An expression system suitable for short-chain non-specific peroxidase and its application

Abstract

The application discloses an expression system suitable for short-chain non-specific peroxidase and application thereof, and belongs to the technical field of enzyme engineering and genetic engineering. The expression system disclosed by the application connects the nucleotide sequence of short-chain non-specific peroxidase with a SUMO and 8HIS label to a pET28a vector to obtain a recombinant vector, and the recombinant vector is used to transform Escherichia coli BL21 (DE3) to obtain a genetically engineered bacterium; the genetically engineered bacterium is cultured in a ZYM5052 liquid culture medium by using a two-stage temperature control method, and 5ALA is added to the culture medium. The application uses an Escherichia coli self-induction system, can make shake flask culture grow to a high density in a short time, and the culture medium contains rich nutrients, and the cell density does not need to be monitored and IPTG does not need to be added during the expression process. In addition, a hemin precursor 5ALA is added to the Escherichia coli self-induction culture medium, and finally, short-chain non-specific peroxidase with high activity is obtained.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and genetic engineering, and more specifically to an expression system suitable for short-chain non-specific peroxidases and its applications. Background Technology

[0002] Unspecific peroxidases (UPOs) can catalyze a wide range of reactions across a broad substrate spectrum, including epoxidation, hydroxylation, heteroatom oxidation, and halogenation. However, unlike P450 enzymes, UPOs utilize hydrogen peroxide (H₂O₂) instead of NAD(P)H and molecular oxygen as oxygen donors and oxidants, directly catalyzing monooxygenation reactions of various organic substrates without the need for any cofactors. This characteristic makes UPOs highly promising biocatalysts, with their unique catalytic capabilities and simplified coenzyme requirements showing broad application prospects in green chemistry, biomedicine, and environmental remediation. However, the main limitation restricting the application of UPOs to date remains the difficulty of heterologous expression. Since the first UPO (AaeUPO) was discovered in the agaric bacterium *Agrocybe aegerita* in 2004, over 4000 putative UPO enzymes exist in databases, but only about 50 of these can be heterologously expressed.

[0003] Currently, all discovered prokaryotic polymorphic oxygen atoms (UPOs) originate from eukaryotes (mainly basidiomycetes and some ascomycetes). Based on their structure, they are classified as short-chain and long-chain. These UPOs rely on their respective hosts' complex expression systems and post-translational modifications, further limiting the heterologous expression of eukaryotic UPOs. Currently, most expression systems use Pichia pastoris, a typical secretory expression system with naturally occurring secretory signal peptides. However, this is not applicable to all UPOs, and while UPOs themselves possess secretory signal peptides, only a few can be used in Pichia pastoris systems. Compared to eukaryotic expression systems, prokaryotic expression systems offer higher intracellular expression capacity and shorter cycles. Self-induced systems not only provide abundant nutrients, allowing for high-density cell growth in a short time, but also eliminate the need for cell density monitoring and IPTG supplementation.

[0004] Therefore, providing an expression system suitable for short-chain non-specific peroxidases and its application is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides an expression system suitable for short-chain nonspecific peroxidases and its application. Four currently reported active short-chain nonspecific peroxidases are applied to this expression system, and their kinetic properties for 1,2-methylenedioxy-4-nitrobenzene (NBD) are studied.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] To address the current challenge of heterologous expression of short-chain UPO in *E. coli* or the resulting low expression levels, this invention utilizes an *E. coli* self-induction system with modified conditions to enable high-density bacterial growth in a short time, successfully increasing the expression level of short-chain UPO. This invention employs an *E. coli* self-induction system, which offers the advantages of enabling rapid high-density growth of shake-flask cultures, providing a nutrient-rich culture medium, and eliminating the need for cell density monitoring and IPTG addition during expression.

[0008] This invention is based on the heme cofactor, which is essential for the catalytic active center of UPO. It utilizes the pathway of Escherichia coli to metabolize glucose carbon source into heme and increases the amount of heme produced by adding trace amounts of 5-aminolevulinate (5ALA), thus successfully obtaining highly active UPO.

[0009] An expression system suitable for short-chain nonspecific peroxidases is provided. The nucleotide sequence of a short-chain nonspecific peroxidase tagged with SUMO and 8HIS is ligated into the pET28a vector. The resulting recombinant vector is transformed into *Escherichia coli* BL21(DE3) to obtain a genetically engineered bacterium. The genetically engineered bacterium is then cultured in ZYM5052 liquid medium using a two-stage temperature-controlled process, with 600 µmol / L of heme precursor 5ALA added to the medium.

[0010] The two-stage temperature-controlled culture is as follows: the first stage is cultured at 37℃ and 180 rpm on a shaker until the OD reaches 1.0-1.5; the second stage is cultured at a low temperature of 16℃ and 180 rpm on a shaker to induce protein expression.

