CN111909921A

CN111909921A - Arginine deiminase mutant and coding gene and application thereof

Info

Publication number: CN111909921A
Application number: CN202010750669.8A
Authority: CN
Inventors: 吴志革; 金志华; 金庆超; 杨郁; 潘霞; 李松浩
Original assignee: Zhejiang University of Science and Technology ZUST
Current assignee: Ningbo University of Technology; Zhejiang University of Science and Technology ZUST
Priority date: 2020-07-30
Filing date: 2020-07-30
Publication date: 2020-11-10
Anticipated expiration: 2040-07-30
Also published as: CN111909921B

Abstract

The invention discloses an arginine deiminase mutant and a coding gene and application thereof, belonging to the technical field of molecular biology. The amino acid sequence of the arginine deiminase mutant is shown as SEQ ID NO.1, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2. The invention adopts error-prone PCR technology to transform wild type Lactobacillus brevis arginine deiminase, and obtains high-enzyme-activity mutant H265R by screening, the catalytic activity of the mutant is obviously improved, and the mutant can be used for in vitro biological preparation of citrulline; the mutant provided by the invention has obvious anti-tumor cell growth activity, has good stability under physiological pH conditions, and has potential application prospects.

Description

Arginine deiminase mutant and coding gene and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to an arginine deiminase mutant and a coding gene and application thereof.

Background

Arginine deiminase (EC 3.5.3.6, ADI) is an Arginine degrading enzyme present in microorganisms such as mycoplasma, unicellular chlorella, bacteria, etc., and catalyzes Arginine to produce citrulline and ammonia in vivo. Earlier, ADI was mainly used to transform arginine to produce citrulline. Recent researches prove that arginine deiminase has obvious antitumor activity, has strong inhibiting effect on the growth of cancer cells such as liver cancer, melanoma, prostate cancer and the like with arginine-succinate synthetase expression defects, and has better potential in the field of antitumor drug development.

At present, ADI of various microbial sources has been found to have certain anticancer activity, but ADI of different sources has great difference in amino acid composition, enzymology property and antitumor type, and the amino acid homology of ADI from Mycoplasma arginni and Pseudomonas plecoglossicida is only 27.1%.

At present, most of arginine deiminase is from mycoplasma, and ADI from mycoplasma can obviously inhibit the growth of various cancer cells such as HCC, melanoma, leukemia, prostate cancer cells, pancreatic cancer cells, renal cancer cells and the like in vivo. The effectiveness and the universality of the ADI from mycoplasma source for inhibiting tumors are closely related to the higher affinity (the Mie constant can reach 0.2mmol/L) of the ADI and arginine under neutral physiological conditions. However, mycoplasma is a parasitic and pathogenic microorganism, and has potential safety hazard as a source for using drugs in human body. Therefore, the discovery of probiotic-derived arginine deiminase is becoming one of the areas of hot spots.

In the existing report, an ADI high-producing strain (enterococcus faecalis NJ402) is obtained from plum blossom, etc. and Mn with proper concentration is found²⁺、Mg²⁺And Co²⁺Has a great promotion effect on the ADI catalytic activity (the research on the enzymology property of enterococcus faecalis arginine deiminase, microbiological report 2008, 35 (6): 846-.

Lactobacillus brevis (Lactobacillus brevis) is very common in animal and plant fermentation products and the digestive system of warm-blooded animals, and is generally considered as a safe food-grade microorganism. ADI from lactic acid bacterium ATCC7962 was found to inhibit the proliferation of SNU-L gastric cancer cells with a Km of 8.7mmol/L, much greater than 0.2mmol/L for the Mycoplasma source ADI.

Disclosure of Invention

The invention aims to modify arginine deiminase of Lactobacillus brevis (Lactobacillus brevis), obtain a mutant with improved enzyme catalysis performance, develop food-grade arginine deiminase and improve the industrial application value of arginine deiminase.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention takes Lactobacillus brevis (Lactobacillus brevis) wild type arginine deiminase coding gene as a template, error-prone PCR amplification is carried out, a mutation library is constructed, and a recombinant vector is constructed by amplified fragments to obtain a mutation gene library; transforming the mutant gene library into competent cells, constructing a mutant library, and inducing the library to express by using an inducer such as lactose or IPTG (isopropyl-beta-thiogalactoside) to obtain an expression library; and (3) carrying out enzyme activity analysis and sequence determination on the expression library, and screening to obtain a lactobacillus brevis arginine deiminase mutant with improved catalytic performance, namely that the 265 th histidine (H) of the amino acid sequence is mutated into arginine (R).

