CN115896076A

CN115896076A - Arginine deiminase mutant, recombinant thereof and application of mutant in catalytic production of citrulline

Info

Publication number: CN115896076A
Application number: CN202211368760.9A
Authority: CN
Inventors: 范超; 齐佳琨; 洪皓; 刘军; 刘明; 吴文忠
Original assignee: Innobio Corp ltd
Current assignee: Innobio Corp ltd
Priority date: 2022-11-03
Filing date: 2022-11-03
Publication date: 2023-04-04
Anticipated expiration: 2042-11-03
Also published as: CN115896076B

Abstract

The invention discloses an arginine deiminase mutant, a recombinant thereof and application thereof in catalytic production of citrulline. The mutant has wide tolerance pH range and is subjected to fusion expression with active enzyme, and the enzyme subjected to fusion expression has catalytic activity domain and immobilization capacity.

Description

Arginine deiminase mutant, recombinant thereof and application of mutant in catalytic production of citrulline

Technical Field

The invention relates to the technical field of biological enzyme immobilization, in particular to an arginine deiminase mutant, a recombinant thereof and application thereof in catalytic production of citrulline.

Background

The catalyst is found to accelerate the industrial production, and the biological enzyme as a natural polymer catalyst has the characteristics of high catalytic efficiency, strong specificity, mild reaction condition and no organic reagent pollution. However, due to the unstable physicochemical properties of the free biological enzyme, the biological enzyme cannot maintain the catalytic activity for a long time in the practical application process, and thus cannot exert the maximum catalytic performance. The appearance of the biological enzyme immobilization technology not only realizes the recycling of the enzyme, but also can improve the catalytic performance and activity of the enzyme.

Citrulline (Citrulline), also known as carbamoylornithine (carbamylornithin), is a non-protein alpha-amino acid involved in a series of metabolic processes, such as the urea cycle, the arginine-Citrulline cycle, etc.; has the physiological functions of eliminating free radicals, maintaining normal cholesterol level, affecting nitrogen balance in human body, regulating immunity of cardiovascular system, helping brain cell store and restore information, etc. Citrulline is added into sports functional beverages produced by canadian companies because of the functions of resisting aging, enhancing immunity, improving muscle strength and endurance of athletes and the like.

There are three main international methods for citrulline production: fermentation, chemical synthesis and enzymatic methods. The chemical synthesis method has high production control difficulty and causes more production wastewater. The current production cost of fermentation methods is high. Compared with the former two methods, the enzyme method has more excellent properties, does not need to add other substances or carry out other reactions in the production process, and has the advantages of simple process steps, easy operation and short production period.

Arginine deiminase (ADI, EC 3.5.3.6) can catalyze hydrolase for producing citrulline from Arginine, and has application prospect in enzymatic synthesis of citrulline. The above enzymatic processes are used in patents CN201510412709, CN201510230669, CN201510101489, and CN201510712520, but the substrate concentration is low, the culture period of the strain is relatively long, and the process is disadvantageous in the promotion of industrialization. Random mutation of the coding region of the gene can change the structure of the enzyme, thereby improving thermodynamic stability or kinetic stability, and further improving the catalytic activity of the enzyme. Patents CN201510012021 and CN201710119423 obtain mutants with improved activity, but the whole process has limitation in industrialization. Nowadays, various means for immobilizing enzymes have been developed, and the immobilization techniques of the embedding method have been widely used for immobilizing enzymes in the latter stage with the development of the techniques, since the first means for immobilizing enzymes are immobilized by the interaction force between a carrier and enzymes or the formation of covalent bonds. The patents CN200610145655 and CN111394295A establish the immobilization process of enzyme, but the related process steps are complicated or have uncertainty, and although the conversion rate is higher, the substrate concentration is not very high.

In summary, there is a need in the art for a new technology that can reduce production cost, improve economic benefits, achieve recycling of enzymes, and solve the problems of difficult enzyme recovery and high cost.

