CN111534493A - Purine nucleoside phosphorylase mutant, gene and application - Google Patents

Abstract

The invention discloses a purine nucleoside phosphorylase mutant, a gene and application thereof. The purine nucleoside phosphorylase mutant is obtained by mutating methionine at position 167 to arginine and phenylalanine at position 168 to alanine of purine nucleoside phosphorylase of Escherichia coli. The invention utilizes the site-directed saturation mutagenesis technology to improve the catalytic performance of Purine Nucleoside Phosphorylase (PNP) of Escherichia coli (Escherichia coli), and the specific activity of the screened PNP mutant enzyme can reach 310U/mg, is improved by about 47.6 percent compared with wild enzyme, and has better thermal stability.

Description

Purine nucleoside phosphorylase mutant, gene and application

Technical Field

The invention relates to the technical field of biological engineering, in particular to a purine nucleoside phosphorylase mutant, a gene and application thereof.

Background

Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) catalyzes a reversible phospholysis reaction of purine nucleosides. They are mainly divided into two categories: low molecular weight homotrimers (80-100 kDa); high molecular weight homo-hexamers (110-160 kDa). The latter enzyme has wide substrate specificity, is especially important in synthesis application, and especially has good application value and prospect in the field of medicine.

Nucleoside analogs as monomers have been widely used in cancer and antiviral therapy. Nucleosides act as antiviral agents by inhibiting replication of the viral genome, while nucleosides act as anticancer compounds by inhibiting replication and repair of cellular DNA, e.g., by inhibiting polymerase activity or acting as chain terminators in RNA or DNA synthesis. Furthermore, C2' -fluorinated nucleosides work better as components of antisense oligonucleotides and small interfering RNAs. Various strategies have been devised to enable the design of effective, selective and non-toxic drugs. These methods involve modifications to the natural nucleosides, particularly changes in the carbohydrate moiety.

Traditionally, 2 '-fluoro-2' -deoxyadenosine is prepared by various chemical methods, but the chemical synthesis of modified nucleosides is challenged by steric and regioselective requirements, as well as the need to protect and deprotect sensitive functional groups. In contrast, thymine phosphorylase (TP; EC 2.4.2.4) and purine nucleoside phosphorylase can be used as biocatalysts, and it is more advantageous to synthesize nucleoside analogs efficiently under environment-friendly conditions, but wild-type purine nucleoside phosphorylase has lower activity and is insufficient for production requirements.

The invention patent application with the publication number of CN101629169A discloses a purine nucleoside phosphorylase modified by molecules and a preparation method thereof. The full-length purine nucleoside phosphorylase fragment is amplified from Streptococcus thermophilus (Streptococcus thermophilus) by adopting a PCR technology, pET-43.1b (+) plasmid is used for constructing pET-43.1b (+) -PNP recombinant vector, the 230 th cysteine of the fragment is mutated into alanine by using a site-directed mutagenesis method, Escherichia coli BL21(DE3) is used for converting the fragment into engineering bacteria, and the obtained mutant enzyme can improve the thermal stability.

The invention patent application with publication number CN103468656A discloses a mutant purine nucleoside phosphorylase of Pseudoalteromonas (Pseudoalteromonas), compared with the wild-type amino acid sequence, the 97 th Asp in the amino acid sequence of the mutant purine nucleoside phosphorylase is mutated into Tyr, and the highest specific activity of the mutant reaches 45U/mg.

For industrial applications, catalytic performance of purine nucleoside phosphorylases is still not yet in demand.

Disclosure of Invention

Aiming at the problems of poor thermal stability and activity of purine nucleoside phosphorylase in the prior art, the invention provides a purine nucleoside phosphorylase mutant, a gene and application thereof.

A purine nucleoside phosphorylase mutant is obtained by mutating methionine to arginine at position 167 and phenylalanine to alanine at position 168 of purine nucleoside phosphorylase of Escherichia coli. Preferably, the amino acid sequence of the purine nucleoside phosphorylase mutant is shown as SEQ ID No. 4.

