An arginine decarboxylase mutant and its immobilization for preparing agmatine salt

Abstract

The present invention relates to the field of biotechnology, and in particular to an arginine decarboxylase mutant and its immobilized application in preparing agmatine salts. The present invention provides a highly active arginine decarboxylase mutant, the amino acid sequence of which is obtained by site-directed mutagenesis of at least one of positions 151, 188, 193, 202, 257, 348, 387, and 740 of the wild-type arginine decarboxylase sequence as shown in SEQ ID NO: 1. The present invention purifies and immobilizes the arginine decarboxylase mutant, thereby improving the reusability and stability of the arginine decarboxylase mutant; and utilizes the immobilized arginine decarboxylase mutant to catalyze the preparation of agmatine salts (e.g., agmatine sulfate, hydrochloride, and nitrate, etc.), establishing a process with a simple preparation process, reusability, and low cost, which is of great significance for the industrial production of agmatine salts.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to an arginine decarboxylase mutant and an application of the mutant in preparing agmatine salt by immobilization. Background Art

[0002] Agmatine is an important biogenic amine substance that is widely distributed in the stomach, intestines, spleen, skeletal muscle and brain of mammals. Agmatine is one of the important metabolites of L-arginine and is produced by the decarboxylation of L-arginine catalyzed by L-arginine decarboxylase.

[0003] As a neurotransmitter, agmatine is a key regulator of the metabolism of L-arginine into nitric oxide and biogenic amines in the body and a variety of neural receptors. It has rich physiological functions, can affect the release of hormones or transmitters, and has the effects of lowering blood sugar, dilating blood vessels, lowering blood pressure, and reducing inflammation.

[0004] Agmatine, a derivative of L-arginine, possesses superior physiological activity compared to arginine. Reported benefits include improved endurance, accelerated recovery, enhanced performance, a surprising and sustained energy supply, increased muscle mass, and reduced fat. As an endogenous cationic ammonium substance, agmatine can effectively alleviate the side effects of drug stimulation of NMDA (N-methyl-D-aspartate) receptors, such as memory impairment and mental disorders. It can effectively reduce and prevent drug dependence, making it a highly promising drug-detoxification drug and therefore widely used in the pharmaceutical and health product markets.

[0005] Agmatine salts include agmatine sulfate, agmatine hydrochloride, and agmatine nitrate, and are salts generated by agmatine under acidic conditions. In recent years, there have been reports of some preparations of agmatine salts both at home and abroad, with chemical methods being the main method. Reports of utilizing biological methods to prepare agmatine salts are rare. In the prior art utilizing biological methods to prepare agmatine salts, there are problems such as low output and low conversion rate of agmatine salts that are unfavorable for the industrialized production of agmatine salts.

[0006] Chinese patent CN105861529A discloses an arginine decarboxylase and its application. Under 37°C conditions, the conversion cycle is 24 hours, and the agmatine sulfate production can reach 61-71g / L, with a conversion rate of 68-82%.

[0007] Jiangnan University has published a highly efficient arginine decarboxylase from Shewanella putrefaciens. By exploring the optimal conversion conditions for L-arginine and conducting tank tests, the maximum yield of agmatine sulfate reached 52.66 g / L, with a conversion rate of 78.27% (Sun Anran, Enzymatic Conversion of L-arginine to Agmatine [D]. Jiangnan University, 2017).

[0008] Chinese patent CN104911223A discloses a method for biologically preparing agmatine sulfate using crude arginine decarboxylase enzyme solution. The crude enzyme solution is directly discarded after a batch of substrates is converted, which is not conducive to reducing costs.

[0009] Chinese patent CN110257448A discloses a method for preparing agmatine sulfate using whole bacterial cells. This method uses whole bacterial cells to transform and produce agmatine sulfate. The whole bacterial cells can also only be used once and cannot be reused.

[0010] Therefore, there is an urgent need to develop a new arginine decarboxylase to increase the concentration of the product agmatine salt, improve the conversion rate of the substrate arginine, and a method and application of preparing agmatine salt using the enzyme. Summary of the Invention

[0011] The present invention aims to solve, at least to a certain extent, one of the technical problems in the related art. To this end, one object of the present invention is to provide a highly active arginine decarboxylase mutant that can be fermented and expressed using Escherichia coli as a host, and can be used to prepare agmatine salts, such as agmatine sulfate, agmatine hydrochloride, and agmatine nitrate. The arginine decarboxylase mutant is immobilized to improve its reusability and stability. Finally, the immobilized arginine decarboxylase mutant is used to catalyze the preparation of agmatine salts. Establishing a process that is simple, reusable, and low-cost is of great significance for the industrial production of agmatine salts.

[0012] To this end, the first aspect of the present invention provides an arginine decarboxylase mutant. According to an embodiment of the present invention, the amino acid sequence of the arginine decarboxylase mutant is obtained by performing site-directed mutagenesis on at least one of positions 151, 188, 193, 202, 257, 348, 387, and 740 of the wild-type arginine decarboxylase sequence from Salmonella enterica as shown in SEQ ID NO:1.

[0013] According to an embodiment of the present invention, the amino acid sequence of the arginine decarboxylase mutant contains the following mutations in the amino acid sequence of arginine decarboxylase shown in SEQ ID NO: 1:

[0014] (1) Serine at position 151 is replaced by alanine, or

[0015] (2) the arginine at position 188 is replaced by alanine, or

[0016] (3) Leucine at position 202 is replaced by alanine, or

[0017] (4) Aspartic acid at position 348 is replaced by alanine, or

[0018] (5) Isoleucine at position 193 is replaced by glycine, or

[0019] (6) Glutamic acid at position 740 is replaced by glycine, or

[0020] (7) Lysine at position 257 is replaced by leucine, or

[0021] (8) Lysine at position 387 is replaced by leucine, or

[0022] (9) Lysine at position 257 is substituted with leucine and serine at position 151 is substituted with alanine, or

[0023] (10) Lysine at position 257 is substituted with leucine and Arginine at position 188 is substituted with alanine, or

[0024] (11) Lysine at position 257 is substituted with leucine and leucine at position 202 is substituted with alanine, or

[0025] (12) Lysine at position 257 is substituted with leucine and aspartic acid at position 348 is substituted with alanine, or

[0026] (13) Lysine at position 387 is substituted with leucine and serine at position 151 is substituted with alanine, or

[0027] (14) Lysine at position 387 is substituted with leucine and arginine at position 188 is substituted with alanine, or

[0028] (15) Lysine at position 387 is substituted with leucine and leucine at position 202 is substituted with alanine, or

[0029] (16) Lysine at position 387 is substituted with leucine and aspartic acid at position 348 is substituted with alanine, or

[0030] (17) Lysine at position 257 is substituted with leucine, arginine at position 188 is substituted with alanine, and isoleucine at position 193 is substituted with glycine, or

[0031] (18) Lysine at position 257 is substituted with leucine, arginine at position 188 is substituted with alanine, and glutamic acid at position 740 is substituted with glycine, or

[0032] (19) Lysine at position 257 is substituted with leucine, leucine at position 202 is substituted with alanine, and isoleucine at position 193 is substituted with glycine, or

[0033] (20) Lysine at position 257 is substituted with leucine, Leucine at position 202 is substituted with alanine, and Glutamic acid at position 740 is substituted with glycine, or

[0034] (21) Lysine at position 387 is substituted with leucine, arginine at position 188 is substituted with alanine, and isoleucine at position 193 is substituted with glycine, or

[0035] (22) Lysine at position 387 is substituted with leucine, arginine at position 188 is substituted with alanine, and glutamic acid at position 740 is substituted with glycine, or

[0036] (23) Lysine at position 387 is substituted with leucine, Leucine at position 202 is substituted with alanine, and Isoleucine at position 193 is substituted with glycine, or

[0037] (24) Lysine at position 387 is substituted with leucine, Leucine at position 202 is substituted with alanine, and Glutamic acid at position 740 is substituted with glycine, or

[0038] (25) Lysine at position 257 is substituted with leucine, Leucine at position 202 is substituted with alanine, Isoleucine at position 193 is substituted with glycine, and Arginine at position 188 is substituted with alanine, or

[0039] (26) Lysine at position 257 is substituted with leucine, Leucine at position 202 is substituted with alanine, Isoleucine at position 193 is substituted with glycine, Arginine at position 188 is substituted with alanine, and Serine at position 151 is substituted with alanine, or

[0040] (27) Lysine at position 257 is substituted with leucine, Leucine at position 202 is substituted with alanine, Isoleucine at position 193 is substituted with glycine, Arginine at position 188 is substituted with alanine, Serine at position 151 is substituted with alanine, and Aspartic acid at position 348 is substituted with alanine, or

[0041] (28) Lysine at position 257 is substituted with leucine, Leucine at position 202 is substituted with alanine, Isoleucine at position 193 is substituted with glycine, Arginine at position 188 is substituted with alanine, Serine at position 151 is substituted with alanine, Aspartic acid at position 348 is substituted with alanine, and Lysine at position 387 is substituted with leucine, or

[0042] (29) Lysine at position 257 was substituted with leucine, leucine at position 202 was substituted with alanine, isoleucine at position 193 was substituted with glycine, arginine at position 188 was substituted with alanine, serine at position 151 was substituted with alanine, aspartic acid at position 348 was substituted with alanine, lysine at position 387 was substituted with leucine, and glutamic acid at position 740 was substituted with glycine.