Disclosure of Invention

Aiming at the defects in the prior art, the invention constructs the secreted fusion enzyme by the fusion expression of the arginine deiminase mutant gene and the gene of the auxiliary infection protein residue mutant, the optimal temperature of the enzyme is improved by 10 ℃ compared with the original temperature, and the enzyme can be combined with the production cells of the enzyme at proper pH and temperature to complete the one-step purification-immobilization process, form the enzyme with improved stability and continuous catalytic production, realize the application of the enzyme in the catalytic production of citrulline and provide an effective way for the enzymatic industrial production. The mutant has wide tolerance pH range and is subjected to fusion expression with active enzyme, and the enzyme subjected to fusion expression simultaneously has catalytic activity range and immobilization capacity.

The invention provides an arginine deiminase mutant, and the amino acid sequence of the arginine deiminase mutant is shown in SEQ ID NO. 2.

The invention provides an arginine deiminase recombinant plasmid on the other hand, wherein the recombinant plasmid is obtained by recombining the gene of the auxiliary infection protein residue mutant and the gene of the arginine deiminase mutant; wherein, the coded amino acid sequence of the gene of the auxiliary infection protein residue mutant is shown in SEQ ID NO. 4; the coded amino acid sequence of the gene of the arginine deiminase mutant is shown as SEQ ID NO. 2.

The second aspect of the invention provides an secreted arginine deiminase recombinant bacterium containing the recombinant plasmid; the arginine deiminase recombinant thallus is obtained by carrying out fusion expression on a gene of an auxiliary infection protein residue mutant and a gene of an arginine deiminase mutant, wherein the amino acid sequence of the auxiliary infection protein residue mutant is shown as SEQ ID NO. 4; the amino acid sequence of the arginine deiminase mutant is shown as SEQ ID NO. 2.

Further, the host of the recombinant bacteria is selected from gram-positive bacteria such as Corynebacterium glutamicum, escherichia coli, bacillus subtilis, or lactic acid bacteria.

Furthermore, the mutant arginine deiminase protein can be secreted and expressed in the fermentation process of recombinant bacteria, is distributed in fermentation liquor, and does not influence the nutrient intake and metabolism space of cells after mass production; and the thalli after fusion expression has wider pH stable conditions (pH 5.0-6.0) relative to the original strain.

The third aspect of the invention provides an application of the recombinant strain for the exo-arginine deiminase in the catalytic production of citrulline; comprises the self-immobilization process of the arginine deiminase on the production strain thereof, wherein the arginine deiminase is obtained by fermenting the arginine deiminase recombinant bacteria.

Further, the substrate solution in the fermentation process is an L-arginine solution or an L-arginine hydrochloride solution with the concentration of 50-650g/L and the pH of 5.0-6.0.

Further, the concentration of the substrate solution in the fermentation process is 600-650g/L.

Further, the application also comprises an arginine deimination immobilized enzyme circulation process: carrying out fermentation culture after arginine deiminase immobilized carriers are obtained by self-immobilization of arginine deiminase obtained by fermentation of the recombinant bacteria on production strains, wherein the concentration of the arginine deiminase immobilized carriers in the fermentation process is 30-80g/50L, after the fermentation culture is finished, centrifugally separating insoluble substances from a reaction solution by regulating the temperature to room temperature and under the condition of neutral pH (25-30 ℃ and pH 7.5), and putting the insoluble substances into a new substrate solution again to repeat the immobilization process for a new round of fermentation; the circulation is not less than 22 times, and the fermentation time is not more than 6h. The method is a purification-immobilization continuous catalytic process, has wide application range and good industrial application value.

Further, the fermentation conditions of the application are 35-55 ℃ and pH 5.0-7.0; more preferably 40-50 deg.C and pH5.0-6.0.

