentioned furandicarboxylate decarboxylase HmfF-N10.

[0008] Based on the above technical solution, further, the nucleotide sequence of the gene is shown as SEQ ID NO.2.

[0009] In a third aspect, the present invention provides a recombinant expression vector into which the above-mentioned gene is inserted.

[0010] Based on the above technical solution, further, an isopropyl transferase gene is inserted into the vector.

[0011] Based on the above technical solution, further, the vector includes the pACYCDuet-1 expression vector.

[0012] In a fourth aspect, the present invention provides a recombinant engineered bacterium carrying the above-mentioned recombinant expression vector and capable of expressing furandicarboxylate decarboxylase HmfF-N10.

[0013] Based on the above technical solution, further, the recombinant engineered bacteria is Escherichia coli.

[0014] In a fifth aspect, the present invention provides use of the above-mentioned furandicarboxylate decarboxylase HmfF-N10, recombinant expression vector or recombinant engineered bacteria in catalyzing the decarboxylation reaction of terephthalic acid.

[0015] Based on the above technical solution, further, the decarboxylation reaction is carried out in a conventional buffer solution, the pH of the buffer solution is 6.5-8.5, and the reaction temperature is 30-80°C, preferably 40-60°C.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The furandicarboxylate decarboxylase HmfF-N10 obtained by the present invention using ancestral sequence reconstruction technology has good thermal stability and a melting temperature of up to 86.83°C, which greatly improves the practicality of the enzyme.

[0018] 2. The furandicarboxylate decarboxylase HmfF-N10 obtained in the present invention using ancestral sequence reconstruction technology not only naturally catalyzes the decarboxylation reaction of furandicarboxylate, but also has catalytic activity in the decarboxylation process of terephthalic acid, thereby enhancing the application potential of this enzyme in the decomposition of terephthalic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the phylogenetic tree of furandicarboxylate decarboxylase HmfF.

[0020] Figure 2 The cavities volumes and predicted melting temperatures (Tm) of the HmfF ancestral enzyme N9, N10, and N13 are shown.

[0021] Figure 3 This is the electrophoresis diagram of HmfF-N10 expressed in E. coli during the purification process.

[0022] Figure 4 Comparison of thermal stability curves of HmfF-N10 and PtHmfF.

[0023] Figure 5 This is a curve of the catalytic rate of HmfF-N10 changing with temperature.

[0024] Figure 6 This is the specific activity diagram of HmfF-N10 and PtHmfF catalyzing terephthalic acid. DETAILED DESCRIPTION

[0025] In order to better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below. It should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to enable the present invention to be fully conveyed to those skilled in the art.

[0026] Example 1: Ancestral sequence reconstruction

[0027] A variety of known Prenylated flavinmononucleotide (prFMN)-dependent furandicarboxylate decarboxylase (HmfF) homologous protein sequences were retrieved and screened from the UniProt protein database. To ensure the representativeness and accuracy of the analysis, sequences were selected to cover multiple related species, and the sequence similarity was within a certain range, which not only avoided high redundancy but also ensured evolutionary diversity. The MAFFT sequence analysis tool was used to align the selected 93 sequences of different species, and the selected sequence similarity spanned from 26% to 90%. The maximum likelihood method was used to construct a phylogenetic tree of furandicarboxylate decarboxylase homologous sequences. The developmental tree is as follows: Figure 1 As shown, this reflects the evolutionary relationships and ancestral nodes of enzyme sequences across species. This phylogenetic tree divides the enzymes into five clusters based on their developmental relationships, revealing that sequence similarity increases as one moves clockwise along the tree. The first two clusters exhibit relatively low inter- and intra-cluster sequence similarity, with most similarities around 35% and a few exceeding 60%. The third cluster exhibits inter- and intra-cluster sequence similarity around 50%, while approximately three-quarters of the sequences in the last two clusters exhibit similarity above 50%. To identify ancestral enzymes with promising evolutionary potential, we sought a relatively moderate level of overall similarity with modern enzymes from their respective branches, which would facilitate further rational analysis of the active pocket. Based on the above sequence analysis, we narrowed our search to the third cluster. Based on the sequence relationships of modern enzymes and their evolutionary relationships within the phylogenetic tree, we initially selected three ancestral enzymes: N9 (amino acid sequence shown in SEQ ID NO:3), N10 (amino acid sequence shown in SEQ ID NO:1), and N13 (amino acid sequence shown in SEQ ID NO:4). The bioinformatics online websites (Cavity Plus and DeepSTABp) were used to predict the cavity volumes and melting temperatures (Tm) of the three ancestral enzymes N9, N10, and N13. The results are as follows: Figure 2 As shown. When the volume of the substrate binding pocket cavity of the enzyme becomes larger, although its capacity to accommodate a wide spectrum of substrates (especially non-natural substrates with larger volumes) can be improved, when the space is too large, it will cause the binding between the enzyme and the substrate to loosen, thereby leading to a decrease in catalytic activity. Compared with N13, the predicted Tm values ​​of N9 and N10 are above 80°C, and have the potential for improved thermal stability. Taking all the above into consideration. The reconstruction object of the ancestral enzyme was determined to be N10, which has an intermediate cavity volume and potential thermal stability, and was named HmfF-N10. The amino acid sequence is shown in SEQ ID NO: 1.