[0043] The second aspect of the present invention provides a nucleic acid. According to an embodiment of the present invention, the nucleic acid encodes the arginine decarboxylase mutant described in the first aspect.

[0044] The third aspect of the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises at least one of the nucleic acid sequences described in the second aspect.

[0045] The fourth aspect of the present invention provides a host cell, wherein the host cell comprises the expression vector described in the third aspect.

[0046] A fifth aspect of the present invention provides an immobilized enzyme. According to an embodiment of the present invention, the immobilized enzyme comprises:

[0047] The arginine decarboxylase mutant according to the first aspect; and

[0048] An immobilized carrier, on which the arginine decarboxylase mutant is immobilized.

[0049] An immobilized arginine decarboxylase mutant is obtained by combining an arginine decarboxylase mutant with an immobilized carrier through a covalent bond. The immobilized arginine decarboxylase mutant is used in the biotransformation of agmatine salt, thereby improving the reusability and stability of the arginine decarboxylase mutant.

[0050] According to an embodiment of the present invention, the immobilization carrier is selected from an amino carrier and / or an epoxy carrier.

[0051] The sixth aspect of the present invention provides use of the arginine decarboxylase mutant described in the first aspect and / or the immobilized enzyme described in the fifth aspect in the preparation of agmatine salt.

[0052] A seventh aspect of the present invention provides a method for preparing an agmatine salt. According to an embodiment of the present invention, the preparation method comprises:

[0053] mixing the immobilized enzyme described in the fifth aspect with a substrate solution to obtain a crude agmatine product;

[0054] Acidifying the crude agmatine product with an acid to obtain a crude agmatine salt product;

[0055] The substrate solution includes arginine and pyridoxal phosphate;

[0056] The acid includes common acids such as dilute sulfuric acid, dilute hydrochloric acid and dilute nitric acid.

[0057] The existing method of preparing agmatine salt by bioconversion method utilizes disclosed arginine decarboxylase to prepare agmatine sulfate, while the preparation of other agmatine salts (such as agmatine hydrochloride and agmatine nitrate) has not been reported, and the output and conversion rate of the agmatine sulfate obtained by this method are both low, which cannot meet the demand for industrialized production of agmatine salt. Based on this, the inventors found a first aspect of the arginine decarboxylase mutant, which has higher catalytic activity than the wild-type arginine decarboxylase, and the action temperature range and action pH range of the arginine decarboxylase mutant are wider than the wild-type, and the enzyme activity of the immobilized arginine decarboxylase mutant provided by the present invention is higher than that of the wild-type arginine decarboxylase. After the arginine decarboxylase mutant provided by the present invention is immobilized, it can be applied to a biotransformation reaction with arginine as a substrate, by respectively adding dilute sulfuric acid, dilute hydrochloric acid and dilute nitric acid to maintain the pH of the reaction system within the optimum pH range of the arginine decarboxylase mutant, thereby efficiently generating agmatine sulfate, agmatine hydrochloride and agmatine nitrate. On the other hand, the immobilized enzyme provided by the present invention is used to react with the substrate arginine for a decarboxylation reaction, which can improve the reaction efficiency of the decarboxylation reaction and increase the conversion rate. In addition, the immobilized enzyme has high reusability and stability.

[0058] In particular, the arginine decarboxylase mutant M29 (K257L / L202A / I193G / R188A / S151A / D348A / K387L / E740G) provided by the present invention has an enzyme activity increased by 60.61%, an agmatine sulfate yield of 322.63 g / L, and a conversion rate of 99.23%, and is an ideal catalyst for the preparation of agmatine sulfate.

[0059] According to an embodiment of the present invention, the mass ratio of the immobilized enzyme to arginine is 0.03:1 to 0.06:1.

[0060] According to an embodiment of the present invention, the concentration of arginine is 100-340 g / L.

[0061] According to an embodiment of the present invention, the concentration of the pyridoxal phosphate is 0.01 to 0.6 g / L.

[0062] Pyridoxal phosphate acts as a coenzyme to enhance the catalytic activity of arginine decarboxylase.

[0063] According to an embodiment of the present invention, the substrate solution further includes α-ketoglutarate.

[0064] α-Ketoglutaric acid is used to promote the decarboxylation reaction to save the decarboxylation reaction time.

[0065] According to an embodiment of the present invention, the concentration of the α-ketoglutaric acid is 0.2-1.0 g / L.

[0066] According to an embodiment of the present invention, the pH value of the substrate solution is 5-8.

[0067] During the preparation of agmatine salt, the pH of the reaction system will continue to increase as the reaction proceeds, and the increase in pH will affect the activity of arginine decarboxylase. Therefore, acid needs to be continuously added to maintain the pH of the reaction system. The pH of the substrate solution is maintained at 5 to 8, which is conducive to the continuous and efficient synthesis of agmatine salt.

[0068] According to an embodiment of the present invention, the mixing temperature is 30-50° C. and the mixing time is 8-12 hours.

[0069] According to an embodiment of the present invention, the preparation method further comprises the step of decolorizing the crude agmatine salt product.

[0070] According to an embodiment of the present invention, the crude agmatine salt product is decolorized using activated carbon to obtain a decolorized agmatine salt product.

[0071] According to an embodiment of the present invention, the preparation method further comprises concentrating the decolorized agmatine salt product and then dropwise adding ethanol to precipitate agmatine salt crystals.

[0072] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0074] Figure 1 The relationship between the enzyme activity of wild-type and mutant arginine decarboxylase and temperature is shown in the examples;

[0075] Figure 2 The relationship between the enzyme activity of wild-type and mutant arginine decarboxylase and pH in the examples;

[0076] Figure 3 (a) shows the relative enzyme activity of the amino-support-immobilized arginine decarboxylase mutants at different cycle numbers;

[0077] Figure 3 (b) shows the relative enzyme activity of the arginine decarboxylase mutants immobilized on the epoxy support at different cycle numbers;

[0078] Figure 4 (a) shows the relative enzyme activity of wild-type arginine decarboxylase immobilized on amino carrier at different cycle numbers;

[0079] Figure 4 (b) shows the relative enzyme activity of wild-type arginine decarboxylase immobilized on epoxy support at different cycle numbers; DETAILED DESCRIPTION

[0080] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0081] The amino acid sequence of the wild-type arginine decarboxylase from Salmonella enterica is shown in SEQ ID NO: 1, and the nucleic acid sequence encoding the wild-type arginine decarboxylase is shown in SEQ ID NO: 2.

[0082] The sequence of SEQ ID NO.1 is as follows:

[0083]

[0084] SEQ ID NO:.2 sequence:

[0085]

[0086] In some embodiments of the present invention, pET28a is used as the expression vector, and other commonly used expression vectors may also be used.

[0087] In some embodiments of the present invention, Escherichia coli BL21 (DE3) is used as a host.

[0088] In some embodiments of the present invention, the method for constructing a recombinant strain expressing the wild type or mutant of the above-mentioned arginine decarboxylase comprises the steps of:

[0089] (1) Synthesize wild-type or mutant genes of arginine decarboxylase;

[0090] (2) ligating the wild-type or mutant arginine decarboxylase gene to the expression vector pET28a to obtain a ligation product;

[0091] (3) The ligation product was transformed into Escherichia coli BL21 (DE3).

[0092] In some specific embodiments of the present invention, the method for immobilizing arginine decarboxylase comprises:

[0093] (1) Preparation of arginine decarboxylase concentrate:

[0094] The engineered strain after the vector is transferred is cultured in LB medium at 37°C and 180 rpm for a period of time for activation, and then transferred to LB liquid medium at a ratio of 1% for expansion culture. After the OD600 value of the culture solution reaches 1.5-2.5, an IPTG inducer with a final concentration of 0.1-1.5 mmol / L is added, and the culture is continued overnight in a constant temperature shaking incubator at 25°C and 180 rpm. After centrifugation in a low-temperature centrifuge, the supernatant is discarded, and the bacteria are collected. The bacteria are resuspended in PBS buffer and then ultrasonically disrupted. The supernatant obtained after centrifugation is the arginine decarboxylase concentrate.

[0095] (2) Nickel-containing resin purification:

[0096] When constructing the arginine decarboxylase expression vector, pET28a with a histidine tag was selected as a tool vector to facilitate the purification of the arginine decarboxylase mutant using nickel-containing resin. The target protein was purified by nickel-containing resin. Polyhistidine can bind to a variety of transition metals and transition metal chelates, so the arginine decarboxylase mutant with an exposed 6X His-tag can bind to the nickel-containing resin. 2+ The five-membered imidazole ring of the histidine residue is the binding site of the protein to the Ni 2+When eluted with a high concentration of imidazole solution, imidazole competes with the imidazole ring of the his-tag fusion protein and eventually elutes the fusion protein.