Compared with the prior art, the invention has the following beneficial effects:

1. a design scheme for assisting in fusion expression of an IAP (infection protein) residue mutant and a mutant arginine deiminase protein can simultaneously realize a catalytic process and purification and immobilization of an exoenzyme;

2. different from surface display, the secreted arginine deiminase is expressed in the culture process and distributed in the fermentation liquor, and the nutrition intake and metabolism space of cells cannot be influenced after mass production;

3. the catalytic process of the immobilized enzyme can be amplified and circulated, and the catalytic activity can be still maintained under the circulation times of 22 times; the product concentration is not reduced.

4. The immobilized enzyme can efficiently catalyze high-concentration arginine (600 g/L) to generate citrulline.

5. Comparative example and example data comparative analysis of the present application shows that: the obtained secreted protein can be immobilized under specific circumstances (binding around ph7.5, room temperature) on a non/active matrix derived from genetically engineered cells producing the protein, in the example host cells for the secreted protein.

Drawings

FIG. 1 shows the comparison of the binding effect of immobilized enzymes at different pH values;

FIG. 2 comparison of binding effects of immobilized enzymes at different temperatures

FIG. 3 shows a comparison of the effect of thermal stability of immobilized enzymes;

fig. 4 citrulline yield after amplification of the catalytic process.

Detailed Description

Specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments.

In the present invention, unless otherwise specified, all experimental methods used are conventional methods, and materials, reagents and the like used are commercially available.

And (3) measuring the thermal stability: the concentration of the cells expressing the active enzyme was diluted to 30g/L with PBS, incubated at 40 ℃ and 50 ℃ and the residual enzyme activity was measured at different times of incubation.

Half-life detection: t is t _1/2 The value is the corresponding time when the residual enzyme activity is 50% after the enzyme is treated for a period of time at a specific temperature. The specific determination method is as follows: taking the activity of arginine deiminase which is not subjected to heat treatment as 100%, respectively measuring and calculating the residual enzyme activity of the enzyme after the enzyme is treated at 40 ℃ and 50 ℃ for different time. The treatment time is used as the abscissa and Ln (% residual enzyme activity) is used as the ordinate, a curve of time-Ln (% residual enzyme activity) is drawn, and t is calculated according to the graph _1/2 ＝Ln2/K _d ，K _d Is the slope of the graph.

Example 1

1. Plasmid and Strain construction

The codon-optimized coding gene (SEQ ID NO: 1) of arginine deiminase is subjected to synthesis, xbaI and EcoRI enzyme cutting sites are introduced into two ends of the coding gene and are connected into pXMJ19 plasmid to obtain pXMJ-ADI plasmid. The plasmid is transferred into corynebacterium glutamicum after being verified to be correct by colony PCR and sequencing to obtain the C.g-pXMJ-ADI strain.

ADI gene and a vector pXMJ-IAP for expressing a coding gene of an auxiliary infection protein IAP residue (SEQ ID No. 3) are subjected to fusion expression, after sequencing verification, mutation is found, the gene is transferred into Corynebacterium glutamicum, and strains with catalytic activity are respectively named as C.g-pXMJ-IAP-ADI-SS and C.g-pXMJ-iapV69H-ADI-SS. The ADI-SS gene (SEQ ID NO: 2) was expressed separately according to the conventional procedure to obtain a strain named C.g-pXMJ-ADI-SS.

2. Expression of proteins

Inoculating C.g-pXMJ-ADI and C.g-pXMJ-ADI-SS engineering bacteria into BHI culture medium containing 20mg/L chloramphenicol, overnight culturing at 35 deg.C and 200rpm, inoculating into fresh BHI culture medium containing 50mg/L according to 10% inoculum size, culturing at 35 deg.C and 200rpm to OD ₅₆₂ After 0.6 to 0.8 hours, 0.2mM IPTG was added for induction, and cells were collected after 12 hours of induction.