[0028] To achieve gene synthesis and heterologous expression of the ancestral enzyme, the amino acid sequence was reverse-translated to obtain the corresponding nucleotide sequence. Taking into account the codon usage preferences of the target expression host (e.g., E. coli), the nucleotide sequence was optimized and adjusted to improve gene expression efficiency. The resulting coding gene sequence is shown in SEQ ID NO: 2.

[0029] Example 2: Construction of HmfF-N10 gene

[0030] The HmfF-N10 gene (nucleotide sequence shown in SEQ ID NO. 2) and the gene for the isopropyltransferase UbiX from Pseudomonas aeruginosa were cloned into the Bam I / Hind III and Nde I / Xho I restriction sites of the expression vector pACYCDuet-1, respectively, to construct recombinant plasmids. HmfF belongs to the UbiD family, and enzymes in this family are typically co-expressed with UbiX for catalytic activity. UbiX synthesizes the cofactor prFMN required for UbiD, which is then transferred to UbiD family enzymes such as HmfF and Fdc1 to activate them and exert their catalytic decarboxylation activity (Archives of Biochemistry and Biophysics, 2017, 632, 209-221). The amino acid sequence of the isopropyltransferase UbiX from Pseudomonas aeruginosa (amino acid sequence shown in SEQ ID NO. 5) can be obtained from protein databases such as UniProt. The pACYCDuet-1 expression vector is a conventional vector used in gene cloning and can be obtained through public purchase, etc. The cloning method is a conventional molecular cloning method, and the recombinant plasmid is constructed and sequenced and verified by a nucleic acid sequencing company.

[0031] Example 3: HmfF-N10 expression and whole cell catalyst acquisition

[0032] The recombinant plasmid constructed in Example 2 was transformed into Escherichia coli competent cells BL21 (DE3) by heat shock transformation, and then coated on an LB solid plate containing 50 μg / mL chloramphenicol resistance. The plate was inverted and cultured at 37°C for 12 hours. The single clone on the plate was transferred to 5 mL of sterile LB liquid culture medium containing 50 μg / mL chloramphenicol. After overnight culture at 37°C, 1 mL was inoculated into 100 mL of sterile LB liquid culture medium containing 50 μg / mL chloramphenicol. After further culture at 37°C for 8 hours, 10 mL was inoculated into 1 L of sterile LB liquid culture medium containing 50 μg / mL chloramphenicol. After culture for about 3 hours, the OD 600 When the pH value reached 0.6-0.8, IPTG was added at a final concentration of 0.4 mM for induction, and cultured overnight at 16°C. The HmfF-N10 bacteria, i.e., the whole-cell catalyst, were obtained by centrifugation.

[0033] Example 4: HmfF-N10 purification method

[0034] The cells obtained by culture in Example 3 were resuspended in 50 mL of buffer A (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM MnCl2, 5% glycerol), and then 50 μL of β-mercaptoethanol was added for ultrasonic disruption for 15 min at an ultrasonic power of 300 W. After ultrasonication, the cells were centrifuged at 4°C and 12,000 rpm for 30 min to obtain a supernatant containing the target protein. The supernatant was first subjected to Ni 2 + affinity chromatography purification, wherein the imidazole concentrations in the wash and elution were 32 mM and 350 mM, respectively. The collected eluate was concentrated to 5 mL by ultrafiltration and then purified using an AKTA pure protein purification system combined with a Superdex200 gel filtration column. The protein collected from the gel chromatography column was concentrated to a certain volume and stored in a -80°C refrigerator. During this process, a sample was retained and the purity of the purified protein was verified by SDS-PAGE. The electrophoresis diagram is shown below. Figure 3 As shown, a protein band appeared around 50 kDa, which was consistent with the molecular weight calculated based on the sequence, indicating that the purified HmfF-N10 protein was successfully obtained.

