CN112094837B

CN112094837B - Recombinant arginine deiminase mutant, preparation method and application thereof

Info

Publication number: CN112094837B
Application number: CN202010885339.XA
Authority: CN
Inventors: 夏李群; 陈祎; 李恭会
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-08-28
Filing date: 2020-08-28
Publication date: 2022-05-17
Anticipated expiration: 2040-08-28
Also published as: CN112094837A

Abstract

The invention discloses a recombinant arginine deiminase mutant, a preparation method and application thereof. The invention modifies the recombinant arginine deiminase gene (the amino acid sequence is shown as SEQ ID NO. 1) through directed evolution, screens beneficial mutations from 2 ten thousand clones, and further obtains an optimal mutant M15 through combined mutation. The amino acid sequence of the optimal mutant M15 is shown in SEQ ID NO. 3. The enzyme activity of the recombinant arginine deiminase mutant prepared by the invention is greatly improved, and the catalytic efficiency is greatly increased. Can be used for preparing anti-tumor (anti-hepatocarcinoma, melanoma, and prostatic cancer) medicines.

Description

Recombinant arginine deiminase mutant, preparation method and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and relates to a recombinant arginine deiminase mutant, a preparation method thereof and application thereof in the aspect of tumor resistance.

Background

Arginine deiminase (ADI, EC 3.5.3.6), which is capable of degrading Arginine, is considered to be a very potent novel anticancer agent for the treatment of Arginine-deficient tumors. At present, the research reports that arginine deiminase has obvious curative effect on Hepatocellular carcinoma (HCC) and Melanoma (Melanoma). Arginine in vivo can be synthesized from citrulline, and normal cells can catalyze citrulline by Arginine Succinate Synthase (ASS) and Arginine Succinate Lyase (ASL) through urea cycle to obtain arginine; arginine deficient tumor cells lack arginine succinate synthase and are unable to self-synthesize arginine. Arginine is an important amino acid in the body and is involved in the synthesis of peptides and proteins and in a number of metabolic pathways in the cell. Since large amounts of arginine are required for tumor cell survival, arginine must be obtained from serum, and thus, the deprivation of exogenous arginine is one of the therapeutic approaches for arginine-deficient tumors.

Cancer is the first killer endangering the life and health of modern people, liver cancer usually occupies the first five times of the global cancer mortality, and the cancer is the second in the rank of the cancer mortality in 2018 in China. Prostate cancer is one of the most common male cancers worldwide, and 1600000 new cases of prostate cancer are reported annually, while the mortality rate of prostate cancer in urban men in china is 4.52/10 ten thousand (2018). A common method of treating cancer is chemotherapy, which typically causes great damage to normal cells while killing tumor cells. Therefore, there is a need for a new method for treating tumor, which can specifically treat tumor without damaging normal cells.

ADI was first discovered in 1933 by f.horns in pseudomonas aeruginosa (Bacillus pyocyaneus), and subsequently reported in mycoplasma, yeast, streptococcus lactis, and pseudomonas. ADI-PEG-20, developed by the E.phoenix company, is a PEG-modified arginine deiminase that has the potential to treat hepatocellular carcinoma, melanoma, while ADI-PEG-20 has also been investigated for the treatment of other types of cancer, such as leukemia, small cell lung cancer, prostate cancer, and the like. Despite its potential therapeutic potential, the use of ADI as an antitumor drug has been hampered by its low activity on arginine under physiological conditions (arginine concentration 0.1-0.12mM, pH7.4), mainly due to its michaelis constant (K)_m) The values are generally an order of magnitude higher than the concentration of arginine in plasma (0.1-0.12 mM). It would therefore be desirable to increase the substrate affinity (i.e., decrease the K) of arginine deiminase_mValue) to improve its activity and enzymatic properties on arginine under physiological conditions.

Disclosure of Invention

The invention mainly aims at the problem of low enzyme activity of arginine deiminase under physiological conditions, and provides a recombinant arginine deiminase mutant, a preparation method and application thereof. The invention excavates an ADI gene from Pseudomonas putida (Pseudomonas putida) through gene comparison, synthesizes and recombinates the ADI coding gene, and adopts protein engineering to reform the activity and the enzymology property of recombinant arginine deiminase, thereby obtaining a plurality of recombinant ADI mutants with improved enzyme activity under physiological conditions.

