Alkaline-resistant arginine deiminase

Abstract

The application provides an arginine deiminase mutant with alkali resistance, and the amino acid sequence of the mutant is SEQ ID NO:3. When L-arginine is used as a substrate to synthesize L-citrulline, the pH value of a reaction system can be as high as pH 11 or higher, and hydrochloric acid neutralization is not needed, so that the cost of product separation and purification and waste water treatment is significantly reduced.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically, it relates to an alkali-resistant arginine deiminase for the synthesis of L-citrulline. Background Technology

[0002] L-citrulline is an α-amino acid, first isolated from watermelon in 1914 by Japanese researchers Yotaro Koga and Ryo Odake, hence its name. To date, citrulline has been successfully isolated from watermelon juice, wild watermelon leaves, walnut kernels, walnut seedlings, and seeds. L-citrulline is a non-protein amino acid and a key intermediate in the urea cycle, in which mammals excrete ammonia by converting it into urea.

[0003]

[0004] Foreign scientists, particularly in Japan, the United States, and Europe, have conducted extensive research on the physiological activities and production of citrulline. Regarding its physiological functions, citrulline has been found to possess several important pharmacological functions, such as free radical scavenging, which is of great significance in human health; and vasodilatory effects, which hold promise for treating endotoxemia and sepsis. Japanese scientists have developed a new citrulline-containing polypeptide and are exploring its potential as an anti-AIDS drug. Citrulline is also considered a highly effective antioxidant, a function already applied in cosmetics, pharmaceuticals, and health foods. Recent international research has demonstrated that citrulline possesses many important physiological functions, such as free radical scavenging, acting as an indicator of foreign body rejection, vasodilatory effects, blood pressure stabilization, diagnosis of rheumatoid arthritis, and antioxidant properties, indicating a very broad application prospect.

[0005] There are three main methods for preparing L-citrulline: chemical method, fermentation method, and enzymatic catalysis method. The chemical method involves hydrolyzing L-arginine under alkaline conditions to obtain L-citrulline. However, process control is difficult, the product contains the enantiomer D-citrulline, affecting product quality, and the process generates large amounts of wastewater, polluting the environment. Therefore, it has been gradually phased out. The fermentation method has been reported extensively, but its production scale is small. Currently, the mainstream production process involves preparing L-citrulline from L-arginine using arginine deiminase. L-arginine is a basic amino acid, and its aqueous solution is highly alkaline. High-concentration arginine solutions typically reach a pH above 11, generally requiring neutralization with hydrochloric acid or sulfuric acid before enzymatic catalysis can proceed. Otherwise, the enzyme will have little or no catalytic activity, significantly increasing production and post-processing costs. Summary of the Invention

[0006] To overcome the aforementioned shortcomings of existing enzyme catalysis technologies, the inventors attempted to screen for mutations in arginine deiminase (ADI), which catalyzes the L-arginine reaction. This resulted in a mutant resistant to alkaline environments (pH 9 and even pH 11 and above), capable of catalyzing the oxidation of the imine group in L-arginine without altering the stereoconfiguration of the chiral center (α-position). Through multiple rounds of directed evolution, a highly pH-tolerant arginine deiminase was obtained, capable of directly catalyzing the reaction in aqueous solutions with arginine concentrations not less than 100 g / L, significantly reducing production costs. Specifically, this invention includes the following technical solutions.

[0007] An arginine deiminase mutant, which is a polypeptide selected from the following group:

[0008] (1) A polypeptide with the amino acid sequence SEQ ID NO:3;

[0009] (2) A polypeptide having more than 90% homology with SEQ ID NO:3, preferably more than 92%, more preferably more than 95%, more preferably more than 97%, more preferably more than 98%, and more preferably more than 99% homology, and whose enzyme activity is increased compared with SEQ ID NO:3 in an environment with a pH value of 11 or higher.

