Artificial metalloenzyme based on azurin scaffold as well as chemical synthesis method and application of artificial metalloenzyme

Abstract

The invention discloses an artificial metalloenzyme based on an azurin scaffold and a chemical synthesis method and application thereof. A mutant azurin is used as an artificial metalloenzyme protein skeleton, a BPG-NHS module is obtained through organic synthesis, then the BPG-NHS is coupled with Cys-Azurin obtained through biological expression, and the novel artificial metalloenzyme with a tridentate N ligand structure at the N terminal is obtained. The metalloenzyme has certain lysing polysaccharide monooxygenase activity. According to the invention, the limitation of introducing specific non-natural amino acids by a biological recombinant expression technology is broken through, and the modification range and channel of the artificial metalloenzyme are widened.

Description

Technical Field

[0001] The present invention relates to the fields of protein chemical synthesis and biotechnology, and in particular to an artificial metalloenzyme based on an azurin scaffold, and a chemical synthesis method and application thereof. Background Art

[0002] Polysaccharides, as the main components of biomass such as starch, cellulose, and chitin, are an important source of carbohydrates and can be used to produce various value-added products such as biofuels, organic acids, aromatic compounds, polyphenols, and sugar alcohols. [1,2] Due to the well-organized lattice in polysaccharides, traditional glycoside hydrolases (GH) are unable to effectively break them down. Enzyme mixtures can effectively break down biomass, but these enzyme mixture products are often very expensive and require long processing times. [3] .

[0003] The discovery of lytic polysaccharide monooxygenases (LPMOs) provides new possibilities for the efficient utilization of polysaccharides. They use unique and powerful oxidative chemistry to cleave the β1-4 glycosidic bonds in the crystalline regions of polysaccharides. [4] The unique ability of LPMO to break down polysaccharides and other complex carbohydrates allows researchers to explore new applications for enzymes to create more efficient bioenergy and high-value-added products. [5,6] However, the relatively low expression yield of natural LPMO enzymes and the difficult purification process have restricted the research and related application progress of LPMOs.

[0004] With the in-depth study of the structure of enzyme active centers, the use of chemical methods to synthesize biomimetic model compounds of enzyme active centers has become a new research hotspot. Artificial metalloenzymes are biological macromolecules that mimic the catalytic function of biological enzymes. They are prepared by introducing suitable transition metal complexes as metal cofactors into the protein backbone.

[0005] Azurin is derived from Pseudomonas aeruginosa and is a type I copper protein composed of 128 amino acids that is involved in electron transport. [7,8] Due to its small size (~14kDa) and stable structure, azurin has been developed and modified as a protein scaffold for artificial metalloenzymes. [9,10] .

[0006] Here, a novel artificial metalloenzyme was constructed using a mutant azurin as an artificial metalloenzyme scaffold and introducing a 2,2'-dipicolylamine (DPA) moiety at its N-terminus to mimic the active center of the LPMO enzyme. This artificial metalloenzyme exhibits limited LPMO activity and can catalyze the cleavage of 4-nitrophenyl-β-D-pyranoside (PNPG).

[0007] References:

[0008] [1] Singhvi M, Kim B S. Current Developments in Lignocellulosic Biomass Conversion into Biofuels Using Nanobiotechology Approach[J / OL]. 2020, 13(20): 10.3390 / en13205300

[0009] [2] Hoell I A, Gustav V-K, And Eijsink V G H. Structure and function of enzymes acting on chitin and chitosan[J]. Biotechnology and Genetic Engineering Reviews, 2010, 27(1): 331 - 366.

[0010] [3] Vermaas J V, Crowley M F, Beckham G T, et al. Effects of Lytic Polysaccharide Monooxygenase Oxidation on Cellulose Structure and Binding of Oxidized Cellulose Oligomers to Cellulases[J]. The Journal of Physical Chemistry B, 2015, 119(20): 6129 - 6143.

