DNA sequence for coding arginine deiminase and application thereof

Abstract

The invention provides a DNA (Deoxyribose Nucleic Acid) sequence for coding arginine deiminase and application of the DNA sequence. The sequence is comprised in SEQ ID NO: 1. In addition, the invention also provides an expression vector containing the DNA sequence, and a microorganism which is subjected to genetic engineering modification and can express arginine deiminase (ADI). Furthermore, the invention provides a method for producing L-citrulline by using the genetically engineered bacterium. The genetically engineered bacterium shows the capability of efficiently recombining and expressing arginine transiminase (ADI), and also has relatively high performance of catalyzing the conversion of L-arginine into L-citrulline, and the production and application cost is effectively reduced in the industrial application process.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a DNA molecule encoding arginine deiminase. In addition, the present invention also includes the vector construction of the DNA molecule, and the specific application and use of the DNA molecules and vectors in the process of producing L-citrulline. Background Art

[0002] L-citrulline, also known as carbamoyl ornithine, is easily soluble in water, but poorly soluble in methanol and ethanol. In the human body, it can be synthesized from ornithine in the urea cycle, or ingested from foods such as watermelon and pumpkin. L-citrulline has a variety of physiological functions, including participating in the urea cycle, improving cardiovascular function, enhancing immunity, and promoting muscle recovery and growth. L-citrulline is widely used in the fields of food, cosmetics, and medicine. It can be used as a nutritional enhancer, improve skin conditions, anti-aging, and has pharmacological effects such as preventing and treating prostate diseases.

[0003] The main production methods of citrulline include extraction, chemical synthesis, microbial fermentation and enzyme conversion. Enzyme conversion is gradually becoming the mainstream technology due to its advantages. However, in practical applications, when using engineered bacteria to express arginine deiminase, the problem of enzyme protein accumulation in the form of inactive inclusion bodies is often faced, which limits the efficiency of the enzyme conversion method. Although some researchers have used chemical molecules such as urea for renaturation, it increases production costs and is not suitable for large-scale applications.

[0004] As a single enzyme, arginine deiminase has higher requirements for temperature and pH stability, soluble expression and activity. Its optimum temperature is mostly concentrated between 35 and 40°C, and actual production has strict requirements on enzyme heat resistance. Enzymes with good thermal stability can increase reaction rate, simplify processes and maintain stable activity.

[0005] The pH value of arginine aqueous solution is usually maintained above 10, while the optimal pH range of arginine deiminase is 6 to 7. Concentrated hydrochloric acid is required to adjust the pH during enzymatic reactions, but as the substrate concentration increases, the amount of hydrochloric acid increases, which may affect the enzyme activity or even inactivate it. The industry has a high demand for enzyme tolerance to pH stability.

[0006] In addition, when the concentration of arginine substrate exceeds a certain threshold, it will have a negative impact on the enzyme catalytic effect and cause substrate excess. Therefore, there is an urgent need for arginine deiminase with strong thermal stability and pH stability, which is both a research hotspot and an important factor in promoting industrial development. Summary of the invention

[0007] In view of the limitations of the prior art, the present invention provides a DNA molecule of a codon-optimized arginine deiminase gene, which significantly improves the soluble expression of arginine deiminase in Escherichia coli. In addition, the obtained arginine deiminase exhibits good thermal stability and pH stability, and efficiently catalyzes the conversion of arginine into L-citrulline. In addition, the present invention also introduces a method for catalyzing the production of L-citrulline using the enzyme.

[0008] The objective of the present invention is achieved through the following technical solutions:

[0009] On the one hand, the present invention provides a DNA molecule encoding arginine deiminase, and its nucleotide sequence is shown in SEQ ID NO: 1. In particular, the DNA molecule is preferably composed of the nucleotide sequence shown in SEQ ID NO: 1. The DNA molecule encoding arginine deiminase of the present invention is designed and optimized based on the sequence information of arginine deiminase from Bacillus sp.UMB0728. The amino acid sequence of the arginine deiminase is derived from the NCBI database, and its amino acid sequence is specifically shown in SEQ ID NO: 2. This amino acid sequence contains a total of 409 amino acids, the protein molecular weight is 46.36kDa, and the isoelectric point pI is 5.43. The sequence information is shown below.