[0010] MNNGINVNSEIGKLKSVLLHRPGAEVENITPDTMKQLLFDDIPYLKIAQKEHDFFAQTLRDNGAETVYIENLATEVFEKSSETKEEFLSHLLHEAGYRPGRTYDGLTEYLTSMSTKDMVEKIYAGVRKNE F D N KRTALSDMAGSDA K NYFYLNPLPNAYFTRDPQASMGVGMTINKMTFPARQPESLITEYVMANHPRFKDTPIWRDRNHTTRIEGGDELILNKTTVAIGVSERTSSKTIQNLAKELFANPLSTFDTVLAVEIPHNHAMMHLDTVFTMINHDQFTVFPGIMDGAGNINVFILRPGKDDEVEIEHLTDKAALKKVLNLSELDLIECGAGDPIAAPREQWN N GSNTLAIAPGEIVTYDRNYVTVELLKEHGIKVHEILSSELGRGRGGARCMSQPLWREDL (SEQ ID NO: 3).

[0011] The mutant SEQ ID NO:3 is a mutant of the wild-type enzyme SEQ ID NO:1 (GenBank:CP010050.1) where L (leucine) at position 131 is replaced with F (phenylalanine), I (isoleucine) at position 133 is replaced with N (asparagine), E (glutamate) at position 147 is replaced with K (lysine), and D (aspartic acid) at position 351 is replaced with N (asparagine).

[0012] MNNGINVNSEIGKLKSVLLHRPGAEVENITPDTMKQLLFDDIPYLKIAQKEHDFFAQTLRDNGAETVYIENLATEVFEKSSETKEEFLSHLLHEAGYRPGRTYDGLTEYLTSMSTKDMVEKIYAGVRKNELDIKRTALSDMAGSDAENYFYLNPLPNAYFTRDPQASMGVGMTINKMTFPARQPESLITEYVMANHPRFKDTPIWRD RNHTTRIEGGDELILNKTTVAIGVSERTSSKTIQNLAKELFANPLSTFDTVLAVEIPHNHAMMHLDTVFTMINHDQFTVFPGIMDGAGNINVFILRPGKDDEVEIEHLTDLKAALKKVLNLSELDLIECGAGDPIAAPREQWNDGSNTLAIAPGEIVTYDRNYVTVELLKEHGIKVHEILSSELGRGRGGARCMSQPLWREDL(SEQ ID NO:1).

[0013] A second aspect of the invention provides a gene encoding the above-mentioned arginine deiminase mutant.

[0014] Preferably, the gene encoding the above-mentioned arginine deiminase mutant SEQ ID NO:3 can be a polynucleotide as shown in the nucleotide sequence SEQ ID NO:4, or a polynucleotide having 90% or more, preferably 92% or more, preferably 95% or more, preferably 97% or more, preferably 98% or more, more preferably 99% or more homology with SEQ ID NO:4.

[0015] The present invention also provides plasmids containing the above-mentioned genes. The plasmids may be pSH plasmids or pET vectors such as pET22b, pET24a, and pET28a.

[0016] The present invention also provides microorganisms expressing the above-mentioned arginine deiminase mutant, such as SEQ ID NO:4, whose genome integrates the gene encoding the above-mentioned arginine deiminase mutant SEQ ID NO:3, or engineered bacteria transformed with the above-mentioned plasmid.

[0017] Another aspect of the present invention provides a microorganism for expressing the above-mentioned arginine deiminase mutant, wherein the genome integrates the coding gene of the above-mentioned mutant, or is transformed with the above-mentioned plasmid.

[0018] In one embodiment, the gene of the mutant can be integrated into the genome of a host microbial cell using gene editing technology, wherein the gene editing technology is selected from the group consisting of: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, and MuGENT.

[0019] Preferably, the microorganism is Escherichia coli, such as Escherichia coli BL21(DE3).

[0020] Another aspect of the present invention provides a method for synthesizing L-citrulline, comprising the following steps: using L-arginine as a substrate, catalyzing with the above-mentioned arginine deiminase mutant, such as SEQ ID NO:3 or its enzyme-enhanced derivative peptide, to obtain the target product L-citrulline.

[0021]

[0022] The aforementioned arginine deiminase mutants can be in enzyme form or in the form of the microbial cells in which they express.

[0023] The reaction system can be an alkaline environment with a pH value above pH 9, such as above pH 11, without the need for neutralization with acids (such as hydrochloric acid, sulfuric acid, phosphoric acid, etc.).