[0011] Furthermore, the composition of the ZYM5052 liquid culture medium is as follows: ZY 50mL, 50×M 1mL, 50×5052 1mL, 1M MgSO4 0.1mL, 1000×Trace metals mixture 0.1mL;

[0012] ZY: 10g / l tryptone, 5g / l yeast extract;

[0013] 50×M: 446g / l Na2HPO4·12H2O, 170g / l KH2PO4, 133.76g / l NH4Cl, 35.4g / lNa2SO4;

[0014] 50×5052: 250g / l glycerol, 25g / l glucose, 100g / l α-lactose;

[0015] 1000×Trace metals mixture: 54mg / l FeCl3·6H2O, 6.4mg / l CaCl2·2H2O, 4mg / l MnCl2·4H2O, 6mg / l ZnSO4·7H2O, 1mg / l CoCl2·6H2O, 0.8mg / l CuCl2·2H2O, 1mg / lNiCl2·6H2O, 1mg / l Na2MoO4·2H2O, 0.8mg / l Na2SeO3, 0.4mg / l H3BO3.

[0016] Furthermore, the short-chain nonspecific peroxidase is TteUPO, CviUPO, DcaUPO, or MroUPO, and its amino acid sequence is shown in SEQ ID NO.1-4.

[0017] Furthermore, the amino acid sequence of the SUMO is shown in SEQ ID NO.5.

[0018] Furthermore, the application of the aforementioned expression system suitable for short-chain nonspecific peroxidases in high-density, high-activity expression of short-chain nonspecific peroxidases.

[0019] This expression system is a self-induced expression system; the heme precursor 5ALA is additionally added to the culture medium; the culture method employs a two-stage temperature control approach, first using high temperature to achieve dense bacterial growth, and then using low temperature for induction. This expression system is suitable for the expression of a class of short-chain non-specific peroxidases. It lays the foundation for validating a new source of short-chain UPOs and for further research on the catalytic performance of short-chain UPOs, establishing a research basis for the discovery and in-depth study of short-chain UPOs.

[0020] As can be seen from the above technical solution, compared with the prior art, this invention discloses an expression system suitable for short-chain non-specific peroxidases and its application. This system uses an *E. coli* self-induction system, with the culture medium being ZYM5052. Based on the importance of heme for the catalytic active site of UPO, the system utilizes glucose as a carbon source to metabolize heme via a pathway. By adding 600 µmol / L of the metabolic intermediate 5ALA, the final heme production was measured to be 14157.3 ng / ml. Finally, through a two-stage temperature-controlled culture, a high temperature of 37°C resulted in rapid and dense bacterial growth, followed by a low temperature induction at 16°C for 48 hours to obtain highly active soluble proteins. The proteins involve four reported short-chain UPOs. Using the pET-28a(+) *E. coli* protein expression plasmid as a vector, and incorporating the fusion tag small molecule ubiquitin-like modified protein SUMO, with *E. coli* BL21(DE3) as the host, the active expression of UPOs was successfully achieved. 4-Nitrocatechol was generated from 1,2-methylenedioxide-4-nitrobenzene (NBD) (absorbance A425). The Kcat / Km values ​​of TteUPO, CviUPO, DcaUPO, and MroUPO were 157.78 mM. -1 ·s -1 90.77mM -1 ·s -1 24.47mM -1 ·s -1 20.62mM -1 ·s -1 The expression levels were 28 mg / L, 15.6 mg / L, 13.4 mg / L, and 12.2 mg / L, respectively. This expression system lays a solid foundation for the subsequent screening of novel UPO enzymes and further research. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 This is the reaction formula with NBD as the substrate.

[0023] Figure 2 The plasmid map of pET28a-SUMO-TteUPO-8HIS.

[0024] Figure 3 The plasmid map of pET28a-SUMO-CviUPO-8HIS.

[0025] Figure 4 The plasmid map of pET28a-SUMO-DcaUPO-8HIS.

[0026] Figure 5 The plasmid map of pET28a-SUMO-MroUPO-8HIS.

[0027] Figure 6 DNA agarose gel plot; where M: 10000bp marker; Dca: DcaUPO; Tte: TteUPO; Cvi: CviUPO; Mro: MroUPO.

[0028] Figure 7 This is a diagram of protein purification.

[0029] Figure 8 The standard curve for 4-nitrocatechol.

[0030] Figure 9 The standard concentration curves for heme ranged from 0.5 to 50 ng / ml.