The technical scheme adopted by the invention is as follows:

a recombinant arginine deiminase has an amino acid sequence shown as SEQ ID NO. 1. The construction method of the recombinant arginine deiminase gene engineering bacteria comprises the following steps: connecting the gene with the amino acid sequence of SEQ ID NO.1 to an expression plasmid pET42b (+) with the restriction enzyme cutting site of Xho I-Nco I to obtain a recombinant plasmid pET42b (+) -adi; the recombinant plasmid pET42b (+) -adi is transformed into E.coli BL21(DE3) competent cells to obtain the recombinant arginine deiminase gene engineering bacteria.

In the technical scheme, further, the nucleotide sequence of the gene for coding the recombinant arginine deiminase is SEQ ID NO. 2.

Further, the recombinant arginine deiminase mutant is obtained by carrying out single mutation or multiple mutation on the 30 th position, the 37 th position, the 38 th position and the 405 th position of the amino acid sequence shown in SEQ ID NO. 1. The enzyme activity of the obtained recombinant arginine deiminase mutant is obviously improved, the Michaelis constant of arginine is obviously reduced, and the catalytic efficiency is obviously improved.

Still further, the recombinant arginine deiminase mutant is one of the following: (1) methionine at position 30 was mutated to arginine (M30R); (2) cysteine at position 37 was mutated to lysine (C37K); (3) aspartic acid at position 38 was mutated to histidine (D38H); (4) histidine at 405 was mutated to arginine (H405R); (5) including any 2-4 mutation sites in (1) - (4). The mutant with all mutations at 4 sites (methionine at position 30 is mutated into arginine, cysteine at position 37 is mutated into lysine, aspartic acid at position 38 is mutated into histidine, and histidine at position 405 is mutated into arginine (M30R/C37K/D38H/H405R) is recombinant arginine deiminase mutant M15.

Furthermore, the amino acid sequence of the recombinant arginine deiminase mutant M15(M30R/C37K/D38H/H405R) is shown as SEQ ID NO. 3. Mutant M15 was the best mutant of all recombinant arginine deiminase mutants. The recombinant arginine deiminase mutant M15 has the enzyme activity improved by about 10 times compared with that of wild ADI-WT under physiological conditions (pH 7.4), and K_mThe value dropped to 0.27. + -. 0.02mM, with an optimum pH of 7.0. Half inhibitory concentration IC on cell growth inhibition of tumor cells₅₀Is greatly reduced compared with wild type, and can be used for preparing antitumor drugs.

Due to the specificity of the amino acid sequence, any fragment of the peptide protein containing the amino acid sequence shown in SEQ ID NO.3 or its variants, such as conservative variants, bioactive fragments or derivatives thereof, as long as the homology of the fragment of the peptide protein or the variant of the peptide protein with the aforementioned amino acid sequence is above 90%, falls within the protection scope of the present invention. Particular such alterations may include deletions, insertions or substitutions of amino acids in the amino acid sequence; where conservative changes to a variant are made, the substituted amino acid has a chemical structure or chemical properties similar to the original amino acid, e.g., replacement of isoleucine with leucine, and the variant may also have non-conservative changes, e.g., replacement of glycine with tryptophan.

The invention also provides application of the recombinant arginine deiminase mutant, which can be used for preparing medicines for treating melanoma, liver cancer and prostate cancer. The recombinant arginine deiminase mutant has an in vitro anti-tumor effect on melanoma cells, liver cancer cells and prostate cancer cells, and IC50 is less than 1 ng/mL.

The preparation method of the recombinant arginine deiminase mutant comprises the steps of centrifuging fermentation liquor obtained by fermenting and culturing engineering bacteria containing recombinant arginine deiminase mutant genes, collecting wet bacteria, carrying out ultrasonic crushing on the wet bacteria to obtain crude enzyme liquid containing the recombinant arginine deiminase mutant, and carrying out separation and purification to obtain the recombinant arginine deiminase mutant.

The engineering bacteria containing the recombinant arginine deiminase mutant gene comprise single-mutation gene engineering bacteria and multi-mutation gene engineering bacteria; the construction method of the single mutation gene engineering bacterium comprises the following steps: taking the coding gene of the recombinant arginine deiminase as a template, designing a primer, and obtaining a mutant gene by adopting an error-prone PCR (polymerase chain reaction) technology; transferring the mutant gene into E.coli BL21(DE3) competent cells to obtain a mutant library; screening to obtain the single mutation gene engineering bacteria.