The invention provides an arginine deiminase mutant, the amino acid sequence of which is shown as SEQ ID NO. 1.

The invention also provides a gene for coding the arginine deiminase mutant, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

The coding gene of the arginine deiminase mutant can be obtained by a site-directed mutagenesis method, and specifically comprises the following steps: using lactobacillus brevis genome as template, and utilizing the site-directed mutagenesis primer to carry out PCR to obtain a site-directed mutagenesis PCR product. The site-directed mutagenesis primer is as follows: an upstream primer: 5'-CCATTAAGATTCCACGTAACCATGCCATGATGC-3', respectively; a downstream primer: 5'-GCATCATGGCATGGTTACGTGGAATCTTAATGG-3' are provided.

The invention also provides an expression cassette, a recombinant plasmid and a transformant containing the gene.

The promoter of the expression cassette is T7 promoter, lac promoter or araBAD promoter. Under the action of the promoters, the arginine deiminase mutant can directly realize intracellular soluble expression in an Escherichia coli host cell.

The original vectors of the recombinant plasmids are plasmids pET21a, pET28a and pET30 a.

The host cell of the transformant is escherichia coli (e.coli) cell BL 21.

Compared with wild arginine deiminase, the mutant H265R provided by the invention has higher catalytic activity, the activity is improved by 41.6%, the thermal stability and the pH tolerance are enhanced, and the conversion rate is more than 90% when the mutant H265R is used for the biological preparation of citrulline.

The invention provides application of the arginine deiminase mutant in producing citrulline by converting arginine.

The application comprises the following steps: the method comprises the steps of carrying out catalytic reaction at 37 ℃ and 300rpm by taking enzyme obtained by breaking wet thalli obtained by fermenting and culturing engineering bacteria containing arginine deiminase mutant coding genes as a catalyst, taking arginine as a substrate and taking sodium phosphate buffer solution (0.2M, pH6.0) as a reaction medium, and after the reaction is finished, separating and purifying the reaction solution to obtain the citrulline.

The engineering bacteria are escherichia coli BL21 containing arginine deiminase mutant plasmid pET28a (+) -ADI (H265R), and the fermentation culture method comprises the following steps: inoculating the engineering bacteria into LB culture medium containing kanamycin, and culturing at 37 ℃ to OD₆₀₀When the concentration is 0.6-1, isopropyl-beta-D-thio is added to the solution to a final concentration of 1mMAnd (3) performing induced culture on the galactopyranoside at 30 ℃ for 10-12h, centrifuging, and collecting thalli to obtain the wet thalli.

In the catalytic reaction system, the substrate concentration is 10-150mM, preferably 100mM, and the enzyme is added in an amount of 10mg/mL in terms of wet cells.

The invention also provides application of the arginine deiminase mutant in preparing medicines for treating liver cancer, cervical cancer, melanoma or prostate cancer. The mutant has stronger inhibiting effect on HepG2 and HeLa cells.

The invention has the following beneficial effects:

the invention adopts error-prone PCR technology to transform wild type Lactobacillus brevis arginine deiminase, and obtains high-enzyme-activity mutant H265R by screening, the catalytic activity of the mutant is obviously improved, and the mutant can be used for in vitro biological preparation of citrulline; the mutant provided by the invention has obvious anti-tumor cell growth activity, has good stability under physiological pH conditions, and has potential application prospects.

Drawings

FIG. 1 shows single-restriction electrophoresis detection of EcoRI plasmid pET28a-H265R, M is DNA marker, and Lane 1 shows single-restriction pET28a-H265R plasmid.

FIG. 2 shows the electrophoretic detection of purified arginine deiminase mutant H265R protein, M is protein marker, and Lane 1 shows purified arginine deiminase mutant H265R protein.

FIG. 3 is a liquid phase diagram of an arginine derivative.

FIG. 4 is a standard curve plotted with the concentration of arginine as the abscissa and the peak area corresponding to each concentration as the ordinate.