The invention also provides a gene for coding the purine nucleoside phosphorylase mutant. Preferably, the nucleotide sequence of the gene is shown as SEQ ID No. 3.

The invention also provides a recombinant expression vector containing the gene. The backbone plasmid of the recombinant expression vector can be selected from any plasmid capable of expressing the corresponding purine nucleoside phosphorylase mutant after insertion of the gene. Preferably, the recombinant expression vector is obtained by inserting the gene into a PET-28a (+) plasmid.

The invention also provides a genetic engineering bacterium containing the recombinant expression vector. The host cell used by the genetic engineering bacteria can be escherichia coli, yeast cells, CHO cells and the like which are commonly used for exogenous gene expression. Preferably, the host cell of the genetically engineered bacterium is escherichia coli BL 21.

The invention also provides application of the purine nucleoside phosphorylase mutant in preparation of 2 '-fluoro-2' -deoxyadenosine.

The invention also provides a method for preparing 2 '-fluoro-2' -deoxyadenosine, 2 '-fluoro-2' -deoxyuridine, adenine, thymine phosphorylase and the purine nucleoside phosphorylase mutant are added into a reaction system,

the uridine on the 2 '-fluoro-2' -deoxyuridine is replaced by phosphate under the catalysis of thymine phosphorylase to obtain 2 '-fluoro-2' -deoxyribose-1 '-phosphate, and then the phosphate of the 2' -fluoro-2 '-deoxyribose-1' -phosphate is replaced by adenine under the catalysis of purine nucleoside phosphorylase mutant to obtain the 2 '-fluoro-2' -deoxyadenosine.

The purine nucleoside phosphorylase mutant of the present invention is a mutant which is obtained by synthesizing a purine nucleoside phosphorylase mutant according to the spatial structure of wild-type purine nucleoside phosphorylase of E.coli (Koellner G,

m, Shugar D, Saenger W, Bzowska A. journal of molecular biology,1998,280(1): 153-66; bennett EM, Li C, Allan PW, Parker WB, Ealick SE. the journal of Biological Chemistry,2003,278(47):47110-8.), and the purine nucleoside phosphorylase gene of the wild Escherichia coli is obtained by carrying out multi-site saturation mutation of the DNA sequence and high-throughput screening of the mutant enzyme, and the purine nucleoside phosphorylase coded by the mutant enzyme has better activity and thermostability.

The invention utilizes the site-directed saturation mutagenesis technology to improve the catalytic performance of Purine Nucleoside Phosphorylase (PNP) of Escherichia coli (Escherichia coli), and the specific activity of the screened PNP mutant enzyme can reach 310U/mg, is improved by about 47.6 percent compared with wild enzyme, and has better thermal stability.

Drawings

FIG. 1 is an electrophoresis diagram of PCR products of purine nucleoside phosphorylase mutants.

FIG. 2 is a diagram showing the results of purification of purine nucleoside phosphorylase enzyme solution.

Detailed Description

EXAMPLE 1 Synthesis of purine nucleoside phosphorylase and thymine phosphorylase genes

The nucleotide sequence of the coding gene of the wild-type purine nucleoside phosphorylase is shown as SEQ ID NO.1 and is synthesized by Shanghai Jieli bioengineering GmbH.

The nucleotide sequence of the coding gene of thymine phosphorylase from escherichia coli is shown as SEQ ID NO.2 and is synthesized by Shanghai Czeri bioengineering GmbH.

The nucleotide sequence of the mutant coding gene of the purine nucleoside phosphorylase is shown in SEQ ID NO.3, and the amino acid sequence of the coded purine nucleoside phosphorylase mutant is shown in SEQ ID NO. 4.

Example 2 construction of expression vectors for wild-type purine nucleoside phosphorylase and thymine phosphorylase.