The multi-mutation genetic engineering bacteria comprise: taking a single mutant gene of the recombinant arginine deiminase mutant as a template, designing a primer, and obtaining a double mutant, a triple mutant and a quadruple mutant by adopting combined mutation; screening to obtain site-directed mutant multi-mutant gene engineering bacteria.

The invention modifies the recombinant arginine deiminase gene (the amino acid sequence is shown as SEQ ID NO. 1) by directed evolution, and the steps are as follows: adopting an error-prone PCR technology, taking a recombinant plasmid pET42b (+) -adi as a template, designing a primer, and obtaining a mutant gene; performing double digestion on the error-prone PCR product and pET42b (+) plasmid by using Xho I and Nco I, and connecting the double digested product by using T4 DNA ligase; transferring the ligation product into E.coli BL21(DE3) competent cells to obtain a mutation library; and (3) screening mutants with enzyme activity improved by more than 2 times compared with that of wild type under physiological conditions by adopting a high-throughput screening method.

The invention provides a high-throughput screening method, which is coupling screening on a microtiter plate and is used for screening recombinant arginine deiminase mutants with improved enzyme activity, and the specific operations are as follows: the content of ammonia generated by the reaction of arginine deiminase and arginine is detected on a microtiter plate, so that the enzyme activity is measured. 20. mu.L of ADI (crude enzyme solution after lysozyme cleavage) and 5.55U of glutamate dehydrogenase solution (GDH, purchased from Sigma, cat # G2626) were added to microtiter plates, 0.4mM of arginine, 5mM of co-substrate alpha-ketoglutarate and 0.4mM of coenzyme NADH were added, and the rate of NADH consumption (v.sub.dH) was measured at 340nm under physiological conditions (PBS buffer, 37 ℃, pH7.4)_ΔA) Thereby determining the velocity v of ammonia production_ΔCCalculated from the formula (1), wherein the absorption coefficient ε of NADH is 6200M^-1cm^-1The liquid height b in the wells of the microtiter plate was 0.59cm (total volume of reactants in each well was 200. mu.L). By v_ΔCFurther, the initial reaction rate of arginine deiminase was obtained.

v_ΔA＝ε×b×v_ΔC (1)

In the detection system, the concentration of the substrate is 0.2-0.5mM, preferably 0.4mM, the addition amount of the coenzyme NADH is 0.2-0.6mM, preferably 0.4mM, and the addition amount of the cosubstrate is 2-5mM, preferably 5 mM. The amount of ADI crude enzyme solution added is 15-25. mu.L, preferably 20. mu.L, and the amount of Glutamate Dehydrogenase (GDH) solution added is 4-6U, preferably 5.55U.

In the invention, the recombinant arginine deiminase gene engineering bacteria and the engineering bacteria containing the recombinant arginine deiminase mutant gene are inoculated, transferred, induced and recovered, and the culture medium can be any culture medium which can enable bacteria to grow and produce the invention in the field, preferably LB culture medium: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of NaCl, and dissolving in distilled water, and adjusting the pH value to 7.0. The culture method and the culture conditions are not particularly limited, and may be appropriately selected according to the type of host and factors such as the culture method, and the like, according to ordinary knowledge in the art.

Compared with the prior art, the invention has the main beneficial effects that:

the invention obtains a recombinant ADI mutant through error-prone PCR and site-directed mutagenesis. Wild-type ADI-WT is due to lower enzymatic activity and higher K under physiological conditions_mThe recombinant arginine deiminase mutant prepared by the invention has the advantages of obviously improved enzyme activity, obviously reduced Michaelis constant on arginine, obviously improved catalytic efficiency, and in-vitro anti-tumor effect on melanoma cells, liver cancer cells and prostate cancer cells, and IC50 is less than 1 ng/mL. The recombinant arginine deiminase mutant M15 is the optimal one of all the mutants, the enzyme activity of the mutant under physiological conditions (pH 7.4) is obviously improved compared with that of wild ADI-WT, and the K of the mutant is_mThe value is reduced to 0.27 +/-0.02 mM, the optimal pH is 7.0, and the method can be used for research of antitumor drugs.

