Photocatalytic biological enzyme and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a photocatalytic biological enzyme and application thereof. The photocatalytic bio-enzyme is obtained through optimization and screening, and the photocatalytic bio-enzyme is obtained by catalyzing the fusion protein and phycocyanobilin through phycocyanin lyase. The photocatalytic biological enzyme and the preparation method are simple, rapid, green and environment-friendly, efficient catalytic reaction can be carried out under mild reaction conditions, and meanwhile, a new thought is provided for preparing clean energy by splitting water, reducing atmospheric carbon dioxide and relieving increasingly serious environmental pollution problems.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to photocatalytic bioenzymes and their applications. Background Technology

[0002] Plants, algae, and cyanobacteria can perform photosynthesis, using water and atmospheric carbon dioxide as raw materials and renewable sunlight as energy to synthesize high-value-added organic matter. Photon capture and electron transfer in the light-dependent reactions are two key steps in achieving efficient photosynthesis. Photon absorption and transfer in organisms mainly rely on photosynthetic pigments and pigment-protein complexes. Photons are transferred to photosynthetic system II (PSII), composed of the P680 chlorophyll complex, to initiate the light-dependent reactions. During this process, the P680 pigment complex is excited and transitions to its excited state (P680...). * It is further converted into cationic free radical P680 through charge separation. ·+ It possesses a high redox potential (approximately 1.25 eV) and exhibits strong oxidizing properties. When coupled with photosynthetic system I (PSI), it forms a typical Z-shaped linear electron transport chain, capable of simultaneously oxidizing water molecules and NAD+. + (NADP + The reduction of oxygen evolution by photocatalysis is also accompanied by proton transmembrane transport, promoting ATP formation and thus providing energy for the carbon fixation reaction in the photosynthetic system.

[0003] The biomimetic study of oxygen evolution photosynthesis is highly attractive. Therefore, researchers have designed and developed various photocatalytic materials to mimic the photosynthetic reactions of plants, including semiconductor materials such as metal oxides and quantum dots, as well as noble metal catalysts. These catalytic materials can rapidly generate charge carriers under light, forming electron-hole pairs to participate in the catalytic reaction. However, due to their complex preparation processes and high costs, they are not truly green and efficient artificial photocatalytic materials. Therefore, exploring new green and efficient artificial photosynthetic materials is urgently needed. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide photocatalytic bioenzymes and their applications.

[0005] This invention provides a fusion protein comprising: formate dehydrogenase and phycobiliprotein connected sequentially from the N-terminus to the C-terminus;

[0006] The phycobiliproteins include: the α subunit of phycocyanin and / or the α subunit of allophycocyanin;

[0007] The amino acid sequences of the α subunit of phycocyanin and / or the α subunit of allophycocyanin are highly homologous. In a specific embodiment of the present invention, the α subunit of allophycocyanin is used. The α subunit of allophycocyanin is derived from Picosynechococcus sp. PCC 7002, and the amino acid sequence of the α subunit of allophycocyanin is shown in SEQ ID NO:17.

[0008] The formate dehydrogenase is derived from Candida botrytis cinerea. In the examples, the formate dehydrogenase derived from Candida botrytis cinerea is written as cbFDH; the amino acid sequence of cbFDH is shown in SEQ ID NO:19.

[0009] In a specific embodiment of the present invention, the amino acid sequence of the fusion protein cbFDH-APC-α is shown in SEQ ID NO:5.

[0010] In a specific embodiment of the present invention, the formate dehydrogenase and phycobiliprotein are fused and expressed. Specifically, their encoding nucleic acids are cloned into expression vectors with typical T7 promoters, such as pET28a, pET3a, and pET15b, and then expressed directly or under the action of other proteins or enzymes.

[0011] In this invention, the fusion protein has a histidine tag at its N-terminus for protein expression identification and protein purification.

[0012] This invention provides a photocatalytic bioenzyme, which is obtained by catalysis of the fusion protein and phycocyanin described in this invention via phycocyanin lyase.

[0013] The phycocyanin lyase includes phycocyanin lyase CpcS and phycocyanin lyase CpcU; the phycocyanin lyase can also be called phycocyanin lyase.

[0014] In the photocatalytic bioenzyme of the present invention, the phycocyanin is obtained by expressing heme oxygenase and biliverdin reductase in Escherichia coli.

[0015] Furthermore, the amino acid sequence of the heme oxygenase is shown in SEQ ID NO:1; the amino acid sequence of the biliverdin reductase is shown in SEQ ID NO:2; the amino acid sequence of the phycocyanin lyase CpcS is shown in SEQ ID NO:3; and the amino acid sequence of the phycocyanin lyase CpcU is shown in SEQ ID NO:4.

[0016] In this invention, the phycobiliprotein in the fusion protein is bound to phycocyanin, and the phycobiliprotein is fused with formate dehydrogenase through genetic engineering to form the photocatalytic bioenzyme described in this invention. The photocatalytic bioenzyme utilizes the naturally bound phycocyanin phycobiliprotein as a photosensitizing molecule and formate dehydrogenase with carbon dioxide reduction activity as a catalytic molecule to realize a recyclable biophotocatalytic system.

[0017] In the preparation of the photocatalytic bioenzyme, the present invention has made several optimizations, specifically including:

[0018] (1) The connection mode of formate dehydrogenase and phycobiliprotein in the fusion protein was investigated. Rigid or flexible linkers were added between them. The results showed that after adding the linker, the photocatalytic enzyme was not expressed normally or the phycobiliprotein synthesis rate was reduced, so that the photocatalytic enzyme (fusion protein) did not have photocatalytic activity. Preferably, the linker sequence was removed and the fusion expression was carried out directly. In this way, during the fusion expression process, the spatial conformation of phycocyanin and formate dehydrogenase is similar and the distance between the two active regions is small. It facilitates electron transfer and forms an ordered arrangement that is conducive to protein crystal growth;

[0019] (2) The phycobiliprotein in the fusion protein was truncated. The truncated photocatalytic enzyme could not be expressed normally and had no photocatalytic activity.