[0031] Figure 10 This is a liquid chromatography-mass spectrum of heme. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Currently, more than 4,000 non-specific peroxidase sequences from fungi have been discovered, but only about 50 UPOs have been successfully expressed heterologously. Among them, there is relatively little research on the modification of their catalytic and enzymatic functions. Regarding the heterologous expression of UPOs, the current focus is mainly on the use of eukaryotic expression systems, such as Saccharomyces cerevisiae and Pichia pastoris. UPOs in eukaryotic systems are mainly expressed through secretion. However, eukaryotic expression systems still have the following problems for the heterologous expression of UPOs: (1) The universality of the system needs to be considered. Further screening of secretory signal peptides is required to obtain active UPOs. Moreover, the expression level of UPOs that have been successfully expressed in Pichia pastoris is not high, all below 20 mg / L. (2) The culture time is long, the culture cost is high, and the stability is poor, which is not conducive to subsequent high-throughput protein screening. Based on the above problems, it is very important to develop a simple and easy-to-operate heterologous expression system that is applicable to most UPOs.

[0034] Therefore, the first aspect of this invention provides an Escherichia coli self-induced expression system capable of heterologous expression of short-chain nonspecific peroxidase (UPO) activity. Simultaneously, 600 µmol / L of the heme precursor 5ALA is additionally added to the Escherichia coli self-induced culture medium. The culture medium ZYM5052 used for the expression system is shown in Table 1.

[0035] Table 1. Culture medium ZYM5052

[0036]

[0037] In some embodiments of the present invention, the Escherichia coli self-induced expression system involves three carbon sources: glucose is responsible for the large-scale growth of bacteria in the early stage; α-lactose is used to induce the expression of the short chain UPO of the target protein in the later stage; and glycerol is an auxiliary carbon source, which, through the synergistic effect of "energy-osmotic pressure-metabolism" in the expression system, becomes the key link "connecting bacterial growth and protein induction" in the Escherichia coli self-induced expression system.

[0038] In some embodiments of the present invention, a heme porphyrin ring with cysteine ​​ligand coordination is involved in the active center of a short-chain UPO ring, in which ferric ions play an important role, and the main substance of trace metal ions is ferric ions.

[0039] In a second aspect, the present invention utilizes the Escherichia coli metabolic pathway, in which glucose is used as a carbon source to generate heme through the Escherichia coli metabolic system. The metabolic process involves the intermediate product 5ALA, which is then used to generate heme. The active center of UPO is composed not only of ferric ions, but more importantly, of the role of heme. The present invention adds an appropriate amount of 5ALA to the expression system to increase the generation of heme.

[0040] In some embodiments of the present invention, the amount of 5ALA added is 200-1000 µmol / L, preferably 600 µmol / L. The amount of heme generated is 14157.3 ng / ml.

[0041] The third aspect of this invention relates to currently reported short-chain UPO enzymes, namely, the UPO of Thievia terrestris (TteUPO), the UPO of Collariella virescens (CviUPO), the UPO of Daldinia caldariorum (DcaUPO), and the UPO of Marasmius rotula (MroUPO); their amino acid sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. Further, a recombinant plasmid is provided, containing the amino acid sequences of four target proteins, a lysis-promoting tag SUMO (amino acid sequence shown in SEQ ID NO.5), and an eight-histidine selection tag added to the C-terminus.

[0042] In some embodiments of the present invention, four nucleotide sequences (as shown in SEQ ID NO. 6-9) were synthesized by Beijing BGI Genomics. Using these synthesized nucleotide sequences as templates, primers were designed to amplify the genes SUMO-TteUPO-8HIS, SUMO-CviUPO-8HIS, SUMO-DcaUPO-8HIS, and SUMO-MroUPO-8HIS. The target genes were then constructed into the pET28a vector by enzyme digestion and ligation to obtain recombinant plasmids pET28a-SUMO-TteUPO-8HIS, pET28a-SUMO-CviUPO-8HIS, pET28a-SUMO-DcaUPO-8HIS, and pET28a-SUMO-MroUPO-8HIS.

[0043] In some embodiments of the present invention, the genetically engineered bacteria are recombinant Escherichia coli strains using pET28a-SUMO-TteUPO-8HIS, pET28a-SUMO-CviUPO-8HIS, pET28a-SUMO-DcaUPO-8HIS, and pET28a-SUMO-MroUPO-8HIS protein expression plasmids as vectors and Escherichia coli BL21(DE3) (Beijing TransGen Biotech Co., Ltd.) as the host bacterium, denoted as BL21(DE3) / pET28a-SUMO-TteUPO-8HIS, BL21(DE3) / pET28a-SUMO-CviUPO-8HIS, BL21(DE3) / pET28a-SUMO-DcaUPO-8HIS, and BL21(DE3) / pET28a-SUMO-MroUPO-8HIS.

[0044] In a fourth aspect, the above-mentioned genetically engineered bacteria are heterologously expressed in Escherichia coli. The expression process employs a two-stage temperature-controlled culture to achieve heterologous expression of UPO in Escherichia coli.