Detailed Description

The present invention will be further described with reference to the following specific examples.

Example 1

First, the acquisition of the mutated gene of the arginine deiminase of Lactobacillus brevis

Taking a cloned fragment of an arginine deiminase coding gene of lactobacillus brevis as a template to carry out error-prone PCR amplification, wherein the PCR system is as follows: 10 × buffer(Mg²⁺free), 5. mu.l; forward primer (10. mu.M concentration), 1. mu.l; downstream primer (concentration 10. mu.M), 1. mu.l; dNTP (2.5mM each), 4. mu.l; MgCl₂(25mM)，14μl；MnCl₂(5mM), 1.5. mu.l; template, 1. mu.l, DNA polymerase, 1. mu.l; ddH₂O, 21.5. mu.l; the total volume was 50. mu.l.

The upstream primer used: 5' -CTAGAATTCATGACAAGTCCGATTCAC-3' (EcoRI cleavage sites are underlined);

the downstream primers used were: 5' -CGCAAGCTTTTAAAGGTCTTCTCGAACTA-3' (HindIII cleavage sites are underlined);

the PCR conditions used were: after denaturation at 94 ℃ for 5min, amplification cycle is carried out, namely denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, total cycle is carried out for 30 times, and finally extension at 72 ℃ for 10 min.

The fragment obtained by the above amplification and pET28a (+) vector were subjected to double digestion with EcoRI and HindIII, and the desired fragment was recovered and ligated with Takara DNA ligation kit ver.2.1 to obtain a mutant gene library.

The mutant gene library was introduced into competent cells of Escherichia coli BL21(DE3), spread on 15% agar LB plate (containing 50. mu.g/ml kanamycin), and cultured at 37 ℃ for 14 hours to obtain a mutant strain library.

Second, screening of mutants

The single clone was picked up to 12-well plates and cultured, each well containing 1ml of medium, and induced to culture at 30 ℃ for 12 hours with 1mM IPTG to obtain an expression library. Collecting thallus, resuspending thallus with PBS, ultrasonicating, transferring 20 μ L supernatant to 12-well plate, adding 380 μ L substrate (10mmol/L L-arginine, 0.2mol/L sodium phosphate buffer solution, pH6.0), reacting at 37 deg.C for 30min, adding 0.36ml acid-iron reagent (7% H)₃PO₄，15.68％H₂SO₄，0.37mM FeCl₃) 0.12ml of DAM-TSC solution (1% DAM, 0.03% TSC) was added, boiling water bath was carried out for 10min, and the intensity of the enzyme activity was judged by the shade of the color reaction or by the absorbance at 490 nm.

The Lactobacillus brevis arginine deiminase mutant H265R, in which the Codon (CAT) encoding histidine was mutated to arginine (CGT), was thus selected. The result shows that the activity of the mutant is obviously improved compared with that of the wild type.

Thirdly, obtaining mutant H265R by site-directed mutagenesis

Mutant H265R can also be obtained by site-directed mutagenesis. Carrying out PCR amplification by taking a cloned fragment of an arginine deiminase encoding gene of lactobacillus brevis as a template, wherein the PCR system is as follows: 10 × buffer (Mg)²⁺free), 5. mu.l; forward primer (10. mu.M concentration), 1. mu.l; downstream primer (concentration 10. mu.M), 1. mu.l; dNTP (2.5mM each), 4. mu.l; MgCl₂(25mM), 14. mu.l; template, 1. mu.l, DNA polymerase, 1. mu.l; ddH₂O, 21.5. mu.l; the total volume was 50. mu.l.

The upstream primer used:

5’-CCATTAAGATTCCACGTAACCATGCCATGATGC-3’；

the downstream primers used were:

5’-GCATCATGGCATGGTTACGTGGAATCTTAATGG-3’。

the PCR conditions used were: after denaturation at 94 ℃ for 5min, amplification cycle is carried out, namely denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 7min, and the cycle is repeated for 20 times.

Digesting the template gene by the obtained site-directed mutagenesis PCR product through DpnI to obtain a mutagenesis gene library; the obtained library of mutant genes was transformed into chemically competent cells of Escherichia coli BL21(DE3), spread on LB agar plates containing 50. mu.g/ml kanamycin, and cultured at 37 ℃ for 14 hours. Single colonies were picked from the plates and cultured overnight in 3ml tubes of LB liquid medium (containing 50. mu.g/ml kanamycin) at 37 ℃ and 200 rpm. DNA sequencing is carried out on the bacterial liquid to obtain the Lactobacillus brevis arginine deiminase H265R mutant.

