Arginine Deiminase Mutant with Improved Enzyme Activity and Temperature Stability and Application Thereof

Abstract

An arginine deiminase mutant with improved enzyme activity and temperature stability and application thereof were provided, belonging to the technical field of genetic engineering and enzyme engineering. The arginine deiminase mutant is proline, namely Gly292 Pro, mutated from glycine near an enzyme active center. A wild-type arginine deiminase arcA coding gene is molecularly modified by a site-directed mutation technique to obtain a mutant enzyme ADIG292P, which has glycine at position 292 of an amino acid sequence of the wild type arginine deiminase mutated to proline. The arginine deiminase, modified by site-directed mutation, of the present invention has 1.5 times of increase in enzyme activity and 5.43 times of increase in half-life period at 40 C. compared with the wild-type enzyme, which solves the problems of low catalytic ability and temperature stability during the catalytic synthesis of citrulline using arginine deiminase, and lays a foundation for industrial production of efficient synthesis of citrulline and medication application.

Claims

1. An arginine deiminase mutant, comprising an amino acid sequence set forth in SEQ ID NO: 1.

2. A recombinant cell line capable of expressing the arginine deiminase mutant of claim 1.

3. The recombinant cell line of claim 2, comprising fungi or bacteria cells.

4. An application of an arginine deiminase mutant, wherein the arginine deiminase mutant comprises an amino acid sequence set forth in SEQ ID NO:1, wherein the application comprises making an expression vector for expressing the arginine deiminase mutant, and obtaining a protein form of the arginine deiminase mutant.

5. The application of claim 4, comprising preparing a medicament for inhibiting arginine-deficient tumors, breast cancer or liver cancer cells.

6. The application of claim 4 comprising preparing a medicament for the treatment of leukemia.

7. The application of claim 4, comprising studying an antitumor activity and related pharmacological activities of medicines.

8. The application of claim 4, comprising transferring the expression vector into Escherichia coli BL21(DE3), and constructing a recombinant Escherichia coli that express the arginine deiminase mutant.

9. A nucleotide sequence of a gene encoding an arginine deaminase set forth in SEQ ID NO: 2.

10. The application of claim 4, comprising using the arginine deiminase mutant in citrulline production.

11. The application of claim 10, comprising using the arginine deiminase mutant as biocatalyst in catalyzing arginine to produce citrulline.

12. The application of claim 4, comprising using cells that express the arginine deiminase mutant in catalyzing arginine to produce citrulline.

13. The application of claim 12, wherein the cells is a recombinant strain that express the arginine deiminase mutant.

14. The application of claim 13, comprising constructing the recombinant strain by following steps: 1) designing a primer according to a gene sequence of arginine deiminase arcA of Enterococcus faecalis SK32.001; by taking Enterococcus faecalis SK32.001 comprising an arginine deiminase sequence as a template, obtaining a gene segment comprising arginine deiminase arcA by a PCR method to construct a recombinant plasmid with a connection to an expression vector pET-28a; 2) using B-FITTER software to recognize key amino acid residues that have adverse effects on temperature stability in enzyme molecules; then using SWISS-MODEL software to perform protein structure simulation on parent arginine deaminase, so as to obtain a tertiary structure of an arginine deaminase; through a comparative analysis, determining an amino acid site to be mutated is glycine at position 292; 3) designing a mutation primer, using a one-step PCR method to perform site-directed mutation on a nucleotide sequence of arginine deaminase, and replacing the amino acid at position 292 to obtain a recombinant vector comprising an arginine deaminase mutant gene sequence; and 4) enabling the recombinant vector comprising the arginine deaminase mutant gene sequence to enter competent cells of Escherichia coli E. coli BL21(DE3), inducing expression, collecting thalli, and using Ni-NTA for protein separation and purification after ultrasonication on cells to obtain the arginine deaminase mutant.

Description

BRIEF DESCRIPTION OF FIGURES

[0022] FIG. 1: Construction map of recombinant plasmid pET-28a-ADIG292P

[0023] FIG. 2: Temperature stability of wild enzyme and mutant enzyme at 40 C.