[0045] In some embodiments of the present invention, the first stage is culture at 37°C and 180 rpm, which is conducive to rapid bacterial growth. After the bacterial count OD reaches 1.2-1.5, the second stage of low-temperature induction culture is started; the temperature is controlled between 16-25°C (preferably 16°C), and expression is induced for 24-72 hours (preferably 48 hours) before expression is stopped. The bacterial cells are collected and purified to obtain pure UPO enzyme. 4-Nitrocatechol (absorbance A425) is generated using 1,2-methylenedioxy-4-nitrobenzene as a substrate. Figure 1 ), to conduct enzyme kinetics studies on UPO enzyme catalysis in vitro.

[0046] The amino acid sequence of TteUPO:

[0047] AGFDSWHPPAPGDRRGPCPMLNTLANHGFLPHNGRNITKEITVNALNSALNVNKTLGELLFNFAVTTNPQPNATFFDLDHLSRHNILEHDASLSRADYYFGHDDHTFNQTVFDQTKSYWKTPII SEQ ID NO.1.

[0048] The amino acid sequence of CviUPO:

[0049] MELDFSKWKTRQPGEFRAPCPAMNSLANHGFIPRDGRNITVAMLVPVLQEVFHLSPELAQTISTLGLFTAQDPSKGVFTLDDLNRHNLFEHDASLSREDYYFHKDASTFRPEVFKKFMSHFKGKEYVTLEDA ASARYAMVQESRKKNPTFTYTVQQRITSYGETIKYFRTIVEPATGKCPVAWIKILFEQERLPYNEGWRPPKAELSGFSMASDVLELALVTPEKLIDKPCEGKQCPQARGIHGYFGMLLPITAQELAVK; SEQ ID NO.2.

[0050] The amino acid sequence of DcaUPO:

[0051] MGSSHHHHHHSSGLVPRGSHMAPWKAPGPDDVRGPCPMLNTLANHGFLPHDGKNIDVNTTVNALSSALNLDDELSRDLHTFAVTTNPQPNATWFSLNHLSRHNVLEHDASLSRQDAYFGPPDVFNAAVFNETKAYWTGDIINFQMAANALTARLMTSNLTNPEFSMSQLGRGFGLGETVAYVTILGSKETRTVPKAFVEYLFENERLPYELGFKKMKSALTEDELTTMMGEIYSLQHLPESFTKPFAKRSEAPFEKRAEKRCPFH; SEQ ID NO.3.

[0052] Amino acid sequence of MroUPO:

[0053] ASAHPWKAPGPNDSRGPCPGLNTLANHGFLPRNGRNISVPMIVKAGFEGYNVQSDILILAGKIGMLTSREADTISLEDLKLHGTIEHDASLSREDVAIGDNLHFNEAIFTTLANSNPGADVYNISSAAQVQHDRLADSLARNPNVTNTDLTATIRSSESAFFLTVMSAGDPLRGEAPKKFVNVFFREERMPIKEGWKRSTTPITIPLLGPIIERITELSDWKPTGDNCGAIVLSPELS; SEQ ID NO.4.

[0054] Amino acid sequence of small ubiquitin-like modifier (SUMO):

[0055] MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG; SEQ ID NO.5.

[0056] Nucleotide sequence of SUMO-TteUPO-8HIS:

[0057] atgtcggactcagaagtcaatcaagaagct accaccaccac cac ;SEQ ID NO.6.

[0058] In SEQ ID NO.6, 1-294bp is the nucleotide sequence of SUMO; 295-1026bp is the nucleotide sequence of TteUPO; 1027-1035bp is the linker connecting the target gene and the 8HIS purification tag (the core function of the linker is to ensure that the tag functions normally and does not interfere with the target protein); and 1036-1059bp is the nucleotide sequence of 8HIS.

[0059] Nucleotide sequence of SUMO-CviUPO-8HIS:

[0060] atgtcggactcagaagtcaatcaagaagct accaccaccac ;SEQ ID NO.7.

[0061] In SEQ ID NO.7, 1-294bp is the nucleotide sequence of SUMO; 295-1074bp is the nucleotide sequence of CviUPO; 1075-1083bp is the linker connecting the target gene and the 8HIS purification tag (the core function of the linker is to ensure that the tag functions normally and does not interfere with the target protein); and 1084-1107bp is the nucleotide sequence of 8HIS.

[0062] Nucleotide sequence of SUMO-DcaUPO-8HIS:

[0063] atgtcggactcagaagtcaatcaagaagct ACCACCACCACCACCAC ; SEQ ID NO.8.