[0011] [4] Westereng B, Arntzen M H, et al. Analyzing Activities of Lytic Polysaccharide Monooxygenases by Liquid Chromatography and Mass Spectrometry[M] / / ABBOTT D W, ZANDBERG W F. Carbohydrate-Protein Interactions: Methods and Protocols. New York, NY; Springer US. 2023: 27 - 51.

[0012] [5]Beeson W T,Vu V V,Span E A,et al.Cellulose Degradation byPolysaccharide Monooxygenases[J].2015,84(Volume 84,2015):923-946.

[0013] [6]Sabbadin F,Urresti S,Henrissat B,et al.Secreted pectinmonooxygenases drive plant infection by pathogenic oomycetes[J].Science,2021,373(6556):774-779.

[0014] [7]Garner D K,Vaughan M D,Hwang H J,et al.Reduction Potential Tuningof the Blue Copper Center in Pseudomonas aeruginosa Azurin by the AxialMethionine as Probed by Unnatural Amino Acids[J].Journal of the AmericanChemical Society,2006,128(49):15608-15617.

[0015] [8]Wilson T D,Yu Y,Lu Y.Understanding copper-thiolate containingelectron transfer centers by in corporation of unnatural amino acids and theCuA center into the type 1copper protein azurin[J].Coordination ChemistryReviews,2013,257(1):260-276.

[0016] [9]Liu Y, Harnden KA, Van Stappen C, et al.A designed Copper Histidine-brace enzyme for oxidative depolymerization of polysaccharides as a model oflytic polysaccharide monooxygenase[J]. Proceedings of the National Academy of Sciences, 2023,120(43):e2308286120.

[0017]

[10] Luo J, He C. Chemical protein synthesis enabled engineering of saccharide oxidative cleavage activity in artificial metalloenzymes[J]. International Journal of Biological Macromolecules, 2024, 256: 128083. Summary of the Invention

[0018] In order to overcome some problems existing in the prior art, one of the objectives of the present invention is to provide an azurin scaffold (mutant azurin).

[0019] A second object of the present invention is to provide an artificial metalloenzyme based on the above-mentioned azurin scaffold.

[0020] A third object of the present invention is to provide a method for preparing the above-mentioned artificial metalloenzyme.

[0021] A fourth object of the present invention is to provide applications of the artificial metalloenzyme.

[0022] In order to achieve the above object, the technical solution adopted by the present invention is:

[0023] An azurin scaffold having the amino acid sequence shown below:

[0024] BPG-CAECSVDIQGNDQMQFNTNAITVDKSCKQFTVNLSHPGNLPKNVMGHNWVLSTAADMQGVVTDGMASGLDKDYLKPDDSRVIAHTKLIGSGEKDSVTFDVSKLKEGEQYMFFCTFPGHSALMKGTLTLK; wherein BPG represents N,N-bis(pyridin-2-ylmethyl)glycine, which is linked to the N-terminal amino group of the protein fragment through an amide bond; the amino acid sequence of the protein fragment is the wild-type azurin sequence with a cysteine residue (Cys-Azurin) extended from the N-terminus.

[0025] An artificial metalloenzyme based on an azurin scaffold comprises the azurin scaffold and a Cu (II) active center.

[0026] The method for preparing the artificial metalloenzyme is to add CuSO4 to the ammonium acetate solution of the azurin scaffold, incubate and purify to obtain the artificial metalloenzyme.

[0027] Preferably, the composition of the ammonium acetate solution is: 50±10mmol / L NH4OAc, pH 5.0±0.1.

[0028] Preferably, the concentration of the azurin scaffold in the ammonium acetate solution is 1 to 3 mg / mL.

[0029] Preferably, the CuSO4 is obtained by dissolving anhydrous copper sulfate powder at a concentration of 200±10 mmol / L in deionized water whose pH has been adjusted to 5.0 with dilute hydrochloric acid.

[0030] Preferably, the incubation condition is stirring at 4°C for 16±2h.

