Reasonable copolymerization strategy for improving oligomeric structure stability of acid-induced high-order oligomeric decarboxylase AdiA in neutral to alkaline environment and application of rational copolymerization strategy

Abstract

The invention discloses a rational copolymerization strategy for improving the stability of an oligomeric structure of acid-induced high-order oligomeric decarboxylase AdiA in a neutral to alkaline environment and application of the rational copolymerization strategy, and belongs to the field of bioengineering. According to the strategy, rational charge overturning transformation is carried out on a pentamer radial oligomeric interface and a dimer weft-wise oligomeric interface at the same time, and stable assembly and efficient catalysis of a decamer under the condition that the pH value is from 7.0 to 9.0 from neutral to alkaline are achieved. Wherein positive charges are introduced into a meridian interface to weaken electrostatic repulsion, and negative charges are introduced into a latitudinal interface to enhance dimer compactness and substrate transfer efficiency. The specific enzyme activity of the representative double mutant AdiAD471K / E467K / H736E is 45.5 times that of a wild type when the pH value is 8.0, and the representative double mutant AdiAD471K / E467K / H736E keeps a complete decamer state when the pH value is 7.0-9.0. The yield of butanediamine is up to 156.5 g / L by using the mutant to catalyze whole cells.

Description

Technical Field

[0001] This invention relates to a rational copolymerization strategy for improving the stability of the oligomeric structure of acid-induced higher-order oligomeric decarboxylase AdiA in neutral to alkaline environments and its application, belonging to the field of bioengineering. Background Technology

[0002] Homo oligomerization of proteins is a common phenomenon in natural evolution, with about half of all proteins existing in the form of homooligomers. These homooligomers have wide applications in physiological regulation, medicine, and chemical engineering, such as alkaline amino acid decarboxylases and tyrosine tRNA synthetases. Homooligomeric proteins are usually predominantly dimers or tetramers, while higher-order oligomers are relatively rare. Their oligomerization is crucial for structural stability and functional activity, and is maintained through covalent or non-covalent bonds at the monomer interfaces.

[0003] Arginine decarboxylase AdiA is a crucial member of the *E. coli* acid stress response system. It is a typical decameric high-order homologous oligomeric protein, consisting of five dimers forming a bilayer pentacyclic structure. This enzyme catalyzes the decarboxylation of L-arginine to produce guanidine, a key enzyme in the biosynthesis of putrescine (an important nylon monomer). However, the assembly of the AdiA decameric structure is highly pH-dependent, remaining stable only under acidic conditions (pH < 6.0). This is because its surface is rich in acidic amino acids, which protonate in acidic environments to neutralize the charge and promote oligomerization. Under neutral and alkaline conditions, the acidic amino acids deprotonate, generating electrostatic repulsion, causing the decameric to depolymerize into dimers or monomers, resulting in a significant loss of enzyme activity. This pH dependence severely limits the whole-cell catalytic application of AdiA in neutral or alkaline environments. Since the intracellular environment in industrial fermentation is typically neutral, and the putrescine product is alkaline, conventional AdiA cannot be efficiently used for the large-scale biosynthesis of putrescine.

[0004] Currently, various engineering strategies have been developed to promote protein oligomerization, such as regulating interfacial residue complementarity, introducing disulfide bonds or electrostatic attraction, and improving interfacial hydrophobicity. These strategies are easily implemented in dimer and tetramer proteins. However, for higher-order decadal proteins like AdiA, due to the complex interfacial interactions (involving both meridional and latitudinal interfaces) and the difficulty in molecular dynamics simulations, existing strategies struggle to effectively achieve oligomeric structural stability under moderately alkaline conditions. Furthermore, other acid-inducible decarboxylases, such as lysine decarboxylase CadA, also exhibit similar pH-regulating mechanisms, limiting the efficient biosynthesis of polyamine compounds.

[0005] Therefore, there is an urgent need to develop a new rational engineering strategy to precisely modify the interface of higher-order oligomeric decarboxylases, so as to shift the pH adaptability of the enzyme towards neutral and alkaline conditions, thereby improving the catalytic efficiency of the enzyme and promoting the green and large-scale production of polyamine compounds such as butanediamine. Summary of the Invention

[0006] The purpose of this invention is to solve the problem of inhibited arginine decarboxylase (AdiA) activity under near-neutral conditions, and thus to address the low synthesis rate of butanediamine in near-neutral environments. This invention provides an arginine decarboxylase mutant with optimal pH upregulation, wherein the combined mutant AdiA... D471K / E467K / H736E The enzyme activity at pH 7.0 was 45.5 times higher than that of AdiA, making the mutant AdiA the one with the best enzyme activity. D471K / E467K / H736E It was applied to the biosynthesis of butanediamine, resulting in a butanediamine yield of 156.5 g / L.