[0064] In SEQ ID NO.8, 1-294bp is the nucleotide sequence of SUMO; 295-1089bp is the nucleotide sequence of DcaUPO; 1090-1098bp is the linker connecting the target gene and the 8HIS purification tag (the core function of the linker is to ensure that the tag functions normally and does not interfere with the target protein); and 1099-1122bp is the nucleotide sequence of 8HIS.

[0065] Nucleotide sequence of SUMO-MroUPO-8HIS:

[0066] atgtcggactcagaagtcaatcaagaagctaagccagaggtcaagccagaagtcaagcctgagactcacatcaatttaaaggtgtccgatggatcttcagagatcttcttcaagatcaaaaagaccactcctttaagaaggctgatggaagcgttcgctaaaagacagggtaaggaaatggactccttaagattcttgtacgacggtattagaattcaagctgatcagacccctgaagatttggacatggaggataacgatattattgaggctcacagagaacagattggtggaGCTTCTGCTCACCCATGGAAGGCTCCAGGTCCAAACGATTCTAGAGGTCCATGTCCAGGTTTGAACACTTTGGCTAACCACGGTTTCTTGCCAAGAAACGGTAGAAACATTTCTGTTCCAATGATTGTTAAGGCTGGTTTCGAGGGTTACAACGTTCAATCTGACATTTTGATCTTGGCTGGAAAGATTGGTATGTTGACTTCTAGAGAGGCTGACACTATTTCTTTGGAGGATTTGAAGTTGCACGGTACTATTGAGCACGACGCTTCTTTGTCTAGAGAGGACGTTGCTATTGGTGACAACTTGCACTTCAACGAGGCTATTTTCACTACTTTGGCTAACTCTAACCCAGGTGCTGACGTTTACAACATTTCTTCTGCTGCTCAAGTTCAACACGACAGATTGGCTGATTCCTTGGCTAGAAACCCAAACGTTACTAACACTGACTTGACTGCTACTATTAGATCTTCTGAGTCTGCTTTCTTCTTGACTGTTATGTCTGCTGGTGACCCATTGAGAGGTGAGGCTCCAAAGAAGTTCGTTAACGTTTTCTTCAGAGAGGAGAGAATGCCAATTAAGGAGGGTTGGAAGAGATCTACTACCCCAATTACTATTCCATTGTTGGGTCCAATTATTGAACGCATAACTGAGTTGAGTGACTGGAAACCAACCGGGGATAACTGCGGGGCGATAGTGCTTAGCCCTGAATTGAGTactcgagcacaccacc accaccaccaccac ;SEQ ID NO.9.

[0067] In SEQ ID NO.9, 1-294bp is the nucleotide sequence of SUMO; 295-1008bp is the nucleotide sequence of MroUPO; 1009-1017bp is the linker connecting the target gene and the 8HIS purification tag (the core function of the linker is to ensure that the tag functions normally and does not interfere with the target protein); 1018-1041bp is the nucleotide sequence of 8HIS.

[0068] Example 1

[0069] Construction of protein expression vectors pET28a-SUMO-TteUPO-8HIS, pET28a-SUMO-CviUPO-8HIS, pET28a-SUMO-DcaUPO-8HIS, and pET28a-SUMO-MroUPO-8HIS.

[0070] Using the gene sequences SEQ ID NO. 6-9 of SUMO-TteUPO-8HIS, SUMO-CviUPO-8HIS, SUMO-DcaUPO-8HIS, and SUMO-MroUPO-8HIS as templates, the target genes were amplified by PCR using primers 28a-SUMO-UPO-8HIS-F / R. The primer sequences are as follows:

[0071] 28a-SUMO-UPO-8HIS-F:gagatatacc atgtcggactcagaagtcaatcaagaagct ; SEQ ID NO.10.

[0072] 28a-SUMO-UPO-8HIS-R: accaccaccaccac tgagatccgg; SEQ ID NO.11.

[0073] Using pET28a plasmid as a template, the linear vector Vector-28a was amplified by PCR using primers 28a-F / R. The primer sequences are as follows:

[0074] 28a-F: ccaccaccac tgagatccggctgctaaca ; SEQ ID NO.12.

[0075] 28a-R: ataattttgtttaactttaagaaggagatatacc atgtcggact; SEQ ID NO.13.

[0076] The total PCR amplification reaction volume was 50 μL, with 25 μL of 2×PrimerStar Mix, 20 μL of ddH2O, 2 μL of upstream primer, 2 μL of downstream primer, and 1 μL of template added. The reaction conditions were: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, and 72℃ extension for 2 min, for a total of 35 cycles; a final extension at 72℃ for 10 min; and finally, storage at 4℃. After the PCR reaction, the amplified fragment was verified by agarose gel electrophoresis and then recovered from the gel. The recovered SUMO-UPO-8HIS fragment was ligated to the vector fragment Vector-28a using the Gibson homologous recombination assembly method. The ligation system consisted of 1 µL Vector-28a, 4 µL SUMO-UPO-8HIS, and 5 µL 2×Basic Seamless ligase, incubated at 45℃ for 15 min.