(1) The coding genes of Purine Nucleoside Phosphorylase (PNP) and Thymine Phosphorylase (TP) are synthesized by a manual synthesis method, and EcoR I and Not I restriction endonuclease recognition site sequences are added at two ends of the coding gene sequences of the purine nucleoside phosphorylase and the thymine phosphorylase in the synthesis process.

(2) And (2) carrying out double enzyme digestion (enzyme digestion sites are EcoR I and Not I) on the coding genes of the purine nucleoside phosphorylase and the thymine phosphorylase in the step (1) and a plasmid vector pET-28a (+), and identifying, purifying and connecting enzyme digestion products of the two.

(3) And transforming the E.coil DH5 alpha clone strain with the ligation product, and obtaining expression vectors pET-28a (+) -PNP and pET-28a (+) -TP after colony PCR identification, plasmid extraction and sequencing identification.

Example 3 preparation of genetically engineered host cells.

(1) Carrying out SalI linearization treatment on the expression vectors pET-28a (+) -PNP and pET-28a (+) -TP constructed in the implementation 2 respectively;

(2) and (2) respectively introducing the expression vectors pET-28a (+) -PNP and pET-28a (+) -TP obtained in the step (1) into the host cell E.coli BL21(DE3) by using a calcium chloride heat shock method. And coating the transformant on an LB (Luria Bertani) plate culture medium containing kanamycin (Kan) for culture and screening, and obtaining an expression host after sequencing and identification.

Example 4 preparation of host cells for purine nucleoside phosphorylase mutants.

(I) design of site-directed saturation mutagenesis primer

Primer Premier software is selected to design a Primer, a gene sequence of wild purine nucleoside phosphorylase is used as an original template, directional saturation mutation is carried out on amino acid on the sequence, the detailed design scheme of the Primer is shown in table 1, wherein N represents any one of A, T, C, G. The following primers were synthesized by Shanghai Czeri bioengineering, Inc.

TABLE 1 design scheme for site-directed saturation mutagenesis primers

(II) PCR amplification

Wild purine nucleoside phosphorylase plasmid is used as a template, and site-directed saturated mutation is carried out on the wild purine nucleoside phosphorylase plasmid by adopting a site-directed mutation primer. The site-directed saturation mutagenesis PCR reaction system and the PCR reaction procedure are shown in Table 2 and Table 3, respectively. DNApolymerase used in this chapter was Super Pfx DNA Polymerase from Kangji century.

TABLE 2 PCR reaction System

PCR reaction reagent	Volume/mass
		ddH2O	30.8μL
dNTP	3.2μL
		DMSO	2.5μL
5×HF Buffer	10μL
		Plasmids	1μL
Upstream primer	1μL
		Downstream primer	1μL
DNA Polymerase	0.5μL

TABLE 3 PCR reaction procedure

After the PCR reaction, 5. mu.L of the product was subjected to agarose gel electrophoresis, and the total length of the mutated gene was 6089bp, as shown in FIG. 1, the target band was correctly positioned.

(III) digestion and purification of PCR products

(1) Taking 50 mu L of the PCR product of the second step (4), adding 1 mu L of Dpn I, standing and putting in a water bath at 37 ℃ for digestion for 2 h;

(2) purifying the PCR product by using a Gel Extraction Kit, and transferring the digested PCR product into a collection tube of the Kit;

(3) adding 700 mu L of Spw Wash Buffer, centrifuging at 13000rpm for 1min, and discarding the waste liquid;

(4) repeating the operation (3) once again;

(5) centrifuging the tube at 13000rpm for 2 min;

(6) discarding the sleeve below the collecting pipe, sleeving a 1.5mL EP pipe, adding 35-50 μ l of Elutionbuffer into the collecting pipe, and standing for 2 min;

(7) centrifuging at 13000rpm for 1min, collecting filtrate, and storing in refrigerator at-20 deg.C.