[0020] (3) The codons encoding the nucleic acids of formate dehydrogenase and phycobiliprotein in the fusion protein are optimized. Before the codon optimization, the expression level of the photocatalytic enzyme is reduced.

[0021] (4) The photocatalytic enzyme has no photocatalytic activity when it only has the fusion protein or only has phycocyanin synthase and lyase;

[0022] (7) The induction temperature was adjusted, indicating that if the induction temperature is too high, the enzyme activity of the photocatalytic biological enzyme will be lost.

[0023] (8) The phycobiliproteins in the fusion protein were screened. Among the fusion proteins containing APC-α and APC-β, the fusion protein containing APC-β had low purity after purification and no photocatalytic activity.

[0024] (9) The order of the fusion protein was optimized and adjusted, indicating that in the fusion protein composed of formate dehydrogenase and phycobiliprotein, the fusion protein with formate dehydrogenase located at the N-terminus does not form inclusion bodies and can be expressed normally.

[0025] In addition, this includes truncating up to 20 amino acid residues at the C-terminus of the formate dehydrogenase sequence and the N-terminus of the APC-α sequence to different lengths, and detecting the fusion expression level; screening expression conditions during the exogenous expression of FDH-APC, including culture medium, culture temperature, induction temperature, and induction time; and screening and optimizing the purification method of FDH-APC protein, including buffer type, salt ion concentration, and pH value. Preferably, the optimal expression temperature of the recombinant protein in the BL21(DE3) host is 20℃, and the optimal induction time is 12h. Preferably, the optimal buffer for the fusion protein purification process is 50mM Tris, and the solution pH is 7.5. Preferably, the visible light wavelength used for the photocatalytic experiment is 620nm.

[0026] This invention provides a method for preparing the aforementioned photocatalytic bioenzyme, characterized by comprising the following steps:

[0027] Step 1: Construct recombinant vector 1 containing the fusion protein described in this invention, and construct recombinant vector 2 containing heme oxygenase, biliverdin reductase, phycocyanin lyase CpcS and phycocyanin lyase CpcU;

[0028] Step 2: Co-transform the recombinant vector 1 and recombinant vector 2 into host cells, and then culture and induce them to obtain the photocatalytic bioenzyme of the present invention.

[0029] This invention provides a biomaterial, characterized in that it comprises at least one of the following: i) to v)

[0030] i) Nucleic acids encoding the fusion protein and / or the photocatalytic enzyme described in this invention;

[0031] ii) Expression units containing the nucleic acids described in i);

[0032] iii) Recombinant vectors integrating nucleic acids as described in i) and / or expression units as described in ii);

[0033] iv) Transfecting or transforming host cells with the recombinant vector described in iii);

[0034] v) Products obtained by culturing and / or inducing host cells as described in iv).

[0035] Furthermore, the nucleotide sequence of the nucleic acid encoding the phycobiliprotein is shown in SEQ ID NO:15; the nucleotide sequence of the nucleic acid encoding the formate dehydrogenase is shown in SEQ ID NO:16.

[0036] The induction temperature is 18–22°C; in a specific embodiment of the present invention, it is 20°C.

[0037] In the described biomaterials, the recombinant vector's carrier backbone includes pET28a, pCDF-Duet series, pET3a, pRSF-Duet series, and / or pET15b. Those skilled in the art can choose suitable carriers in obtaining the photocatalytic enzymes described in this invention, and this invention does not limit this choice. Specifically, in the expression of heme oxygenase, biliverdin reductase, phycocyanin lyase CpcS, and phycocyanin lyase CpcU, the carrier used is pCDF-Duet-1 or pRSF-Duet-1.

[0038] The nucleic acid encoding the recombinant antigen described in this invention can be DNA, RNA, cDNA, or PNA. In embodiments of this invention, the nucleic acid is in the form of DNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The nucleic acid can be topologically linear or circular. The nucleic acid can be part of a vector (e.g., an expression or cloning vector) or a fragment thereof. The nucleic acid can be obtained directly from a natural source or can be prepared with the assistance of recombinant, enzymatic, or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0039] The present invention also provides an expression unit for the recombinant antigen, wherein the expression unit refers to a DNA sequence from the start of the promoter to the end of the terminator. Regulatory fragments may also be included on either side of or between the promoter and terminator. These regulatory fragments may include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operatively linked to the nucleic acid sequence, such as enhancers of promoters, poly(A) signals, etc.

[0040] The recombinant vector described in this invention refers to a recombinant nucleic acid vector, a recombinant DNA molecule containing the desired coding sequence and suitable nucleic acid sequences or elements essential for the expression of an operatively linked coding gene in a specific host organism. Prokaryotic cells are known to utilize promoters, enhancers, and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" are sometimes used interchangeably because plasmids are currently the most commonly used form of vector. However, this invention intends to include other forms of expression vectors that perform equivalent functions and are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors, and / or simply potential genomic inserts. In specific embodiments, the nucleic acid encoding the fusion protein provided by this invention can be constructed in various prokaryotic expression vectors.

[0041] The transformation methods include chemical transformation and electroconversion; the transfection methods include calcium phosphate co-precipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, and lentivirus transfection.

[0042] The host cells provided by this invention can be derived from plants, animals, bacteria, fungi, bacteriophages, or viruses; specifically, the bacteria include, but are not limited to, *Escherichia coli*, which includes, but is not limited to, BL21(DE3); the fungi include, but are not limited to, yeast, and this invention does not limit them. This invention uses a vector constructed using recombinant DNA technology to transform or transfect host cells, thereby enabling the transformed host cells to replicate the protein-encoding vector or express the desired protein.

[0043] This invention provides the application of at least one of the following (a) to (c) in biophotocatalysis:

[0044] a) Encoding the fusion protein described in this invention;

[0045] b) The photocatalytic bioenzyme described in this invention;

[0046] c) The biomaterials described in this invention.

