Non-heme chloroperoxidase and use thereof

Abstract

This invention provides a non-heme chloride peroxidase and its application, belonging to the field of genetic engineering. The non-heme chloride peroxidase... Ro CPO, whose amino acid sequence is SEQ ID NO.2; the non-heme chlorperoxidase Ro The nucleotide sequence of the CPO encoding gene is SEQ ID NO.1; the non-heme chlorperoxidase... Ro Application of CPO in the preparation of 5-hydroxymethyl-2-furanic acid and / or 2,5-furanic acid.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to a non-heme chloroperoxidase. Ro CPO and its applications. Background Technology

[0002] 2,5-Furfurandicarboxylic acid (FDCA) is an important bio-based platform compound. FDCA is a key monomer in the synthesis of polyethylene 2,5-furandicarboxylate (PEF), a renewable polyester material that outperforms traditional polyethylene terephthalate (PET) in terms of gas barrier properties and biodegradability, showing broad application prospects. Furthermore, FDCA can be used to produce other high-performance polymers, plasticizers, coatings, and fragrances. With increasing global focus on sustainable development and environmental protection, the market demand for FDCA as a bio-based chemical is growing rapidly.

[0003] Biocatalysis (such as enzyme catalysis or whole-cell catalysis) has become a research hotspot due to its advantages such as mild reaction conditions (ambient temperature and pressure), high selectivity, and environmental friendliness. The biocatalytic synthesis of FDCA mainly uses 5-hydroxymethylfurfural (HMF) as the starting substrate and involves multiple oxidation steps. A common oxidation pathway is as follows: HMF is first oxidized to 5-hydroxymethyl-2-furanoic acid (HMFCA), then HMFCA is further oxidized to 5-formyl-2-furanoic acid (FFCA), and finally FFCA is further oxidized to FDCA (…). Picture 1 (Path A).

[0004] In the biosynthetic pathway of FDCA, few enzymes have been reported that can catalyze the oxidation of HMF and FFCA. Therefore, discovering novel and efficient oxidases is of great significance for achieving efficient biotransformation of HMF to FDCA and for expanding the existing enzyme resource library for FDCA biocatalysis. Non-heme peroxidases are a class of redox enzymes that use hydrogen peroxide as an oxidant and do not require a heme cofactor. They are mainly used to catalyze halogenation, oxidation, and oxygen transfer reactions, but there are no reports of their application in catalyzing the synthesis of FDCA from HMF. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a non-heme chloride peroxidase and its applications.

[0006] This invention, through the Ro CPO was heterologously expressed and purified, and then subjected to in vitro enzyme catalysis, which for the first time confirmed... Ro CPO can catalyze the oxidation of HMF and FFCA, producing HMFCA and FDCA, respectively.

[0007] The non-heme chloride peroxidase described in this invention Ro CPO is derived from *Rauvolfia micrantha* (ornithine-derived bacteria). Raoultella ornithinolytica BF60).

[0008] The technical solution of the present invention is as follows: A non-heme chloride peroxidase Ro CPO, whose amino acid sequence is SEQ ID NO.2.

[0009] A gene encoding the aforementioned non-heme chloride peroxidase. Ro CPO.

[0010] Preferably, the nucleotide sequence of the encoding gene is SEQ ID NO.1.

[0011] A recombinant plasmid containing the aforementioned non-heme chloride peroxidase Ro The gene that encodes CPO.

[0012] A recombinant bacterium containing the aforementioned recombinant plasmid.

[0013] Preferably, the host bacteria of the recombinant bacteria are E. coli BL21(DE3) was expressed using the pACYCDuet-1 plasmid.

[0014] The above non-heme chloride peroxidase Ro The use of CPO, encoding genes, recombinant plasmids or recombinant bacteria in the preparation of 5-hydroxymethyl-2-furanic acid and / or 2,5-furandicarboxylic acid.

[0015] The above non-heme chloride peroxidase Ro A method for preparing 5-hydroxymethyl-2-furanoic acid or 2,5-furandicarboxylic acid from CPO includes the following steps: Add to Ro CPO enzyme solution, substrate HMF or FFCA final concentration 1-20 mM. Ro The CPO enzyme concentration was 0.02-0.1 mg / mL, and the reaction was carried out in sodium phosphate buffer at a pH of 5.0-9.0 for 1-48 h at 20-37℃.

[0016] Beneficial effects This invention identifies for the first time a non-heme chloride peroxidase from the whole genome of *Rauvolobacterium ornithine-derived* that can catalyze the oxidation of HMF and FFCA to generate HMFCA and FDCA, respectively. Ro CPO provides a novel enzyme resource for the biosynthesis of FDCA. This discovery enriches the enzyme library available for FDCA biosynthesis and also provides a reference for the enzymatic synthesis of other furan analogs. Attached Figure Description

[0017] Picture 1 This is a process route diagram for the biocatalytic synthesis of FDCA from HMF.