[0097] (3) Immobilization:

[0098] 1) Amino carrier immobilization

[0099] a) Carrier pretreatment

[0100] 10 g of amino carrier was added into 100 mL of 0.1 M phosphate buffer solution with a pH of 5.5, and the mixture was shaken at 60 rpm for 1 h at a temperature of 25°C, and then filtered and dried.

[0101] b) Glutaraldehyde cross-linking

[0102] Add 50 mL of 2% glutaraldehyde phosphate buffer to 10 g of the amino carrier treated in a), shake slowly at 60 rpm for 1 h on a shaker controlled at 25° C., filter, and wash the carrier with deionized water until the water is clear.

[0103] c) Enzyme and carrier immobilization

[0104] 2 mL of the purified arginine decarboxylase from step (2) and 10 g of the cross-linked amino carrier were added to 50 mL of 0.1 M phosphate buffer (pH 5.5) and shaken at 60 rpm for 12 h on a shaker controlled at 25°C. After centrifugation, the solid was collected to obtain the amino carrier-immobilized arginine decarboxylase.

[0105] 2) Epoxy-based carrier immobilization

[0106] a) Carrier pretreatment

[0107] 10 g of epoxy carrier was added with 100 mL of 0.1 M phosphate buffer at pH 5.5, and the mixture was shaken at 60 rpm for 1 h at 25°C, and then filtered and dried.

[0108] b) Enzyme and carrier immobilization

[0109] 2 mL of the purified arginine decarboxylase from step (2) and 10 g of the treated epoxy support were added to 50 mL of 0.1 M phosphate buffer (pH 5.5) and shaken at 60 rpm for 12 h on a shaker controlled at 25°C. After centrifugation, the solid was collected to obtain the epoxy support-immobilized arginine decarboxylase.

[0110] In some embodiments of the present invention, conversion experiments are conducted using immobilized arginine decarboxylase and the substrate arginine at a mass ratio of 0.03:1 to 0.06:1. The initial concentrations of the components of the substrate solution are: 100-340 g / L arginine, 0.01-0.6 g / L pyridoxal phosphate, and 0.2-1.0 g / L α-ketoglutarate. The pH of the substrate solution is controlled at 5-8 using acid. Conversion is performed at 30-50°C for 8-12 hours.

[0111] After the conversion is completed, the substrate solution is centrifuged to collect the immobilized arginine decarboxylase, activated carbon is added to the centrifuged substrate solution, and after decolorization is completed, the supernatant is centrifuged to collect.

[0112] Collect the resulting supernatant and concentrate it on a rotary evaporator. After concentration, cool the concentrate to 10°C. Begin adding ethanol dropwise and continue stirring at 200 rpm for 2 hours before filtering. Dry the resulting solid in a vacuum drying oven at 60°C and -0.09 MPa.

[0113] The following definitions are used in the present invention:

[0114] 1) Amino acid residues are referred to using the generally accepted IUPAC nomenclature, using either three-letter abbreviations or single-letter symbols. DNA sequences are referred to using the generally accepted IUPAC nomenclature.

[0115] 2) Mutant Identification: The mutated amino acid in the arginine decarboxylase mutant is represented by "amino acid substituted at the original amino acid position." For example, S151A indicates that the amino acid at position 151 is substituted from Ser in the wild-type arginine decarboxylase to Ala. The position numbering corresponds to the amino acid sequence numbering of the arginine decarboxylase in SEQ ID NO: 1. For example, S151A / R188A indicates that both amino acids at positions 151 and 188 are mutated.

[0116] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0117] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0118] The immobilized carrier used in the embodiment of the present invention is an amino carrier of Xi'an Lanxiao LX-1000EA and epoxy-based carrier LX-1000EP.

[0119] Example 1 Arginine decarboxylase activity detection method

[0120] Preparation of reaction substrate: 0.2 M sodium acetate buffer, pH = 5.5, 10% arginine, 0.02% pyridoxal phosphate, and 0.3% α-ketoglutaric acid dissolved in sodium acetate buffer.

[0121] Reaction conditions: Add 0.002 mL of enzyme solution or 0.01 g of immobilized enzyme to 10 mL of reaction substrate solution, react in a 37°C water bath for 10 min, and then boil and cool.

[0122] After the reaction was completed, 1 mL of the reaction solution was diluted 25 times and then tested by HPLC.

[0123] The enzyme activity was defined as the amount of enzyme required to generate 1 μmol of agmatine per minute at pH 5.5 and 37°C.

[0124] Preparation of agmatine salt standard curve: Accurately weigh 10 mg of agmatine salt reference substance and place it in a 100 mL volumetric flask. Add mobile phase to dissolve and dilute to the scale, shake well, then take 1, 2, 3, 4, and 5 mL of the solution and dilute to 10 mL with mobile phase, shake well, and use these as reference solution. The reference solution was subjected to HPLC detection to prepare the agmatine salt standard curve.

[0125] HPLC determination of agmatine salts: Determination was performed using a ThermoFisher high-performance liquid chromatograph. Column: C18, 4.6 mm × 250 mm, 5 μm; Detector: UV detector; Mobile phase: 0.1% perchloric acid: acetonitrile = 95:5; Detection wavelength: 195 nm; Flow rate: 0.8 mL / min; Column temperature: 30°C; Injection volume: 20 μL.

[0126] Example 2 Construction of wild-type and mutant arginine decarboxylase genetically engineered bacteria

[0127] 1. Source of reagents

[0128] Plasmid pET28a was purchased from Wuhan Miaoling Biotechnology Co., Ltd., and Escherichia coli BL21 (DE3) is commonly used in the field. DNA polymerase (Q5 High-Fidelity DNA Polymerase) was purchased from Gene Co., Ltd.; restriction enzymes (XbaI, BamHI), DNA markers, plasmid miniprep kit, and DNA gel recovery and purification kit were all purchased from Takara Bioengineering (Dalian) Co., Ltd.; the OneStep clonning kit was purchased from NEB Beijing; and kanamycin sulfate was purchased from Biosharp. Plasmid extraction procedures were referred to the instructions for the plasmid miniprep kit; DNA fragment recombination and ligation procedures were referred to the instructions for the OneStep clonning kit.

[0129] 2. List of single mutations and combined mutants, as shown in Table 1:

[0130] Table 1 List of single and combined mutants

[0131]

[0132]

[0133] Mutation primers for the K257L / L202A / I193G / R188A / S151A / D348A / K387L / E740G mutation sites were designed, as shown in Primer List 2:

[0134] Table 2 Mutation primers at mutation sites

[0135]

[0136] The amino acid sequence of wild-type arginine decarboxylase is shown in SEQ ID NO: 1. The nucleotide sequence expressing this amino acid sequence was codon-optimized based on the codon preference of Escherichia coli. XbaI and HindIII restriction sites were added at both ends of the gene and then sent to Jinkairui Biotechnology Co., Ltd. for artificial synthesis (SEQ ID NO: 2).

[0137] Plasmid pET28a and the synthesized AdiA gene fragment were double-digested with XbaI / HindIII, respectively. The reaction system consisted of: 1 μg of plasmid or AdiA gene fragment, 5 μL of 10× buffer, 1 μL of XbaI, 1 μL of HindIII, and water added to 50 μL. The digestion was incubated at 37°C for 5 h. The fragment was analyzed by 1% agarose gel electrophoresis, and a 5.4 kb linear fragment was recovered using a DNA gel purification kit. The double-digested product (i.e., XbaI / BamHI double-digested plasmid pET28a) and the AdiA gene were recombined using a Onestep cloning kit. The recombinant product was transformed into Escherichia coli BL21, plated on plates containing 50 μg / mL of kanamycin sulfate, and cultured overnight at 37°C. Transformants were selected for sequencing. Transformants that correctly sequenced were identified as wild-type arginine decarboxylase-producing strain W1.

[0138] Single point mutation: AdiA FP / S151A RP, AdiA FP / R188A RP, AdiA FP / L202A RP, AdiA FP / D348A RP, AdiA FP / I193G RP, AdiA FP / E740G RP, AdiA FP / K257L RP, and AdiA FP / K387L RP were used as primers, AdiA-pET28a plasmid was used as template, and high-fidelity Q5 High-Fidelity DNA Polymerase was used to amplify the left fragment of each mutation site; S151A FP / AdiA RP, R188A FP / AdiA RP, L202A FP / AdiA RP, D348A FP / AdiA RP, I193G FP / AdiA RP, E740G FP / AdiA RP, K257L FP / AdiARP, and K387L FP / AdiA RP were used as primers, and AdiA-pET28a was used as a template to amplify the fragment to the right of the mutation site. Then, AdiA FP / AdiA RP was used as primers, and overlap extension PCR was performed with the left and right fragments corresponding to the amplified mutation as templates to amplify the full-length AdiA single mutation fragments M1 to M8.