[0007] The arginine decarboxylase derived from Escherichia coli of this invention can also be derived from... Hafnia alvei or Kosakonia calanthe s or ‌Salmonella choleraesuis or Vibrio marinisediminis ; The present invention also provides a source from Hafnia alvei The arginine decarboxylase mutant is obtained by mutating aspartic acid at position 471 to lysine and glutamic acid at position 467 of the arginine decarboxylase with NCBI number WP_111330920.1; or by mutating aspartic acid at position 471 to lysine and glutamic acid at position 467 of the arginine decarboxylase with NCBI number WP_111330920.1 to lysine and histidine at position 736 to glutamic acid.

[0008] The present invention also provides a source from Kosakonia calanthe The arginine decarboxylase mutant of s is obtained by mutating asparagine at position 471 to lysine and simultaneously mutating glutamic acid at position 467 to lysine in the arginine decarboxylase with NCBI number WP_342322096.1; or by mutating asparagine at position 471 to lysine and glutamic acid at position 467 to lysine in the arginine decarboxylase with NCBI number WP_342322096.1, and simultaneously mutating histidine at position 736 to glutamic acid.

[0009] The present invention also provides a source from Salmonella choleraesuis The arginine decarboxylase mutant is obtained by mutating glutamic acid at position 471 to lysine and simultaneously mutating glutamic acid at position 467 to lysine in the arginine decarboxylase with NCBI number WP_408603515.1.

[0010] The present invention also provides a source from Vibrio marinisediminisThe arginine decarboxylase mutant is obtained by mutating aspartic acid at position 479 to lysine and glutamic acid at position 475 to lysine in the arginine decarboxylase with NCBI number WP_182106049.1; or by mutating aspartic acid at position 479 to lysine and glutamic acid at position 475 to lysine in the arginine decarboxylase with NCBI number WP_182106049.1, and histidine at position 744 to glutamic acid.

[0011] This invention provides an arginine decarboxylase mutant, wherein the arginine decarboxylase mutant is obtained by mutating aspartic acid at position 471 of the arginine decarboxylase as shown in SEQ ID NO.1 to lysine, and simultaneously mutating glutamic acid at position 467 to lysine. Alternatively, it can be obtained by mutating aspartic acid at position 471 of the arginine decarboxylase shown in SEQ ID NO.1 to lysine, and simultaneously mutating histidine at position 736 to glutamic acid. Alternatively, it can be obtained by mutating aspartic acid at position 471 to lysine, glutamic acid at position 467 to lysine, and histidine at position 736 to glutamic acid in the arginine decarboxylase shown in SEQ ID NO.1.

[0012] AdiA D471K / E467K / H736E Sequence (SEQ ID NO.3) MKVLIVESEFLHQDTWVGNAVERLADALSQQNVTVIKSTSFDDGFAILSSNEAIDCLMFSYQMEHPDEHQNVRQLIGKLHERQQNVPVFLLGDREKALAAMDRDLLELVDEFAWIL EDTADFIAGRAVAAMTRYRQQLLPPLFSALMKYSDIHEYSWAAPGHQGGVGFTKTPAGRFYHDYYGENLFRTDMGIERTSLGSLLDHTGAFGESEKYAARVFGADRSWSVVVGTSGS NRTIMQACMTDNDVVVVDRNCHKSIEQGLMLTGAKPVYMVPSRNRYGIIGPIYPQEMQPETLQKKISESPLTKDKAGQKPSYCVVTNCTYDGVCYNAKEAQDLLEKTSDRLHFDEA WYGYARFNPIYADHYAMRGEPGDHNGPTVFATHSTHKLLNALSQASYIHVREGRGAINFSRFNQAYMMHATTSPLYAICASNDVAVSMMDGNSGLSLTQEVIDEAVDFRQAMARLYK K FTA K GSWFFKPWNKEVVTDPQTGKTYDFADAPTKLLTTVQDCWVMHPGESWHGFKDIPDNWSMLDPIKVSILAPGMGEDGELEETGVPAALVTAWLGRHGIVPTRTTDFQIMFLFSMGVTRGKWGTLVNTLCSFKR HYDANTPLAQVMPELVEQYPDTYANMGIHDLGDTMFAWLKENNPGARLNAAYSGLPVAEVTPREAYNAIVDNNVELVSIENLPGRIAANSVIPYPPGIPMLLSGENFGDKNSPQVSYLRSLQSWDHHFPGFE E ETEGTEIIDGIYHVMCVKA The present invention also provides a gene encoding the above-mentioned arginine decarboxylase mutant or a recombinant vector carrying the gene.

[0013] The present invention also provides a recombinant cell expressing the above-mentioned mutant, the above-mentioned gene, or the recombinant vector.