[0018] Picture 2 For pACYCDuet-1 plasmid and Ro Agarose gel electrophoresis image of the CPO gene fragment; In the image: (A) is the electrophoresis diagram of the pACYCDuet-1 plasmid, and (B) is... Ro Electrophoresis diagram of the CPO gene.

[0019] Picture 3 for Ro SDS-PAGE image of CPO purified enzyme; In the diagram: M stands for Marker, and lane 1 is empty. Ro CPO gene blank control, lane 2 is the supernatant after lysis, lane 3 is 10% elution buffer, lane 4 is... Ro CPO pure enzyme solution.

[0020] Picture 4 for Ro CPO catalyzed using HMF as a substrate.

[0021] Picture 5 for Ro CPO catalyzed using FFCA as a substrate. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental methods involved in the embodiments are carried out in accordance with the prior art; unless otherwise specified, the drugs and reagents involved in the embodiments are all commercially available products.

[0023] The culture media involved in the examples are as follows: LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and water as the remainder.

[0024] TB medium: tryptone 12 g / L, yeast extract 24 g / L, dipotassium hydrogen phosphate 12.54 g / L, potassium dihydrogen phosphate 2.31 g / L, glycerol 4 mL / L, balance water.

[0025] The protein purification solutions involved in the examples are as follows: Binding / washing buffer (pH 7.4): 7.75 mM NaH2PO4, 12.25 mM Na2HPO4, 0.5 M NaCl, 0.02 M imidazole. After preparation, filter twice through a 0.22 μm filter membrane.

[0026] Elution buffer (pH 7.4): 7.75 mM NaH2PO4, 12.25 mM Na2HPO4, 0.5 M NaCl, 0.5 M imidazole. After preparation, filter twice through a 0.22 μm filter membrane.

[0027] Sodium phosphate buffer (pH 7.4): 7.75 mM NaH2PO4, 12.25 mM Na2HPO4. After preparation, filter twice through a 0.22 μm filter membrane.

[0028] High-performance liquid chromatography (HPLC) methods for the determination of HMF, HMFCA, FFCA and FDCA: After the sample is diluted a certain factor, 12000× g Centrifuge for 10 min, filter through a 0.22 μm aqueous filter membrane, and then perform analysis. Detection conditions: Shimadzu LC-20A, VWD detector; detection wavelength 268 nm; Aminex HPX-87H column (300 × 7.8 mm); mobile phase: 10 mM H2SO4 solution; flow rate: 0.6 mL / min; column temperature: 60℃; injection volume: 20 μL.

[0029] Example 1 Ro Construction of CPO expression strains Plasmids containing pACYCDuet-1 (a commercially available product) were extracted from strains preserved in the laboratory. Using pACYCDuet-1 as a template and pACYCDuet-1-F and pACYCDuet-1-R as primers, a linear pACYCDuet-1 plasmid fragment was amplified by PCR. The agarose gel electrophoresis results are shown below. Picture 2 A. Using *Rauvolfia pulmonalis* (ornithine-releasing bacterium) preserved in the laboratory. Raoultella ornithinolytica Using the BF60 genome as a template, Ro CPO-F and Ro The target gene fragment was obtained by PCR amplification using CPO-R primers (containing a 20 bp homologous sequence). Ro CPO (nucleotide sequence SEQ ID NO.1, amino acid sequence SEQ ID NO.2), its agarose gel electrophoresis is shown in [image missing]. Picture 2 B. The above-mentioned target gene fragment Ro CPO was recombinantly ligated with a linear pACYCDuet-1 plasmid fragment using a one-step cloning kit (C112-02, Nanjing Novizan Biotechnology Co., Ltd.), and the ligation product was then transferred into... E. coliIn BL21(DE3), single colonies with normal morphology were selected and subjected to colony PCR amplification. Preliminary verification was then performed by gel electrophoresis. Bacterial solutions with band sizes consistent with theoretical values ​​were then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing verification, ultimately yielding the correctly constructed recombinant bacteria. E. coli / pACYCDuet-1- Ro CPO.

[0030] The primer sequences used above are as follows: pACYCDuet-1-F is shown in SEQ ID NO.3; pACYCDuet-1-R is shown in SEQ ID NO.4; Ro CPO-F is shown in SEQ ID NO. 5; Ro CPO-R is shown in SEQ ID NO.6.

[0031] The PCR reaction system consisted of 1 μL each of forward and reverse primers, 25 μL of 2×Phanta Max Master Mix, 1 μL of template, and double-distilled water to a final volume of 50 μL.