Drawings

FIG. 1ADI cascades with GDH;

FIG. 2 is an agarose gel electrophoresis of an error-prone PCR product, lane M shows the molecular weight of a standard DNA, and

lanes

1, 2 and 3 show the gene fragments of the recombinant arginine deiminase mutant;

FIG. 3 SDS-PAGE patterns of recombinant arginine deiminase wild-type and mutant, M: a standard protein molecule; lane 1 is ADI-WT supernatant; lane 2 is ADI-WT pellet; lane 3 is ADI-M15 supernatant; lane 4 is ADI-M15 pellet;

FIG. 4 optimum pH of recombinant ADI-WT (A), ADI-M15 (B);

FIG. 5 morphology of tumor cells after 6 days of treatment with ADI-WT and ADI-M15.

Detailed Description

The invention is further described below with reference to specific examples.

Example 1: construction of recombinant arginine deiminase ADI gene engineering bacteria

(1) Amplification of target Gene

An arginine deiminase gene (Uniport ID Q88P52) derived from Pseudomonas putida (ATCC 47054) was synthesized by Hangzhou Otsugaku Biotechnology Ltd. Designing a primer:

an upstream primer: 5' -GCTCCATGGATGTCCACCGAAAACCTGCAGC-3’

A downstream primer: 5' -ATCTCGAGTTAATAGTCCACCGGGTCACGG-3’

The gene is used as a template to carry out PCR amplification to obtain an amplification product. And carrying out agarose gel electrophoresis detection on the PCR amplification product.

TABLE 1 PCR amplification of Gene of interest reaction System

And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 56-58 ℃ for 30s, and extension at 72 ℃ for 2min (30 cycles); extension at 72 ℃ for 10 min.

(2) Construction of recombinant expression vector pET42b (+) -adi

Cutting the gel and recovering PCR amplification products. The purified gene fragment was designated as adi (amino acid sequence SEQ ID NO.1, nucleotide sequence SEQ ID NO. 2). The pET42b (+) plasmid and adi were double-digested with Nco I-Xho I, respectively.

TABLE 2 double digestion reaction System

The enzyme was cleaved at 37 ℃ and 200rpm for 6h, and the cleaved product was recovered. And performing connection recombination reaction on the target gene subjected to double enzyme digestion and a linearized pET-42b vector.

TABLE 3 ligation reaction System

The linker system was incubated at 37 ℃ for 30 min. Immediately after the reaction was completed, it was taken out and ice-cooled for 5 min. A recombinant expression vector pET42b (+) -adi was constructed. The recombinant expression vector was transformed into e.coli BL21(DE3), plated on LB plates containing 50 μ g/mL kanamycin and positive clones were selected, sequenced and stored e.coli BL21(DE3)/pET42b (+) -adi.

Example 2: construction of mutant libraries

Error-prone PCR (epPCR) introduces erroneous bases into a gene by primarily altering the PCR reaction conditions to increase the frequency of mutations during PCR, i.e., to reduce the fidelity of the DNA polymerase. The occurrence of the wrong base causes the corresponding amino acid to mutate, thereby generating a mutant. The mutation frequency during amplification is usually changed by increasing the magnesium ion concentration, adding manganese ions, changing the concentration of four dNTPs in the system, or using low-fidelity DNA polymerase and the like. Too high mutation frequency can cause most mutations to be harmful mutations, so that the mutants lose enzyme activity and beneficial mutations cannot be screened; too low a mutation frequency may result in all wild-type clones in the library, reducing the efficiency of the screening.

The primers used were: an upstream primer: 5'-GCTCCATGGATGTCCACCGAAAACCTGC-3', downstream primer: 5'-ATCTCGAGTTAATAGTCCACCGGGTCA-3' are provided. By changing the manganese ion concentration, the mutation frequency was increased. The concentration of manganese ions is 0.04-0.1mM, preferably 0.08 mM.

TABLE 4 error-prone PCR reaction System

And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 30s, annealing at 55-58 ℃ for 30s, and extension at 72 ℃ for 2min (25 cycles); extension at 72 ℃ for 10 min.

The PCR amplification products were subjected to agarose gel electrophoresis and the results are shown in FIG. 2. Cutting the gel and recovering PCR amplification products. The pET42b (+) plasmid and error-prone PCR product were digested simultaneously with Nco I-Xho I under the same conditions as in example 1. The error-prone PCR product after double digestion was ligated to the linear plasmid, and the ligation reaction system and conditions were the same as in example 1. Coli BL21(DE3) was transformed with the ligation product, spread on LB plates containing 50. mu.g/mL kanamycin, and cultured overnight at 37 ℃ to obtain a library of mutations.