[0010]

[0011] MKHPLNVTSEIGELKSVLLHRPGKEVENLTPQYLKRLLFDDIPYLPAIQKEHDYFAGVLSNRGIEVLYLDKLMTETLAQPEIRSRFTEQILMESQSNVNGSFEAVSEYLNSLPADELVRKVMAGVRKNEIDLKEKVHLHEIMADSYPFFLDPMPNLYFTRDPAAVIGKGITINNMNEPARRRESIFMDYIIKHHPRFKEHDIPVFF NRDEHYTLEGGDELVLSREVAAIGVSARTSAQGIEKLARELFARQEDIKKVVAVEIPKSRAFMHLDTVFTMIDHDKFTYHPAIENSDGKMKIYILEQGNSGEL KITEKDSLVETLKEVLNLPELVLLPCGGGDVIAAAREQWNDGSNTLAIAPGVVVTYDRNYVSNDMMRQNGIEVLEIMSSELSRGRGGPRCMSMPIVRGEL(SEQ ID NO:2)

[0012] On the other hand, the present invention provides an expression vector comprising the aforementioned DNA molecule. Preferably, the expression vector is a microbial expression vector, more preferably selected from the pBE-S series or pBE-XK99E series or pGAPZ series or pET series vectors, wherein the pET28α expression vector is further preferred.

[0013] Furthermore, the present invention also provides a genetically engineered bacterium for expressing arginine deiminase. The genetically engineered bacterium comprises the DNA molecule or the aforementioned expression vector of the present invention. In terms of bacterial species selection, Bacillus subtilis, Corynebacterium glutamicum, yeast or Escherichia coli are preferred; more specifically, recombinant Escherichia coli is a better choice, and recombinant Escherichia coli BL21 (DE3) is further preferred.

[0014] The present invention further provides the use of a DNA molecule, an expression vector or a genetically engineered bacterium encoding arginine deiminase in the preparation of L-citrulline.

[0015] A specific embodiment of the present invention describes in detail a method for preparing a genetically engineered bacterium for expressing arginine deiminase. The preparation method mainly comprises the following steps:

[0016] According to the codon preference of Escherichia coli cells, a nucleotide sequence encoding arginine deiminase is designed and synthesized. Preferably, the nucleotide sequence is as shown in SEQ ID NO: 1;

[0017] The nucleotide sequence encoding arginine deiminase was constructed into the pET28a expression vector, and the pET28a-ADI recombinant plasmid was successfully obtained; the pET28a-ADI recombinant plasmid was transformed into Escherichia coli BL21 (DE3); and the stable recombinant strain BL21 (DE3) / pET28a-ADI was screened and obtained.

[0018] The present invention also provides a method for preparing arginine deiminase, which utilizes the aforementioned expression vector or genetically engineered bacteria to express the arginine deiminase gene. Preferably, the amino acid sequence of the expressed arginine deiminase is shown in SEQ ID NO:2.

[0019] In addition, the present invention also provides a method for preparing L-citrulline, which is characterized by comprising the following steps:

[0020] The genetically engineered bacteria of the present invention are induced to express arginine deiminase, and then the bacteria are collected and crushed to obtain arginine deiminase; the obtained arginine deiminase is used to convert L-arginine into L-citrulline. In the 5L tank fermentation of Escherichia coli BL21 (DE3) / pET28a-ADI, the induction conditions specifically include: ① seed culture at 37°C and pH 7.0; ② adding an inducer and fermenting at a temperature range of 23-33°C; preferably, the inducer is IPTG (isopropyl-β-D-thiogalactoside) or lactose.

[0021] Furthermore, the culture medium used for seed culture preferably contains: 5-15 g / L peptone, 1-10 g / L yeast extract and 5-15 g / L NaCL; more preferably, the culture medium composition is: 10 g / L peptone, 5 g / L yeast powder and 10 g / L NaCL.

[0022] Preferably, the culture medium used for seed culture is adjusted to a pH range of 6.8-7.2 with an aqueous ammonia solution and sterilized; more preferably, before inoculation, a sterile filtered aqueous solution of sodium kanamycin is added to the culture medium to a final concentration of 30 to 70 μg / mL.