[0014] In one embodiment of the present invention, the recombinant cells are bacteria or fungi as host cells.

[0015] The present invention also provides a recombinant enzyme catalyst containing the above-mentioned arginine decarboxylase mutant, which is any one of the following forms: (1) Culture recombinant expression transformants containing the arginine decarboxylase mutant and isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme; (2) Cultivate recombinant expression transformants containing the arginine decarboxylase mutant, isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme, and break the transformant cells containing the recombinant arginine decarboxylase mutant enzyme to obtain cell lysate. (3) Cultivate recombinant expression transformants containing the arginine decarboxylase mutant, isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme, break the transformant cells containing the recombinant arginine decarboxylase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant arginine decarboxylase mutant enzyme to obtain lyophilized enzyme powder.

[0016] This invention also provides a method for improving the enzyme activity of arginine decarboxylase under neutral and alkaline conditions or for increasing the yield of butanediamine prepared by arginine decarboxylase. The method is that the arginine decarboxylase mutant is formed by mutating aspartic acid at position 471 of the arginine decarboxylase as shown in SEQ ID NO.1 to lysine, and simultaneously mutating glutamic acid at position 467 to lysine. Alternatively, the aspartic acid at position 471 of the arginine decarboxylase, as shown in SEQ ID NO.1, may be mutated to lysine, and the histidine at position 736 may be mutated to glutamic acid. Alternatively, the amino acid sequence of arginine decarboxylase, as shown in SEQ ID NO.1, may be modified by mutating aspartic acid at position 471 to lysine, glutamic acid at position 467 to lysine, and histidine at position 736 to glutamic acid.

[0017] The present invention also provides the application of the above-mentioned mutant, recombinant cell, or recombinase catalyst in the preparation of butanediamine, wherein the reaction system in the preparation of butanediamine includes pyridoxal phosphate (PLP) and arginine.

[0018] The present invention also provides a genetically engineered bacterium that expresses the above-mentioned arginine decarboxylase mutant, arginine decarboxylase SpeA (SEQ ID NO.6), and guanidine aminoase SpeB (SEQ ID NO.4).

[0019] In one embodiment of the present invention, the genetically engineered bacteria include, but are not limited to, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and yeast.

[0020] The present invention also provides a method for synthesizing butanediamine, wherein the method comprises using arginine as a substrate and employing the above-mentioned genetically engineered bacteria or its lysate or fermentation broth as a catalyst to catalytically convert and synthesize butanediamine.

[0021] In one embodiment of the present invention, the amount of arginine added is 300~400 g / L, and the amount of genetically engineered bacteria added is 80~100 g wet weight / L. In one embodiment of the present invention, the reaction system further contains PLP at a concentration of 0.08~0.15 mmol / L; In one embodiment of the present invention, the reaction conditions are a temperature of 35~45℃, a rotation speed of 200~400 r / min, and an initial pH of 7.0.

[0022] The present invention also provides the application of the above-mentioned genetically engineered bacteria or the above-mentioned method in the preparation of butanediamine or products containing butanediamine or in the preparation of nylon products.

[0023] The nylon products include, but are not limited to, nylon 46, nylon 410, and nylon 4T.

[0024] Beneficial effects Following the rational copolymerization modification strategy described in this invention, the meridional interface and zonal channel interface of arginine decarboxylase were rationally modified, either separately or simultaneously, resulting in a series of mutants. The optimal mutant, AdiA, obtained using the dual-interface synergistic modification method of this invention is... D471K / E467K / H736E The specific enzyme activity at pH 8.0 reached 45.5 times that of the wild type, and maintained high activity throughout the entire neutral-alkaline range of pH 7.0-9.0. The optimal pH of the arginine decarboxylase mutant obtained in this invention is increased, and the enzyme activity stability in the neutral pH range is improved, making it more suitable for the conditions required for industrial microbial fermentation and laying the foundation for the efficient synthesis of butanediamine. Attached Figure Description

[0025] Figure 1 Arginine is converted to guanidine by arginine decarboxylase. Detailed Implementation

[0026] The culture media involved in the following examples: LB solid culture: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and 2 g / L agar powder.

[0027] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0028] The detection methods involved in the following embodiments are as follows: The method for detecting arginine decarboxylase activity is as follows: Prepare a 200 µL enzyme activity reaction system according to Table 1 to determine the arginine decarboxylase activity. The reaction system was precisely reacted at 37°C for 10 min, and the reaction was terminated by adding 20 µL of 40% trichloroacetic acid. The reaction solution was then cooled in an ice-water bath. After centrifugation at 12000 r / min for 10 min, the supernatant was collected, derivatized, and used for HPLC analysis to determine the butanediamine yield. The unit enzyme activity was defined as U / g = 1 mol butanediamine / min / g protein.