[0139] Combined mutation: Using the amplified single mutation DNA gene fragment as a template, refer to the above primer combination method, first amplify the fragment on the left side of the mutation site, then amplify the fragment on the right side of the mutation site, and then use AdiA FP / AdiA Using RP as primer, overlap extension PCR was performed with the left and right fragments corresponding to the amplified mutations as templates, respectively, to amplify full-length AdiA double mutation fragments M9 to M16; similarly, using the amplified double mutation DNA gene fragment as a template, full-length AdiA triple mutation fragments M17 to M24 were amplified; similarly, using the amplified triple mutation DNA gene fragment as a template, full-length AdiA quadruple mutation fragment M25 was amplified; similarly, using the amplified quadruple mutation DNA gene fragment as a template, full-length AdiA pentamutation fragment M26 was amplified; similarly, using the amplified pentamutation DNA gene fragment as a template, full-length AdiA hexamutation fragment M27 was amplified; similarly, using the amplified hexamutation DNA gene fragment as a template, full-length AdiA heptamutation fragment M28 was amplified; similarly, using the amplified heptamutation DNA gene fragment as a template, full-length AdiA octamutation fragment M29 was amplified.

[0140] The PCR reaction system (50 μL) is as follows: 10 μL of 5×Q5 reaction buffer, 1 μL of 10 mM dNTP, 2.5 μL of each primer, template depending on the sample concentration, 0.5 μL of Q5 enzyme, and water to 50 μL.

[0141] The final PCR product was detected by 1% agarose gel electrophoresis and purified using a DNA gel recovery kit.

[0142] Plasmid pET28a was digested with XbaI / HindIII using the following reaction system: 1 μg of plasmid, 5 μL of 10× buffer, 1 μL of XbaI, 1 μL of HindIII, and water added to 50 μL. Digestion was incubated at 37°C for 5 h. A 5.4 kb linear fragment was recovered using a DNA gel purification kit and analyzed by 1% agarose gel electrophoresis. The linear fragment and the amplified mutant fragment were reconstituted using the Onestep clonning kit. The recombinant products were transformed into Escherichia coli BL21(DE3) and plated on plates containing 50 μg / mL of kanamycin sulfate. After overnight incubation at 37°C, transformants were selected for sequencing. Transformants that correctly sequenced were identified as genetically engineered strains expressing arginine decarboxylase mutants M1 to M29.

[0143] Example 3 Inducing wild-type arginine decarboxylase genetically engineered bacteria and mutant arginine decarboxylase genetically engineered bacteria to produce enzymes and purify them

[0144] The wild-type arginine decarboxylase genetically engineered bacteria W1 and mutant genetically engineered bacteria M1-M29 in Example 2 were streaked onto LB plates containing 50 μg / mL of kanamycin and cultured at 37°C for 12 h. Single colonies were picked and transferred into 5 mL of LB liquid culture medium containing 50 μg / mL of kanamycin and cultured at 37°C, 180 rpm for 12 h. The inoculum size of 1% was transferred to 100 mL of LB liquid culture medium (10 g / L peptone, 5 g / L yeast powder, 10 g / L sodium chloride) containing 50 μg / mL of kanamycin and cultured at 37°C, 180 rpm until an OD600 value of 1.5-2.5 was achieved. Isopropylthiogalactoside (IPTG) was added to the culture to a final concentration of 0.1-1.5 mmol / L and the culture was continued with shaking at 25°C, 180 rpm for 12 h.

[0145] At the end of the culture, 1 mL of the culture was aspirated and the final OD600 value was determined. The cells were then collected by centrifugation at 5000 rpm for 10 min. The cells were washed and resuspended with 20 mL of 0.1 M PBS buffer at a pH of 7.4 and then disrupted using an ultrasonic cell disruptor. After disruption, the disrupted liquid was collected and centrifuged at 12000 rpm for 10 min. The supernatant was collected to obtain crude enzyme solutions of wild-type arginine decarboxylase and arginine decarboxylase mutants.

[0146] The crude wild-type arginine decarboxylase enzyme solution and the crude arginine decarboxylase mutant enzyme solution were fully mixed with nickel-containing resin at 4° C., filtered, and then eluted with 250 mM imidazole solution to obtain purified wild-type arginine decarboxylase enzyme solution and arginine decarboxylase mutant enzyme solution.

[0147] Example 4 Enzyme activity detection of wild-type arginine decarboxylase and arginine decarboxylase mutants

[0148] The wild-type arginine decarboxylase W1 and arginine decarboxylase mutants M1-M29 purified in Example 3 were tested for enzyme activity according to the arginine decarboxylase activity detection method in Example 1. The results are shown in Table 3.

[0149] Table 3 Enzyme activities of wild-type arginine decarboxylase and arginine decarboxylase mutants

[0150] Arginine decarboxylase Enzyme activity (U / mg) Arginine decarboxylase Enzyme activity (U / mg) W1 538.16±8.61 M15 627.54±11.92 M1 557.45±9.48 M16 608.31±12.17 M2 569.36±10.25 M17 661.35±13.89 M3 576.97±10.96 M18 647.44±14.24 M4 554.74±11.09 M19 670.96±15.43 M5 573.37±12.04 M20 658.05±15.79 M6 559.46±12.31 M21 657.14±16.43 M7 591.94±13.61 M22 643.23±14.15 M8 585.73±14.06 M23 659.75±13.20 M9 605.23±15.13 M24 648.84±11.68 M10 633.14±13.93 M25 737.16±11.79 M11 630.75±12.62 M26 766.45±13.80 M12 615.52±11.08 M27 793.03±17.45 M13 608.02±9.73 M28 830.61±19.10 M14 614.93±11.07 M29 864.32±21.61

[0151] All enzyme activity assays were performed in triplicate, and data were analyzed using SPSS 22.0 for Windows.

[0152] The results of enzyme activity detection showed that the enzyme activity of different mutants was improved, among which M29 had the highest enzyme activity of 864.32±21.61U / mg, which was 60.61% higher than that of wild-type W1 (538.16±8.61U / mg), indicating that the enzyme catalytic activity was improved by modifying the enzyme through site-directed mutagenesis.

[0153] Example 5 Determination of the optimum temperature and optimum pH of wild-type arginine decarboxylase and arginine decarboxylase mutants M4, M9, M22, M25 and M29

[0154] Since there are many mutants, several representative mutants were selected for experiments, including single mutation, double mutation, triple mutation, mutant with the lowest enzyme activity among more than three mutations, and mutant with the highest enzyme activity, namely mutants M4, M9, M22, M25 and M29.

[0155] The wild-type arginine decarboxylase W1 and arginine decarboxylase mutants M4, M9, M22, M25 and M29 purified in Example 3 were incubated at 20°C, 25°C, 30°C, 37°C, 40°C, 45°C, 50°C, 55°C and 60°C for 30 min, respectively. The enzyme activity was then detected according to the arginine decarboxylase activity detection method of Example 1. The relative enzyme activity at other temperatures was calculated with the maximum enzyme activity as 100%. The results are shown in FIG. Figure 1As shown, the optimal temperature for arginine decarboxylase mutants M4, M9, M22, M25, and M29 is the same as that for wild-type W1, all at 37°C. When the temperature is below or above 37°C, the arginine decarboxylase mutants M4, M9, M22, M25, and M29 exhibit higher catalytic activity than wild-type arginine decarboxylase W1. Since the selected mutants all had the lowest enzymatic activity among single, double, triple, and more than three mutations, it can be inferred that the mutants have a wider temperature range of activity than wild-type W1.

[0156] The wild-type arginine decarboxylase W1 and arginine decarboxylase mutants M4, M9, M22, M25 and M29 purified in Example 3 were placed in 0.1 M PBS buffer at pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 for 30 min, and then the enzyme activity was detected according to the arginine decarboxylase activity detection method in Example 1. The relative enzyme activity at other pH values was calculated with the maximum enzyme activity as 100%. The results are shown in FIG. Figure 2 As shown, the optimal pH of the arginine decarboxylase mutants is the same as that of wild-type W1, at 5.5. When the pH is below or above 5.5, the arginine decarboxylase mutants M4, M9, M22, M25, and M29 exhibit higher catalytic activity than wild-type arginine decarboxylase W1. Since the selected mutants all had the lowest enzymatic activity among single, double, triple, and three or more mutations, it can be inferred that the mutants have a wider pH range of activity than wild-type W1.

[0157] Example 6 Immobilization of wild-type W1 and mutants M4, M9, M22, M25 and M29 arginine decarboxylase and detection of enzyme activity after immobilization

[0158] Immobilization of arginine decarboxylase:

[0159] 1) Amino carrier immobilization

[0160] a) Carrier pretreatment

[0161] 10 g of amino carrier was added into 100 mL of 0.1 M phosphate buffer solution with a pH of 5.5, and the mixture was shaken at 60 rpm for 1 h at a temperature of 25°C, and then filtered and dried.

[0162] b) Glutaraldehyde cross-linking

[0163] Add 50 mL of 2% glutaraldehyde phosphate buffer to 10 g of the amino carrier treated in a), shake slowly at 60 rpm for 1 h on a shaker controlled at 25° C., filter, and wash the carrier with deionized water until the water is clear.