[0023] In addition, preferably, in a 250 mL seed bottle, the volume of the culture medium is 40-60 mL; and in a 500 mL conical flask, the volume of the culture medium is 80-120 mL.

[0024] Preferably, the culture medium used in the shake flask fermentation culture contains 3-6 g / L glycerol, 10-15 g / L peptone, 20-28 g / L yeast extract, 10-15 g / L K2HPO4, and 2-4 g / L KH2PO4.

[0025] Preferably, the culture medium used for fermentation culture needs to be adjusted to a pH range of 6.8 to 7.2 by an aqueous ammonia solution and sterilized. More preferably, before inoculation, a sterile filtered aqueous solution of sodium kanamycin is added to the culture medium to a final concentration of 30 to 70 μg / mL.

[0026] Furthermore, for use in fermentation culture, the liquid volume of the culture medium should be set to 1000mL / 5000mL fermenter.

[0027] According to a particularly preferred embodiment of the present invention, the culture medium used in the preparation process is prepared in the following manner:

[0028] Seed medium: contains 10 g / L peptone, 5 g / L yeast extract and 10 g / L sodium chloride. The pH is adjusted to a range of 6.8 to 7.2 using an aqueous ammonia solution, and then sterilized at 121°C for 20 minutes. Before inoculation, a sterile filtered ampicillin sodium aqueous solution is added to a final concentration of 50 μg / mL.

[0029] Fermentation medium: contains (NH4)2HPO4 4g / L, KH2PO4 13.5g / L, citric acid H2O1.7g / L, MgSO4.7H2O 1.4g / L, trace elements 10mL, peptone 1g / L, yeast powder 2g / L and glucose 10g / L, pH is adjusted to the range of 6.8 to 7.2. Sterilization is also performed at 121°C for 20 minutes. Before inoculation, a sterile filtered sodium kanamycin aqueous solution is added to a final concentration of 30 to 70μg / mL.

[0030] In the method for producing L-citrulline by enzymatic conversion of 6.1 of the L-citrulline preparation method of the present invention, the specific conditions for the conversion are as follows: the concentration of the substrate L-arginine is controlled at 180-280 g / L, the conversion temperature is maintained at 40-50° C., the pH value is adjusted to 5.0-8.0, and the conversion time is 4-12 h.

[0031] In addition, the present invention also provides L-citrulline prepared by the above method. Compared with the prior art, the technical solution of the present invention exhibits the following significant advantages:

[0032] The DNA molecule provided by the present invention has been codon-optimized, effectively avoiding various factors that affect transcription and translation efficiency, and significantly improving the soluble expression of the enzyme.

[0033] The genetically engineered bacteria expressing arginine deiminase constructed by the present invention greatly improve the expression efficiency, thereby effectively reducing the production and use costs.

[0034] The arginine deiminase provided by the present invention has excellent thermal stability and pH stability, can tolerate high salt concentration, and has extremely high activity in catalyzing arginine to generate L-citrulline.

[0035] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0037] Figure 1 : In Example 1, PCR was used to obtain the arginine deiminase gene, and the ADI gene size was 1230 bp.

[0038] Figure 2 : SDS-PAGE protein gel image of arginine deiminase in Example 3, 0 expresses empty load, 1 expresses ADI.

[0039] Figure 3 : Detection of the optimum temperature of arginine deiminase in Example 4.

[0040] Figure 4 : Detection of the optimum pH for arginine deiminase in Example 4.

[0041] Figure 5 : Detection of the heat resistance of arginine deiminase in Example 4.

[0042] Figure 6 : pH tolerance detection of arginine deiminase in Example 4. DETAILED DESCRIPTION

[0043] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0044] Culture medium used in the present invention

[0045] LB medium: yeast powder 5g / L, peptone 10g / L, NaCl 10g / L.

[0046] TB medium: glycerol 5 g / L, peptone 12 g / L, yeast extract 24 g / L, dipotassium hydrogen phosphate 12.54 g / L, potassium dihydrogen phosphate 2.31 g / L.

[0047] 5-L fermentation medium: (NH4)2HPO4 4g / L, KH2PO4 13.5g / L, citric acid H2O 1.7g / L, MgSO4.7H2O 1.4g / L, trace elements 10mL, peptone 1g / L, yeast powder 2g / L, glucose 10g / L, pH 7.2.