[0031] Preferably, the purification is size exclusion chromatography (SEC) purification.

[0032] More preferably, the buffer solution for size exclusion chromatography purification is ammonium acetate solution, and the composition of the ammonium acetate solution is: 50±10 mmol / L NH4OAc, pH 5.0±0.1.

[0033] Preferably, the azurin scaffold is prepared by the following method: obtaining the BPG-succinimidyl ester module BPG-NHS by organic synthesis; obtaining the mutant azurin Cys-Azurin by biological recombinant expression, and then coupling BPG-NHS with Cys-Azurin.

[0034] Preferably, the coupling comprises the following steps: dissolving the mutant azurin Cys-Azurin in an ammonium acetate solution, then adding tris(2-carboxyethyl)phosphine (TCEP) to the protein solution, shaking, and then sequentially adding imidazole and BPG-NHS for reaction.

[0035] More preferably, the composition of the ammonium acetate solution is: 50±10 mmol / L NH4OAc, pH 5.0±0.1.

[0036] More preferably, the concentration of the mutant Cys-Azurin in the ammonium acetate solution is 1±0.2 mM.

[0037] More preferably, the amount of tris(2-carboxyethyl)phosphine added is calculated so that the final concentration is 5±0.5 mol / L.

[0038] More preferably, the amount of imidazole added is calculated so that the final concentration is 1±0.5 mol / L.

[0039] More preferably, the added amount of BPG-NHS is calculated as 2.5±0.5 equivalents of the mutant azurin Cys-Azurin.

[0040] Preferably, the BPG-NHS is prepared by the following method: adding glycine ethyl ester, pyridine-2-carboxaldehyde, and sodium triacetoxyborohydride to dichloroethane (DCE) to carry out a reductive amination reaction to obtain N,N-di(pyridin-2-ylmethyl)glycine ethyl ester; hydrolyzing N,N-di(pyridin-2-ylmethyl)glycine ethyl ester and condensing it with N-hydroxysuccinimide (NHS) to obtain the BPG-NHS.

[0041] More preferably, in the reductive amination reaction, the ratio of dichloroethane, glycine ethyl ester, pyridine-2-carboxaldehyde, and sodium triacetoxyborohydride is 40 mL: 1.86±0.1 g: 3.00±0.2 g: 7.06±0.5 g.

[0042] More preferably, after the reductive amination reaction is completed, the mixture is washed with saturated brine, the organic phase is dried over anhydrous sodium sulfate, filtered, and the solvent is removed by rotary evaporation to obtain N,N-di(pyridin-2-ylmethyl)glycine ethyl ester.

[0043] More preferably, in the hydrolysis system, the ratio of N,N-di(pyridin-2-ylmethyl)glycine ethyl ester, methanol, and sodium hydroxide is 2.0 g: 20-30 mL: 2.0±0.2 g.

[0044] More preferably, after the hydrolysis is completed, the methanol is removed by rotary evaporation, the residue is dissolved in water, extracted with dichloromethane, the organic phase is dried over anhydrous sodium sulfate, filtered, concentrated by rotary evaporation, slurried with ether, and recrystallized from isopropanol to obtain N,N-di(pyridin-2-ylmethyl)glycine.

[0045] More preferably, in the condensation system, the ratio of N,N-di(pyridin-2-ylmethyl)glycine, anhydrous tetrahydrofuran, NHS, and DCC is 0.2 g: 7-10 mL: 0.09±0.01 g: 0.21±0.02 g.

[0046] More preferably, during the condensation, NHS is added and then the mixture is cooled to 0°C in an ice bath; after adding DCC, the mixture is transferred to room temperature and stirred overnight under a N2 atmosphere; after the condensation, the solid is filtered off and the solvent is removed from the filtrate to obtain the final product.

[0047] An artificial metalloenzyme is obtained by the above chemical synthesis method.

[0048] The application of the artificial metalloenzyme is to catalyze the cleavage of 4-nitrophenyl-β-D-pyranoglucoside.