[0047] This invention obtains a photocatalytic bioenzyme through optimization and screening. The photocatalytic bioenzyme is obtained by catalysis of the fusion protein and phycocyanin described in this invention via phycocyanin lyase. The photocatalytic bioenzyme has a simple, rapid, green and environmentally friendly preparation method. It can carry out efficient catalytic reactions under mild reaction conditions, and at the same time provides a new approach for producing clean energy from water splitting, reducing atmospheric carbon dioxide, and alleviating the increasingly serious environmental pollution problem. Attached Figure Description

[0048] Figure 1 The expression and light absorption of the fusion protein in the artificial photocatalytic system of Example 1 are shown; where a is the SDS-PAGE result of the purified fusion protein; b is the native-PAGE result of the purified fusion protein; and c is the absorption spectrum of the fusion protein in the ultraviolet-visible range.

[0049] Figure 2 The image shows the photoinduced NADH regeneration and the ultraviolet spectra before and after photoinduced NADH regeneration of the artificial photocatalytic system in Example 1; a is the result of photoinduced NADH regeneration; b is the change in ultraviolet spectrum before and after photoinduced NADH regeneration.

[0050] Figure 3 The fusion protein in the artificial photocatalytic system of Example 1 was subjected to in vitro artificial photosynthetic reaction activity detection: where a is the activity test of the fusion protein in reducing carbon dioxide to formic acid, and b is the activity test of the fusion protein in NAD+. + And oxygen production under light conditions;

[0051] Figure 4 The expression and light absorption of fusion proteins containing cbFDH-APC-α and cbFDH-APC-β are shown. In this paper, a represents the expression of fusion proteins containing cbFDH-APC-α (FDH-α) and cbFDH-APC-β (FDH-β), respectively. b and c represent the solution characteristics of fusion proteins containing cbFDH-APC-α and cbFDH-APC-β under fluorescent and ultraviolet lamps, respectively.

[0052] Figure 5 The UV-Vis spectral scan results show the fusion protein containing cbFDH-APC-α and the fusion protein containing cbFDH-APC-β. Detailed Implementation

[0053] This invention provides photocatalytic bioenzymes and their applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0054] Organic pigment molecules derived from natural plants possess excellent light absorption properties and can be used as photosensitizers, showing considerable application potential in the construction of artificial photocatalytic systems. The photocatalytic bioenzyme constructed in this invention possesses complete photocatalytic activity, including promoting the splitting of water molecules into oxygen, the reduction of carbon dioxide into formic acid, and the cyclic regeneration of the cofactor NADH. By constructing a photocatalytic bioenzyme molecule through fusion, expression, and covalent binding, a sustainable photocatalytic reaction system is formed, which is not found in existing similar bioenzyme systems. Furthermore, this system is a purely biological system, exhibiting higher biocompatibility compared to other metal nanoparticle systems or bio-semi-biological hybrid systems.

[0055] The photosensitizing proteins involved in this patent are mainly phycobiliproteins that bind to phycocyanin, including allophycocyanin (α / β subunit) and phycocyanin (α / β subunit), while the formate dehydrogenase selected is an NADH-dependent formate dehydrogenase. Allophycocyanin and phycocyanin have high structural similarity, so allophycocyanin was chosen for expression.

[0056] The photocatalytic bioenzyme described in this invention, compared to existing photocatalytic materials and technologies, is a fused photoenzyme based entirely on green and environmentally friendly protein elements. Each enzyme molecule possesses two functional domains: allophycocyanin enables the cleavage of water molecules and the regeneration of cofactors, ensuring the carbon dioxide reduction by formate dehydrogenase. Under visible light irradiation, this enzyme molecule can perform the three major light reactions of traditional photosynthesis. This system overcomes the drawback of existing cadmium sulfide-based catalysts failing to cleave water to produce oxygen. Furthermore, the preparation method of this photocatalytic material is simple and rapid, enabling highly efficient catalytic reactions under mild reaction conditions. This invention provides a new approach for cleaving water to produce clean energy, reducing atmospheric carbon dioxide, and alleviating the increasingly serious environmental pollution problem.

[0057] The overall technical solution of this invention is as follows:

[0058] Selection and design of fusion protein photoenzymes: A protein with phycocyanin as a ligand was selected as the photosensitizing domain, and a formate dehydrogenase catalyzing the directed reduction of carbon dioxide was selected as the catalytic domain. The primary sequences of the proteins were fused using genetic engineering techniques to construct the recombinant protein. This involved codon optimization of the gene encoding the fusion protein and optimization of the primary sequence of the fusion protein.

[0059] Phycobilin synthesis: Phycobilin synthesis in vivo requires the catalysis of enzymes, and the genes involved include Ho1 and PcyA, which mediate phycobilin synthesis, and CpcS and CpcU, which catalyze the covalent linkage of phycobilin to a protein scaffold.

[0060] Construction of fusion photoenzyme recombinant expression vector: The genes encoding formate dehydrogenase and pigment protein were sequentially synthesized into an expression vector with a T7 promoter to construct a recombinant plasmid for fusion protein expression; the Ho1 and PcyA gene pairs and the CpcS and CpcU gene pairs were cloned into two multiple cloning sites of a dual promoter vector to construct a recombinant plasmid for phycocyanin synthesis.

[0061] Fusion protein exogenous expression: Two recombinant plasmids were co-transformed into E. coli for protein expression.

[0062] Photocatalytic activity verification: Photocatalytic experiments were conducted at the protein's maximum absorption wavelength to detect oxygen production and formic acid generation.