[0077] The ligation product was introduced into Trans10 competent cells (Beijing TransGen Biotech Co., Ltd.) and transformed using the E. coli chemical transformation method, as follows:

[0078] (1) Take Trans 10 competent cells and place them on ice to melt. Add all 10 μL of the above ligation product and incubate on ice for 30 min.

[0079] (2) Heat shock at 42℃ for 30 seconds, followed immediately by ice bath for 2 minutes.

[0080] (3) Add 500 μL of antibiotic-free LB medium that has been pre-chilled and thawed at 37°C and 180 rpm for 1 h.

[0081] (4) After the resuscitation is completed, centrifuge at 6000 rpm for 2 min, remove the supernatant, keep 100 μL, resuspend the bacterial cells, and spread them all on a plate containing Kana resistance (100 µg / ml).

[0082] (5) Incubate overnight at 37℃ for approximately 12 hours. Randomly select 10 uniform single colonies from the cultured plates as the original plasmids for colony PCR. After PCR, the band sizes of the products are checked by 1% agarose gel electrophoresis. After extraction of the recombinant plasmids, first-generation sequencing confirms correct sequencing and suitability for subsequent protein expression. The plasmid maps for pET28a-SUMO-TteUPO-8HIS, pET28a-SUMO-CviUPO-8HIS, pET28a-SUMO-DcaUPO-8HIS, and pET28a-SUMO-MroUPO-8HIS are shown below. Figure 2-5 .

[0083] Example 2 Protein Expression and Purification

[0084] 1) Protein expression

[0085] (1) The recombinant expression vectors pET28a-SUMO-TteUPO-8HIS, pET28a-SUMO-CviUPO-8HIS, pET28a-SUMO-DcaUPO-8HIS, and pET28a-SUMO-MroUPO-8HIS constructed in Example 1 were transformed into *Escherichia coli* BL21(DE3). Positive clones were selected using kanamycin-resistant plates (Kana, 100 µg / ml), and the bacteria were cultured overnight at 37°C to obtain recombinant bacteria. Colony PCR was performed after obtaining the recombinant strains. Universal primers were used for pET28a sequencing.

[0086] 5' Sequencing primers and sequence: T7: 5'-TAATACGACTCACTATAGGG-3'; SEQ ID NO.14.

[0087] 3' Sequencing primers and sequence: T7t: 5'-GCTAGTTATTGCTCAGCGG-3'; SEQ ID NO.15.

[0088] Verification was performed using agarose gel electrophoresis, and the results are shown below. Figure 6 The presence of target bands at 732bp, 780bp, 795bp, and 714bp indicates that the recombinant plasmid was successfully introduced and can be used for subsequent protein expression.

[0089] (2) Inoculate the single clones on the plate into 5 mL of LB liquid medium (Kana, 50 µg / mL) and culture in a shaker at 37 °C and 180 r / min for 12 h to obtain seed liquid.

[0090] (3) The seed culture obtained in step (2) was inoculated into 100 mL of ZYM5052 liquid medium with a Kana resistance concentration of 50 µg / mL at a ratio of 1:50. At the same time, 600 µmol / L of 5ALA was added to the medium. The first stage was cultured at 37℃ and shaken at 180 rpm until the OD reached 1.2. The second stage was cultured at a low temperature of 16℃ and shaken at 180 rpm for 48 h to induce protein expression.

[0091] (4) After the induction of expression was completed, the bacterial culture expressed in step (3) was centrifuged at 4500 rpm for 20 min at 4℃. After centrifugation, the supernatant was discarded, the bacterial cells were collected, and the bacterial count was determined. The bacterial cells were resuspended in 100 ml of pre-cooled cell lysis buffer (50 mM phosphate buffer, 300 mM NaCl, 5 mM β-ME, pH=7.5) to obtain a resuspension with an OD of 8.0. Lysozyme (final concentration 0.2 mg / mL) and protease inhibitor (PMSF) (final concentration 0.17 mg / mL) were added respectively. Lysozyme and protease inhibitor were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0092] (5) The cell suspension obtained in step (4) was disrupted (amplifier power 40%) for 40 min, and centrifuged at 4℃ and 12000 rpm. The supernatant obtained was 100 ml of crude enzyme solution required for the experiment (containing a large amount of target protein and other miscellaneous proteins).

[0093] 2) Purification of four UPO proteins

[0094] (1) Chromatography column treatment: 5 mL of nickel column packing material was rinsed with 20 mL of deionized water, and then rinsed with buffer (50 mM phosphate buffer, 150 mM sodium chloride, pH=7.5) for 20 mL.