[0049] Preferably, the application comprises the following steps: dissolving the artificial metalloenzyme in phosphate buffer, and sequentially adding a reducing agent, hydrogen peroxide and 4-nitrophenyl-β-D-pyranoglucoside to obtain 4-nitrophenol.

[0050] The composition of the phosphate buffer is: 50±10mmol / L NaH2PO4, pH 7.4±0.1.

[0051] The artificial metalloenzyme is dissolved in phosphate buffer at a concentration of 2.5±0.1 μmol / L.

[0052] The reducing agent is ascorbic acid, which is dissolved in phosphate buffer and then added to a final concentration of 20±1 mmol / L.

[0053] The hydrogen peroxide was dissolved in phosphate buffer and then added to a final concentration of 5.0±0.1 mmol / L.

[0054] The 4-nitrophenyl-β-D-pyranoglucoside is dissolved in phosphate buffer and then added to a final concentration of 20±1 mmol / L.

[0055] The reaction conditions are 25±2°C, 10 to 20 hours.

[0056] The principle of the invention is as follows: a cysteine residue is extended to the N-terminus of wild-type azurin, and the synthesized BPG-NHS is coupled with the obtained protein fragment under 1M imidazole and pH 6.5 to obtain a chemically synthesized novel protein scaffold, which is finally coordinated with divalent copper ions to obtain a novel artificial metalloenzyme based on the azurin scaffold.

[0057] The present invention has the following advantages and effects compared to the prior art:

[0058] This invention uses azurin as the protein scaffold. This method is flexible and efficient, addressing the limitation of biorecombinant expression for the unrestricted introduction of specific non-natural amino acid ligands to modify metalloproteins. Using water as the reaction medium aligns with the principles of green chemistry.

[0059] The novel LPMO-like artificial metalloenzyme constructed based on the azurin scaffold has been modified compared to the wild-type by introducing unnatural amino acids as active centers, broadening the avenues for ligand modification. Furthermore, this study provides a deeper understanding of the protein scaffold, providing valuable guidance for the construction of other artificial enzymes based on the azurin scaffold.

[0060] The method of the present invention introduces non-natural amino acid ligands onto the azurin scaffold through protein chemical modification to construct an artificial azurin metalloenzyme with the novel ligand coordination, which is then used to catalyze the cleavage reaction of 4-nitrophenyl-β-D-glucopyranoside. This method expands the scope of artificial metalloenzyme modification based on the azurin scaffold.

[0061] The present invention expands the scope and approach of ligand modification, and uses the cleavage reaction of 4-nitrophenyl-β-D-pyranoglucopyranoside as an application to facilitate the study of the structure-activity relationship of artificial metalloenzymes.

[0062] The method of the present invention is modular, and the protein fragment only needs to retain the N-terminal cysteine to synthesize similar types of ligands and introduce them using the same method.

[0063] In summary, the present invention uses a mutant azurin as the artificial metalloenzyme protein backbone, obtains a BPG-NHS module through organic synthesis, and then couples BPG-NHS with biologically expressed Cys-Azurin to produce a novel artificial metalloenzyme with a tridentate N-ligand structure at the N-terminus. This metalloenzyme exhibits moderate lytic polysaccharide monooxygenase activity. This invention overcomes the limitations of biological recombinant expression technology for the introduction of specific non-natural amino acids, broadening the scope and avenues for the modification of artificial metalloenzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Schematic diagram of the three-dimensional structure of the azurin artificial metalloenzyme BPG(Cu)-Cys-Azurin in Example 3.

[0065] Figure 2 Schematic diagram of the chemical synthesis route of the azurin scaffold BPG-Cys-Azurin (left) and liquid phase monitoring of the synthesis of the azurin scaffold BPG-Cys-Azurin (right).