[0048] The culture media mentioned in this patent are all sterilized in an autoclave at a temperature of 121°C for 20 minutes; the culture media are all added with filtered and sterilized kanamycin sodium at a final concentration of 50 μg / mL.

[0049] Example 1 Construction of recombinant plasmid

[0050] The arginine deiminase (ADI) gene sequence from Bacillus sp.UMB0728, such as SEQ ID NO: 1, was codon-optimized by Genewise and the nucleotide sequence was synthesized on the plasmid pET28a, named pET28a-ADI.

[0051] The arginine deiminase (ADI) gene sequence used in this application is derived from Bacillus sp.UMB0728. The gene sequence was synthesized by Jinweizhi Co., Ltd. on the plasmid pUC to obtain the plasmid pUC-ADI. The nucleotide sequence of the arginine deiminase gene is shown in SEQ ID NO: 1, as follows. The amino acid sequence of the enzyme expressed by the arginine deiminase gene is shown in SEQ ID NO: 2, as follows. PCR was used to obtain the target arginine deiminase gene. The upstream primer ADI-NdeI-F (GGAATTCC CATATG AAACATCCGCTGAACGTG, SEQ ID NO: 3), downstream primer ADI-hindⅢ-(CCC AAGCTT CAGTTCGCCGCGCACAATCG, SEQ ID NO: 4) is the primer, the underlined part is the restriction site, and the PCR amplification target arginine deiminase gene reaction system is as follows: upstream primer ADI-NdeI-F 2μL, downstream ADI-hindⅢ-R 2μL, template pUC-ADI (100ng / μL) 0.5μL, DNA polymerase 2×Prime STAR Max DNA 25μL, and sterile water to 50μL. The PCR reaction program is as follows: PCR amplification program: 98℃ pre-denaturation for 3min, 98℃ denaturation for 10s, 58℃ annealing for 5sec, 72℃ extension for 60s, 30 cycles, and finally 72℃ extension for 5min. Take 3μL of ADI enzyme gene PCR product for nucleic acid verification. The target band size is 1230bp, such as Figure 1 The size shown is correct and the remaining PCR products were stored at 12°C.

[0052] The linear plasmid pET28a was obtained by PCR, and the upstream primer pET28a-F (CCC AAGCTT GCGGCCGCACTCGAG, SEQ ID NO: 5), downstream primer pET28a-R (GGAATTCC CATATG GCTGCCGCGCGGCAC, SEQ ID NO: 6). The reaction system for PCR amplification to obtain the target fragment is as follows: upstream primer pET28a-F 2μL, downstream pET28a-R 2μL, template pET28a (100ng / μL) 0.5μL, DNA polymerase 2×Prime STAR Max DNA 25μL, and sterile water to 50μL. The PCR reaction program is as follows: PCR amplification program: 98℃ pre-denaturation for 3min, 98℃ denaturation for 10s, 58℃ annealing for 5sec, 72℃ extension for 80s, 30 cycles, and finally 72℃ extension for 5min, and 12℃ storage of PCR products.

[0053] The above-obtained PCR products, arginine deiminase gene fragments and pET28a linear fragments were detected by 1% agarose gel nucleic acid electrophoresis and recovered by gel cutting with the OMEGA gel recovery kit. First, the two recovered PCR products were digested with NdeI and hindⅢ restriction endonucleases for 30 minutes and purified. Then, the arginine deiminase gene fragment and the pET28a fragment were connected at 16°C for 2 hours by T4 ligase. Subsequently, the ligation products of the two were transformed into DH5α competent cells, and after heat shock at 42°C for 60 seconds, 1mL LB liquid culture medium was added, and after culturing for 45 minutes in a shaker at 37°C and 200rpm, they were evenly coated on LB solid culture medium containing 50μg / mL kanamycin sodium, and inverted overnight in a 37°C incubator. Pick a single clone, verify the colony PCR and send it to the company for sequencing, pick the strain with correct sequencing for culture, extract the plasmid, and obtain the recombinant plasmid pET28a-ADI.