[0164] c) Enzyme and carrier immobilization

[0165] 2 mL of purified arginine decarboxylase mutant enzyme solution and 2 mL of wild-type arginine decarboxylase enzyme solution were added to 50 mL of 0.1 M phosphate buffer (pH 5.5) along with 10 g of the cross-linked amino support. The mixture was shaken at 60 rpm for 12 hours at 25°C. After centrifugation, the solids were collected. The resulting solids were the amino support-immobilized arginine decarboxylase mutant and wild-type arginine decarboxylase.

[0166] 2) Epoxy-based carrier immobilization

[0167] a) Carrier pretreatment

[0168] 10 g of epoxy carrier was added with 100 mL of 0.1 M phosphate buffer at pH 5.5, and the mixture was shaken at 60 rpm for 1 h at 25°C, and then filtered and dried.

[0169] b) Enzyme and carrier immobilization

[0170] 2 mL of purified mutant arginine decarboxylase enzyme solution and 2 mL of wild-type arginine decarboxylase enzyme solution were added to 50 mL of 0.1 M phosphate buffer (pH 5.5) and shaken at 60 rpm for 12 hours at 25°C. After centrifugation, the solids were collected to form the epoxy-supported immobilized arginine decarboxylase mutant and wild-type arginine decarboxylase.

[0171] The obtained immobilized enzyme was washed three times with 0.1 M pH 5.5 phosphate buffer, a small amount of 0.1 M pH 5.5 phosphate buffer was added to immerse the immobilized enzyme, and the enzyme was stored at 4° C. for later use.

[0172] The wild-type arginine decarboxylase and immobilized arginine decarboxylase mutants M4, M9, M22, M25 and M29 were tested for enzyme activity according to the arginine decarboxylase enzyme activity detection method of Example 1. The enzyme activities were 527.37 U / mg, 542 U / mg, 598.76 U / mg, 639.19 U / mg, 732 U / mg and 859.86 U / mg, respectively. The enzyme activities of the immobilized mutants M4, M9, M22, M25 and M29 were all higher than those of the immobilized wild-type arginine decarboxylase, and the influence of immobilization on the enzyme activity was less.

[0173] Since the immobilized arginine decarboxylase mutant M29 had the highest enzyme activity, the immobilized arginine decarboxylase mutant M29 was selected for use in subsequent examples.

[0174] Example 7 Detection of enzyme activity after repeated use of immobilized arginine decarboxylase

[0175] The immobilization steps in Example 5 were performed on the arginine decarboxylase mutant M29 and the wild-type arginine decarboxylase W1, respectively, and the enzyme activities of the immobilized enzymes after reuse were detected.

[0176] After 35 reuses, the enzyme activities of amino-support immobilized arginine decarboxylase mutant M29 and epoxy-support immobilized arginine decarboxylase mutant M29 decreased to 35.3% and 48.6% of the initial enzyme activities, respectively. Figure 3 shown.

[0177] After 35 reuses, the enzyme activities of amino-supported immobilized wild-type arginine decarboxylase W1 and epoxy-supported immobilized wild-type arginine decarboxylase W1 decreased to 31.3% and 40.4% of the initial enzyme activities, respectively. Figure 4 shown.

[0178] In view of the good reusability and stability of amino-support immobilized arginine decarboxylase mutants and epoxy-support immobilized arginine decarboxylase mutants, and the relatively better reusability of epoxy-support immobilized arginine decarboxylase mutants, epoxy-support immobilized arginine decarboxylase mutant M29 was selected to catalyze the preparation of agmatine salt from arginine.

[0179] Example 8: Preparation of Agmatine Sulfate from Arginine Catalyzed by an Epoxy-Based Carrier-Immobilized Arginine Decarboxylase Mutant

[0180] The epoxy-supported immobilized arginine decarboxylase mutant M29 obtained in Example 6 was subjected to a conversion experiment with the substrate arginine in a mass ratio of 0.05:1. The initial concentrations of the components of the substrate solution were: arginine 330 g / L, pyridoxal phosphate 0.4 g / L, and α-ketoglutaric acid 0.7 g / L. The temperature was controlled at 37°C, and the pH of the substrate solution was controlled to 5.5 using dilute sulfuric acid. The conversion was completed when the conversion time was 11 hours. After the conversion was completed, the reaction volume was increased due to the addition of dilute sulfuric acid to control the pH. The concentration of agmatine sulfate detected by HPLC was 322.63 g / L, and the conversion rate was 99.23%.

[0181] The conversion solution was filtered to collect the epoxy-supported immobilized arginine decarboxylase mutant, and activated carbon was added to the filtered conversion solution in an amount of 1.5% of the weight of agmatine sulfate in the conversion solution. After decolorization, the supernatant was filtered and collected for concentrated crystallization.

[0182] 300 mL of the collected supernatant was collected, and the theoretical weight of agmatine sulfate was 97.54 g. After concentration on a rotary evaporator, the temperature was lowered to 10° C. Ethanol was added dropwise, and stirring was continued at 200 rpm for 2 hours before filtration. The agmatine sulfate solid was then dried in a vacuum drying oven at 60° C. and -0.09 MPa. 95.26 g of agmatine sulfate solid was obtained, and the calculated agmatine sulfate crystal yield was 97.66%, with a purity of 99.69% for the agmatine sulfate crystals.

[0183] Example 9: Preparation of Agmatine Hydrochloride from Arginine Catalyzed by Epoxy-Based Carrier-Immobilized Arginine Decarboxylase Mutant

[0184] The epoxy-supported immobilized arginine decarboxylase mutant M29 obtained in Example 6 was subjected to a conversion experiment with the substrate arginine in a mass ratio of 0.05:1. The initial concentrations of the components of the substrate solution were: arginine 330 g / L, pyridoxal phosphate 0.4 g / L, and α-ketoglutaric acid 0.7 g / L. The temperature was controlled at 37°C, and the pH of the substrate solution was controlled to 5.5 with dilute hydrochloric acid. The conversion was completed when the conversion time was 11 hours. After the conversion was completed, the reaction volume increased due to the addition of dilute hydrochloric acid to control the pH. The concentration of agmatine hydrochloride detected by HPLC was 286.95 g / L, and the conversion rate was 99.15%.

[0185] The conversion solution was filtered to collect the epoxy-supported immobilized arginine decarboxylase mutant, and activated carbon was added to the filtered conversion solution in an amount of 1.5% of the weight of agmatine hydrochloride in the conversion solution. After decolorization, the supernatant was filtered and collected for concentrated crystallization.

[0186] 300 mL of the collected supernatant was collected, and the theoretical weight of agmatine hydrochloride was 86.82 g. After concentration on a rotary evaporator, the temperature was lowered to 10° C. Ethanol was added dropwise, and stirring was continued at 200 rpm for 2 hours before filtration. The agmatine hydrochloride solid was then dried in a vacuum drying oven at 60° C. and -0.09 MPa. 82.17 g of agmatine hydrochloride solid was obtained, and the calculated agmatine hydrochloride crystal yield was 94.64%, and the purity of the agmatine hydrochloride crystals reached 99.16%.

[0187] Example 10: Preparation of Agmatine Nitrate from Arginine Catalyzed by an Epoxy-Based Carrier-Immobilized Arginine Decarboxylase Mutant

[0188] The epoxy-supported immobilized arginine decarboxylase mutant M29 obtained in Example 6 was subjected to a conversion experiment with the substrate arginine in a mass ratio of 0.05:1. The initial concentrations of the components of the substrate solution were: arginine 330 g / L, pyridoxal phosphate 0.4 g / L, and α-ketoglutaric acid 0.7 g / L. The temperature was controlled at 37°C, and the pH of the substrate solution was controlled to 5.5 using dilute nitric acid. The conversion was completed when the conversion time was 11 hours. After the conversion was completed, the reaction volume was increased due to the addition of dilute nitric acid to control the pH. The agmatine nitrate concentration was 360.59 g / L by HPLC sampling, and the conversion rate was 98.78%.

[0189] The conversion solution was filtered to collect the epoxy-supported immobilized arginine decarboxylase mutant, and activated carbon was added to the filtered conversion solution in an amount of 1.5% of the weight of agmatine nitrate in the conversion solution. After decolorization, the supernatant was filtered and collected for concentrated crystallization.

[0190] 300 mL of the collected supernatant was collected, and the theoretical weight of agmatine nitrate was 109.51 g. After concentration on a rotary evaporator, the temperature was lowered to 10° C. Ethanol was added dropwise, and stirring was continued at 200 rpm for 2 hours before filtration. The agmatine nitrate solid was then dried in a vacuum drying oven at 60° C. and -0.09 MPa. 105.60 g of agmatine nitrate solid was obtained, and the calculated agmatine nitrate crystal yield was 96.43%, with agmatine nitrate crystal purity of 99.18%.

[0191] As shown in Examples 8-10, the immobilized arginine decarboxylase mutant provided by the present invention can be used to catalyze arginine to efficiently prepare agmatine salts (sulfate, hydrochloride, nitrate), and the yield and concentration of the agmatine salt product are both high.