Inoculating C.g-pXMJ-iap-ADI-SS and C.g-pXMJ-iapV69H-ADI-SS engineering bacteria into BHI culture medium containing 20mg/L chloramphenicol, culturing at 35 deg.C and 200rpm overnight, inoculating into fresh BHI culture medium containing 50mg/L chloramphenicol according to 10% inoculum size, culturing at 35 deg.C and 200rpm to OD ₅₆₂ After 0.6 to 0.8 hours, 0.2mM IPTG was added for induction for 12 hours.

3. Purification immobilization of fusion enzymes

Adjusting the fermentation liquid of C.g-pXMJ-iap-ADI-SS and C.g-pXMJ-iapV69H-ADI-SS to 25-30 ℃, combining for 1H under the pH value of 7.5, and washing once to obtain the pure enzyme fixed on the cell surface, namely the immobilized enzyme.

Example 2

Effect of different pH on arginine deiminase and its mutant Effect

The reaction system is as follows: 50g/L arginine solution, adjusting pH = 5.0-7.0 with 30% hydrochloric acid/20% sulfuric acid, and adding 2g/L C.g-pXMJ-ADI-SS and C.g-pXMJ-ADI; reacting the cells of C.g-pXMJ-iap-ADI-SS and C.g-pXMJ-iapV69H-ADI-SS at 50 ℃ for 30min, sampling, stopping the reaction by 10% trichloroacetic acid in equal proportion, centrifuging at 8000rpm for 5min, and detecting the concentration of the citrulline product and the residue of arginine as a substrate by HPLC.

In the above reaction system, the influence of pH on the reaction effect was compared. As can be seen from fig. 1, the mutation of arginine deiminase did not affect its optimum pH, both pH =5.0, and the optimum pH was shifted after fusion expression of the sequence of the helper invasion residue. Optimal pH of c.g-pXMJ-iap-ADI-SS =6.5-7.0, optimal pH of c.g-pXMJ-iapV69H-ADI-SS =5.0-6.0. And it can be seen that the catalytic effect of C.g-pXMJ-iap-ADI-SS is worse than that of C.g-pXMJ-ADI-SS and C.g-pXMJ-iapV69H-ADI-SS at the same time

Example 3

Effect of different temperatures on the bonding Effect

The reaction system is as follows: 50g/L arginine solution, using 30% hydrochloric acid/20% sulfuric acid to adjust to respective optimum pH, adding 0.5g/L C.g-pXMJ-ADI-SS, C.g-pXMJ-ADI; reacting the cells of C.g-pXMJ-iap-ADI-SS and C.g-pXMJ-iapV69H-ADI-SS at 35-55 ℃, sampling every 10-30 min, stopping the reaction by 10% trichloroacetic acid in equal proportion, centrifuging at 8000rpm for 5min, and detecting the concentration of the citrulline product and the residue of arginine serving as a substrate by HPLC.

In the above reaction system, the influence of temperature on the reaction effect was compared. As can be seen from FIG. 2, the mutation of arginine deiminase increased the optimum temperature of C.g-pXMJ-ADI-SS from 40 ℃ to 50 ℃ and increased the reaction rate, compared to C.g-pXMJ-ADI; after fusion expression of the sequences of the auxiliary infection residues, the optimal temperatures of C.g-pXMJ-iap-ADI-SS and C.g-pXMJ-iapV69H-ADI-SS are both 50 ℃.

The cells were incubated at 40 ℃ and 50 ℃ respectively, and the residual enzyme activity at different incubation times was determined with the enzyme activity at 100% without incubation. As can be seen from FIG. 3, the mutant C.g-pXMJ-ADI-SS has significantly improved thermal stability compared to C.g-pXMJ-ADI, and after fusion expression, has no adverse effect on thermal stability, and C.g-pXMJ-iapV69H-ADI-SS has a certain improvement. Half-life was calculated according to the curve, and it can be seen that the half-life of the arginine deiminase mutant C.g-pXMJ-ADI-SS was significantly extended, 4.1 times that of the original enzyme at 40 ℃ and more 6.8 times that of the original enzyme at 50 ℃. In addition, the thermostability of the mutant is not obviously reduced after the fusion expression of the auxiliary infection protein, and the thermostability of the mutant enzyme produced by C.g-pXMJ-iapV69H-ADI-SS is slightly increased.