[0029] Table 1: Reaction system for arginine decarboxylase activity detection

[0030] The 15 mmol / L potassium sodium phosphate buffer solution is prepared as follows: the pH value of 15 mmol / L KH2PO4 is adjusted to the corresponding pH value using 15 mmol / L Na2HPO4.

[0031] HPLC detection of butanediamine 1) Sample derivatization and extraction The pre-column derivatization procedure for dansyl chloride is as follows: After centrifuging the fermentation broth or reaction solution at 12000 r / min for 10 min, take 500 μL of the supernatant as the sample. Add 500 μL of saturated NaHCO3 solution and internal standard (5 μL of 10 g / L heptamethylenediamine), mix thoroughly, and adjust the pH to 10 with saturated NaOH solution. Then add 1 mL of the derivatization reagent dansyl chloride (5 g / L, soluble in acetone). Incubate the mixture in a light-protected water bath at 60°C for 30 min, add 2 mL of anhydrous diethyl ether, and extract for 10 min. Collect the upper organic phase. Repeat the extraction operation twice, mix the two organic phases, and dry them with nitrogen to remove the diethyl ether. Dissolve the derivatized compound in 500 μL of acetonitrile solution, filter through a 0.22 μm filter membrane, and use for HPLC detection.

[0032] 2) HPLC chromatographic determination Chromatographic conditions: High-performance liquid chromatography (HPLC) separation of diamine dansyl chloride derivatives was performed on a C18 column at a separation temperature of 30°C and a UV detection wavelength of 254 nm. The injection volume was 10 μL. Mobile phase A was ultrapure water, and mobile phase B was HPLC-grade acetonitrile. Both mobile phases were filtered through a 0.22 μm filter before use. The gradient elution program was set as follows: 0–4 min, 55%–70% B; 4–6 min, 70% B; 6–11 min, 70% B; 11–12 min, 95% B; 12–13 min, 95% B; 13–16 min, 55% B. The total flow rate was set to 0.7 mL / min.

[0033] Example 1: Determination of the AdiA mutation site in arginine decarboxylase The specific steps are as follows: Arginine decarboxylase AdiA catalyzes the synthesis of guanidine from arginine and is a key enzyme in the butanediamine ADC synthesis pathway. Figure 1 Its catalytic reaction requires the participation of pyridoxal phosphate (PLP) to assist in decarboxylation and proton transfer in the reaction, but does not consume PLP. The effective quaternary structure of AdiA is a decameric, but the decameric can only form in an environment with pH < 6, resulting in its optimal reaction pH being slightly acidic (pH 5.4). Based on the crystal structure of the AdiA decameric (PDB ID: 2VYC), interface analysis was performed using PyMOL 2.5 software. The AdiA decameric consists of a bilayer pentacyclic structure composed of five dimers, exhibiting centrosymmetry. The meridional interface is located between the pentacyclic monomers, mainly involving the acidic amino acid enrichment region, and generates significant electrostatic repulsion under moderately alkaline conditions. The latitudinal interface is located between the dimers and simultaneously forms substrate channels. The substrate channels were predicted using the CAVER tool, directly affecting the binding and transport of the substrate arginine. The AdiA surface is rich in acidic amino acids, resulting in the decameric being stable only at pH < 6.0. Figure 1 ).

[0034] Based on the above research, D471, G205, H736, and E467 were identified as mutation sites for arginine decarboxylase AdiA.

[0035] Example 2: Preparation of Arginine Decarboxylase AdiA Mutant The specific steps are as follows: Through primers adiA -F and adiA -R from E. coli Amplification of arginine decarboxylase gene in the BL21(DE3) genome adiA ,use Nde I and Xho I performed double digestion of the PCR product and pETDuet-1 plasmid, and after purification, the product was ligated using T4 DNA ligase. adiA The ligation product was introduced into E. coli JM109 and screened by colony PCR and Sanger sequencing to finally obtain the recombinant plasmid pETDuet-adiA.