[0192] Example 11: Preparation of Agmatine Sulfate from Arginine Catalyzed by an Epoxy-Based Carrier-Immobilized Arginine Decarboxylase Mutant

[0193] The epoxy-supported immobilized arginine decarboxylase mutant M29 obtained in Example 6 was subjected to a conversion experiment with the substrate arginine in a mass ratio of 0.05:1. The initial concentrations of the components of the substrate solution were: arginine 200 g / L, pyridoxal phosphate 0.4 g / L, and α-ketoglutaric acid 0.7 g / L. The temperature was controlled at 37°C, and the pH of the substrate solution was controlled to 5.5 using dilute sulfuric acid. The conversion was completed when the conversion time was 10 hours. After the conversion was completed, the reaction volume increased due to the addition of dilute sulfuric acid to control the pH. The concentration of agmatine sulfate detected by HPLC was 216.76 g / L, and the conversion rate was 99.25%.

[0194] The conversion solution was filtered to collect the epoxy-supported immobilized arginine decarboxylase mutant, and activated carbon was added to the filtered conversion solution in an amount of 1.5% of the weight of agmatine sulfate in the conversion solution. After decolorization, the supernatant was filtered and collected for concentrated crystallization.

[0195] 300 mL of the resulting supernatant was collected, yielding a theoretical agmatine sulfate weight of 65.52 g. The mixture was concentrated on a rotary evaporator and cooled to 10° C. Ethanol was added dropwise, and stirring continued at 200 rpm for 2 hours before filtration. The agmatine sulfate solid was then dried in a vacuum drying oven at 60° C. and −0.09 MPa to obtain 63.97 g of solid, yielding agmatine sulfate crystals of 97.63% (calculated). The purity of the agmatine sulfate crystals was 99.70%.

[0196] Example 12: Preparation of Agmatine Sulfate from Arginine Catalyzed by an Epoxy-Based Carrier-Immobilized Arginine Decarboxylase Mutant

[0197] The epoxy-supported immobilized arginine decarboxylase mutant M29 obtained in Example 6 was subjected to a conversion experiment with the substrate arginine in a mass ratio of 0.05:1. The initial concentrations of the components of the substrate solution were: 150 g / L arginine, 0.4 g / L pyridoxal phosphate, and 0.7 g / L α-ketoglutaric acid. The temperature was controlled at 37°C, and the pH of the substrate solution was controlled to 5.5 with dilute sulfuric acid. The conversion was completed when the conversion time was 9 hours. After the conversion was completed, the reaction volume was increased due to the addition of dilute sulfuric acid to control the pH. The concentration of agmatine sulfate detected by HPLC was 169.66 g / L, and the conversion rate was 99.27%.

[0198] The conversion solution was filtered to collect the epoxy-supported immobilized arginine decarboxylase mutant, and activated carbon was added to the filtered conversion solution in an amount of 1.5% of the weight of agmatine sulfate in the conversion solution. After decolorization, the supernatant was filtered and collected for concentrated crystallization.

[0199] 300 mL of the resulting supernatant was collected, yielding a theoretical agmatine sulfate weight of 51.27 g. The mixture was concentrated on a rotary evaporator and cooled to 10° C. Ethanol was added dropwise, and stirring continued at 200 rpm for 2 hours before filtration. The agmatine sulfate solid was then dried in a vacuum drying oven at 60° C. and −0.09 MPa to obtain 50.04 g of solid, yielding a 97.60% agmatine sulfate crystal yield. The purity of the agmatine sulfate crystals was 99.72%.

[0200] Example 13: Preparation of Agmatine Sulfate from Arginine Catalyzed by Epoxy-Carrier-Immobilized Arginine Decarboxylase Mutant

[0201] The epoxy-supported immobilized arginine decarboxylase mutant M29 obtained in Example 6 was subjected to a conversion experiment with the substrate arginine in a mass ratio of 0.05:1. The initial concentrations of the components of the substrate solution were: arginine 240 g / L, pyridoxal phosphate 0.4 g / L, and α-ketoglutaric acid 0.7 g / L. The temperature was controlled at 37°C, and the pH of the substrate solution was controlled to 5.0 using dilute sulfuric acid. The conversion was completed when the conversion time was 12 hours. After the conversion was completed, the reaction volume was increased due to the addition of dilute sulfuric acid to control the pH. The concentration of agmatine sulfate detected by HPLC was 244.54 g / L, and the conversion rate was 96.42%.

[0202] The conversion solution was filtered to collect the epoxy-supported immobilized arginine decarboxylase mutant, and activated carbon was added to the filtered conversion solution in an amount of 1.5% of the weight of agmatine sulfate in the conversion solution. After decolorization, the supernatant was filtered and collected for concentrated crystallization.

[0203] 300 mL of the collected supernatant was collected, and the theoretical weight of agmatine sulfate was 76.08 g. After concentration on a rotary evaporator, the temperature was lowered to 10° C. Ethanol was added dropwise, and stirring was continued at 200 rpm for 2 hours before filtration. The agmatine sulfate solid was then dried in a vacuum drying oven at 60° C. and -0.09 MPa. 73.44 g of agmatine sulfate solid was obtained, and the calculated agmatine sulfate crystal yield was 96.53%, with a purity of 98.21% for the agmatine sulfate crystals.

[0204] Example 14: Preparation of Agmatine Sulfate from Arginine Catalyzed by an Epoxy-Based Carrier-Immobilized Arginine Decarboxylase Mutant

[0205] The epoxy-supported immobilized arginine decarboxylase mutant M29 obtained in Example 6 was subjected to a conversion experiment with the substrate arginine in a mass ratio of 0.05:1. The initial concentrations of the components of the substrate solution were: 240 g / L arginine, 0.4 g / L pyridoxal phosphate, and 0.7 g / L α-ketoglutaric acid. The temperature was controlled at 37°C, and the pH of the substrate solution was controlled to 7.0 using dilute sulfuric acid. The conversion was completed when the conversion time was 12 hours. After the conversion was completed, the reaction volume was increased due to the addition of dilute sulfuric acid to control the pH. The concentration of agmatine sulfate detected by HPLC was 220.00 g / L, and the conversion rate was 86.74%.

[0206] The conversion solution was filtered to collect the epoxy-supported immobilized arginine decarboxylase mutant, and activated carbon was added to the filtered conversion solution in an amount of 1.5% of the weight of agmatine sulfate in the conversion solution. After decolorization, the supernatant was filtered and collected for concentrated crystallization.

[0207] 300 mL of the collected supernatant was collected, and the theoretical weight of agmatine sulfate was 76.08 g. After concentration on a rotary evaporator, the temperature was lowered to 10° C. Ethanol was added dropwise, and stirring was continued at 200 rpm for 2 hours before filtration. The agmatine sulfate solid was then dried in a vacuum drying oven at 60° C. and -0.09 MPa. 71.98 g of agmatine sulfate solid was obtained, and the calculated agmatine sulfate crystal yield was 94.61%, with a purity of 89.55% for the agmatine sulfate crystals.

[0208] Comparative Example 1

[0209] The epoxy-supported immobilized wild-type arginine decarboxylase W1 obtained in Example 6 was subjected to a conversion experiment with the substrate arginine in a mass ratio of 0.05:1. The initial concentrations of the components of the substrate solution were: arginine 330 g / L, pyridoxal phosphate 0.4 g / L, and α-ketoglutaric acid 0.7 g / L. The temperature was controlled at 37°C, and the pH of the substrate solution was controlled to 5.5 with dilute sulfuric acid. The conversion was completed when the conversion time was 11 hours. After the conversion was completed, the reaction volume was increased due to the addition of dilute sulfuric acid to control the pH. The concentration of agmatine sulfate detected by HPLC was 226.15 g / L, and the conversion rate was 68.53%.

[0210] Comparison of the conversion experiments performed under the same conditions between the immobilized wild-type arginine decarboxylase and the immobilized arginine decarboxylase mutant M29 in Example 8 revealed that the immobilized arginine decarboxylase mutant had a higher conversion rate than the immobilized wild-type arginine decarboxylase.