Example 3: mutant library screening

Screening dominant mutants of the obtained mutants, which specifically comprises the following steps: a single colony of the ADI mutation library of example 2 was picked up in a 96-well plate containing 150mL of LB medium containing 50. mu.g/mL of kanamycin per well, and cultured at 37 ℃ for 16 hours in a 900rpm incubator, and 75mL of the culture solution per well was added to a second 96-well plate. 75mL of 30% glycerol was added to the first plate and stored in a-80 ℃ freezer. The second plate was incubated with 75mL of fresh LB medium (containing 50. mu.g/mL kanamycin and 0.4mM IPTG) at 37 ℃ for 12 hours in a 900rpm constant temperature shaker, centrifuged to remove the supernatant, and the cells were cryopreserved. To each well containing the frozen cell pellet, 80. mu.L of lysozyme (final concentration: 80. mu.g/mL) was added, the cells were lysed, gently pipetted, the cells were resuspended, and incubated at 37 ℃ for 20 min. Centrifuge at 4000rpm for 15min at 4 ℃ and take 20. mu.L of supernatant per well to a new well plate. In a new well plate, 0.4mM NADH, 5mM alpha-ketoglutarate, 5.55U GDH pure enzyme solution was added per well, after incubation for 10min at 37 ℃, 80. mu.L arginine (final concentration 0.4mM) was added per well to initiate the cascade reaction, and the absorbance value at 340nm was recorded using a microtiter plate reader.

The ammonia production velocity v is calculated from the formula (1)_ΔCWherein the absorption coefficient ε of NADH is 6200M^-1cm^-1The liquid height b in the wells of the microtiter plate was 0.59cm (total volume of reactants in each well was 200. mu.L). By v_ΔCFurther, the initial reaction rate of arginine deiminase was obtained (see FIG. 1).

v_ΔA＝ε×b×v_ΔC (1)

The size of the primarily screened mutant library is 2 ten thousand mutant strains, 900 mutant strains with 2-3 times of enzyme activity improvement compared with wild type and 180 mutant strains with 3-4 times of enzyme activity improvement are screened out altogether. And (3) carrying out secondary screening by using 0.2mM and 0.3mM substrates to finally obtain four mutants of M1(M30R), M2(C37K), M3(D38H) and M4(H405R), wherein the enzyme activities of the four mutants are respectively improved by 3.1 times, 2.9 times, 3.0 times and 3.5 times compared with the wild type.

TABLE 5 enzymatic Activity of ADI and its mutants

Example 4: site-directed mutagenesis and screening

Taking the plasmid of the dominant single mutant strain as a template, designing primers as shown in table 4, and performing combined mutation by adopting a full-plasmid PCR method to respectively obtain a double mutant, a triple mutant and a quadruple mutant, wherein the specific operations are as follows: the 30 th, 37 th, 38 th and 405 th positions of the amino acid sequence of the arginine deiminase ADI shown in SEQ ID NO.1 are mutated into corresponding amino acids by taking the plasmid pET28a (+) -ADI as a template through PCR, and are transformed and coated with a plate. PCR reaction (50. mu.L): 1 μ L of the forward primer (100 μ M), 1 μ L of the reverse primer (100 μ M), 25 μ L of 2 XPPhanta Max buffer, 1 μ L of dNTP mix (10 mM each), 1 μ L of plasmid template, 1 μ L of LDNA polymerase and 20 μ L of ddH₂And O. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 56-58 ℃ for 30s, and extension at 72 ℃ for 8.0min (30 cycles); extension at 72 ℃ for 10 min. And carrying out agarose gel electrophoresis detection on the PCR product, cutting the gel and recovering the PCR product. mu.L of the PCR-recovered product that was positive in the electrophoretic detection was removed, 1. mu.L of Dpn I enzyme and 1. mu.L of cutmarst buffer were added, the original template was digested at 37 ℃ and 160rpm for 15min, the product was transformed into E.coli BL21(DE3), spread on an LB plate containing 50. mu.g/mL kanamycin, and cultured overnight at 37 ℃. Single colonies were randomly picked to extract plasmids for sequencing. And (4) after sequencing, carrying out bacterium preservation on the mutated positive bacterial colony and determining the enzyme activity.