TABLE 1 half-lives of arginine deiminase, mutants thereof, and immobilized enzyme

	40℃	50℃
			C.g-pXMJ-ADI	7.57h	3.61h
C.g-pXMJ-ADI-SS	30.94h	24.67h
			C.g-pXMJ-iap-ADI-SS	29.12h	20.63h
C.g-pXMJ-iapV69H-ADI-SS	32.39h	24.58h

Example 4

Amplification of catalytic processes

In a 50L reaction system, 600g/L arginine solution is adjusted with 30% hydrochloric acid/20% sulfuric acid to have pH =5.0-6.0, 30-80g immobilized enzyme C.g-pXMJ-iapV69H-ADI-SS is respectively added, reaction is carried out at 50 ℃, samples are taken at intervals of 2-4H, reaction is stopped by 10% trichloroacetic acid in equal proportion, and after centrifugation at 8000rpm for 5min, the concentration of the product citrulline and the residue of substrate arginine are detected by HPLC. As can be observed in FIG. 4, the amplified reaction reaches the end point within 2-8h, the citrulline concentration of the product reaches 608g/L, and the conversion rate reaches 99.5%.

Under a 50L reaction system, the input amount of immobilized enzyme is within the range of 30-80g, 600g/L arginine can be completely catalyzed to generate citrulline, and less enzyme is insufficient to complete the catalysis process within 8 h.

Example 5

Test group-recycle of immobilized enzymes

40g of immobilized enzyme C.g-pXMJ-iapV69H-ADI-SS is put into 600g/L of L-arginine with pH =5.0-6.0, the reaction volume is 50L, and the L-citrulline with 600 +/-5 g/L can be generated after 4H of reaction at 50 ℃.

After the reaction is finished, adjusting the pH =7.5 of the solution, reducing the temperature to 25-30 ℃, centrifugally separating insoluble substances from the reaction solution, adding the insoluble substances into an L-arginine solution with the pH =5.0-6.0 at the concentration of 600g/L, reacting at the temperature of 50 ℃, sampling every 1h, and detecting the product concentration and the substrate residue; the product concentration is not reduced after 22 times of circulation, and the reaction time of each time can be ensured to be within 4-6 h.

Comparative group 1

After the reaction is finished, centrifugally separating insoluble substances from a reaction solution, adding the insoluble substances into an L-arginine solution with the concentration of 600g/L and the pH =5.0-6.0 again, reacting at the temperature of 50 ℃ in a volume of 50L, and sampling every 1h to detect the product concentration and the substrate residue; the number of such circulations is reduced, the concentration of the product is not reduced under the condition of reaching 16 circulations at most, but the reaction time of each circulation is gradually prolonged, even 1-2 hours is prolonged, the last circulation is finished within 8 hours, and the prolonged reaction time is not beneficial to the practical application in large-scale production.

Comparative group 2

40g of immobilized enzyme C.g-pXMJ-iap-ADI-SS is put into 600g/L of L-arginine with pH =6.5-7.0, the reaction volume is 50L, L-citrulline with the concentration of 540 +/-3 g/L can be generated after 6h of reaction at 50 ℃, the reaction is continued, and L-citrulline with the concentration of 590 +/-5 g/L can be detected after 8h of reaction.

After the reaction is finished, adjusting the pH =7.5 of the solution, reducing the temperature to 25-30 ℃, centrifugally separating insoluble substances from the reaction solution, adding the insoluble substances into an L-arginine solution with the pH =5.0-6.0, the reaction volume is 50L, reacting at 50 ℃, sampling every 2h, and detecting the product concentration and the substrate residue; although the pH is adjusted to the optimum recovery condition each time, the longer reaction time can only maintain the product concentration not to be reduced for 12 times of circulation, and the reaction time is prolonged each time, and at the 12 th circulation, the reaction time exceeds 16h, so the method is obviously not suitable for scale production.