[0024] FIG. 3: Conversion rate of biosynthesized citrulline of wild enzyme and mutant enzyme

DETAILED DESCRIPTION

[0025] The invention is further illustrated below by examples, which are for the purpose of illustration and are not intended to limit the scope of the invention.

[0026] Materials and reagents: restriction enzyme, Solution I ligase, PCR reagents and the like used herein were all purchased from TaKaRa Bio Inc.; plasmid extraction kit, genome extraction kit, agarose purification kit, escherichia coli DH5, BL21 (DE3) strains and primers were all purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; other reagents were all analytical pure reagents purchased at home or abroad.

Example 1: Construction of Recombinant Plasmid

[0027] Enterococcus faecalis 5K32.001 was cultured to an exponential growth metaphase, and 2 mL of a bacteria solution was centrifuged at 10000 r/min for 10 min. Supernatant was discarded, and lysozyme treatment was performed for 30 min. Genomic DNA was extracted according to kit instructions.

[0028] The following primers were designed for the amplification of arcA:

TABLE-US-00001 FADI-2: 5-CGCGGATCCATGAGTCATCCAATTAATGT-3 (containingBamHIrestrictionenzymecutting sites), RADI-2: 5-CCGCTCGAGTTAAAGATCTTCACGGT-3 (containingXhoIrestrictionenzymecutting sites).

[0029] PCR amplification conditions: 3 min of denaturation at 95 C., 30 cycles (95 C. 30 s, 55 C. 30 s, 72 C. 210 s), at last 2 min of extension at 72 C.

[0030] After purification on an amplification product, a PCR product and a vector pET-28a-c (+) were double-digested with BamH I and Xho I, and the digested products were respectively recovered and joint with Solution I ligase for 2 h at 16 C. for heat shock transformation into DH5 cells. When transformants grew on a plate, single colonies were picked into a liquid medium, and the plasmid was extracted. A recombinant plasmid pET-28a-ADI was verified through enzyme digestion. The recombinant plasmid was transformed into BL21 (DE3) cells to obtain BL21 (DE3)/pET-28a-ADI engineered bacteria.

Example 2: Determination of Arginine Deiminase Mutation Sites

[0031] Software B-FITTER was used to calculate a temperature factor (B-factor) of parent arginine deiminase amino acid residues to obtain amino acid residues with the highest temperature factor in an enzyme molecule; SWISS-MODEL software was used to simulate an arginine deiminase protein structure to obtain a tertiary structure model of arginine deiminase; Discovery Studio software was used for analyzing a spatial structure of and distance between the amino acid residues with the highest temperature factor and an enzyme catalytic activity center; and an amino acid site to be mutated was then determined to be glycine at position 292.

Example 3: Site-Directed Mutation

[0032] The primer design was performed based on a coding gene encoding arcA in Enterococcus faecalis SK23.001.

TABLE-US-00002 G292P-forwardprimer: 5-CATCCAGAAATCGAACCTGGCTTGGTTGTTTT-3, and G292P-reverseprimer: 5-TTCGATTTCTGGATGAATCGTAAATTTATCATA-3,

[0033] wherein an underlined part represents a codon corresponding to glycine at position 292, encoded by a mutant gene.

[0034] PCR Amplification System:

TABLE-US-00003 10 Reaction Buffer 5 L dNTP mix 1 L Forward primer (100 ng/L) 1.25 L Reverse primer (100 ng/L) 1.25 L Template pET-28a-ADI (10 ng) 2 L pfuTurbo DNA polymerase 1 L (2.5 U/L) ddH.sub.2O 38.5 L

[0035] After PCR amplification, 1 L of Dpn I restriction enzyme (10 U/L) was added into the reaction solution and was thermally insulated at 37 C. for 1 hour to eliminate a template. A PCR product was transferred into Escherichia coli DH5 cells to coat the plate. Single colonies were picked to a liquid medium. A plasmid was extracted, and the correct mutant plasmid was obtained by sequencing. The successfully constructed mutant plasmid was transferred into Escherichia coli BL21 (DE3) to obtain a mutant strain BL21 (DE3)/pET-28a-ADIG292P.