TABLE 6 arginine deiminase site-directed mutagenesis primer design

The results of the experiment are shown in Table 7.

TABLE 7 enzymatic Activity of ADI and its mutants

Through combined mutation, a mutant M15 with greatly improved enzyme activity compared with the wild type is obtained, and the relative activity is 980%.

Example 5: purification of wild-type arginine deiminase and mutants

Coli BL21(DE3)/pET42b (+) -adi of example 1 and the arginine deiminase-optimized mutant strains M15 and M1, M2, M3, M4 obtained by mutation in example 4 were inoculated into LB liquid medium containing kanamycin to a final concentration of 50. mu.g/mL, cultured at 37 ℃ for 10 hours, inoculated into fresh LB liquid medium containing kanamycin to a final concentration of 50. mu.g/mL in an inoculum size of 2.0% (v/v) by volume, cultured at 37 ℃ and 180rpm for 2 hours, added with IPTG to a final concentration of 0.20mM, cultured at 28 ℃ for 12 hours, and centrifuged at 4 ℃ and 8000rpm for 10 minutes to obtain wet cells. The obtained cell produces corresponding protein and can be used for preparing protein pure enzyme solution.

The obtained wet cells were centrifuged at 8000rpm at 4 ℃ for 10min and collected, and washed twice with 0.9% (w/v) saline. Adding the bacterial strain into PBS buffer solution with pH value of 7.0 and 100mM according to the amount of 25g/L of the total bacterial strain, carrying out ultrasonic disruption on an ice-water mixture for 6min, wherein the ultrasonic disruption conditions are as follows: the power is 400W, crushing for 1s and pausing for 1s, and crude enzyme solutions of wild arginine deiminase, mutant strains M15, M1, M2, M3 and M4 are obtained. The supernatant was collected by centrifugation at 8000rpm at 4 ℃ for 10min, and after microfiltration through a 0.45 μm membrane, the mutant protein was purified using a Super-Q anion exchange resin and desalted with PD-10, and the protein concentration was determined using the BCA method.

Example 6: determination of kinetic parameters and optimum pH of wild-type arginine deiminase and mutants M1, M2, M3, M4 and M15

20. mu.L of the wild-type arginine deiminase and the mutant pure enzyme solutions prepared in example 5 and 80. mu.L of the substrate solutions (at concentrations of 0.1mM, 0.2mM, 0.5mM, 0.8mM, and 1mM, respectively) were subjected to the reaction at 37 ℃ according to the procedure for screening in a microtiter plate in example 3 to determine the enzyme kinetic parameters.

Definition of enzyme activity: the amount of enzyme required per minute per 1. mu. mol of ammonia produced under standard conditions is defined as one enzyme activity unit U.

Specific enzyme activity definition: the number of enzyme activity units per mg of protein is recorded as U/mg.

The optimum pH of the enzyme was measured by the procedure of screening in a microtiter plate in example 3 using 20. mu.L of the wild-type arginine deiminase prepared in example 5 and 80. mu.L of the substrate solution (concentration: 0.1mM each) and 80. mu.L of the substrate solution (pH 5.0 to 6.5 acetic acid-sodium acetate buffer, pH 6.0 to 8.0 sodium phosphate buffer, pH 7.5 to 8.5Tris-HCl buffer) as shown in FIG. 4. The pH optimum for wild-type ADI was 6.5, while the pH optimum for M15 was 7.0.

From Table 8, the K of the mutant can be seen_mThe value is obviously reduced compared with the wild type, and the catalytic efficiency is obviously improved.

TABLE 8 kinetic parameters of arginine deiminase and mutant M15 for arginine

Example 7: in vitro anti-tumor effects of wild type arginine deiminase and mutant M15

Respectively culturing melanoma cells, liver cancer cells and prostate cancer cells, and specifically operating as follows: melanoma cell line SK-MEL-28, G361: adding 100 mu g/mL streptomycin and 100 mu g/mL penicillin into DEME culture medium containing 10% fetal bovine serum; human liver cancer cells HepG2, SSMC-7721: DEME culture medium containing 10% fetal bovine serum; prostate cancer cell line PC 3: RPMI 1640 medium containing 10% fetal bovine serum, CWR22RV1 cells: DMEM containing 10% fetal calf serum was supplemented with 100. mu.g/mL streptomycin and 100. mu.g/mL penicillin. The cells were cultured separately.