[0032] PCR conditions were as follows: pre-denaturation at 95℃ for 5 min; followed by 30 cycles (95℃ for 15 s, 55℃ for 15 s, 72℃ for 2 min), extension at 72℃ for 5 min; and finally storage at 4℃.

[0033] Example 2 Ro Cultivation and protein purification of CPO recombinant strains The recombinant bacteria successfully constructed in Example 1 E. coli / pACYCDuet-1- Ro CPO was streaked in a zigzag pattern on LB solid medium, then incubated upside down at 37°C for 12 h. Single colonies of the recombinant strain were picked and inoculated into liquid LB medium, and cultured at 37°C and 220 rpm for 12 h to obtain seed culture. The seed culture was then transferred to TB medium at a 1% (v / v) inoculation rate and cultured at 37°C and 220 rpm until OD reached. 600 When the concentration of the protein was 0.8, IPTG was added to induce protein expression. The final concentration of IPTG was 0.2 mM, and the mixture was cultured at 25°C for another 12 h.

[0034] After cultivation, bacterial cells were collected by centrifugation at 12,000 rpm for 5 min. The cells were washed twice with pH 7.4 phosphate buffer and resuspended. Cell disruption was then performed using an ultrasonic cell disruptor, followed by centrifugation at 12,000 rpm and 4°C for 10 min. The supernatant was filtered through a 0.22 μm filter to obtain crude enzyme solution. Protein purification of the crude enzyme solution was performed using a nickel column. First, the instrument tubing and nickel column were moistened with binding buffer. Then, the crude enzyme solution was loaded onto the column. The tubing and nickel column were then moistened again with binding buffer. Next, elution buffer was used to remove contaminating proteins, and finally, elution buffer was used to remove the target protein. The resulting purified enzyme solution was then desalted using a desalting column. First, the instrument tubing and desalting column were rinsed with sodium phosphate buffer. Then, the purified enzyme solution was loaded onto the column, and sodium phosphate buffer was used to elute the target protein, yielding a liquid purified enzyme solution.

[0035] Purified Ro CPO performs SDS-PAGE analysis, such as Picture 3 As shown.

[0036] Example 3 Ro CPO catalyzes the formation of HMFCA from HMF. To add Ro CPO pure enzyme solution was used as the experimental group, with no additives. Ro CPO pure enzyme solution served as a blank control group. The enzyme was added to a final concentration of 0.1 mg / mL, the catalytic temperature was 30℃, and the substrate HMF (purchased from Shanghai Rongli Chemical Technology Co., Ltd.) concentration was 5 mM. The total catalytic system volume was adjusted to 3 mL using sodium phosphate buffer. After 48 h of catalytic reaction, samples were taken for HPLC analysis. The HMFCA yield was determined to be 4.79 mM, with a molar conversion rate of 95.8% (e.g., ...). Picture 4 (As shown).

[0037] Example 4 Ro CPO catalyzes the conversion of FFCA to FDCA To add Ro CPO pure enzyme solution was used as the experimental group, with no additives. Ro CPO purified enzyme solution served as a blank control group. The enzyme was added to a final concentration of 0.1 mg / mL, the catalytic temperature was 30℃, and the substrate FFCA (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) concentration was 5 mM. The total catalytic system volume was adjusted to 3 mL using sodium phosphate buffer. After 48 h of catalytic reaction, samples were taken for HPLC analysis. The FDCA yield was determined to be 2.58 mM, with a molar conversion of 51.6% (e.g., ...). Picture 5 (As shown).

Claims

1. A non-heme chloride peroxidase Ro The use of CPO, a coding gene, a recombinant plasmid or a recombinant bacterium in the preparation of 5-hydroxymethyl-2-furan carboxylic acid and / or 2,5-furandicarboxylic acid; The nonheme chlorperoxidase Ro CPO, whose amino acid sequence is SEQ ID NO.2; The encoding gene encodes the nonheme chlorperoxidase. Ro CPO; The recombinant plasmid contains the encoding gene; The recombinant bacteria contains the recombinant plasmid.

2. The application as described in claim 1, characterized in that, The nucleotide sequence of the encoded gene is SEQ ID NO.

1.

3. The application as described in claim 1, characterized in that, The host bacteria of the recombinant bacteria are E. coli BL21(DE3) was expressed using the pACYCDuet-1 plasmid.

4. A non-heme chloride peroxidase Ro A method for preparing 5-hydroxymethyl-2-furanoic acid or 2,5-furandicarboxylic acid from CPO includes the following steps: Add to Ro CPO enzyme solution, substrate HMF or FFCA final concentration 1-20 mM. Ro The CPO enzyme concentration was 0.02-0.1 mg / mL, and the reaction was carried out in sodium phosphate buffer at pH 5.0-9.0 for 1-48 h at 20-37℃. The nonheme chlorperoxidase Ro CPO, whose amino acid sequence is SEQ ID NO.2.

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