[0036] The amino acid sequence (SEQ ID NO.1) of the wild-type arginine decarboxylase is as follows: MKVLIVESEFLHQDTWVGNAVERLADALSQQNVTVIKSTSFDDGFAILSSNEAIDCLMFSYQMEHPDEHQNVRQLIGKLHERQQNVPVFLLGDREKALAAMDRDLLELVDEFAWILEDTADFIAGRAVAAMTRYRQQLLPPLFSALMKYSDIHEYSWAAPGHQGGVGFTKTPAGRFYHDYYGENLFRTDMGIERTSLGSLLDHTGAFGESEKYAARVFGADRSWSVVVGTSGSNRTIMQACMTDNDVVVVDRNCHKSIEQGLMLTGAKPVYMVPSRNRYGIIGPIYPQEMQPETLQKKISESPLTKDKAGQKPSYCVVTNCTYDGVCYNAKEAQDLLEKTSDRLHFDEAWYGYARFNPIYADHYAMRGEPGDHNGPTVFATHSTHKLLNALSQASYIHVREGRGAINFSRFNQAYMMHATTSPLYAICASNDVAVSMMDGNSGLSLTQEVIDEAVDFRQAMARLYKEFTADGSWFFKPWNKEVVTDPQTGKTYDFADAPTKLLTTVQDCWVMHPGESWHGFKDIPDNWSMLDPIKVSILAPGMGEDGELEETGVPAALVTAWLGRHGIVPTRTTDFQIMFLFSMGVTRGKWGTLVNTLCSFKRHYDANTPLAQVMPELVEQYPDTYANMGIHDLGDTMFAWLKENNPGARLNAAYSGLPVAEVTPREAYNAIVDNNVELVSIENLPGRIAANSVIPYPPGIPMLLSGENFGDKNSPQVSYLRSLQSWDHHFPGFEHETEGTEIIDGIYHVMCVKA The nucleotide sequence encoding the arginine decarboxylase (SEQ ID NO.2) is as follows: catcatc atcatcatcattaa The arginine decarboxylase gene is expressed in Escherichia coli BL21 with a 6×His tag at the N-terminus. The bold underlined part in the above nucleotide sequence is the histidine tag.

[0037] pETDuet-adiA was subjected to full-plasmid PCR using primers D471K-F and D471K-R. The PCR product was purified and then used. Dpn The bacteria were digested with enzyme I, and the digested product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain adiA. D471K Mutant recombinant plasmid pETDuet-adiA D471K .

[0038] pETDuet-adiA was subjected to full-plasmid PCR using primers D471H-F and D471H-R. The PCR product was purified and then used. Dpn The bacteria were digested with enzyme I, and the digested product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain adiA. D471H Mutant recombinant plasmid pETDuet-adiA D471H .

[0039] pETDuet-adiA was induced using primers G205E-F and G205E-R. D471K Perform whole-plasmid PCR, and use the PCR products after purification. Dpn The bacteria were digested with enzyme I, and the digested product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain adiA. D471K / G205E Mutant recombinant plasmid pETDuet-adiA D471K / G205E .

[0040] pETDuet-adiA was induced using primers H736E-F and H736E-R. D471K Perform whole-plasmid PCR, and use the PCR products after purification. Dpn The bacteria were digested with enzyme I, and the digested product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain adiA. D471K / H736E The mutant recombinant plasmid pETDuet- adiA D471K / H736E .

[0041] pETDuet-adiA was induced using primers E467K-F and E467K-R. D471KPerform whole-plasmid PCR, and use the PCR products after purification. Dpn The bacteria were digested with enzyme I, and the digested product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain adiA. D471K / E467K The mutant recombinant plasmid pETDuet- adiA D471K / E467K .

[0042] pETDuet-adiA was induced using primers E467K-F and E467K-R. D471K / H736E Perform whole-plasmid PCR, and use the PCR products after purification. Dpn The bacteria were digested with enzyme I, and the digested product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain adiA. D471K / E467K / H736E Mutant recombinant plasmid pETDuet-adiA D471X / E467K / H736E .

[0043] Table 2: Primer Sequence Listing

[0044] Example 3: Expression of mutant arginine decarboxylase and purification of AdiA The specific steps are as follows: The recombinant plasmids expressing wild-type AdiA and its mutant genes, and the recombinant plasmids expressing guanidine aminotransferase SpeB gene, prepared in Example 2, were introduced into Escherichia coli BL21 to prepare recombinant strains. The AdiA and its mutant genes, and the guanidine aminotransferase SpeB gene were expressed in Escherichia coli BL21 in the form of an N-terminus with a 6×His tag.

[0045] All expression strains were cultured in 500 mL SOB medium at 37°C for 3 hours, followed by induction of protein expression with 0.1 mmol / L IPTG at 30°C for 12 hours, and then the bacterial cells were collected. The cultured bacterial cells were collected by freezing centrifugation at 8000 rpm for 5 min, resuspended in 50 mL Tris-HCl buffer, and then sonicated. The supernatant was purified by affinity chromatography using a Ni-NTA Superflow resin column.