[0063] In the process of two genes fusion expression in prokaryotes, it is necessary to remove the stop codon of the nucleic acid encoded by the first gene so that the two genes are in the same open reading frame for transcription and translation;

[0064] The amino acid sequence of Ho1 is: MSVNLASQLREGTKKSHSMAENVGFVKCFLKGVVEKNSYRKLVGNLYFVYSAMEEEMAKFKDHPILSHIYFPELNRKQSLEQDLQFYYGSNWRQEVKISAAGQAYVDRVRQVAATA PELLVAHSYTRYLGDLSGGQILKKIAQNAMNLHDGGTAFYEFADIDDEKAFKNTYRQAMNDLPIDQATAERIVDEANDAFAMNMKMFNELEGNLIKAIGIMVFNSLTRRRSQGSTEVGLATSEG(SEQ ID NO:1);

[0065] The amino acid sequence of PcyA is: MAVTDLLSLTNSSLMPTLNPMIQQLALAIAASWQSLPLKPYQLPEDLGYVEGRLEGEKLVIENRCYQTPQFRKMHLELAKVGKGLDILHCVMFPEPLYGLPLFGCDIVAGPGGVSAAIADL SPTQSDRQLPAAYQKSLAELGQPEFEQQRELPPWGEIFSEYCLFIRPSNVTEEERFVQRVVDFLQIHCHQSIVAEPLSEAQTLEHRQGQIHYCQQQKNDKTRRVLEKAFGEAWAERYMSQVLFDVIQ(SEQ ID NO:2);

[0066] The amino acid sequence of CpcS is: MQSFADAKEFFQYSAGQWQSRRVTHHLPFRRAESGGSNIQVETLEKDDPRIIEICQMHDMDASLSVGGSYVTWAGTMQWDKDDENHEGSTVFALIPDADNPRQGKLLRERGYAEIVPVAGEYHLDHEDGLVLTTEYETMTIYERFWFANPDLRLRTSTVKRFGGFNTTTFCMEERIQTSPVTATAAAETNPLYAISGW (SEQ ID NO:3);

[0067] The amino acid sequence of CpcU is: MDINAFIHQSAGNWFAQRTFYQAHHPEPDNGKANLAFELLPLDHPEVSRFAAAIAQAPNEHWRVFQSSWDTSVDWGKPKAVGSSLFAFVINPDQPTQGQAFSLDERTLAQGQYRLGEDQILIVTLEAGEVKIIERQWFGNENLRLRTNIVTGKTGVLQTAFYSEIRRIIEPEKTAEVTAEASN (SEQ ID NO:4);

[0068] The amino acid sequence of cbFDH-APC-α is: MKIVLVLYDAGKHAADEEKLYGCTENKLGIANWLKDQGHELITTSDKEGGNSVLDQHIPDADIIITTPFHPAYITKERIDKAKKLKLVVVAGVGSDHIDLDYINQTGKKISVLEVTGSNVVSVAEHVLMTMLVLVRNFVPAHEQIINHDWEVAAIAKDAYDIEGKTIATIGAGRIGYRVLERLVPFNPKELLYYDYQALPKDAEEKVGARRVENIEELVAQADIVTINAPLHAGTKGLINKELLSKFKKGAWLVNTARGAICVAEDVAAALESGQLRGYGGDVWFPQPAPKDHPWRDMRNKYGAGNAMTPHYSGTTLDAQTRYAEGTKNILESFFTGKFDYRPQDIILLNGEYITKAYGKHDKKSIVTKSIVNADAEARYLSPGELDRIKAFVTSGESRLRIAETLTGSRERIIKSAGDALFQKRPDVVSPGGNAYGEEMTATCLRDMDYYLRLITYGVVAGDVTPIEEIGLVGVREMYKSLGTPVDAVAQAVREMKAVATGMMSGDDAAEAGAYFDYVIGAME (SEQ ID NO:5);

[0069]

[0070] The amino acid sequence of linker 1 is: GGGS (SEQ ID NO:7);

[0071] The amino acid sequence of Linker 2 is: GSGSGS (SEQ ID NO:8);

[0072] The amino acid sequence of linker 3 is: GGGSGGGSGGGS (SEQ ID NO:9);

[0073] The amino acid sequence of linker 4 is: APAPAP (SEQ ID NO:10);

[0074] The amino acid sequence of linker 5 is: PAPAP (SEQ ID NO:11);

[0075] The amino acid sequence of linker 6 is: APAPA (SEQ ID NO:12);

[0076] The unoptimized nucleotide sequence of the gene encoding the α subunit of allophycocyanin (APC-α) is: agcatcgtgaccaagtcgatcgtgaacgcggacgccgaggcgcgctacctgagccccggtgagctggaccgcatcaaggccttcgtgacctcgggcgagtcccgcctgcgcatcgccgagaccctgacgggctcgcgcgagcgcattatcaagagcgcgggcgacgccctgttccagaagcgcccggacgtggtgagccccggcggcaacgcttacggcgaggaga tgaccgccacctgcctgcgcgacatggactactacctgcgactgatcacctacggcgtggtggccggcgacgtgaccccgatcgaggagatcggcctggtcggcgtgcgcgagatgtacaagtccctggg cacccccgtggacgccgtggcccaggcggtgcgcgagatgaaggctgtggccaccggcatgatgtccggcgacgacgcggccgaggccggcgcttacttcgactacgtgatcggcgccatggagtaa(SEQ ID NO:13);

[0077]

[0078] The nucleotide sequence after codon optimization of the gene encoding allophycocyanin α subunit (APC-α) is: agcatcgttactaaatctatcgtgaacgcagacgctgaggcccgctatctgtctccgggtgaactggaccgtatcaaggcgttcgtgactagcggtgaatctcgcctgcgtatcgctgaaaccctgactggctctcgcgaacgcatcatcaaatccgctggtgatgccctgttccagaaacgcccggatgttgttagcccgggtggtaacgcttacggcgaagagatgaccgcaacctgcctgcgcgacatggactactacctgcgcctgatcacttatggcgtggtagctggcgacgttactccgatcgaagagattggtctggtcggcgttcgtgaaatgtacaaatccctgggcactcctgtcgatgccgtggcgcaggcggtacgtgaaatgaaagcggttgcgaccggtatgatgagcggtgacgacgcagctgaggctggtgcatacttcgactacgtgattggcgcgatggagtaa (SEQ ID NO:15);

[0079]