[0066] Figure 3 ESI-MS characterization images of Cys-Azurin (left) and BPG-Cys-Azurin (right). DETAILED DESCRIPTION

[0067] The present invention discloses a novel artificial metalloenzyme and describes its preparation and application. Those skilled in the art can refer to the present disclosure and appropriately modify the process parameters to achieve the objectives. The methods and products of the present invention have been specifically described through preferred embodiments. It is obvious that those skilled in the art can modify, alter, and combine the methods described herein to implement the present invention without departing from the content, spirit, and scope of the present invention.

[0068] To further understand the present invention, the following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0070] Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available products and can be purchased through commercial channels.

[0071] The basic plasmid used for the expression of Cys-Azurin in the following examples was pET-26b(+) (product of Nanjing GenScript Biotech Co., Ltd.), the restriction enzyme sites used were NcoI and XhoI, and the inserted nucleotide sequence was as follows:

[0072] TGCGCTGAATGTTCAGTAGATATACAAGGAAACGATCAGATGCAATTCAACACCAATGCGATCACGGTCGATAAGTCTTGTAAACAGTTCACCGTGAACTTGTCCCATCCGGGTAATCTCCCGAAAAACGTGATGGGCCACAACTGGGTTCTGTCGACCGCTGCCGATATGCAAGGTGTTGTGACTGACGGCATG GCGAGCGGTCTGGACAAGGACTACCTGAAGCCAGATGACAGCCGTGTTATCGCGCATACCAAATTAATTGGTTCCGGCGAGAAAGACAGCGTTACGTTTGATGTGAGCAAACTGAAAGAAGGTGAGCAGTATATGTTTTTCTGCACCTTTCCGGGCCACAGCGCACTGATGAAGGGCACCTTGAACCTGAAGTAA.

[0073] The structural formula of BPG-NHS used in the following examples is shown below:

[0074]

[0075] The LB medium in the following examples was prepared as follows: 20 g of tryptone, 20 g of NaCl, and 10 g of yeast extract were weighed and dissolved in 2 L of water. After sufficient dissolution, the mixture was sterilized by autoclaving at 120° C. for 30 min, and the sterilized mixture was cooled to room temperature.

[0076] The agar solid medium in the following examples was prepared as follows: 2.5 g of tryptone, 1.25 g of NaCl, and 4 g of yeast extract were weighed and dissolved in 250 mL of water. After sufficient dissolution, the mixture was autoclaved at 120°C for 30 min. The sterilized mixture was cooled to about 60°C, 25 mg of kanamycin was added, dissolved and mixed, and then evenly spread into 100 mm cell culture dishes (each dish containing 20-25 mL). After cooling and solidification, it was sealed in a 4°C refrigerator.

[0077] The names and abbreviations of the reagents used in the following examples are:

[0078] Tris: Tris(hydroxymethyl)aminomethane

[0079] NaCl: sodium chloride

[0080] NH4OAc: ammonium acetate

[0081] NaH2PO4: sodium dihydrogen phosphate

[0082] IPTG: Isopropyl-β-D-thiogalactopyranoside

[0083] TCEP: tris(2-carboxyethyl)phosphine

[0084] DCM: dichloromethane

[0085] NHS: N-hydroxysuccinimide

[0086] DCC: N,N'-dicyclohexylcarbodiimide

[0087] MeOH: methanol

[0088] THF: Tetrahydrofuran

[0089] NMP: N-methylpyrrolidone

[0090] The amino acid sequence of wild-type azurin is as follows:

[0091] AECSVDIQGNDQMQFNTNAITVDKSCKQFTVNLSHPGNLPKNVMGHNWVLSTAADMQGVVTDGMASGLDKDYLKPDDSRVIAHTKLIGSGEKDSVTFDVSKLKEGEQYMFFCTFPGHSALMKGTLTLK