[0046] The method for preparing the recombinant plasmid expressing the guanidine aminotransferase SpeB gene is as follows: First, the guanidine amino acid enzyme SpeB gene was ligated into the pETDuet vector by double digestion (restriction sites: NdeI, XhoI) to construct the guanidine amino acid enzyme SpeB overexpression plasmid pETDuet-SpeB. The amino acid sequence of SpeB (SEQ ID NO.4) is as follows: MSTLGHQYDNSLVSNAFGFLRLPMNFQPYDSDSDWVITGVPFDMATSGRAGGRHGPAAIRQVSTNLAWEHNRFPWNFDMRERLNVVDCGDLVYAFGDAREMSEKLQAHAEKLLAAGKRMLSFGGDHFVTLPLLRAHAKHFGKMALVHFDAHTD TYANGCEFDHGTMFYTAPKEGLIDPNHSVQIGIRTEFDKDNGFTVLDACQVNDRSVDDVIAQVKQIVGDMPVYLTFDIDCLDPAFAPGTGTPVIGGLTSDRAIKLVRGLKDLNIVGMDVVEVAPAYDQSEITALAAATLALEMLYIQAAKKGE The nucleotide sequence (SEQ ID NO.5) encoding the guanidine amino acid enzyme SpeB is shown below: atgagcaccttaggtcatcaatacgataactcactggtttccaatgcctttggttttttacgcctgccgatgaacttccagccgtatgacagcgattcagactgggtgattactggcgtgccgttcgatatggccacttctggtcgtgcgggtggtcgccacggtccggcagcgatccgtcaggtttcgacgaatctggcctgggaacacaaccgcttcccgtggaatttcgacatgcgtgagcgtctgaacgtcgtggactgcggcgatctggtatatgcctttggcgatgcccgtgagatgagcgaaaagctgcaggcgcacgccgagaagctgctggctgccggtaagcgtatgctctctttcggtggtgaccactttgttacgctgccgctgctgcgtgctcatgcgaagcatttcggcaaaatggcgctggtacactttgacgcccacaccgatacctatgcgaacggttgtgaatttgaccacggcactatgttctataccgcgccgaaagaaggtctgatcgacccgaatcattccgtgcagattggtattcgtaccgagtttgataaagacaacggctttaccgtgctggacgcctgccaggtgaacgatcgcagcgtggatgacgttatcgcccaagtgaaacagattgtgggtgatatgccggtttacctgacttttgatatcgactgcctggatcctgcttttgcaccaggcaccggtacgccagtgattggcggcctgacctccgatcgcgctattaaactggtacgcggcctgaaagatctcaacattgttgggatggacgtagtggaagtggctccggcatacgatcagtcggaaatcactgctctggcagcggcaacgctggcgctggaaatgctgtatattcaggcggcgaaaaagggcgag catcatcatcatcatcattaa The gene for the guanidine amino acid enzyme SpeB is expressed in Escherichia coli BL21 with a 6×His tag at the N-terminus. The bold underlined part in the above nucleotide sequence is the histidine tag.

[0047] Pure enzyme solutions of wild-type AdiA, AdiA mutant, and guanidine amino acid enzyme SpeB were prepared.

[0048] Example 4: Enzyme activity assay of wild-type and mutant enzymes at different pH values The specific steps are as follows: Following the reaction system in Table 1, i.e., to a potassium sodium phosphate buffer solution containing dithiothreitol, EDTA, pyridoxal phosphate (PLP), MgSO4, arginine, and guanidine aminotransferase SpeB pure enzyme solution (prepared in Example 3), the arginine decarboxylase AdiA or mutant pure enzyme solution prepared in Example 3 was added to the reaction system, with the final concentration shown in Table 1. The reaction system was reacted at 37°C for 10 min, and the reaction was terminated by adding 20 µL of 40% trichloroacetic acid. The reaction solution was cooled in an ice-water bath. After centrifugation at 12000 r / min for 10 min, the supernatant was derivatized and used for HPLC detection of butanediamine yield. The unit enzyme activity was defined as U / g = 1 mole of butanediamine / min / g protein.

[0049] The results are shown in Table 3 below: Table 3: Enzyme activity under different pH conditions

[0050] The results show: HPLC analysis revealed that the mutant AdiA D471X / E467K / H736E At pH 8.0, the enzyme activity was 45.5 times that of the wild type, and it remained elevated in the pH range of 7.0-9.0.

[0051] Example 5: Whole-cell catalytic production of butanediamine by recombinant bacteria containing mutant arginine decarboxylase The specific steps are as follows: 1. Construction of recombinant strains Through primers speA -F and speA -R amplifies the gene from the genome of Escherichia coli BL21(DE3). ​ .use ​ I and ​ I performed double digestion of the PCR product and pETDuet-1 plasmid, and after purification, the product was ligated using T4 DNA ligase. ​ The ligation product was ligated into pETDuet-1. The ligation product was introduced into E. coli JM109, and screened by colony PCR and Sanger sequencing to finally construct the plasmid pETDuet-speA.

[0052] Through primers ​ -F and ​ -R amplifies the gene from the genome of Escherichia coli BL21(DE3). ​ .