[0024] The reaction temperature of the above reaction system is 25-50℃, preferably 28-48℃, preferably 30-45℃, preferably 32-42℃, preferably 35-40℃, for example, around 37℃.

[0025] Optionally, a quaternary ammonium salt, such as hexadecyltrimethylammonium bromide (CTAB), can be added to the above reaction system to increase the reaction rate.

[0026] This invention involves random mutation of arginine deiminase (ADI) from Lactococcus lactis. Through high-throughput screening, a mutant with high enzyme activity and high pH tolerance is obtained. This mutant can catalyze the production of L-citrulline in a solution with an L-arginine concentration of 100 g / L (pH 11) without the need for neutralization with hydrochloric acid, greatly reducing the cost of separation, purification, and wastewater treatment, and providing a guarantee for the industrial-scale production of L-citrulline. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the structure of plasmid pET24a-lacADI. Detailed Implementation

[0028] The inventors discovered that the arginine deiminase SEQ ID NO:1 (GenBank:CP010050.1, referred to as LacADI in this paper) derived from Lactococcus lactis has the ability to catalyze the reaction of L-arginine to L-citrulline. This enzyme performs well in a neutral environment (below pH 8.0), with high enzyme activity and stereoselectivity. However, its enzyme activity is significantly reduced in an alkaline environment with a pH value above pH 9. It is pH sensitive and requires hydrochloric acid to regulate the pH of the reaction system during the catalytic reaction, which has insurmountable drawbacks in terms of economy and environmental protection.

[0029] To reduce the separation and purification process of L-citrulline, the product of the enzyme reaction, and lower wastewater treatment costs, it is necessary to modify the enzyme. The inventors attempted combined site-directed mutagenesis at multiple sites in wild-type arginine deiminase SEQ ID NO:1, hoping to modify it through amino acid sequence mutations, thereby improving the enzyme's tolerance to alkaline environments and even enhancing its activity while maintaining stereoselectivity.

[0030] In some implementations, the term “(alkaline tolerance and / or enzyme activity) improvement” can mean an improvement of at least 10% compared to a reference level, such as an improvement of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100%, or any improvement between 10% and 100%, or an improvement of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any improvement between 2 times and 10 times or more compared to a reference level.

[0031] The term "mutation" includes, but is not limited to, the substitution, deletion, insertion, or chemical modification of amino acid residues, preferably positive mutations, i.e., mutations that increase tolerance to alkaline environments (pH 10 and above) and / or enzyme activity. The substitution can be a non-conservative substitution, a conservative substitution, or a combination of both. A "conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and therefore generally includes the substitution of amino acids in polypeptides with amino acids from the same or similar amino acid definition categories. However, as used herein, if a conserved mutation can alternatively be an aliphatic to aliphatic, nonpolar to nonpolar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or restriction residue to restriction residue substitution, then a conserved mutation does not include hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue substitution. As is known in this technical field, common examples of conservative substitutions include: substitutions between aromatic amino acids F, W, and Y; substitutions between hydrophobic amino acids L, I, and V; substitutions between polar amino acids Q and N; substitutions between basic amino acids K, R, and H; substitutions between acidic amino acids D and E; and substitutions between hydroxyl amino acids S and T. Furthermore, A, V, L, or I can be conservatively mutated into another aliphatic residue or another nonpolar residue.

[0032] "Non-conservative substitution" refers to the substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitution can be performed between, rather than within, the amino acids defined above. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the substituted region (e.g., proline replacing glycine), (b) charge or hydrophobicity, or (c) side chain volume.

[0033] "Deletion" refers to a modification of a peptide by removing one or more amino acids from a reference peptide. Deletion can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% or up to 20% of the total number of amino acids constituting the reference enzyme, while preserving enzyme activity and / or the modified properties of the engineered aldolase. Deletion can target the interior and / or ends of the peptide. In several embodiments, the deletion can comprise a continuous segment or can be discontinuous.

[0034] "Insertion" refers to a modification of a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, modified engineered aldolases include inserting one or more amino acids into a naturally occurring aldolase and inserting one or more amino acids into other modified aldolase polypeptides. The insertion can be internal to the polypeptide, or at the carboxyl terminus or amino terminus. Insertions as used herein include fusion proteins as known in the art. The insertion can be a continuous amino acid segment or separated by one or more amino acids in a naturally occurring polypeptide.