[0080] The amino acid sequence of APC-α is: SIVTKSIVNADAEARYLSPGELDRIKAFVTSGESRLRIAETLTGSRERIIKSAGDALFQKRPDVVSPGGNAYGEEMTATCLRDMDYYLRLITYGVVAGDVTPIEEIGLVGVREMYKSLGTPVDAVAQAVREMKAVATGMMSGDDAAEAGAYFDYVIGAME (SEQ ID NO: 17);

[0081] The N-terminus of the APC sequence is truncated to 20 amino acid residues: ELDRIKAFVTSGESRLRIAETLTGSRERIIKSAGDALFQKRPDVVSPGGNAY GEEMTATCLRDMDYYLRLITYGVVAGDVTPIEEIGLVGVREMYKSLGTPVD AVAQAVREMKAVATGMMSGDDAAEAGAYFDYVIGAME (SEQ ID NO:18);

[0082] The amino acid sequence of formate dehydrogenase FDH is: MKIVLVLYDAGKHAADEEKLYGCTENKLGIANWLKDQGHELITTSDKEGGNSVLDQHIPDADIIITTPFHPAYITKERIDKAKKLKLVVVAGVGSDHIDLDYINQTGKKISVLEVTGSNVVSVAEHVLMTMLVLVRNFVPAHEQIINHDWEVAAIAKDAYDIEGKTIATIGAGRIG YRVLERLVPFNPKELLYYDYQALPKDAEEKVGARRVENIEELVAQADIVTINAPLHAGTKGLINKELLSKFKKGAWLVNTARGAICVAEDVAAALESGQLRGYGGDVWFPQPAPKDHPWRDMRNKYGAGNAMTPHYSGTTLDAQTRYAEGTKNILESFFTGKFDYRPQDIILLNGEYITKAYGKHDKK(SEQ ID NO:19).

[0083] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0084] Example 1: Preparation of an artificial photocatalytic system

[0085] I. Steps

[0086] (1) After codon optimization, the formate dehydrogenase (cbFDH) and allophycocyanin α subunit (APC-α) encoding genes were used to construct a fusion fragment cbFDH-APC-α (amino acid sequence as shown in SEQ ID NO:5, nucleotide sequence as shown in SEQ ID NO:6) using genetic engineering. The formate dehydrogenase (cbFDH) was located at the N-terminus of the fusion fragment, the N-terminus of the fusion fragment had an HIS tag, and there was no linker between the two genes. Subsequently, the fusion fragment cbFDH-APC-α was cloned into the pET28a vector to construct the pET28a-cbFDH-APC-α recombinant plasmid.

[0087] (2) The coding genes of CpcS and CpcU are fused to obtain the fusion fragment CpcS-CpcU; the coding genes of Ho1 and PcyA are fused to obtain the fusion fragment Ho1-PcyA; the fusion fragments CpcS-CpcU and Ho1-PcyA are inserted into the multiple cloning site 1 (restriction enzyme sites: ScaⅠ and HindⅢ) and the multiple cloning site 2 (restriction enzyme sites: NdeⅠ and XhoⅠ) of pRSF-Duet-1, respectively, to obtain the recombinant vector pRSF-Duet-CpcS-CpcU-Ho1-PcyA;

[0088] (3) The protein was expressed exogenously. The specific implementation plan was as follows: pET28a-cbFDH-APC-α recombinant plasmid and pRSF-Duet-CpcS-CpcU-Ho1-PcyA were co-transformed into BL21(DE3) strain. Positive colonies were obtained by screening on solid plates containing kanamycin and streptomycin. Single positive colonies were picked and cultured in 5 mL LB medium containing antibiotics for seed culture. Subsequently, the culture was expanded in 500 mL LB medium and cultured at 37℃ with shaking until the optical density at 600 nm was 0.8. Then, the culture was induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 20℃ for 10 hours. The strain was harvested by centrifugation.

[0089] (4) Purification of target protein: The bacterial cells were collected by centrifugation, resuspended in 25mM Tris and then broken. The cell lysis supernatant was collected, filtered through a 0.45μm filter membrane, and then purified using a gravity column loaded with nickel column packing. The fusion protein was obtained by elution with 500mM imidazole.

[0090] (5) Purity identification of fusion protein: The purity and subunit composition of the protein were analyzed by SDS-PAGE and native-PAGE. The purified protein solution was dialyzed into 50 mM Tris-HCl pH 7.5 for later use.

[0091] (6) Assay of fusion protein activity and photocatalytic performance: 2 mM NADH was added to the fusion protein solution, carbon dioxide was bubbled through until saturation, and the reaction was carried out at a constant temperature of 37℃. The amount of formic acid produced was then measured. + In the presence of carbon dioxide, the mixture was saturated and then irradiated with 620nm visible light to detect the amount of formic acid produced and the amount of oxygen produced.

[0092] II. Results

[0093] SDS-PAGE and native-PAGE analyses showed that the fusion protein in Example 1 had high purity and was homogeneous in solution (e.g., ...). Figure 1 As shown); the UV spectral scan results of the artificial photocatalytic system in Example 1 before and after photoinduced NADH regeneration are as follows. Figure 2 The phycocyanin domain in the fusion protein is responsive to visible light, enabling rapid reduction and regeneration of NADH under illumination, with a regeneration rate of 80% after 30 minutes of light reaction. With the addition of NDA+, the fusion enzyme system catalyzes its reduction to NADH, which then acts as a functional cofactor for formate dehydrogenase, mediating the reduction of CO2 to formic acid. A 1 mg / mL fusion protein produces 140 μM of formic acid after half an hour of catalytic reaction. In contrast, the formate dehydrogenase control group without phycocyanin... Figure 3 (represented by FDH, with the same concentration as the fusion protein), due to the lack of the cofactor NADH, it cannot produce formic acid (e.g. Figure 3 As shown in the figure, because CpcS-CpcU mediates the binding of cbFDH-APC-α to phycocyanin, and phycocyanin has a very small molecular weight, the actual size of the fusion protein is reflected in the size of cbFDH-APC-α (the fusion protein in the figure is represented by FDH-APC).