[0053] use ​ I and ​ III. The PCR product and pETDuet-speA plasmid were double-digested, and the purified products were then ligated using T4 DNA ligase. ​ Ligation was performed on pETDuet-speA. The ligation product was introduced into *E. coli* JM109, and screened by colony PCR and Sanger sequencing to finally construct the plasmid pETDuet-speA-speB. Primers were then used to... ​ -F and ​ -R from pETduet- ​ D471X / E467K / H736E amplified mutant gene ​ D471X / E467K / H736E ,use ​ I and ​ I performed double digestion of the PCR product and the pETDuet-speA-speB plasmid, and after purification, the product was ligated using T4 DNA ligase. ​ D471X / E467K / H736E The plasmid pETDuet-speA-speB was ligated into pETDuet-speA-speB, and the ligation product was introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to obtain the recombinant plasmid pETDuet-speA-. ​ D471X / E467K / H736E -speB. The recombinant plasmid pETDuet-speA- ​ D471X / E467K / H736E -speB conversion to ​ BL2(DE3) was used to obtain the butanediamine-synthesizing recombinant strain PUT1.

[0054] In the same way, using wild type ​ Gene replacement ​ D471X / E467K / H736E Gene, construct pETDuet-speA-adiA-speB recombinant plasmid, and transform it into ​ BL2(DE3) was used to obtain the butanediamine-synthesizing recombinant strain PUT0 as a control strain.

[0055] The primer sequences involved are as follows: ​ -F: CATG ​ GCTCTGACGACATGTCTATGGGTT; ​ -R:CG ​ TTACTCATCTTCAAGATAAGTATAACCG; ​ -F: GGAATTC ​ ATGAGCACCTTAGGTCATCAATACG; ​ -R:CCG ​ TTACTCGCCCTTTTTCGCC; The amino acid sequence (SEQ ID NO.6) of the arginine decarboxylase SpeA is as follows: MGSDDMSMGLPSSAGEHGVLRSMQEVAMSSQEASKMLRTYNIAWWGNNYYDVNELGHISVCPDPDVPEARVDLAQLVKTREAQGQRLPALFCFPQILQHRLRSINAAFKRARESYGYNGDYFLVYPIKVNQHRRVIESLIHSGEPLGLEAGSKAELMAVLAHAG MTRSVIVCNGYKDREYIRLALIGEKMGHKVYLVIEKMSEIAIVLDEAERLNVVPRLGVRARLASQGSGKWQSSGGEKSKFGLAATQVLQLVETLREAGRLDSLQLLHFHLGSQMANIRDIATGVRESARFYVELHKLGVNIQCFDVGGGLGVDYEGTRSQSDCSV NYGLNEYANNIIWAIGDACEENGLPHPTVITESGRAVTAHHTVLVSNIIGVERNEYTVPTAPAEDAPRALQSMWETWQEMHEPGTRRSLREWLHDSQMDLHDIHIGYSSGIFSLQERAWAEQLYLSMCHEVQKQLDPQNRAHRPIIDELQERMADKMYVNFSLFQ SMPDAWGIDQLFPVLPLEGLDQVPERRAVLLDITCDSDGAIDHYIDGDGIATTMPMPEYDPENPPMLGFFMVGAYQEILGNMHNLFGDTEAVDVFVFPDGSVEVELSDEGDTVADMLQYVQLDPKTLLTQFRDQVKKTDLDAELQQQFLEEFEAGLYGYTYLEDE The nucleotide sequence (SEQ ID NO.7) encoding the arginine decarboxylase SpeA is shown below: 2. Preparation of wet mycelium (1) After the recombinant butanediamine strains PUT0 and PUT1 stored in glycerol tubes were streaked on LB solid plates containing 50 μg / mL kanamycin, single colonies were picked and inoculated into 50 mL LB liquid medium and cultured in a shaker at 37 ℃ and 250 r / min for 12 h to prepare seed culture.

[0056] (2) The prepared seed culture was transferred to 1 L LB medium containing the same antibiotic at an inoculation rate of 2% (v / v) and cultured at 37 °C until OD. 600 The temperature was lowered to 30 °C at ≈15-20 °C, and 0.1 mmol / L IPTG was added to induce expression for 16 h to prepare the fermentation broth.

[0057] (3) After induction, the prepared fermentation broth was centrifuged at 4 ℃ and 8000 r· / min for 20 min to collect the cells. The cells were washed twice with 50 mmol / L phosphate buffer at pH 7.0 and then resuspended to obtain wet cells.

[0058] 3. Whole-cell catalytic preparation of butanediamine Whole-cell catalysis was conducted in a 5 L bioreactor with a working volume of 3 L. The reaction system composition was as follows: total L-arginine concentration of 348.4 g / L (added in batches), and wet cell concentration equivalent to OD0.05. 600 ≈80, PLP 0.1 mmol / L, temperature 42 ℃, rotation speed 300 r / min, initial pH 7.0, closed reaction, reaction time 12 h.