[0092] Example 1: Soluble Expression and Purification of Cys-Azurin

[0093] (1) The plasmid was transformed into Escherichia coli BL21 (DE3) chemically competent cells by chemical transformation. The transformation process was as follows: 50 μL of BL21 (DE3) chemically competent cells stored at -80°C were thawed on ice, 2 μL of plasmid solution dissolved in sterile ultrapure water (final concentration was approximately 50 ng / μL) was added, the mixture was gently stirred and placed on ice for 30 min; then, heat-shocked in a 42°C water bath for 60 seconds (not more than 90 seconds), quickly returned to ice for 2 minutes, 400 μL of sterilized LB culture medium without antibiotics was added, and cultured at 37°C with shaking for 1 hour (1000 rpm); finally, the cell suspension was spread on agar solid medium containing 50 mg / L kanamycin for resistance screening. The successfully screened monoclonal strain was inoculated into 50 ml of LB culture medium containing 50 mg / L kanamycin and cultured with shaking at 37°C and 110 rpm overnight. The next day, 40 mL of overnight bacterial suspension was transferred to 2 L of fresh LB medium (contained in a 5 L shake flask, containing 50 mg / L kanamycin) for large-scale culture and induced expression. The specific steps are as follows: First, culture at 37 ° C, 110 rpm under shaking conditions until OD 600After the pH value reaches 0.5-0.6, IPTG is added to a final concentration of 0.25 mmol / L and shaken at 110 rpm at 18°C. After 16 hours of incubation, the cells are centrifuged at 8000 rpm for 15 minutes. The bacterial precipitate is washed with 0.9% NaCl solution and centrifuged at 8000 rpm for 10 minutes. The cells are then stored in a -25°C refrigerator until use.

[0094] After ultrasonic disruption of 5.5 g of bacterial cells, the target protein was extracted with a buffer solution (0.1 M Tris, 0.1 M NaCl, pH 8.0). The pH was adjusted to 5.0 with hydrochloric acid, followed by centrifugation to remove the precipitate produced during the process. Next, a 10 mM ZnSO4 solution was added to the supernatant, and the mixture was stirred at 4°C overnight. After completion, the mixture was centrifuged and filtered, and initially purified by SEC using an AKTA Protein Purification System (AKTAPurifier 10, GE) using elution buffer (50 mM NH4OAc, pH 5.0). Further purification was performed using a cation exchange column with mobile phases A1 (50 mM NH4OAc, pH 4.2) and B1 (50 mM NH4OAc, pH 6.7).

[0095] Example 2: Synthesis of BPG-NHS

[0096] To 40 mL of DCE, add 1.86±0.1 g of glycine ethyl ester, 3.00±0.2 g of pyridine-2-carboxaldehyde, and 7.06±0.5 g of sodium triacetoxyborohydride. Stir at room temperature for 2–3 hours. After the reaction, wash three times with saturated brine. Dry the organic phase over anhydrous sodium sulfate, filter, and remove the solvent by rotary evaporation to obtain N,N-di(pyridin-2-ylmethyl)glycine ethyl ester.

[0097] To 2.0 g of N,N-bis(pyridin-2-ylmethyl)glycine ethyl ester, add 20-30 mL of methanol and 2.0 ± 0.2 g of solid sodium hydroxide, and stir at room temperature for 2-3 hours. After the reaction, remove the methanol by rotary evaporation, dissolve the residue in 10-15 mL of water, and extract with dichloromethane three to four times. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated by rotary evaporation, slurried with ether, and recrystallized from isopropanol to obtain N,N-bis(pyridin-2-ylmethyl)glycine.

[0098] Dissolve 0.2 g of N,N-bis(pyridin-2-ylmethyl)glycine in 7–10 mL of anhydrous tetrahydrofuran, add 0.09 ± 0.01 g of NHS, cool to 0°C on ice, add 0.21 ± 0.02 g of DCC, transfer the reaction mixture to room temperature, and stir overnight under a nitrogen atmosphere. Filter the solids, and remove the solvent from the filtrate to obtain the final product, BPG-succinimidyl ester, referred to as BPG-NHS.