[0094] Example 2: Preparation of an artificial photocatalytic system

[0095] I. Steps

[0096] (1) After codon optimization, the formate dehydrogenase (cbFDH) and allophycocyanin α subunit (APC-α) encoding genes were used to construct a fusion fragment cbFDH-APC-α (amino acid sequence as shown in SEQ ID NO:5, nucleotide sequence as shown in SEQ ID NO:6) using genetic engineering. The formate dehydrogenase (cbFDH) was located at the N-terminus of the fusion fragment, the N-terminus of the fusion fragment had an HIS tag, and there was no linker between the two genes. Subsequently, the fusion fragment cbFDH-APC-α was cloned into the pET3a vector to construct the pET3a-cbFDH-APC-α recombinant plasmid.

[0097] (2) The coding genes of CpcS and CpcU are fused to obtain the fusion fragment CpcS-CpcU; the coding genes of Ho1 and PcyA are fused to obtain the fusion fragment Ho1-PcyA; the fusion fragments CpcS-CpcU and Ho1-PcyA are inserted into the multiple cloning site 1 (restriction enzyme sites: ScaⅠ and HindⅢ) and the multiple cloning site 2 (restriction enzyme sites: NdeⅠ and XhoⅠ) of pRSF-Duet-1, respectively, to obtain the recombinant vector pRSF-Duet-CpcS-CpcU-Ho1-PcyA;

[0098] (3) The protein was expressed exogenously. The specific implementation plan was as follows: the recombinant plasmids pET3a-cbFDH-APC-α and pRSF-Duet-CpcS-CpcU-Ho1-PcyA were co-transformed into the BL21(DE3) strain. Positive colonies were screened using solid plates containing kanamycin and streptomycin. Single colonies were picked and cultured in 5 mL LB medium containing antibiotics for seed culture. Subsequently, the culture was expanded in 500 mL LB medium and cultured at 37℃ with shaking until the optical density at 600 nm was 0.8. Then, the culture was induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 20℃ for 10 hours. The strain was harvested by centrifugation.

[0099] (4) Purification of target protein: The bacterial cells were collected by centrifugation, resuspended in 25mM Tris and then broken. The cell lysis supernatant was collected, filtered through a 0.45μm filter membrane, and then purified using a gravity column loaded with nickel column packing. The fusion protein was obtained by elution with 500mM imidazole.

[0100] (5) Purity identification of fusion protein: The purity and subunit composition of the protein were analyzed by SDS-PAGE and native-PAGE. The purified protein solution was dialyzed into 50 mM Tris-HCl pH 7.5 for later use.

[0101] (6) Assay of fusion protein activity and photocatalytic performance: 2 mM NADH was added to the fusion protein solution, carbon dioxide was bubbled through until saturation, and the reaction was carried out at a constant temperature of 37℃. The amount of formic acid produced was then measured. + In the presence of carbon dioxide, the mixture was saturated and then irradiated with 620nm visible light to detect the amount of formic acid produced and the amount of oxygen produced.

[0102] II. Results

[0103] SDS-PAGE and native-PAGE analyses showed that the fusion protein in Example 2 had high purity and was homogeneous in solution, similar to that in Example 1. Figure 1 The results shown are similar; the UV spectral scans of the artificial photocatalytic system in Example 1 before and after photoinduced NADH regeneration are also similar to those in Example 1. Figure 2 Similar results were observed; the phycocyanin domain in the fusion protein responded to visible light, enabling rapid reduction and regeneration of NADH under illumination. With the addition of NDA+, the fusion enzyme system catalyzed its reduction to NADH, which then acted as a functional cofactor for formate dehydrogenase, mediating the reduction of CO2 to HCOOH (similar to Example 1). Figure 3 The results shown are similar.

[0104] Example 3: Preparation of an artificial photocatalytic system

[0105] I. Steps

[0106] (1) After codon optimization, the formate dehydrogenase (cbFDH) and allophycocyanin α subunit (APC-α) encoding genes were used to construct a fusion fragment cbFDH-APC-α (amino acid sequence as shown in SEQ ID NO:5, nucleotide sequence as shown in SEQ ID NO:6) using genetic engineering. The formate dehydrogenase (cbFDH) was located at the N-terminus of the fusion fragment, the N-terminus of the fusion fragment had an HIS tag, and there was no linker between the two genes. Subsequently, the fusion fragment cbFDH-APC-α was cloned into the pET28a vector to construct the pET28a-cbFDH-APC-α recombinant plasmid.

[0107] (2) The coding genes of CpcS and CpcU are fused to obtain the fusion fragment CpcS-CpcU; the coding genes of Ho1 and PcyA are fused to obtain the fusion fragment Ho1-PcyA; the fusion fragments CpcS-CpcU and Ho1-PcyA are inserted into the multiple cloning site 1 (restriction enzyme sites: ScaⅠ and HindⅢ) and the multiple cloning site 2 (restriction enzyme sites: NdeⅠ and XhoⅠ) of pCDFDuet-1 to obtain the recombinant vector pCDF-Duet-1-CpcS-CpcU-Ho1-PcyA;

[0108] (3) The protein was expressed exogenously. The specific implementation plan was as follows: the pET28a-cbFDH-APC-α recombinant plasmid and pCDF-Duet-CpcS-CpcU-Ho1-PcyA were co-transformed into the BL21(DE3) strain. Positive colonies were screened using solid plates containing kanamycin and ampicillin. Single colonies were picked and cultured in 5 mL LB medium containing antibiotics to start a small-scale culture. Subsequently, the culture was expanded in 500 mL LB medium and cultured at 37℃ with shaking until the optical density at 600 nm was 0.8. Then, the culture was induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 20℃ for 10 hours. The strain was harvested by centrifugation.

[0109] (4) Purification of target protein: The bacterial cells were collected by centrifugation, resuspended in 25mM Tris and then broken. The cell lysis supernatant was collected, filtered through a 0.45μm filter membrane, and then purified using a gravity column loaded with nickel column packing. The fusion protein was obtained by elution with 500mM imidazole.