Example 6

Expressing iapV69H-ADI-SS fusion enzyme in colibacillus, bacillus subtilis and lactic acid bacteria according to the conventional operation, adjusting the fermentation liquor to 25-30 ℃ after the culture is finished, combining for 1H under the pH value of 7.5, washing once, adjusting the cell concentration to 400g/50L, mixing with arginine solution with certain concentration according to a proper proportion to ensure that the arginine solubility is 600g/L and the cell concentration is 60g/50L, carefully maintaining the pH value of 5.0-6.0 and the reaction volume is 50L in the mixing process, detecting no substrate peak at the temperature of 50 ℃ for 3.2H, 3.1H and 4.8H respectively, and detecting the L-citrulline concentration of 600 +/-4 g/L. After the reaction is finished, adjusting the pH of the solution to be =7.5, reducing the temperature to 25-30 ℃, centrifugally separating insoluble substances from the reaction solution, and adding the insoluble substances into the substrate solution again for reaction, wherein the effective cycle times respectively reach 26 times, 28 times and 18 times.

Comparative example 1

Free enzyme

Inoculating the obtained C.g-pXMJ-ADI-SS engineering bacteria into BHI culture medium containing 20mg/L, culturing at 35 ℃,200rpm overnight, then transferring into fresh BHI culture medium containing 20mg/L according to 10% of inoculum size, culturing at 35 ℃,200rpm for 12h, then adding 0.2mM IPTG for induction, collecting cells after induction for 12h, adjusting the cell concentration to 400g/50L, then crushing, mixing with arginine solution with a certain concentration according to a proper proportion to ensure that the arginine solubility is 600g/L, carefully maintaining the pH =5.0 and the reaction volume is 50L in the mixing process, reacting at 50 ℃, and reacting at 50 ℃ for 4h to generate 600 +/-5 g/L-citrulline. The supernatant has high catalytic activity, but continuous catalysis cannot be realized. Meanwhile, the final reaction solution of the cells has more complex components, which increases the separation cost.

Comparative example 2

Cell surface display

Construction of a C-terminal truncated NCgl1221 protein (SEQ NO: 5) as an anchor protein, fusion expression with ADI gene, ligation to pXMJ19 plasmid, transfer to Corynebacterium glutamicum, construction of a strain named C.g-pXMJ-NCg11221-ADI-SS, inoculation into BHI medium containing 20mg/L chloramphenicol, overnight culture at 35 deg.C and 200rpm, inoculation into fresh BHI medium containing 20mg/L at 10% inoculum size, culture at 35 deg.C and 200rpm to OD ₅₆₂ Adding 0.2mM IPTG for induction when the concentration is 0.6-0.8, and collecting insoluble substances and supernatant after 12h of induction. The insoluble substances are washed once to obtain the surface display cells.

Under the same culture conditions, the biomass of the cells is lower than the enzyme secreted into the culture medium; indicating that the displayed sites on the cell surface may not be completely distributed on the cell surface, or the sites are not beneficial to the metabolism of bacterial substances after being distributed on the surface, thereby influencing the growth.

40g of surface display cells are put into 600g/L of arginine solution with pH =5.0, the reaction volume is 50L, the reaction is carried out at 50 ℃, the disappearance of the substrate can be detected in 7h, the citrulline yield reaches 601g/L, and the catalytic efficiency of the immobilized enzyme is 57.1%.