[0099] Example 3: Acquisition and Copper Coupling of BPG-Cys-Azurin Protein Scaffold

[0100] Prepare a 200mM solution of BPG-NHS in NMP. Dissolve the purified Cys-Azurin protein sample in 50mM NH4OAc buffer at pH 5.0 to a concentration of 1mM. Then, add 5mM TCEP to the protein solution and shake for 2 minutes. Subsequently, add 1M imidazole and 2.5 equivalents of BPG-NHS in sequence. Oscillating reaction at room temperature for 2.5 hours. The 1M imidazole is added by preparing a concentrated imidazole solution at pH 6.6 and a concentration of 5M, and adding the concentrated imidazole solution to the protein solution until the concentration of imidazole in the system is 1M. Due to the strong pH buffering capacity of imidazole, the system pH is approximately 6.5 at this time.

[0101] After the reaction, the protein was purified using SEC with a mobile phase of 50 mM NH4OAc buffer, pH 5.0. The resulting protein solution was concentrated to 1-3 mg / mL, and 1.2 equivalents of CuSO4 were added. The mixture was incubated overnight at 4°C for 16 h. Excess CuSO4 was then removed.

[0102] Example 4: Artificial metalloenzyme BPG(Cu)-Cys-Azurin catalyzes the cleavage of 4-nitrophenyl-β-D-glucopyranoside

[0103] The artificial metalloenzyme was dissolved at a concentration of 2.5 μmol / L in phosphate buffer (50 mmol / L NaH2PO4, pH 7.4). 20 mmol / L ascorbic acid, 5 mmol / L hydrogen peroxide, and 20 mmol / L 4-nitrophenyl-β-D-pyranoglucoside were added sequentially. The mixture was shaken at 25°C for 16 hours to produce 4-nitrophenol. The absorbance of the reaction solution at 400 nm was measured by UV-visible absorption spectroscopy, and the total turnover rate (TTN) of the artificial metalloenzyme in this reaction was calculated to be 57%.

[0104] Figure 1 Schematic diagram of the three-dimensional structure of the azurin artificial metalloenzyme BPG(Cu)-Cys-Azurin in Example 3. It can be seen that the azurin artificial metalloenzyme of the present invention is a new artificial metalloenzyme with a tridentate N ligand structure at the N-terminus.

[0105] Figure 2 A schematic diagram of the chemical synthesis route of the azurin scaffold BPG-Cys-Azurin (left) and liquid phase monitoring of the synthesis of the azurin scaffold BPG-Cys-Azurin (right) show that the BPG-NHS molecule modified the N-terminal cysteine in Cys-Azurin smoothly, achieving complete conversion within 2.5 hours.

[0106] Figure 3 ESI-MS characterization images of Cys-Azurin (left) and BPG-Cys-Azurin (right). It can be seen that BPG was successfully introduced into the N-terminus of the Cys-Azurin protein scaffold, and the target protein scaffold was successfully obtained.

[0107] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An azurin scaffold, characterized in that: Has the following amino acid sequence: BPG-CAECSVDIQGNDQMQFNTNAITVDKSCKQFTVNLSHPGNLPKNVMGHNWVLST AADMQGVVTDGMASGLDKDYLKPDDSRVIAHTKLIGSGEKDSVTFDVSKLKEGEQYMFF CTFPGHSALMKGTLTLK; wherein BPG represents N,N-di(pyridin-2-ylmethyl)glycine.

2. An artificial metalloenzyme based on an azurin scaffold, characterized by: The invention comprises the azurin scaffold as claimed in claim 1 and a Cu(II) active center.

3. The method for preparing the artificial metalloenzyme according to claim 2, characterized in that: CuSO4 is added to the ammonium acetate solution of the azurin scaffold, incubated and purified to obtain the artificial metalloenzyme.

4. The preparation method according to claim 3, wherein: The composition of the ammonium acetate solution is: 50±10mmol / L NH4OAc, pH 5.0±0.1; The concentration of the azurin scaffold in the ammonium acetate solution is 1 to 3 mg / mL; The CuSO4 is obtained by dissolving anhydrous copper sulfate powder at a concentration of 200±10 mmol / L in deionized water whose pH was previously adjusted to 5.0 with dilute hydrochloric acid; The incubation condition is stirring at 4°C for 16±2h.