Induced expression of Lactobacillus brevis arginine deiminase mutant

And streaking and culturing the engineering expression strain of the lactobacillus brevis arginine deiminase and the engineering expression strain of the mutant obtained on a plate. Single clones were picked and cultured overnight in 2m1 test tubes containing kanamycin to a final concentration of 50. mu.g/ml. The overnight culture was transferred to a shaking flask containing 300ml of TB culture (containing 50. mu.g/ml kanamycin) at a temperature of 3Culturing at 7 deg.C and 200rpm to OD₆₀₀The value is about 0.6-1, adding IPTG with the final concentration of 1mM for induction, and inducing overnight under the conditions that the temperature is 30 ℃ and the rotating speed is 200 rpm.

Preparation of crude enzyme solution of Lactobacillus brevis arginine deiminase mutant

After the fermentation is finished, centrifuging at 6000rpm for 10min to collect bacterial liquid, discarding the supernatant, washing the thalli for 1 time by using ice-precooled sodium phosphate buffer (0.2M, pH6.0), resuspending 50ml of sodium phosphate buffer, breaking cells at high pressure, centrifuging at 10000rpm for 10min, and taking the supernatant, namely the crude enzyme liquid.

Purification of arginine deiminase mutant protein

Centrifuging at 6000rpm for 10min to collect the fermentation liquid after induction expression, discarding the supernatant, and using ice-precooled buffer A (50mM NaH) for the thalli₂PO₄-Na₂HPO₄300mM NaCl, pH7.0), sonicated, and then centrifuged at 15,000rpm for 30 minutes at 4 ℃. The supernatant was then added to pre-washed Ni-NTA beads and roller mixed for one hour at 4 ℃. The beads-containing suspension was transferred to a column using buffer B (50mM NaH)₂PO₄-Na₂HPO₄300mM NaCl, 5mM Imidazole, pH7.0) was washed 3 times and then with an Elution Buffer (50mM NaH)₂PO₄-Na₂HPO₄300mM NaCl, 150mM Imidazole, pH7.0), and desalting to obtain arginine deiminase mutant H265R protein, as shown in FIG. 2.

Activity detection of hepta-arginine deiminase mutant protein

10mM arginine, an appropriate amount of arginine deiminase mutant protein or arginine deiminase wild-type protein was added to 5ml of the reaction system, and the reaction was carried out at 37 ℃ and 500rpm in a buffer solution of sodium phosphate buffer (0.2M, pH 6.0). After the reaction is finished, terminating the reaction in boiling water bath for 5min, centrifuging to remove precipitates, taking the reaction liquid for color reaction, and measuring the absorbance value at the wavelength of 490. And drawing a standard curve according to different citrulline concentrations and absorbance at 490 wavelengths, and substituting the measured absorbance values into the standard curve to calculate the activity of the arginine deiminase mutant. The results showed that the mutant had an approximately 41.6% increase in activity over the wild type.

Eighthly, temperature stability and pH tolerance test

Diluting arginine deiminase protein with 0.2M sodium phosphate solution, incubating in water bath at 50 ℃ for 30min, 60min, 90min and 120min, and detecting the residual activity of the Lactobacillus brevis arginine deiminase and the H265R mutant respectively.

The results showed that the residual activities of lactobacillus brevis arginine deiminase wild type and H265R mutant were 54.6% and 65.3% of the initial values, respectively, after 120 minutes.

Arginine deiminase protein is added into buffer solutions with pH values of 4, 5, 6, 7 and 8 respectively, and the catalytic activity of the lactobacillus brevis arginine deiminase and the H265R mutant are detected respectively.

The results showed that the lactobacillus brevis arginine deiminase wild type and the H265R mutant had better catalytic activity in acidic solution, and the residual activities of the lactobacillus brevis arginine deiminase wild type and the H265R mutant were 13.5% and 25.6% of the optimum pH value and 3.1% and 6.3% of the optimum pH value, respectively, in the buffer at pH7.