[0054] Example 2 Construction of a recombinant expression strain of arginine deiminase

[0055] Take 5 μL of the correct recombinant plasmid pET28a-ADI obtained in Example 1 above and transform it into competent cells of Escherichia coli BL21 (DE3), heat shock at 42°C for 60s, and add 1mL LB medium. After culturing at 37°C and 200rpm for 45min, spread it on LB solid medium containing 50 μg / mL kanamycin sodium, and invert and culture it at 37°C overnight for about 10h to obtain recombinant transformant Escherichia coli BL21 (DE3) / for expressing arginine deiminase gene.

[0056] pET28a-ADI monoclonal strain. Take a single clone in LB medium, culture at 37°C, 200rpm for 10-12h, use 50% sterile glycerol to preserve E. coli BL21 (DE3) / pET28a-ADI bacterial liquid, and store it in a -80°C refrigerator for subsequent experiments.

[0057] Example 3 Purification of arginine deiminase

[0058] In the present embodiment, in order to purify arginine deiminase (ADI).It is necessary to carry out shake flask fermentation to obtain enzyme solution, and the specific steps are as follows, first, take 50 μL of glycerol bacteria solution containing Escherichia coli BL21 (DE3) / pET28a-ADI, add it to a 50mL conical flask equipped with 10mL LB liquid culture medium. Subsequently, culture 10h in a shaking table at 37°C temperature and 200rpm. Then, take out the culture solution of 5mL strain as seed culture solution, transfer to a 500mL conical flask containing 50mL TB liquid culture medium, and continue to culture 2h under the same conditions (37°C and 200rpm / min).Afterwards, add a final concentration of IPTG inducer, and the fermentation temperature is reduced to 25°C, to promote the recombinant expression of arginine deiminase, and continue to ferment for 24h.

[0059] The purification step is carried out using a nickel-NTA (Ni-NTA) affinity chromatography column. First, collect the bacterial solution after the fermentation is completed, centrifuge it at 8000g for 20 minutes at 4°C, discard the supernatant, and retain the bacteria. Then, resuspend the bacteria in 0.2moL / L phosphate buffer with a pH of 6.0, and lyse the cells by high-pressure crushing. Subsequently, centrifuge it again at 8000g for 20 minutes at 4°C to separate the cell-free component supernatant containing arginine deiminase. Next, add the above-mentioned cell-free extract to a pre-cooled Ni-NTA chromatography column for purification. The purification process is divided into two elution steps: the first step is to use buffer A containing 50mmoL / L phosphate buffer (PBS) and 100mmol / L imidazole (pH 6.0) for washing to remove non-specifically bound impurities; the second step is to use buffer B containing 50mmoL / L PBS and 300mmoL / L imidazole (pH 6.0) for elution to effectively recover arginine deiminase.

[0060] Finally, the purified enzyme was dialyzed in 0.2 mol / L phosphate buffer with a pH of 6.0, and stored in a -20°C refrigerator after aliquoting. The buffer used in the entire purification process must be precooled to 4°C before use. After the above steps, purified arginine deiminase was successfully obtained. 2 μL of arginine deiminase was taken for protein gel electrophoresis verification. Figure 2As shown, size is correct.

[0061] Example 4 Detection of the enzymatic properties of arginine deiminase

[0062] 4.1 Enzyme activity determination

[0063] The definition of enzyme activity unit (U) is as follows: Under the reaction conditions of pH 6.0 and 37°C, using crude enzyme solution or purified protease, with 100 g / L L-arginine as substrate, the amount of enzyme required to catalyze the production of 1.0 μmol L-citrulline per minute is one enzyme activity unit.

[0064] The specific process is as follows:

[0065] The engineered strain Escherichia coli BL21 (DE3) / pET28a-ADI was fermented to obtain a fermentation broth, and the bacterial cells were obtained by centrifugation. Sodium phosphate buffer (0.2 mol / L, pH 6) was added, and then the cells were lysed by high-pressure crushing. The supernatant liquid after centrifugation at 4°C and 12000g contained a crude enzyme solution of arginine deiminase.