[0035] Starting from the wild enzyme SEQ ID NO:1, the inventors obtained a relatively satisfactory arginine deiminase mutant from the mutant library after multiple rounds of mutation screening.

[0036] In this paper, the terms "wild-type" and "wild-type enzyme" have the same meaning, referring to the arginine deiminase with the amino acid sequence SEQ ID NO:1. Correspondingly, mutants of the wild-type enzyme, such as SEQ ID NO:3, can be called "mutant enzymes". For the sake of convenience, the wild-type arginine deiminase and its mutants are collectively referred to as "arginine deiminase" in this paper.

[0037] The arginine deiminase mutant of the present invention has 410 amino acids with a well-defined sequence, thus those skilled in the art can easily obtain its encoding gene, expression cassettes and plasmids containing these genes, and transformants containing the plasmids. For example, the encoding gene of wild-type arginine deiminase SEQ ID NO:1 can be SEQ ID NO:2.

[0038] These genes, expression cassettes, plasmids, and transformants can be easily obtained by those skilled in the art through well-known genetic engineering construction methods.

[0039] To optimally express the arginine deiminase mutant SEQ ID NO:3 in a microbial host such as Escherichia coli, which is most commonly used in genetic engineering, the expression gene can be codon-optimized.

[0040] Codon optimization is a technique used to maximize protein expression in an organism by increasing the translation efficiency of genes of interest. Different organisms often exhibit a particular preference for one of a set of codons encoding the same amino acid due to mutational predisposition and natural selection. For example, in fast-growing microorganisms such as *E. coli*, optimized codons reflect the composition of their respective genomic tRNA repertoires. Thus, in fast-growing microorganisms, low-frequency codons for amino acids can be replaced with high-frequency codons for the same amino acid. Consequently, the expression of the optimized DNA sequence is improved in fast-growing microorganisms. For example, after codon optimization, the gene encoding the mutant enzyme SEQ ID NO:3 could be SEQ ID NO:4.

[0041] The construction of genetically engineered bacteria expressing the arginine deiminase mutant SEQ ID NO:3 can be carried out by conventional plasmid transformation, i.e., cloning the enzyme gene into a plasmid vector suitable for replication in a cell module, and then transforming it into the cell module by chemical transformation or electroporation; alternatively, the enzyme gene can be cloned into the genome of the cell module by gene editing technology, such as homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT (multiplex genome editing by natural transformation), etc.

[0042] In the reaction system of this invention, arginine deiminase can be in either enzyme form or bacterial cell form. The enzyme form includes free enzymes and immobilized enzymes, including purified enzymes, crude enzymes, fermentation broth, and enzymes immobilized on a carrier.

[0043] In the field of biocatalysis, it is well known that compared with free enzyme methods, the application of immobilized enzyme technology has advantages such as simplified production processes and improved production efficiency. Furthermore, because enzymes can be used multiple times and their stability is improved, the productivity per unit enzyme is effectively increased. Secondly, immobilized enzymes are easily separated from substrates and products, simplifying purification processes, resulting in higher yields and better product quality.

[0044] Optionally, the microorganisms constructed in this invention, such as the engineered Escherichia coli BL21(DE3), can be used as catalysts in the form of bacterial cells, including both live and dead cells. The bacterial cells themselves are a natural form of enzyme immobilization and can be used as an enzyme preparation for catalytic reactions without the need for disruption or even extraction and purification. Since the reaction substrates and products are small molecule compounds, they can easily cross the biological barrier of the bacterial cell—the cell membrane—thus eliminating the need for cell disruption, which is economically advantageous.

[0045] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0046] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0047] Example

[0048] Materials and methods

[0049] In the embodiments, the whole gene synthesis, primer synthesis and sequencing were all completed by Anhui General Biotechnology Co., Ltd.