Example 4: Expression and Purification of Wild Enzyme and Mutant Enzyme

[0036] Single colonies of BL21 (DE3)/pET-28a-ADI and BL21 (DE3)/pET-28a-ADIG292P were picked and put in an LB culture medium containing 0.5 mmol/L kanamycin, were cultured at 37 C. for 12 h at 200 r/min, then were transferred to an LB culture medium containing 0.5 mmol/L kanamycin, were cultured at 37 C. and 200 r/min until OD600 fell into the range of 0.5 to 0.7, and 1 mmol/L IPTG was added at the conditions of 28 C. and 200 r/min for 9 h's induction.

[0037] After fermentation broth was centrifuged at 10000 r/min and 4 C. for 10 min, supernatant was discarded. Then the fermentation broth was washed twice with a phosphate buffer, a 15-20 mL phosphate buffer was added to suspend thalli, and ultrasonication was performed for 15 min (power of 22 W, 2 s of intermittence for each 1 s of ultrasonication). Centrifugation was performed at the conditions of 4 C. and 10000 r/min for 10 min, and supernatant was collected as crude enzyme solution and was filtered through a hydrophilic membrane with the pore diameter of 0.22 m.

[0038] A Binding Buffer was used to pre-balance Ni2+chelating agarose resin column; the crude enzyme solution was added, and then the Binding Buffer and Washing Buffer were used for balancing separately; an Elution Buffer was used to elute the enzyme, and the enzyme was recovered; and the recovered enzyme solution was dialyzed in a dialysis buffer and then was restored in a 4 C. refrigerator.

[0039] Buffer Preparations Involved:

[0040] Phosphate Buffer (PB): 50 mmol/L, pH 5.5;

[0041] Binding Buffer: 50 mmol/L PB, 500 mmol/L NaCl, pH 7.0;

[0042] Washing Buffer: 50 mmol/L PB, 500 mmol/L NaCl, pH 7.0, 50 mmol/L imidazole;

[0043] Elution Buffer: 50 mmol/L PB, 500 mmol/L NaCl, pH 7.0, 500 mmol/L imidazole; and

[0044] the dialysis buffer: 50 mmol/L PB, pH 5.5, 10 mmol/L EDTA.

Example 5: Determination of Enzyme Activity and Temperature Stability of Wild Enzyme and Mutant Enzyme

[0045] Definition of enzyme activity: Under this condition, the amount of enzyme for the catalytic production of 1 mol of citrulline per minute is defined as one enzyme activity unit (U).

[0046] Temperature stability: an enzyme solution was thermally insulated at 40 C., the time-gradient samples were taken out according to a time gradient, was added into a substrate L-arginine, and was placed in 45 C. water for a water bath for 10 min, and reaction was immediately terminated by boiling. Then citrulline yield was measured by HPLC, and relative enzyme activity was calculated. The enzyme activity of the untreated enzyme solution was defined as 100%, and the percentage of relative enzyme activity versus time was plotted to assess the temperature stability of enzyme. Results obtained are shown in FIG. 2: half-life period of the enzyme of a mutant G292P was prolonged to 91.8 min from 16.9 min of the wild enzyme, and was increased by 5.43 times.

Example 6: Efficient Synthesis of Citrulline

[0047] Separately take 1 g of bacteriophage of wild enzyme and mutant enzyme into 50 mL L-arginine solution with a concentration of 100 g/L. Reaction was carried out at 45, 150 r/min and pH 6.0-6.5, and timing sampling and citrulline yield analysis were performed. Results are shown in FIG. 3: 95% or more of arginine was converted into citrulline by wet thalli of the mutant enzyme after 2 h., and 95% or more of arginine was converted into citrulline by wet thalli of the wild enzyme after 5 h.

[0048] The citrulline yield in this example was 95% or more.

[0049] Through the invention, a higher concentration of citrulline can be obtained by enzymatic conversion in a relatively short period of time, which lays a foundation for future industrial application.