[0066] Enzyme activity determination method: Add 1 mL of crude enzyme solution to a sodium phosphate buffer (0.2 mol / L, pH 6) with an arginine concentration of 100 g / L at 37°C and 200 rpm for 10 min. After the reaction is completed, boil in a boiling water bath for 10 min to terminate the reaction, centrifuge to remove the precipitate, and take the reaction solution for liquid phase detection.

[0067] 4.2 Optimal temperature and pH of enzymes

[0068] The optimum temperature and the optimum pH were determined by testing the ADI enzyme activity at different temperatures (30, 40, 50 and 60°C) in a sodium dihydrogen phosphate-citric acid buffer (0.2 mol / L) at pH 7.0, and determining the optimum temperature based on the enzyme activity. At 50°C, the arginine deiminase enzyme activity was tested in sodium dihydrogen phosphate-citric acid buffer (0.2 mol / L / 0.1 mol / L) at pH 4.0-8.0 (4.0, 5.0, 6.0, 7.0 and 8.0), and determining the optimum pH based on the enzyme activity. The maximum enzyme activity was defined as 100%, and the optimum temperature and pH were as follows: Figure 3 and Figure 4 The detection methods of enzyme activity are all referred to 4.1.

[0069] 4.3 Enzyme heat resistance and pH resistance testing

[0070] The purified enzyme solution was incubated at different temperatures (30-60°C) at pH 6.0, and then the enzyme activity was determined according to the enzyme activity detection method. The maximum enzyme activity was taken as 100%, and the relative enzyme activity at other temperatures was calculated. The results are as follows: Figure 5As shown, the enzyme has good heat resistance.

[0071] The purified enzyme solution was incubated at different pH values ​​(4.0-8.0) at 40°C, and then the enzyme activity was determined according to the enzyme activity detection method. The maximum enzyme activity was taken as 100%, and the relative enzyme activity at other pH values ​​was calculated. The results are as follows: Figure 6 As shown, the enzyme has good pH tolerance.

[0072] Example 5 5 L tank fermentation of strain Escherichia coli BL21 (DE3) / pET28a-ADI

[0073] 5.1 Strain activation

[0074] Take 50 μL of glycerol culture of E. coli BL21(DE3) / pET28a-ADI from the -80°C freezer and add it to a 50 mL conical flask containing 10 mL of LB liquid medium. Then, culture it in a shaker at 37°C and 200 rpm for 10 h.

[0075] 5.2 First-level seeds

[0076] 20 mL of the activated E. coli BL21 (DE3) / pET28a-ADI was transferred to a 1000 mL conical flask containing 100 mL of LB medium and cultured in a shaker at 37° C. and 200 rpm for 10 h.

[0077] 5.3 Fermentation in 5L fermenter

[0078] The primary seed Escherichia coli BL21 (DE3) / pET28a-ADI was transferred to a 5L fermenter. After an adaptation period of about 3 hours, the bacteria entered a rapid proliferation stage, at which time the initial pH value of the fermentation broth of 7.0 began to gradually decrease. To maintain pH stability, the system automatically replenished ammonia water to adjust and maintain the pH at around 7. As the density of the bacteria continued to increase, the dissolved oxygen content gradually decreased. Subsequently, the ventilation ratio was increased from 0.5 to 1vvm, and the rotation speed was increased from 200rpm to 500rpm to ensure that the dissolved oxygen content in the fermenter was maintained within the appropriate range of 20% to 30%.

[0079] When the OD of the strain 600 When the value reached about 15-20, a sharp rise in dissolved oxygen was observed, i.e., a sudden change in dissolved oxygen, and glycerol was then added at a rate of 5 g / L / h. 600 When the value increases to 30-40, the temperature of the fermenter is adjusted from 37°C to 30°C, and the rate of glycerol supplementation is increased to 10g / L / h. At this time, IPTG with a final concentration of 0.1mM is added as an inducer to induce the recombinant expression of the target arginine deiminase, and the fermentation temperature is reduced to 25°C to optimize the recombinant expression conditions of the ADI enzyme.

[0080] Generally speaking, the fermentation process is 600 The fermentation ends when the value reaches between 100 and 120. The whole cycle lasts about 40 hours. After the fermentation is completed, the bacteria are collected by centrifugation, the cell wall is broken by a high-pressure homogenizer, and the crude enzyme solution is obtained by centrifugation for subsequent use.