[0050] The molecular biology experiments in the examples, including plasmid construction, enzyme digestion, competent cell preparation, and transformation, were mainly conducted in accordance with *Molecular Cloning: A Laboratory Manual* (3rd Edition), edited by J. Sambrook and DW. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. Specific experimental conditions could be determined through simple experiments if necessary.

[0051] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0052] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2, autoclaved at 121°C for 20 min. (LB solid medium is supplemented with 20 g / L agar powder.)

[0053] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K2HPO4·3H2O, 2.31 g / L KH2PO4, 5 g / L glycerol, pH 7.0-7.5 (TB solid medium is supplemented with 20 g / L agar powder), autoclaved at 121℃ for 20 min.

[0054] 96-well plate colorimetric reagent:

[0055] (1) Ferric acid solution: Slowly add 160ml of 98% concentrated sulfuric acid and 70ml of 85% phosphoric acid to 600ml of water. After cooling to room temperature, add 10ml of 10g / L FeCl3·6H2O and make up to 1L. Seal and store.

[0056] (2) 0.5M diacetyl monooxime (DAM): 50.55g, add water to make up to 1L.

[0057] Taq enzyme: Fermentas.

[0058] DpnI: Thermo Fisher Scientific.

[0059] DNA Gel Recovery Kit: Axygen DNA Gel Recovery Kit AP-GX-50.

[0060] Example 1: Construction of strain overexpressing wild-type arginine deiminase

[0061] 1.1 Construction of plasmids

[0062] Based on the amino acid sequence of wild-type arginine deiminase SEQ ID NO:1 (GenBank:CP010050.1), the following primer pair LacADI-F / LacADI-R was designed:

[0063] Forward primer LacADI-F: gtttaactttaagaaggagatatacatatgaacaatggaattaatgttaac,

[0064] Reverse primer LacADI-R: cggatctcagtggtggtggtggtggtgctcgagttacaaatcttcacgccaaagtg.

[0065] Using the Lactococcus lactis genome (GenBank: CP010050.1) as a template, lacADI was amplified by PCR, and the pET24a-lacADI plasmid was constructed using the Gibson (Novozymes) assembly method (see...). Figure 1 ).

[0066] 1.2 Strain Construction

[0067] The recombinant plasmid pET24a-lacADI was transformed into the competent cells of the host Escherichia coli BL21(DE3) (Invitrogen) by electroporation to obtain recombinant Escherichia coli expressing wild-type arginine deiminase, which was named lacADI.

[0068] Example 2: Directed Evolution of LacADI

[0069] Using pET24a-lacADI plasmid as a template, error-prone PCR was performed using LacADI-F / LacADI-R (as above).

[0070] The 50 μL error-prone PCR reaction system includes: 10 ng plasmid template pET24a-lacADI, 50 pmol LacADI-F and LacADI-R, 1×Taq buffer, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, 1 mM dTTP, 7 mM MgCl2, (0 mM, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM) MnCl2, and 2.5 units of Taq enzyme.

[0071] The PCR reaction conditions were: 95℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 45 s; 30 cycles; 72℃ for 10 min.

[0072] The 0.95 kb PCR fragment was recovered by gel extraction. Using the recovered PCR fragment as a large primer, MegaPrimer PCR was performed using KOD-plus DNA polymerase: 94℃ for 5 min; 98℃ for 10 s, 60℃ for 30 s, 68℃ for 1 min, 25 cycles; 68℃ for 10 min. The plasmid template was digested with DpnI restriction endonuclease, electrotransformed into E. coli BL21(DE3) (Invitrogen) competent cells, and plated on LB agar plates containing 50 μg / mL kanamycin. A random mutant library of over 4000 clones was obtained for screening for alkaline-tolerant mutant enzymes.

[0073] Example 3: High-throughput screening of mutant libraries

[0074] Single-clone transformants were picked from LB agar plates and inoculated into 500 μL of LB liquid medium containing 50 μg / mL kanamycin in a 96-well deep-well plate. The plates were incubated overnight at 37°C. Then, 80 μL of the overnight culture was transferred to 800 μL of LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C for 3 h. Finally, 0.5 mM IPTG was added, the temperature was lowered to 30°C, and the plates were incubated for another 16 h. Then, 100 μL of the bacterial culture was centrifuged at 4000 rpm for 5 min to collect the bacterial cells. A colorimetric reaction was then performed.