[0110] (5) Purity identification of fusion protein: The purity and subunit composition of the protein were analyzed by SDS-PAGE and native-PAGE. The purified protein solution was dialyzed into 50 mM Tris-HCl pH 7.5 for later use.

[0111] (6) Assay of fusion protein activity and photocatalytic performance: 2 mM NADH was added to the fusion protein solution, carbon dioxide was bubbled through until saturation, and the reaction was carried out at a constant temperature of 37℃. The amount of formic acid produced was then measured. + In the presence of carbon dioxide, the mixture was saturated and then irradiated with 620nm visible light to detect the amount of formic acid produced and the amount of oxygen produced.

[0112] II. Results

[0113] SDS-PAGE and native-PAGE analyses showed that the fusion protein in Example 3 had high purity and was homogeneous in solution, similar to that in Example 1. Figure 1 The results shown are similar; the UV spectral scans of the artificial photocatalytic system in Example 1 before and after photoinduced NADH regeneration are also similar to those in Example 1. Figure 2 Similar results were observed; the phycocyanin domain in the fusion protein responded to visible light, enabling rapid reduction and regeneration of NADH under illumination. With the addition of NDA+, the fusion enzyme system catalyzed its reduction to NADH, which then acted as a functional cofactor for formate dehydrogenase, mediating the reduction of CO2 to HCOOH (similar to Example 1). Figure 3 The results shown are similar.

[0114] Comparative Example 1 with Connector 3

[0115] I. Steps

[0116] After codon optimization, a fusion fragment cbFDH-Linker 3-APC-α was constructed using genetic engineering techniques to encode the formate dehydrogenase (cbFDH) and allophycocyanin α subunit (APC) genes. The amino acid sequence of Linker 3 is shown in SEQ ID NO:9. Subsequent steps were the same as in Example 1 to obtain the fusion protein.

[0117] II. Results

[0118] In Comparative Example 1, the expression level of the fusion protein was reduced, and the protein stability was decreased. Under both SDS-PAGE and solution conditions, the fusion protein showed partial depolymerization and no photocatalytic activity.

[0119] Comparative Example 2 with Connector 4

[0120] I. Steps

[0121] After codon optimization, a fusion fragment cbFDH-Linker 4-APC-α was constructed using genetic engineering techniques to encode the formate dehydrogenase (cbFDH) and allophycocyanin α subunit (APC) genes. The amino acid sequence of Linker 4 is shown in SEQ ID NO:10. Subsequent steps were the same as in Example 1 to obtain the fusion protein.

[0122] II. Results

[0123] In Comparative Example 2, the expression level of the fusion protein was reduced, and the synthesis rate of phycocyanin in the protein was decreased. This was due to changes in the three-dimensional structure and conformation of the fusion protein, which had no photocatalytic activity.

[0124] Comparative example 3APC truncation

[0125] I. Steps

[0126] Twenty amino acids were removed from the N-terminus of the α subunit of APC to obtain the APC-α-20 truncated variant, the amino acid sequence of which is shown in SEQ ID NO:18. After codon optimization, the formate dehydrogenase (cbFDH) and APC-α-20 truncated variant gene fusion fragment cbFDH-APC-α-20 was constructed using genetic engineering. Subsequent steps were the same as in Example 1 to obtain the fusion protein.

[0127] II. Results

[0128] The fusion protein constructed in Comparative Example 3 had a low expression level, and the synthesis rate of phycocyanin in the protein was reduced, indicating that sequence truncation affected the three-dimensional conformation of the protein and had no photocatalytic activity.

[0129] Comparative Example 4: Was it codon optimized?

[0130] I. Steps

[0131] The formate dehydrogenase (cbFDH) and allophycocyanin α subunit (APC-α) encoding genes were not codon optimized, and the subsequent steps were the same as in Example 1 to obtain the fusion protein.

[0132] II. Results

[0133] SDS-PAGE and native-PAGE analyses showed that the expression level of the sequence-optimized fusion protein was significantly increased compared to the original codon.

[0134] Comparative Example 5: Photocatalytic system with only cbFDH and APC

[0135] I. Steps

[0136] The pET28a-cbFDH-APC-α recombinant plasmid (construction of pET28a-cbFDH-APC-α as described in Example 1) was transformed into the BL21(DE3) strain. Positive colonies were screened using solid plates containing both kanamycin and streptomycin. Single colonies were picked and cultured in 5 mL LB medium containing antibiotics for seed culture. Subsequently, the culture was expanded in 500 mL LB medium and cultured at 37°C with shaking until the optical density at 600 nm reached 0.8. Then, induction was performed using 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 20°C for 10 hours. The strain was harvested by centrifugation. Subsequent steps were the same as in Example 1.

[0137] II. Results

[0138] The fusion protein in Comparative Example 5 could be expressed normally, but due to the lack of phycocyanin synthase and lyase, an optically active fusion protein could not be obtained.

[0139] Comparative Example 6: Photocatalytic system with only CpcS, CpcU, Ho1, and PcyA

[0140] I. Steps

[0141] Only PCDF-Duet-CpcS-CpcU-Ho1-PcyA (constructed as in Example 1) was transformed into the BL21(DE3) strain. Positive colonies were obtained by screening on solid plates containing both kanamycin and streptomycin. Single positive colonies were picked and cultured in 5 mL LB medium containing antibiotics for seed culture. Subsequently, the culture was expanded in 500 mL LB medium and cultured at 37°C with shaking until the optical density at 600 nm reached 0.8. Then, induction was performed with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 20°C for 10 hours. The strain was harvested by centrifugation. Subsequent steps were the same as in Example 1.

[0142] II. Results

[0143] In this expression system, the four proteins CpcS-CpcU-Ho1-PcyA can be expressed normally, and E. coli can synthesize phycocyanin (the cell lysate after induction is blue). However, due to the lack of the cbFDH-APC-α protein backbone, CpcS and CpcU lyases cannot catalyze the covalent binding of phycocyanin to the fusion protein in the system, so an active biocatalytic enzyme cannot be obtained.