[0211] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0212] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention. SEQUENCE LISTING <110> Wuhan Yuanda Hongyuan Co., Ltd. <120> An arginine decarboxylase mutant and its immobilization for preparing agmatine salt <130> PIDC3205525 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 756 <212> PRT <213> Salmonella enterica <400> 1 Met Met Lys Val Leu Ile Val Glu Ser Glu Phe Leu His Gln Asp Thr 1 5 10 15 Trp Val Gly Asn Ala Val Glu Arg Leu Ala Asp Ala Leu Ser Gln Gln 20 25 30 Asn Val Thr Val Ile Lys Ser Thr Ser Phe Asp Asp Gly Tyr Ala Ile 35 40 45 Leu Ser Ala Asn Glu Ala Ile Asp Cys Leu Met Phe Ser Tyr Gln Met 50 55 60 Glu Gln Pro Asp Glu His Leu Ser Val Arg Gln Leu Ile Gly Lys Leu 65 70 75 80 His Glu Arg Gln Gln Asn Val Pro Val Phe Leu Leu Gly Asp Arg Glu 85 90 95 Lys Ala Thr Ala Ser Leu Asp Arg Asp Leu Leu Glu Leu Val Asp Glu 100 105 110 Phe Ala Trp Ile Leu Glu Asp Thr Ala Asp Phe Ile Ala Gly Arg Ala 115 120 125 Val Ala Ala Met Thr Arg Tyr Arg Gln Gln Leu Leu Pro Pro Leu Phe 130 135 140 Asn Ala Leu Met Thr Ser Ser Ile His His Glu Tyr Ser Trp Ala Ala 145 150 155 160 Pro Gly His Gln Gly Gly Val Gly Phe Thr Lys Thr Pro Ser Gly Arg 165 170 175 Phe Tyr His Asp Tyr Tyr Gly Glu Asn Leu Ala Arg Ser Asp Met Gly 180 185 190 Ile Gly Thr Ser Leu Leu Gly Ser Leu Leu Ala His Thr Gly Ala Phe 195 200 205 Gly Glu Ser Glu Lys Asn Ala Ala Arg Val Phe Gly Ala Asp Arg Ser 210 215 220 Trp Ser Val Val Val Gly Thr Ser Gly Ser Asn Arg Thr Ile Met Gln 225 230 235 240 Ala Cys Met Thr Asp Asn Asp Val Val Val Leu Asp Arg Asn Cys His 245 250 255 Lys Ser Ile Glu Gln Gly Leu Ile Leu Thr Gly Ala Lys Pro Val Tyr 260 265 270 Met Val Pro Ser Arg Asn Arg Tyr Gly Ile Ile Gly Pro Ile Tyr Pro 275 280 285 Gln Glu Met Gln Pro Glu Thr Leu Gln Lys Lys Ile Ser Ala Ser Pro 290 295 300 Leu Thr Lys Thr Lys Ala Gly Gln Lys Pro Ser Tyr Ser Val Val Thr 305 310 315 320 Asn Cys Thr Tyr Asp Gly Val Cys Tyr Asn Ala Lys Glu Ala Gln Asp 325 330 335 Gly Leu Ala Lys Thr Ser Asp Arg Ile His Phe Asp Glu Ala Trp Tyr 340 345 350 Gly Tyr Ala Arg Phe Asn Pro Ile Tyr Cys Asp His Tyr Ala Met Arg 355 360 365 Gly Glu Pro Gly Asp His Asn Gly Pro Thr Val Phe Ala Thr His Ser 370 375 380 Thr His Lys Leu Leu Asn Ala Leu Ser Gln Ala Ser Tyr Ile His Val 385 390 395 400 Arg Glu Gly Arg Gly Ala Val Asn Phe Ser Arg Phe Asn Gln Ala Tyr 405 410 415 Met Met His Ala Thr Thr Ser Pro Leu Tyr Ala Ile Cys Ala Ser Asn 420 425 430 Asp Val Ala Val Ser Met Met Asp Gly Asn Ser Gly Leu Ser Leu Thr 435 440 445 Gln Glu Val Ile Asp Glu Ala Val Asp Phe Arg Gln Ala Met Ala Arg 450 455 460 Leu Tyr Lys Glu Phe Thr Asp Glu Gly Asp Trp Phe Phe Lys Pro Trp 465 470 475 480 Asn Lys Asp Val Val Thr Asp Pro Gln Thr Gly Lys Thr Tyr Asp Phe 485 490 495 Ala Asp Ala Pro Ala Lys Leu Leu Ala Thr Asp Gln Asn Cys Trp Val 500 505 510 Met Arg Pro Gly Glu Thr Trp His Gly Phe Lys Asp Leu Pro Asp Asn 515 520 525 Trp Ser Met Leu Asp Pro Ile Lys Val Ser Ile Leu Ala Pro Gly Met 530 535 540 Gly Asp Asp Gly Glu Leu Glu Ala Ser Gly Val Pro Ala Ala Leu Val 545 550 555 560 Thr Ala Trp Leu Gly Arg His Gly Ile Val Pro Thr Arg Thr Thr Asp 565 570 575 Phe Gln Ile Met Phe Leu Phe Ser Met Gly Val Thr Arg Gly Lys Trp 580 585 590 Gly Thr Leu Ile Asn Thr Leu Cys Ser Phe Lys His His Tyr Asp Ala 595 600 605 Asn Thr Pro Leu Ala Gln Val Met Pro Glu Leu Val Gln Asp Tyr Pro 610 615 620 Asp Thr Tyr Ala Asn Met Gly Ile His Asp Leu Gly Asp Lys Met Phe 625 630 635 640 Ala Trp Leu Arg Glu Asn Asn Pro Gly Ala Arg Leu Asn Ala Ala Tyr 645 650 655 Ser Thr Leu Pro Val Ala Glu Ile Thr Pro Arg Asp Ala Tyr Asn Ala 660 665 670 Ile Val Asn Asn Asn Ile Glu Met Val Ala Ile Glu Asn Leu Pro Gly 675 680 685 Arg Ile Ala Ala Asn Ser Val Ile Pro Tyr Pro Pro Gly Ile Pro Met 690 695 700 Leu Leu Ser Gly Glu Asn Phe Gly Asp Glu Asn Ser Pro Gln Val Gly 705 710 715 720 Tyr Leu Arg Ser Leu Gln Ser Trp Asp His His Phe Pro Gly Phe Gly 725 730 735 His Gly Thr Glu Gly Gly Gly Ile Ala Asp Gly Val Tyr His Val Met 740 745 750 Cys Val Lys Ala 755 <210> 2 <211> 2268 <212> DNA <213> Salmonella enterica <400> 2 atgatgaaag ttctgattgt agaaagcgag ttcctgcacc aggacacctg ggttggtaac 60 gcagtggaac gtctggctga cgcgctgtct cagcagaacg tgactgttat caaatctacc 120 agcttcgacg atggttacgc gatcctgagc gcaaatgaag cgatcgattg cctgatgttc 180 tcttatcaga tggagcagcc ggatgaacac ctgtctgtgc gtcagctgat cggcaaactg 240 cacgagcgtc agcaaaacgt accggttttc ctgctgggtg accgcgaaaa agccactgca 300 tctctggatc gcgacctgct ggaactggtt gacgaattcg cgtggattct ggaagacacc 360 gcagatttta tcgcgggccg tgcagtcgca gcaatgaccc gctatcgtca gcagctgctg 420 ccgccgctgt ttaacgcgct gatgacctct tctatccacc acgagtatag ctgggcagct 480 ccgggccacc agggtggcgt tggtttcact aaaaccccat ctggtcgttt ctatcatgac 540 tactatggtg aaaacctggc tcgcagcgat atgggtatcg gtacctctct gctgggcagc 600 ctgctggcgc acaccggcgc atttggtgaa tctgagaaga acgcggctcg tgtattcggc 660 gcagaccgtt cttggtccgt ggttgtgggt acttccggta gcaaccgtac tatcatgcag 720 gcttgtatga ccgacaacga tgttgtggtc ctggatcgta actgccacaa atccatcgaa 780 cagggcctga tcctgaccgg cgcaaaaccg gtgtacatgg ttccgtcccg taaccgttat 840 ggtatcattg gtccaattta tccgcaggaa atgcagccgg aaaccctgca gaaaaaaatt 900 tccgccagcc cgctgacgaa aaccaaggcg ggtcagaaac cgtcttatag cgttgttact 960 aactgcacgt acgatggcgt gtgttacaac gcaaaagaag cccaggacgg tctggctaag 1020 acctccgatc gcatccactt cgacgaggct tggtatggtt acgcacgttt caatcctatt 1080 tactgcgacc attacgcgat gcgcggtgaa ccgggcgatc ataacggccc aaccgttttc 1140 gcgactcact ctactcataa actgctgaat gcgctgagcc aggcttctta cattcacgta 1200 cgtgaaggcc gtggtgcggt taacttctct cgtttcaacc aggcctacat gatgcatgct 1260 accaccagcc ctctgtatgc aatctgcgct agcaacgatg tcgccgtctc catgatggac 1320 ggtaactctg gtctgtccct gactcaggaa gttattgatg aagctgttga tttccgtcag 1380 gccatggcgc gtctgtacaa agagtttact gatgaaggcg attggttctt caagccgtgg 1440 aacaaagatg ttgtgacgga ccctcaaacc ggtaagacct acgacttcgc ggatgcgccg 1500 gccaaactgc tggcaaccga ccagaactgc tgggtgatgc gcccgggtga aacctggcac 1560 ggctttaaag acctgccgga caactggtct atgctggacc cgatcaaagt gagcatcctg 1620 gccccgggca tgggcgatga cggtgaactg gaggcatctg gcgttccggc agccctggtt 1680 accgcttggc tgggccgcca cggtatcgta ccaacccgca ccaccgattt ccagatcatg 1740 tttctgttca gcatgggtgt aacgcgtggt aaatggggta cgctgatcaa caccctgtgt 1800 agcttcaaac atcactacga cgcgaatacc ccgctggccc aggtgatgcc ggaactggtt 1860 caggattacc cagacactta cgccaacatg ggcattcacg acctgggcga caaaatgttc 1920 gcttggctgc gtgaaaacaa cccgggcgca cgtctgaacg cggcgtactc taccctgccg 1980 gtggcggaga tcactccgcg tgacgcgtat aacgccatcg ttaacaacaa catcgaaatg 2040 gtggcaatcg aaaacctgcc gggtcgtatc gcggcgaaca gcgtgatccc gtatccgccg 2100 ggcattccaa tgctgctgtc tggtgaaaac ttcggtgacg aaaactcccc gcaggtcggc 2160 tacctgcgtt ccctgcagag ctgggatcat cacttcccgg gtttcggtca cggcacggaa 2220 ggcggcggta tcgcggatgg cgtctatcat gttatgtgtg taaaagct 2268 <210> 3 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> AdiA FP <400> 3 cggataacaa ttcccctcta gaatgatgaa agtactga 38 <210> 4 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> AdiA RP <400> 4 agctcgaatt cggatcctgc tttgacgcac ataacgtg 38 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> S151A FP <400> 5 cgcgctgatg acctccgcca ttcaccacga atact 35 <210> 6 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> S151A RP <400> 6 agtattcgtg gtgaatggcg gaggtcatca gcgcg 35 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> R188A FP <400> 7 cggcgaaaac ctggcagcta gcgacatggg tggtg 35 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> R188A RP <400> 8 caccacccat gtcgctagct gccaggtttt cgccg 35 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> L202A FP <400> 9 tctgctgggt tccctggccg ctcatactgg tgcct 35 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> L202A RP <400> 10 aggcaccagt atgagcggcc agggaaccca gcaga 35 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> D348A FP <400> 11 tgaccgtatt cacttcgcgg aagcgtggta cggtt 35 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> D348A RP <400> 12 aaccgtacca cgcttccgcg aagtgaatac ggtca 35 <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> I193G FP <400> 13 agctagcgac atgggtggtg gtacttctct gctgg 35 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> I193G RP <400> 14 ccagcagaga agtaccacca cccatgtcgc tagct 35 <210> 15 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> E740G FP <400> 15 cggataacaa ttcccctcta gaatgatgaa agtactga 38 <210> 16 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> E740G RP <400> 16 gctcgaattc ggatcctgct ttgacgcaca taacgtggta gacgccatcc gcaataccac 60 cgccaccag 69 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> K257L FP <400> 17 ggaccgcaac tgtcacctgt ctatcgaaca aggtc 35 <210> 18 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> K257L RP <400> 18 gaccttgttc gatagacagg tgacagttgc ggtcc 35 <210> 19 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> K387L FP <400> 19 aactcattct actcatctgc tgctgaacgc cctgt 35 <210> 20 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> K387L RP <400> 20 acagggcgtt cagcagcaga tgagtagaat gagtt 35