[0075] The colorimetric steps were as follows: 100 μL of water was added to the 96-well plate for resuspending, followed by 100 μL of 2× reaction solution (200 mM arginine, 8 mM cetyltrimethylammonium bromide (CTAB), pH 11), and the mixture was incubated at 37°C for 2 hours. Then, 200 μL of water was added, and the mixture was centrifuged at 4000 rpm for 10 min. 100 μL of the supernatant was transferred to a new 96-well plate and diluted twice with 100 μL of water. Then, 120 μL of ferric sulfate solution and 40 μL of 0.5 M diacetyl monooxime (DAM) were added, and the mixture was incubated at 55°C for 15 min. The plate was then diluted with Tecan solution, and the OD490 was measured using a microplate reader. A higher colorimetric reading indicates higher enzyme activity of the strain (enzyme) at pH 11.

[0076] Mutant strains with significantly increased enzyme activity compared to the starting strain were selected, and their genomes were sequenced. Specifically, plasmids were extracted using the Axygen AP-MN-P-50 plasmid mini-extraction kit, and Anhui General Biotechnology Co., Ltd. was commissioned to perform gene sequencing on the plasmids to determine the gene sequence of arginine deiminase (mutant) and the amino acid mutation status.

[0077] Using the same method, the plasmids of the obtained alkali-resistant strains with high enzyme activity were used as templates for the second and third rounds of error-prone PCR.

[0078] As the alkali tolerance of directed evolution enzymes gradually increases, the enzyme catalytic reaction time and the amount of bacteria can be changed accordingly, for example, by reducing the amount of bacteria.

[0079] Example 4: Identification of the catalytic performance of the mutant enzyme

[0080] In each round of error-prone PCR screening, 1400 strains with high enzyme activity detected by 96-well plates were selected and cultured in LB shake flasks according to the cell preparation method described in Example 3. The cells were collected for enzyme catalytic performance identification. Simultaneously, plasmids were extracted (using the Axygen mini plasmid preparation kit) and sent to Anhui General Biotechnology Co., Ltd. for sequencing to verify the LacADI mutation target. The results of the three rounds of error-prone PCR are listed in Table 1 below.

[0081] Table 1. Enzyme activity parameters and mutation site confirmation (bacterial cell dosage 20 g / L)

[0082]

[0083] After three rounds of mutation screening, a mutant strain, ADI5, was obtained with ideal enzyme activity in a pH 11 reaction environment. The expressed mutant enzyme was confirmed by sequencing to be a (L131F, I133N, E147K, D351N) mutation, with the amino acid sequence SEQ ID NO:3. Another advantage of this mutant is the absence of substrate / product inhibition. The concentration of the substrate L-arginine can be as high as 100 g / L, and the concentration of the product L-citrulline can be as high as 89 g / L or more. No acid neutralization is required during the reaction, creating favorable conditions for industrial application.

Claims

1. An arginine deiminase mutant, which is a polypeptide with the amino acid sequence shown in SEQ ID NO:

3.

2. A gene encoding the arginine deiminase mutant as described in claim 1.

3. The gene as described in claim 2, characterized in that, The gene is a polynucleotide with a nucleotide sequence as shown in SEQ ID NO:

4.

4. A plasmid containing the gene as described in claim 3.

5. A microorganism for expressing the arginine deiminase mutant as described in claim 1, characterized in that, The genome integrates the gene as described in claim 3, or is transformed with the plasmid as described in claim 4.

6. The microorganism as described in claim 5, characterized in that, The microorganism in question is Escherichia coli.

7. Use of the arginine deiminase mutant as described in claim 1 or the microorganism as described in claim 6 in the production of L-citrulline.

8. The use as described in claim 7, characterized in that, Using L-arginine as a reaction substrate, L-citrulline was obtained by employing the arginine deiminase mutant as described in claim 1 or the microbial catalytic imine oxidation reaction as described in claim 5.

9. The use as described in claim 8, characterized in that, The pH of the reaction system is above pH 9.

10. The use as described in claim 8, characterized in that, Quaternary ammonium salts were also added to the reaction system.

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