[0095] (2) Filter the 100ml crude enzyme solution obtained above through a 0.22μm filter membrane to remove impurities. Add the treated crude enzyme solution into a nickel column and mix it thoroughly. Discard the effluent.

[0096] (3) After the sample loading is completed, rinse the nickel column with 15 mL of buffer solution to remove unsuccessfully loaded proteins and impurities, and discard the eluent.

[0097] (4) Add 20 ml of buffer containing 60 mM imidazole to the column to remove impurities and discard the eluent.

[0098] (6) Add 20 mL of buffer containing 200 mM imidazole to the column, elute the target protein, and collect the effluent.

[0099] (7) Add 20 mL of buffer containing 500 mM imidazole to the column to clean the nickel column by washing away the residual proteins.

[0100] (8) Add 20 mL of buffer solution and deionized water to the nickel column in sequence to clean the nickel column; finally add 15 mL of 20% ethanol to preserve the nickel column.

[0101] (9) Place the protein collected in step (6) into a 30kDa ultrafiltration tube and centrifuge at 4500rpm until 500μL of liquid remains in the tube. Add 15mL of cell lysis buffer to dilute the imidazole concentration in the protein. Continue centrifuging until about 500μL of protein remains in the tube.

[0102] (10) Take 10 μL of sample for SDS-PAGE gel electrophoresis verification.

[0103] The correct expression of the target proteins TteUPO, CviUPO, DcaUPO, and MroUPO was verified by polyacrylamide (SDS) gel electrophoresis.

[0104] According to SDS-PAGE validation and comparison with the marker, distinct specific bands were found at 41.6 KD, 44.0 KD, 44.8 KD, and 41.0 KD. Figure 7 This demonstrates the successful expression of the fusion protein SUMO-UPO. Protein concentrations were determined using a NanoDrop spectrophotometer, with a small sample size (typically 1-2 µL). The results were: TteUPO, CviUPO, DcaUPO, and MroUPO: 28 mg / L, 15.6 mg / L, 13.4 mg / L, and 12.2 mg / L, respectively.

[0105] The UPO protein was finally concentrated to 1.5 mg / mL, flash-frozen in liquid nitrogen, and stored at -80°C.

[0106] Example 3: Determination of UPO enzyme activity and bacterial count (OD)

[0107] UPO enzyme activity assay:

[0108] The maximum characteristic absorption wavelength of the product 4-nitrocatechol is 425 nm. Standard concentration curves were established using the absorbance at 425 nm for different concentrations, and the corresponding calculation formula was obtained to calculate the product concentration. 30 µl of 4-nitrocatechol at different concentrations (0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mmol / L), 1 µl of H₂O₂ dilution solution (30% H₂O₂ diluted tenfold), 30 µl of H₂O, and 139 µl of PBS (pH=7.0) were mixed, with a total volume of 200 µl. The resulting standard curve is shown below. Figure 8 As shown, the calculation formula is y = 0.8788x + 0.0973, R 2 =0.9815, where the y-axis represents absorbance (A425) and the x-axis represents product concentration.

[0109] After establishing the standard concentration curve, an enzyme-catalyzed reaction using NBD as a substrate was performed. The reaction system was as follows: 30 µg of purified protein was mixed with 30 µl of NBD solution (NBD concentration 6.6 mmol / L, dissolved in acetonitrile) in a 2 mL centrifuge tube. 1 µl of H2O2 dilution buffer (30% H2O2 diluted 10 times) was added, and the volume was brought up to 200 µl with cell lysis buffer. The 2 mL centrifuge tube was placed in a 25 °C metal bath and reacted at 200 rpm for 5 min. After centrifugation at 12000 rpm for 2 min, the upper layer was transferred to a 96-well plate and detected using a microplate reader at an absorbance of 425 nm. The product concentration was calculated using the formula above. The enzyme activity (mmol*min) was further calculated based on the reaction time (product concentration mM / reaction time min). -1 The enzyme activity of the four enzymes is calculated based on the reaction results: TteUPO-2070 (U / L), CviUPO-618 (U / L), DcaUPO-1050 (U / L), and MroUPO-307 (U / L).

[0110] Determination of bacterial count (OD): After the bacterial culture was completed, shake well, take 100µl and put it into a cuvette, then add deionized water to make up to 1ml, diluting it tenfold. Prepare a blank control at the same time. Measure OD600 using a spectrophotometer. The OD was 8 for all four UPO enzymes after the culture was completed.