5. The preparation method according to claim 3 or 4, characterized in that: The azurin scaffold is prepared by the following method: obtaining the BPG-succinimidyl ester module BPG-NHS through organic synthesis; obtaining the mutant azurin Cys-Azurin through biological recombinant expression, and then coupling BPG-NHS with Cys-Azurin.

6. The preparation method according to claim 5, characterized in that: The coupling comprises the following steps: dissolving the mutant azurin Cys-Azurin in an ammonium acetate solution, then adding tris(2-carboxyethyl)phosphine to the protein solution, shaking, and then sequentially adding imidazole and BPG-NHS to react; The BPG-NHS is prepared by the following method: adding glycine ethyl ester, pyridine-2-carboxaldehyde, and sodium triacetoxyborohydride to dichloroethane to carry out a reductive amination reaction to obtain N,N-di(pyridin-2-ylmethyl)glycine ethyl ester; and hydrolyzing the N,N-di(pyridin-2-ylmethyl)glycine ethyl ester and condensing it with N-hydroxysuccinimide to obtain the BPG-NHS.

7. The preparation method according to claim 6, characterized in that: The composition of the ammonium acetate solution is: 50±10mmol / L NH4OAc, pH 5.0±0.1; The concentration of the mutant Cys-Azurin in the ammonium acetate solution is 1±0.2 mM; The amount of tris(2-carboxyethyl)phosphine added is calculated based on a final concentration of 5±0.5 mol / L; The amount of imidazole added is calculated based on its final concentration of 1±0.5 mol / L; The amount of BPG-NHS added is calculated based on 2.5±0.5 equivalents of the mutant Cys-Azurin; In the preparation of the BPG-NHS: In the reductive amination reaction, the ratio of dichloroethane, glycine ethyl ester, pyridine-2-carboxaldehyde, and sodium triacetoxyborohydride is 40 mL: 1.86 ± 0.1 g: 3.00 ± 0.2 g: 7.06 ± 0.5 g; In the hydrolysis system, the ratio of N,N-di(pyridin-2-ylmethyl)glycine ethyl ester, methanol, and sodium hydroxide is 2.0 g: 20-30 mL: 2.0 ± 0.2 g; In the condensation system, the ratio of N,N-di(pyridin-2-ylmethyl)glycine, anhydrous tetrahydrofuran, NHS, and DCC is 0.2 g: 7-10 mL: 0.09±0.01 g: 0.21±0.02 g.

8. An artificial metalloenzyme, characterized in that: The method is obtained by the preparation method according to any one of claims 1 to 7.

9. The use of the artificial metalloenzyme according to claim 8, characterized in that: The application is the application of catalyzing the cleavage reaction of 4-nitrophenyl-β-D-pyranoglucoside.

10. The use according to claim 9, characterized in that: The application comprises the following steps: dissolving the artificial metalloenzyme in a phosphate buffer, and sequentially adding a reducing agent, hydrogen peroxide, and 4-nitrophenyl-β-D-pyranoglucoside to obtain 4-nitrophenol; The composition of the phosphate buffer is: 50±10mmol / L NaH2PO4, pH 7.4±0.1; The artificial metalloenzyme is dissolved in phosphate buffer at a concentration of 2.5±0.1 μmol / L; The reducing agent is ascorbic acid, which is dissolved in phosphate buffer and then added to a final concentration of 20±1 mmol / L; The hydrogen peroxide was dissolved in phosphate buffer and then added to a final concentration of 5.0±0.1mmol / L; The 4-nitrophenyl-β-D-pyranoglucoside is dissolved in phosphate buffer and then added to a final concentration of 20±1 mmol / L; The reaction conditions are 25±2°C, 10 to 20 hours.

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