[0081] Example 6 Arginine deiminase conversion to produce citrulline

[0082] The method for catalyzing the conversion of the substrate L-arginine into L-citrulline by arginine deiminase and detecting the enzyme activity is disclosed in the patent authorization announcement number CN 113584054B.

[0083] 6.1 Enzymatic Production of L-Citrulline

[0084] The first step is to establish a reaction system. Add 20g of L-arginine to a 500mL conical flask. Then, add 20mL of crude enzyme solution stored at low temperature to the system, add water to make the volume 100mL, and perform three parallel experiments for enzyme catalysis. The second step is to adjust the pH of the reaction system to 6.0 with hydrochloric acid. The third step is to place the 500mL conical flask in a shaker and adjust the temperature to 45℃ and the speed to 150rpm. The fourth step is to complete the conversion of L-arginine after 6h of reaction. The fifth step is sampling and analysis. Every hour, 1mL of sample is extracted from the reaction system, which is then diluted 20 times and filtered using a microporous filter membrane with a pore size of 0.45 microns. The filtered sample needs to be analyzed by high performance liquid chromatography (HPLC) technology to accurately determine the concentration of L-citrulline and the remaining content of L-arginine.

[0085] 6.2 Detection method of L-citrulline

[0086] HPLC conditions: Use C18 chromatographic column, column flow rate of 1.0 mL / min, detection wavelength of 205 nm, column temperature of 25°C, injection volume of 20 μL; mobile phase preparation: dissolve 13.6 g of potassium dihydrogen phosphate in 1000 mL of purified water, then add 30 mL of acetonitrile, mix well and filter.

[0087] Enzyme activity calculation: According to the HPLC test results, the enzyme activity was calculated using the following formula:

[0088] X(IU / g)=(W1*S*(A1-A0)*n*1000*V) / (W2*A*M*175.19*10)W1 represents the mass of citrulline reference substance (mg); S represents the content of citrulline reference substance; A1 represents the area of ​​citrulline after 15min reaction; A0 represents the area of ​​citrulline after 5min reaction; n represents the dilution factor of the reaction solution; V represents the final volume of the reaction system; W2 represents the weight of protein in the purified liquid enzyme (g); A represents the area of ​​citrulline reference substance; m represents the dilution factor of citrulline in the reference substance; 175.19 represents the molecular weight of citrulline; 10 represents the time interval between two samplings (min); 1000 represents the μM conversion factor. The results show that the enzyme activity of the crude enzyme solution is 370U / mL, and the enzyme activity of the purified enzyme is 12060U / g.

[0089] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A DNA molecule encoding arginine deiminase (ADI), characterized in that Its nucleotide sequence is shown in SEQ ID NO:1, and its corresponding amino acid sequence is shown in SEQ ID NO:

2.

2. An expression vector, characterized in that: The invention comprises the DNA molecule encoding arginine deiminase as claimed in claim 1.

3. The expression vector according to claim 2, wherein The expression vector is an expression system designed for prokaryotic or eukaryotic microorganisms.

4. The expression vector according to claim 3, wherein The expression vector is one of the following vectors: pBE-S, pEB60, pUB18, pEB10, pWB980, pUC, pGAPZ series vectors or pET series vectors.

5. The expression vector according to claim 4, wherein The expression vector is a highly efficient pET28α expression vector.

6. A genetically engineered bacterium for expressing arginine deiminase, characterized in that: The genetically engineered bacteria contains the DNA molecule according to claim 1 or the expression vector according to any one of claims 2-5.

7. The genetically engineered bacterium according to claim 6, wherein The host cell of the genetically engineered bacteria is selected from Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis or yeast.

8. The genetically engineered bacterium according to claim 7, wherein The genetically engineered bacteria are genetically modified Escherichia coli.

9. The genetically engineered bacterium according to claim 8, wherein The genetically engineered bacteria is recombinant Escherichia coli BL21 (DE3), which is optimized to efficiently express arginine deiminase.

10. Use of the DNA molecule encoding arginine deiminase according to claim 1, the expression vector according to claim 2 or the genetically engineered bacterium according to claim 6, particularly in a production process for preparing L-citrulline under different conditions (such as pH value and temperature).

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