Ninthly, the arginine deiminase mutant obviously inhibits the growth of HepG2, HeLa and other tumor cells

Respectively culturing HepG2 cells and HeLa cells in a DMEM medium containing 10% serum, collecting the cells, adjusting the concentration of cell suspension, inoculating the cells into a culture dish with the diameter of 35mm, culturing at 37 ℃ for 24 hours, adding the arginine deiminase mutant protein, setting a control group, and continuing to culture. Every 24 hours, cell counting was performed with a hemocytometer.

The result shows that the arginine deiminase mutant H265R obviously inhibits the growth of HepG2 cells, and the inhibition rate reaches about 48.2%. Meanwhile, the arginine deiminase mutant also obviously inhibits the growth of HeLa cells, and the inhibition rate is about 38.5%.

Ten, preparing citrulline by hydrolyzing arginine by using arginine deiminase mutant

100mM arginine and 10ml crude enzyme solution were added to 50ml of the reaction system, and the reaction was carried out at 37 ℃ and 300rpm for 4 hours in a sodium phosphate buffer (0.2M, pH6.0), and then stopped in a boiling water bath for 5min, and the precipitate was removed by centrifugation.

The reaction solution was taken 100. mu.l and added with 0.5M NaHCO₃Diluting the solution by 50 times, taking 75 mu l of diluted reaction solution and equal volume of 0.5M NaHCO₃The solutions were mixed and dansyl chloride was added in 150. mu.l, 40 ℃,800rmp, 2 hours, then filtered through a 0.45 μm microporous membrane and the arginine content of the solution was determined by HPLC. The liquid phase conditions of HPLC were: chromatographic column Hypersil ODS 2C 18 column (250 mm. times.4.6 mm), detection wavelength 254nm, column temperature 40 deg.C, flow rate 1mL/min, sample size 2. mu.L, mobile phase A methanol, mobile phase B tetrahydrofuran: methanol: 0.05mol/L sodium acetate (pH 6.2) (5:75:420, V/V), gradient elution, mobile phase B ratio in 22min from 80% to 0 gradually, then in the subsequent 3 min to 80% gradually. The concentration of arginine was plotted as abscissa and the peak areas corresponding to different concentrations of arginine were plotted as ordinate to obtain a standard curve, as shown in FIG. 4. The results showed that the conversion of arginine reached 90% or more after 4 hours of reaction.

Sequence listing

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Gln Gln Glu His Asp Asn Phe Ala Glu Thr Leu Arg Gln Asn Gly Thr

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Glu Val Leu Tyr Leu Glu Gln Leu Ser Ala Glu Ala Leu Asp Asp Gly

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Gly Glu Glu Val Lys Leu Asn Phe Leu Glu Gln Met Leu Ala Glu Ser

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Gly Tyr Val Ala Gly Val Thr His Asp Ala Leu Lys Glu Tyr Leu Leu

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Ser Leu Asp Thr Gln Ala Met Val Asn Lys Ile Met Gly Gly Val Arg

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

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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. 1.

2. The gene encoding the arginine deiminase mutant according to claim 1, wherein the nucleotide sequence is set forth in SEQ ID No. 2.

3. An expression cassette comprising the gene of claim 2.

4. The expression cassette of claim 3, wherein the promoter is a T7 promoter, a lac promoter, or an araBAD promoter.

5. A recombinant plasmid comprising the expression cassette of claim 3.

6. A transformant comprising the recombinant plasmid according to claim 5.

7. The transformant according to claim 6, wherein the host cell is an E.coli cell.

8. Use of an arginine deiminase mutant according to claim 1 for the production of citrulline by converting arginine.

9. The application of claim 8, wherein the application comprises: carrying out catalytic reaction at 37 ℃ and 300rpm by using an enzyme obtained by crushing wet thalli obtained by fermenting and culturing engineering bacteria containing arginine deiminase mutant coding genes as a catalyst and arginine as a substrate and using 0.2M sodium phosphate buffer solution as a reaction medium, and after the reaction is finished, separating and purifying the reaction solution to obtain citrulline; the substrate concentration in the reaction system was 100mM, and the amount of enzyme added was 10mg/mL in terms of wet cells.

10. Use of the arginine deiminase mutant according to claim 1 in the preparation of a medicament for treating liver cancer, cervical cancer, melanoma, or prostate cancer.