[0111] Example 5 Heme Detection

[0112] Simultaneously, the target protein was sequenced and transferred to BL21(DE3), and the same procedure was performed on PET28a as a blank control to determine the amount of heme produced with and without 5ALA. 10 ml of the cultured bacterial solution was centrifuged. After centrifugation, the bacterial cells were resuspended in 10 ml of 1 M sodium hydroxide solution and sonicated for 10 min (35% power, 2 s on, 2 s off). The cells were then centrifuged at 14,000 rpm for 20 min, and the supernatant was filtered through a membrane for analysis of intracellular heme content. Intracellular heme was quantified using an Agilent 1260 high-performance liquid chromatograph. The chromatographic column was an XDB-C18 (5 µm, 4.6 × 250 mm, Agilent, USA) reversed-phase column; the mobile phases were: Phase A - ultrapure water containing 0.1% trifluoroacetic acid, and Phase B - methanol containing 0.1% trifluoroacetic acid. The column temperature was 40℃, the flow rate was 0.8 mL / min, and the detection wavelength was 400 nm. The elution program was gradient elution, as detailed in Table 2.

[0113] Table 2

[0114]

[0115] Figure 9 The standard concentration curves for heme ranged from 0.5 to 50 ng / ml. Figure 10 This is a liquid chromatography-mass spectrum of heme.

[0116] After the expression system without 5ALA was cultured, the amount of heme produced was 4737.3 ng / ml, while the amount of heme produced after the expression system with 5ALA was 14157.3 ng / ml. The addition of 5ALA resulted in the production of more heme, thereby enhancing the supply of intracellular heme cofactors and thus obtaining highly active UPO.

[0117] Example 5: Determination of in vitro catalytic kinetic parameters

[0118] Using the method for determining enzyme activity described in Example 3 above, the maximum reaction rate V of four UPO enzymes was first determined. max Measurements were performed: A425 was measured every 2 minutes during the reaction, and Vmax was calculated. K was calculated based on the amount of enzyme added. cat .

[0119] Next, 30 µl of NBD in a concentration gradient of 0.1–6.6 mM was selected as the substrate, and 30 µg of UPO enzyme was added. The reaction was carried out at 25 °C for 5 min, and then 20 µl of 30% HCl was added to terminate the reaction. After centrifugation, the supernatant was transferred to a 96-well plate, and the absorbance was measured using a microplate reader. The absorbance was determined according to the standard concentration curve (…). Figure 8 The product concentration was calculated. The KM of each enzyme was obtained by fitting the KM curve using grphpad, and the results are shown in Table 3.

[0120] Table 3 Enzyme kinetic parameters Km and Kcat

[0121]

[0122] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for expressing short-chain nonspecific peroxidases, characterized in that, The nucleotide sequence of a short-chain nonspecific peroxidase with SUMO and 8HIS tags was ligated into the pET28a vector, and the resulting recombinant vector was transformed into Escherichia coli BL21(DE3) to obtain a genetically engineered bacterium. The genetically engineered bacterium was cultured in ZYM5052 liquid medium in a two-stage temperature-controlled manner, while 600 µmol / L of the heme precursor 5-aminolevulinate was added to the medium. The two-stage temperature-controlled culture is as follows: the first stage is cultured at 37℃ and 180 rpm on a shaker until the OD reaches 1.0-1.5; the second stage is cultured at a low temperature of 16℃ and 180 rpm on a shaker to induce protein expression. The short-chain nonspecific peroxidase is Tte UPO, Cvi UPO, Dca UPO or Mro UPO, whose amino acid sequences are shown in SEQ ID NO.1-4; The amino acid sequence of the SUMO is shown in SEQ ID NO.

5.

2. The expression method for short-chain nonspecific peroxidases according to claim 1, characterized in that, The composition of the ZYM5052 liquid culture medium is as follows: ZY 50mL, 50×M 1mL, 50×5052 1mL, 1MMgSO4 0.1mL, 1000×Trace metals mixture 0.1mL; ZY: 10g / L peptone, 5g / L yeast extract; 50×M: 446g / L Na2HPO4·12H2O, 170g / L KH2PO4, 133.76g / L NH4Cl, 35.4g / L Na2SO4; 50×5052: 250 g / L glycerol, 25 g / L glucose, 100 g / L α-lactose; 1000×Trace metals mixture: 54mg / L FeCl3·6H2O, 6.4mg / L CaCl2·2H2O, 4mg / LMnCl2·4H2O, 6mg / L ZnSO4·7H2O, 1mg / L CoCl2·6H2O, 0.8mg / L CuCl2·2H2O, 1mg / LNiCl2·6H2O, 1mg / L Na2MoO4·2H2O, 0.8mg / L Na2SeO3, 0.4mg / L H3BO3.

3. The application of the expression method for short-chain nonspecific peroxidase as described in claim 1 in the expression of short-chain nonspecific peroxidase.

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