Median Inhibitory Concentration (IC) of ADI-WT and ADI-M15 for cell growth inhibition of melanoma cells, liver cancer cells and prostate cancer cells₅₀) And (3) determination: purified ADI enzyme (0.01, 0.05, 0.1, 0.2, 0.4, 1, 2, 4, 8, 10. mu.g/mL) was filter sterilized through a 0.2. mu.M filter. Cells (2X 10) were removed from 180. mu.L growth medium³) Added to each well of a microtiter plate, and after 24h of incubation, 20. mu.L of sterile enzyme was added to each well. Microtiter plates were incubated at 37 ℃ with 5% CO₂After 6 days in the Cell incubator, 20. mu.L of Cell Titer-Blue dye was added to each well, and after 3h of incubation, the assay was at 560_ExFluorescence of/590 Em. IC (integrated circuit)₅₀ADI enzyme concentration to inhibit cell growth by 50%. Data were analyzed using GraphPad Prism 6. The results are shown in Table 9 and FIG. 5. Table 9 shows that the recombinant arginine deiminase mutant M15 of the present invention has activity of inhibiting melanoma, liver cancer cells, and prostate cancer cells, i.e., has an application of developing drugs for treating three tumors (anti-liver cancer, melanoma, prostate cancer, etc.).

TABLE 9 in vitro antitumor Effect of wild-type arginine deiminase and mutant M15

Sequence listing

<110> Zhejiang university

<120> recombinant arginine deiminase mutant, preparation method and application thereof

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tactggcctg ctcgccgcca agaaactctg ctggttacgg caatctacaa gttccacccg 600

gatttcgcgg ctgcagacgt taaagtttgg tggggcgatc cggacgttga tcacggtagc 660

gcgaccctgg aaggtggtga tgtgatgccg atcggtaatg gcgttgttct gattggcatg 720

ggtgaacgta ctagccatca ggccatcggc caggtggcaa tgtctctgtt taaagctggt 780

gcggctgaac gtgttatcgt tgccgctctg ccgaaatctc gctctgcaat gcacctggac 840

actgttttca gcttctgtga tcgtgacctg gttaccatct ttccggaggt ggttaaacag 900

atcgtagcct tctccctgcg tccggacgaa agcaaaccga acggtatcga cctgcgtcgt 960

gaatctaaac cgtttctgga cgtcgtagca gaggctctga aactgccggc gctgcgtgtt 1020

gtacagaccg gtggtaactc ttttgaggcg gaacgtgaac agtgggacga cggcaataac 1080

gttgttgctc tgcagccggg tgttgtgctg gcgtacgatc gtaacattca taccaatacc 1140

ctgctgcgta aagcaggcgt tgaagtgatt accatctctt ctgcagaact gggtcgtggt 1200

cgtggcggtg gtcactgtat gacctgtccg attatccgtg acccggtgga ctat 1254

<210> 3

<211> 418

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Ser Thr Glu Asn Leu Gln Leu Gly Val His Ser Glu Val Gly Met