[0144] Optimization of induction temperature in Comparative Example 7

[0145] I. Steps

[0146] The recombinant plasmid pET28a-cbFDH-APC-α and PCDF-Duet-CpcS-CpcU-Ho1-PcyA were co-transformed into strain BL21(DE3) (the construction of pET28a-cbFDH-APC-α and PCDF-Duet-CpcS-CpcU-Ho1-PcyA is as described in Example 1). Positive colonies were obtained by screening on solid plates containing both kanamycin and streptomycin. Single positive colonies were picked and cultured in 5 mL LB medium containing antibiotics for seed culture. Subsequently, the culture was expanded in 500 mL LB medium and cultured at 37°C with shaking until the optical density at 600 nm reached 0.8. Then, induction was performed with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C for 10 hours. The strain was harvested by centrifugation. Subsequent steps were the same as in Example 1.

[0147] II. Results

[0148] SDS-PAGE analysis revealed that the protein was expressed as an inclusion body and had no photocatalytic activity.

[0149] Comparative Example 8: Phycocyanin α subunit replaced with β subunit

[0150] I. Steps

[0151] After codon optimization, a fusion fragment cbFDH-APC-β was constructed using genetic engineering techniques to encode the formate dehydrogenase (cbFDH) and allophycocyanin α subunit (APC-β) genes. The formate dehydrogenase (cbFDH) gene was located at the N-terminus of the fusion fragment, which had an HIS tag, and there was no linker between the two genes. The cbFDH-APC-β fusion fragment was then cloned into the pET28a vector to construct the pET28a-cbFDH-APC-β recombinant plasmid. Subsequent steps were the same as in Example 1 to obtain the fusion protein.

[0152] II. Results

[0153] Since the product of CpcS-CpcU-Ho1-PcyA is a small molecule, the actual size of the fusion protein is based on cbFDH-APC-β (cbFDH-APC-α). SDS-PAGE and native-PAGE analyses showed that the fusion protein containing cbFDH-APC-β (FDH-β) had lower purity after purification compared to the fusion protein containing cbFDH-APC-α (FDH-α), and at the same concentration, the protein solution was a lighter blue, indicating insufficient fusion of the pigment molecules and lack of photocatalytic activity. Figure 4 and Figure 5 As shown.

[0154] Comparative Example 9: Connection order of APC-α and cbFDH

[0155] I. Step (1) After codon optimization of the formate dehydrogenase (cbFDH) and allophycocyanin α subunit (APC-α) encoding genes, a fusion fragment APC-α-cbFDH of the formate dehydrogenase (cbFDH) and allophycocyanin α subunit (APC-α) genes was constructed by genetic engineering. The allophycocyanin α subunit (APC-α) is located at the N-terminus of the fusion fragment, the C-terminus of the fusion fragment has an HIS tag, and there is no linker between the two genes. Subsequently, the fusion fragment cbFDH-APC-α was cloned into the pET28a vector to construct the pET28a-cbFDH-APC-α recombinant plasmid.

[0156] II. Results

[0157] SDS-PAGE analysis showed that the fusion protein after the sequence exchange was expressed as an inclusion body and had no photocatalytic activity.

[0158] In addition, the results of protein expression and photocatalytic activity of the fusion proteins in the examples and comparative examples were summarized and compared, and the comparison results are shown in Table 1.

[0159] Table 1. Expression and photocatalytic activity of fusion proteins in examples and comparative examples

[0160]

[0161]

[0162] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fusion protein, characterized in that, include: Formate dehydrogenase and phycobiliprotein are connected sequentially from the N-terminus to the C-terminus; The phycobiliproteins include: the α subunit of phycocyanin and / or the α subunit of allophycocyanin.

2. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the phycobiliprotein is shown in SEQ ID NO:17; the amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO:

19.

3. The fusion protein according to claim 1 or 2, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:

5.

4. A photocatalytic bioenzyme, characterized in that, The fusion protein according to any one of claims 1 to 3 and phycocyanin were obtained by catalysis of phycocyanin lyase. The phycocyanin lyase includes phycocyanin lyase CpcS and lyase CpcU.

5. The photocatalytic bioenzyme according to claim 4, characterized in that, The phycocyanin was obtained by expressing heme oxygenase and biliverdin reductase in Escherichia coli.

6. The photocatalytic bioenzyme according to claim 4 or 5, characterized in that, The amino acid sequence of the heme oxygenase is shown in SEQ ID NO:1; The amino acid sequence of the biliverdin reductase is shown in SEQ ID NO:2; The amino acid sequence of the phycocyanin lysin CpcS is shown in SEQ ID NO:3; The amino acid sequence of the phycocyanin lysin CpcU is shown in SEQ ID NO:

4.

7. A biomaterial, characterized in that, Including at least one of the following: i) to v) i) Nucleic acid encoding the fusion protein of claim 1 or 2 and / or the photocatalytic enzyme of any one of claims 3 to 6; ii) Expression units containing the nucleic acids described in i); iii) Recombinant vectors integrating nucleic acids as described in i) and / or expression units as described in ii); iv) Transfecting or transforming host cells with the recombinant vector described in iii); v) Products obtained by culturing and / or inducing host cells as described in iv).

8. The biomaterial according to claim 7, characterized in that, The nucleotide sequence of the nucleic acid encoding the phycobiliprotein is shown in SEQ ID NO:15; the nucleotide sequence of the nucleic acid encoding the formate dehydrogenase is shown in SEQ ID NO:

16.

9. The biomaterial according to claim 7, characterized in that, The induction temperature is 18–22°C.

10. The application of at least one of the following shown in a) to c) in biophotocatalysis: a) Encoding the fusion protein as described in claim 1 or 2; b) The photocatalytic bioenzyme according to any one of claims 3 to 6; c) The biomaterial according to any one of claims 7 to 9.

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