Comparative example 3

Immobilization by embedding

1) Preparing a sodium alginate carrier: soaking sodium alginate in 15g of glycerol, adding 75mL of deionized water, stirring for dissolving, adding 1% of polyethylene glycol octyl phenyl ether, 0.8% of polyethylene glycol and 0.5mM of dithiothreitol, and stirring for dissolving to obtain a sodium alginate solution with the final concentration of 4.5%; 2) Culturing C.g-pXMJ-ADI-SS cells, collecting, adjusting the cell concentration to 40g/50L, crushing, adding the crushed enzyme solution into the sodium alginate solution prepared in the step (1), and uniformly mixing the crushed enzyme solution and the sodium alginate solution to obtain a sodium alginate enzyme solution; 3) Dropping sodium alginate into 5% CaCl at constant speed by using constant flow pump and external injector needle ₂ Stirring and fixing in the solution; 4) Filtering, washing the gel precipitate with PBS buffer solution repeatedly, and drying to obtainTo granular immobilized arginase.

And (3) putting the immobilized product into 600g/L arginine solution with pH of 5.0, reacting at 50 ℃ in a reaction volume of 50L, and sampling to detect the product concentration and the substrate residue. The disappearance of the substrate can be detected within 9 hours, the citrulline yield reaches 603g/L, the conversion rate is close to 99 percent, and the conversion rate is 44.4 percent of the catalytic efficiency of the immobilized enzyme.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and those skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. An arginine deiminase mutant is characterized in that the amino acid sequence of the arginine deiminase mutant is shown as SEQ ID NO. 2.

2. An arginine deiminase recombinant plasmid is characterized in that the recombinant plasmid is obtained by recombining a gene assisting in infecting a protein residue mutant and a gene assisting in infecting an arginine deiminase mutant; wherein, the coded amino acid sequence of the gene of the auxiliary infection protein residue mutant is shown in SEQ ID NO. 4; the coded amino acid sequence of the gene of the arginine deiminase mutant is shown as SEQ ID NO. 2.

3. An secreted arginine deiminase recombinant bacterium containing the recombinant plasmid of claim 1; the recombinant strain is characterized in that the recombinant strain of the arginine deiminase is obtained by carrying out fusion expression on a gene of an auxiliary infection protein residue mutant and a gene of an arginine deiminase mutant, wherein the amino acid sequence of the auxiliary infection protein residue mutant is shown as SEQ ID No. 4; the amino acid sequence of the arginine deiminase mutant is shown as SEQ ID NO. 2.

4. The recombinant bacterial cell according to claim 3, wherein the host of the recombinant bacterial cell is selected from Corynebacterium glutamicum, escherichia coli, bacillus subtilis, and Lactobacillus.

5. The use of the recombinant bacterial cell of the secreted arginine deiminase according to claim 3 for the catalytic production of citrulline; the method is characterized by comprising a self-immobilization process of arginine deiminase on a production strain of the arginine deiminase obtained by fermenting the arginine deiminase recombinant bacteria.

6. The use according to claim 5, wherein the substrate solution in the fermentation process is an L-arginine solution or an L-arginine hydrochloride solution having a concentration of 50 to 650g/L and a pH of 5.0 to 6.0.

7. Use according to claim 6, wherein the concentration of the substrate solution during the fermentation is 600-650g/L.

8. The use of claim 5, further comprising an arginine deiminase immobilized enzyme recycling process: carrying out fermentation culture after arginine deiminase immobilized carriers are obtained by self-immobilization of the arginine deiminase obtained by fermentation of the recombinant bacteria on production strains, wherein the concentration of the arginine deiminase immobilized carriers in the fermentation process is 30-80g/50L, centrifugally separating insoluble substances from a reaction solution after the fermentation culture is finished by regulating the temperature to room temperature and under the condition of neutral pH, and putting the insoluble substances into a new substrate solution again to repeat the immobilization process for a new round of fermentation; the circulation is not less than 22 times, and the fermentation time is not more than 6h.

9. The use according to claim 5, wherein the fermentation conditions are 35-55 ℃ and pH 5.0-7.0.

10. Use according to claim 9, wherein the fermentation conditions are 40-50 ℃ and pH5.0-6.0.