1 5 10 15

Leu Arg Arg Val Met Val Cys Ser Pro Gly Leu Ala His Arg Arg Leu

20 25 30

Thr Pro Asn Asn Lys His Ser Leu Leu Phe Asp Asp Val Leu Trp Val

35 40 45

Ser Gln Ala Lys Arg Asp His Phe Asp Phe Val Ser Lys Met Arg Glu

50 55 60

Arg Gly Val Asp Val Ile Glu Met His Asn Leu Leu Thr Thr Thr Val

65 70 75 80

Ala Ile Pro Glu Ala Arg Lys Trp Ile Leu Asp Arg Lys Ile Thr Ala

85 90 95

Asp Leu Val Gly Ile Gly Leu Leu Glu Glu Val Arg Ser Trp Leu Asp

100 105 110

Ser Leu Glu Pro Arg His Leu Ala Glu Phe Leu Met Gly Gly Val Val

115 120 125

Ala Pro Asp Leu Pro Ala Asp Thr Pro Ser Glu Val Leu Ser Ile Phe

130 135 140

Arg Glu Asn Phe Gly Asn Ala Ser Phe Ile Leu Pro Pro Leu Pro Asn

145 150 155 160

Thr Gln Phe Thr Arg Asp Thr Thr Cys Trp Ile Tyr Gly Gly Val Thr

165 170 175

Leu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gln Glu Thr Leu Leu Val

180 185 190

Thr Ala Ile Tyr Lys Phe His Pro Asp Phe Ala Ala Ala Asp Val Lys

195 200 205

Val Trp Trp Gly Asp Pro Asp Val Asp His Gly Ser Ala Thr Leu Glu

210 215 220

Gly Gly Asp Val Met Pro Ile Gly Asn Gly Val Val Leu Ile Gly Met

225 230 235 240

Gly Glu Arg Thr Ser His Gln Ala Ile Gly Gln Val Ala Met Ser Leu

245 250 255

Phe Lys Ala Gly Ala Ala Glu Arg Val Ile Val Ala Ala Leu Pro Lys

260 265 270

Ser Arg Ser Ala Met His Leu Asp Thr Val Phe Ser Phe Cys Asp Arg

275 280 285

Asp Leu Val Thr Ile Phe Pro Glu Val Val Lys Gln Ile Val Ala Phe

290 295 300

Ser Leu Arg Pro Asp Glu Ser Lys Pro Asn Gly Ile Asp Leu Arg Arg

305 310 315 320

Glu Ser Lys Pro Phe Leu Asp Val Val Ala Glu Ala Leu Lys Leu Pro

325 330 335

Ala Leu Arg Val Val Gln Thr Gly Gly Asn Ser Phe Glu Ala Glu Arg

340 345 350

Glu Gln Trp Asp Asp Gly Asn Asn Val Val Ala Leu Gln Pro Gly Val

355 360 365

Val Leu Ala Tyr Asp Arg Asn Ile His Thr Asn Thr Leu Leu Arg Lys

370 375 380

Ala Gly Val Glu Val Ile Thr Ile Ser Ser Ala Glu Leu Gly Arg Gly

385 390 395 400

Arg Gly Gly Gly Arg Cys Met Thr Cys Pro Ile Ile Arg Asp Pro Val

405 410 415

Asp Tyr

Claims

1. A recombinant arginine deiminase mutant characterized by: the amino acid sequence of the recombinant arginine deiminase mutant is shown as SEQ ID NO. 3.

2. Use of the recombinant arginine deiminase mutant according to claim 1 for the preparation of a medicament for the treatment of melanoma, liver cancer or prostate cancer.

3. The preparation method of the recombinant arginine deiminase mutant as claimed in claim 1, characterized in that the recombinant arginine deiminase mutant is obtained by centrifuging fermentation broth after fermentation culture of engineering bacteria containing recombinant arginine deiminase mutant genes, collecting wet bacteria, carrying out ultrasonic disruption on the wet bacteria to obtain crude enzyme liquid containing the recombinant arginine deiminase mutant, and carrying out separation and purification.

4. The method for preparing the recombinant arginine deiminase mutant according to claim 3, comprising the following steps:

inoculating the engineering bacteria containing the recombinant arginine deiminase mutant gene into an LB liquid culture medium containing 50 mu g/mL kanamycin at the final concentration, culturing for 10h at 37 ℃, inoculating the engineering bacteria into a fresh LB liquid culture medium containing 50 mu g/mL kanamycin at the final concentration by an inoculation amount with the volume concentration of 1.5-2.0%, culturing for 2h at 37 ℃ and 200rpm, adding isopropyl thiogalactoside at the final concentration of 0.15 mM into the culture solution, culturing for 12h at 28 ℃, and centrifuging for 10min at 4 ℃ and 8000rpm to obtain wet bacteria containing the recombinant arginine deiminase mutant; washed with 0.9% (w/v) saline; adding the bacterial strain into 100mM sodium phosphate buffer solution with pH of 7.0 according to the amount of 25g/L of the total bacterial strain, resuspending the bacterial strain, and carrying out ultrasonic crushing on an ice-water mixture for 6min under the ultrasonic crushing conditions: the power is 400W, the crushing lasts for 1s, and the suspension lasts for 1 s; the obtained supernatant is crude enzyme liquid containing the recombinant arginine deimination mutant, the Super-Q anion exchange resin is used for purifying mutant protein, and PD-10 is used for desalting to obtain the recombinant arginine deimination mutant.