[0059] Samples were taken every 6 hours. After centrifugation at 12,000 r / min, the supernatant was derivatized with dansyl chloride, and the putrescine concentration was detected by HPLC.

[0060] The results are shown in Table 4 below: Table 4: Butanediamine yield of different strains

[0061] Finally, the optimal double mutant AdiA of this invention was utilized. D471K / E467K / H736E The recombinant whole-cell catalyst achieved a putrescine yield of 156.5 g / L within 12 h.

[0062] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An arginine decarboxylase mutant, characterized in that, The arginine decarboxylase mutant is obtained by mutating aspartic acid at position 471 of the arginine decarboxylase shown in SEQ ID NO.1 to lysine, and simultaneously mutating glutamic acid at position 467 to lysine. Alternatively, it can be obtained by mutating aspartic acid at position 471 of the arginine decarboxylase shown in SEQ ID NO.1 to lysine, and simultaneously mutating histidine at position 736 to glutamic acid. Alternatively, it can be obtained by mutating aspartic acid at position 471 to lysine, glutamic acid at position 467 to lysine, and histidine at position 736 to glutamic acid in the arginine decarboxylase shown in SEQ ID NO.

1.

2. The gene encoding the arginine decarboxylase mutant of claim 1 or a recombinant vector carrying the gene.

3. A recombinant cell expressing the mutant of claim 1 or carrying the gene of claim 2 or the recombinant vector, preferably, the recombinant cell is a bacterial or fungal host cell.

4. A recombinant enzyme catalyst containing the arginine decarboxylase mutant of claim 1, characterized in that, It is any of the following forms: (1) Culture recombinant expression transformants containing the arginine decarboxylase mutant and isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme; (2) Cultivate recombinant expression transformants containing the arginine decarboxylase mutant, isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme, and break the transformant cells containing the recombinant arginine decarboxylase mutant enzyme to obtain cell lysate. (3) Cultivate recombinant expression transformants containing the arginine decarboxylase mutant, isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme, break the transformant cells containing the recombinant arginine decarboxylase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant arginine decarboxylase mutant enzyme to obtain lyophilized enzyme powder.

5. A method for improving the enzyme activity of arginine decarboxylase under neutral and alkaline conditions or for increasing the yield of butanediamine prepared from arginine decarboxylase, characterized in that, The method involves mutating aspartic acid at position 471 of the arginine decarboxylase, as shown in SEQ ID NO.1, to lysine, and simultaneously mutating glutamic acid at position 467 to lysine. Alternatively, the aspartic acid at position 471 of the arginine decarboxylase, as shown in SEQ ID NO.1, may be mutated to lysine, and the histidine at position 736 may be mutated to glutamic acid. Alternatively, the amino acid sequence of arginine decarboxylase, as shown in SEQ ID NO.1, may be modified by mutating aspartic acid at position 471 to lysine, glutamic acid at position 467 to lysine, and histidine at position 736 to glutamic acid.

6. The use of the mutant of claim 1, the recombinant cell of claim 3, or the recombinase catalyst of claim 4 in the preparation of butanediamine.

7. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria expressed the arginine decarboxylase mutant, arginine decarboxylase SpeA, and guanidine aminoase SpeB as described in claim 1. Preferably, the genetically engineered bacteria include, but are not limited to, genetically engineered Escherichia coli, genetically engineered Bacillus subtilis, genetically engineered Corynebacterium glutamicum, and genetically engineered yeast. Preferably, the amino acid sequence of the arginine decarboxylase SpeA is shown in SEQ ID NO.6, and the amino acid sequence of the guanidine amino acid enzyme SpeB is shown in SEQ ID NO.

4.

8. A method for synthesizing butanediamine, characterized in that, The method involves using arginine as a substrate and the genetically engineered bacteria described in claim 7, its lysate, or its fermentation broth as a catalyst to catalytically convert and synthesize butanediamine.

9. The method according to claim 8, characterized in that, The amount of arginine added is 300~400 g / L, and the amount of genetically engineered bacteria added is 80~100 g wet weight / L; Preferably, the reaction system further contains 0.08~0.15 mmol / L of PLP; Preferably, the reaction conditions are a temperature of 35~45℃, a rotation speed of 250~300 r / min, and an initial pH of 7.

0.

10. The genetically engineered bacteria of claim 7 or the method of claim 8 or 9 is used in the preparation of butanediamine or products containing butanediamine or in the preparation of nylon products; preferably, the nylon products include, but are not limited to, nylon 46, nylon 410, and nylon 4T.

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