Claims

1. An arginine decarboxylase mutant, characterized in that The amino acid sequence of the arginine decarboxylase mutant is the arginine decarboxylase amino acid sequence shown in SEQ ID NO: 1, with only the following mutations: (1) Serine at position 151 is replaced by alanine, or (2) the arginine at position 188 is replaced by alanine, or (3) Leucine at position 202 is replaced by alanine, or (4) Aspartic acid at position 348 is replaced by alanine, or (5) Isoleucine at position 193 is replaced by glycine, or (6) Glutamic acid at position 740 is replaced by glycine, or (7) Lysine at position 257 is replaced by leucine, or (8) Lysine at position 387 is replaced by leucine, or (9) Lysine at position 257 is substituted with leucine and serine at position 151 is substituted with alanine, or (10) Lysine at position 257 is substituted with leucine and Arginine at position 188 is substituted with alanine, or (11) Lysine at position 257 is substituted with leucine and leucine at position 202 is substituted with alanine, or (12) Lysine at position 257 is substituted with leucine and aspartic acid at position 348 is substituted with alanine, or (13) Lysine at position 387 is substituted with leucine and serine at position 151 is substituted with alanine, or (14) Lysine at position 387 is substituted with leucine and arginine at position 188 is substituted with alanine, or (15) Lysine at position 387 is substituted with leucine and leucine at position 202 is substituted with alanine, or (16) Lysine at position 387 is substituted with leucine and aspartic acid at position 348 is substituted with alanine, or (17) Lysine at position 257 is substituted with leucine, arginine at position 188 is substituted with alanine, and isoleucine at position 193 is substituted with glycine, or (18) Lysine at position 257 is substituted with leucine, arginine at position 188 is substituted with alanine, and glutamic acid at position 740 is substituted with glycine, or (19) Lysine at position 257 is substituted with leucine, leucine at position 202 is substituted with alanine, and isoleucine at position 193 is substituted with glycine, or (20) Lysine at position 257 is substituted with leucine, Leucine at position 202 is substituted with alanine, and Glutamic acid at position 740 is substituted with glycine, or (21) Lysine at position 387 is substituted with leucine, arginine at position 188 is substituted with alanine, and isoleucine at position 193 is substituted with glycine, or (22) Lysine at position 387 is substituted with leucine, arginine at position 188 is substituted with alanine, and glutamic acid at position 740 is substituted with glycine, or (23) Lysine at position 387 is substituted with leucine, Leucine at position 202 is substituted with alanine, and Isoleucine at position 193 is substituted with glycine, or (24) Lysine at position 387 is substituted with leucine, Leucine at position 202 is substituted with alanine, and Glutamic acid at position 740 is substituted with glycine, or (25) Lysine at position 257 is substituted with leucine, Leucine at position 202 is substituted with alanine, Isoleucine at position 193 is substituted with glycine, and Arginine at position 188 is substituted with alanine, or (26) Lysine at position 257 is substituted with leucine, Leucine at position 202 is substituted with alanine, Isoleucine at position 193 is substituted with glycine, Arginine at position 188 is substituted with alanine, and Serine at position 151 is substituted with alanine, or (27) Lysine at position 257 is substituted with leucine, Leucine at position 202 is substituted with alanine, Isoleucine at position 193 is substituted with glycine, Arginine at position 188 is substituted with alanine, Serine at position 151 is substituted with alanine, and Aspartic acid at position 348 is substituted with alanine, or (28) Lysine at position 257 is substituted with leucine, Leucine at position 202 is substituted with alanine, Isoleucine at position 193 is substituted with glycine, Arginine at position 188 is substituted with alanine, Serine at position 151 is substituted with alanine, Aspartic acid at position 348 is substituted with alanine, and Lysine at position 387 is substituted with leucine, or (29) Lysine at position 257 was substituted with leucine, leucine at position 202 was substituted with alanine, isoleucine at position 193 was substituted with glycine, arginine at position 188 was substituted with alanine, serine at position 151 was substituted with alanine, aspartic acid at position 348 was substituted with alanine, lysine at position 387 was substituted with leucine, and glutamic acid at position 740 was substituted with glycine.

2. A nucleic acid, characterized in that The nucleic acid encodes the arginine decarboxylase mutant according to claim 1.

3. An expression vector, characterized in that The expression vector comprises at least one of the nucleic acid sequences of claim 2.

4. A host cell, characterized in that The host cell comprises the expression vector according to claim 3, and the host cell is a microbial cell.

5. An immobilized enzyme, characterized in that include: The arginine decarboxylase mutant according to claim 1; as well as An immobilized carrier, on which the arginine decarboxylase mutant is immobilized.

6. The immobilized enzyme according to claim 5, characterized in that The immobilization carrier is selected from an amino carrier and / or an epoxy carrier.

7. Use of the arginine decarboxylase mutant according to claim 1 and / or the immobilized enzyme according to claim 5 or 6 in the preparation of agmatine salt.

8. A method for preparing an agmatine salt, characterized in that: The method comprises: mixing the immobilized enzyme according to claim 5 or 6 with a substrate solution to obtain a crude agmatine product; Acidifying the crude agmatine product with an acid to obtain a crude agmatine salt product; Wherein, the substrate solution includes arginine and pyridoxal phosphate.

9. The preparation method according to claim 8, characterized in that The mass ratio of the immobilized enzyme to arginine is 0.03:1 to 0.06:

1.

10. The preparation method according to claim 8, characterized in that The concentration of the arginine is 100-340 g / L.

11. The preparation method according to claim 8, characterized in that The concentration of the pyridoxal phosphate is 0.01-0.6 g / L.

12. The preparation method according to claim 8, characterized in that The substrate solution further includes α-ketoglutarate.

13. The preparation method according to claim 12, characterized in that The concentration of the α-ketoglutaric acid is 0.2-1.0 g / L.

14. The preparation method according to claim 8, characterized in that The pH value of the substrate solution is 5-8.

15. The preparation method according to claim 8, characterized in that The mixing temperature is 30-50° C. and the mixing time is 8-12 hours.

16. The preparation method according to claim 8, characterized in that The preparation method further comprises the step of decolorizing the crude agmatine salt product.

17. The preparation method according to claim 16, characterized in that The crude agmatine salt product is decolorized by using activated carbon to obtain a decolorized agmatine salt product.

18. The preparation method according to claim 16, characterized in that The preparation method further comprises concentrating the decolorized agmatine salt product and then dropwise adding ethanol to precipitate agmatine salt crystals.

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