[0035] Example 5: HmfF-N10 enzymatic stability determination

[0036] The purified HmfF-N10 was diluted to 1 mg / mL with buffer A (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM MnCl2, 5% glycerol) and centrifuged at 4 ° C and 12000 rpm for 10 min to remove bubbles and aggregates. The treated protein was aspirated with a capillary tube and the thermal stability of the protein was measured within 25-95 ° C using a high-throughput multi-parameter protein stability analyzer. The experiment was repeated three times independently, and the Tm value was fitted with the Boltzmann function to obtain a value of 86.83 ± 0.30 ° C, which is 15 ° C higher than the Tm value of HmfF (PtHmfF) from Pelotomaculum thermopropionicum reported (ACS Catalysis, 2019, 9, 4, 2854-2865). Figure 4 ).

[0037] The thermal stability of HmfF-N10 was determined by measuring its catalytic rate of decarboxylation of its natural substrate, 2,5-furandicarboxylic acid, at 30-100°C. The decarboxylation reaction was carried out in buffer A (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM MnCl2, 5% glycerol) for 2 h. The reaction system contained 1 mL of 1 mM 2,5-furandicarboxylic acid and 730 μL of whole-cell catalyst. The whole-cell catalyst was obtained by sonicating 100 mL of LB liquid culture medium in 5 mL of buffer A. The decarboxylation yield curve as a function of temperature is shown in Figure 2. Figure 5 As shown in Figure 2, HmfF-N10 reached its maximum catalytic rate at 60-70°C and still maintained 60% relative catalytic activity at 80°C. The above thermal stability experiments show that HmfF-N10 has excellent thermal stability.

[0038] Example 6: HPLC Analysis of Terephthalic Acid Substrate and Products

[0039] Analysis was performed using an Agilent 1260 Infinity II HPLC system using a Z0RBAX SB-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°C. A series of terephthalic acid and benzoic acid standard solutions (0.01-1.5 mM) were prepared. Terephthalic acid and benzoic acid were successfully separated by isocratic elution for 16 minutes using a mobile phase consisting of a mixture of 0.1% trifluoroacetic acid and acetonitrile (v / v = 0.8:0.2).

[0040] Example 7: Determination of the activity of HmfF-N10 in catalytic decarboxylation of terephthalic acid

[0041] The activity of 0.934 mg of purified HmfF-N10 enzyme in catalyzing the decarboxylation of 5 mM terephthalic acid to benzoic acid was measured at pH 7.5 and 50°C. The reaction buffer was 10 mM HEPES (pH 7.5), the reaction system was 1 mL, and the reaction time was 15 h. In the control group, PtHmfF was used instead of HmfF-N10, and the other conditions remained unchanged.

[0042] After the reaction was completed, the reaction was terminated with a four-fold volume of a methanol-acetonitrile mixture (v / v=1:1). The product concentration was confirmed by HPLC using the detection method of Example 6. Calculations showed that PtHmfF had almost no catalytic activity in the decarboxylation of terephthalic acid, and the specific activity of HmfF-N10 was 0.246 U / mg ( Figure 6 ), U is defined as μmol / h.

Claims

1. A furandicarboxylate decarboxylase HmfF-N10, characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding the furandicarboxylate decarboxylase HmfF-N10 according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. A recombinant expression vector into which the gene according to claim 2 or 3 is inserted.

5. The recombinant expression vector according to claim 4, characterized in that The vector includes the pACYCDuet-1 expression vector. A recombinant engineered bacterium carrying the recombinant expression vector according to claim 4 or 5 and capable of expressing furandicarboxylate decarboxylase HmfF-N10.

7. The recombinant engineered bacterium according to claim 6, characterized in that The recombinant engineering bacteria is Escherichia coli.

8. Use of the furandicarboxylate decarboxylase HmfF-N10 according to claim 1, the recombinant expression vector according to claim 4 or 5, or the recombinant engineered bacteria according to claim 6 or 7 in catalyzing the decarboxylation reaction of terephthalic acid.

9. The use according to claim 8, characterized in that The decarboxylation reaction is carried out in a conventional buffer solution with a pH of 6.5 to 8.5 and a reaction temperature of 30 to 80° C., preferably 40 to 60° C.

Images