(IV) transformation of PCR products

(1) Taking 5 mu L of the PCR purified product of the third step (4) and adding the PCR purified product into 100 mu L of competent cells BL 21;

(2) performing ice bath for 30 min;

(3) carrying out water bath heat shock at 42 ℃ for 90 s;

(4) ice-bath for 3 min;

(5) adding 800 μ L of non-antibiotic liquid LB into the competent cell BL21 finished in ice bath, and culturing in a shaker at 37 deg.C and 200rpm for 50 min;

(6) dropwise adding 80mL of the cultured bacterial liquid onto a flat plate containing kanamycin, uniformly coating the bacterial liquid by using an applicator, inverting the coated bacterial liquid, and transferring the bacterial liquid to a constant-temperature constant-humidity incubator at 37 ℃ for overnight culture;

EXAMPLE 5 preparation of thymine phosphorylase enzyme solution

The host cells of thymidine phosphorylase in example 3 were inoculated into a sterile glass tube containing 5mL of LB medium without antibody, 5. mu.L of 40mg/mL kanamycin was added, and the mixture was cultured at 37 ℃ for 12 hours or more on a shaker at 200 rpm.

4mL of the activated thymidine phosphorylase bacterial solution was inoculated into a 1000mL Erlenmeyer flask containing 200mL of LB-free medium, and 200. mu.L of 40mg/mL kanamycin was added thereto. The culture was carried out at 37 ℃ for 3h on a shaker at 200 rpm. 100 μ L of 1mmol/mL IPTG was added to the cell in a clean bench and incubated at 28 ℃ for 12h on a shaker at 200 rpm.

Centrifuging the cultured bacteria liquid at 10000rpm for 15min, removing supernatant, washing off culture medium, adding 10mL100mM phosphate buffer solution, resuspending, and storing in a refrigerator at 4 ℃. Setting the power of an ultrasonic cell breaking instrument at 400W, carrying out ultrasonic treatment for 3s and intermittent treatment for 6s, breaking cells for 20min, centrifuging the broken cell bacteria liquid at 13000rpm for 20min, and taking the supernatant and storing the supernatant in a refrigerator at-20 ℃ for later use.

EXAMPLE 6 preparation of purine nucleoside phosphorylase enzyme solution

The host cells of purine nucleoside phosphorylase in example 3 and the host cells of purine nucleoside phosphorylase mutant in example 4 were inoculated into a sterile glass test tube containing 5mL of LB medium without antibody, 5. mu.L of 40mg/mL kanamycin was added, and the mixture was cultured at 37 ℃ for 12 hours or more on a shaker at 200 rpm.

In a clean bench, 4mL of the activated mutant strain liquid was inoculated into a 1000mL Erlenmeyer flask containing 200mL of LB-free medium, and 200. mu.L of 40mg/mL kanamycin was added. The culture was carried out at 37 ℃ for 2.5h on a shaker at 200 rpm. In a clean bench, 180. mu.L of 1mmol/mL IPTG was added and incubated at 33 ℃ for 12h on a shaker at 200 rpm.

Centrifuging the cultured bacteria liquid at 10000rpm for 15min, removing supernatant, washing off culture medium, adding 10mL100mM phosphate buffer solution, resuspending, and storing in a refrigerator at 4 ℃. Setting the power of an ultrasonic cell breaking instrument at 400W, carrying out ultrasonic treatment for 3s and intermittent treatment for 6s, breaking cells for 20min, centrifuging the broken cell bacteria liquid at 13000rpm for 20min, and taking the supernatant and storing the supernatant in a refrigerator at-20 ℃ for later use.

Example 7 establishment of high throughput screening method for purine nucleoside phosphorylase mutant Strain

High throughput primary screening of purine nucleoside phosphorylase mutant strains

Directly using whole-cell phosphoric acid suspension of the mutant strain to catalyze the reaction, detecting the amount of the generated product through high performance liquid chromatography to compare, and primarily screening the mutant strain with better catalytic effect.

As many single colonies as possible were picked from the plate obtained in example 4 (IV), inoculated into a sterile test tube containing 5mL of liquid LB, added kanamycin to a final concentration of 40mg/L, covered with a rubber stopper, and subjected to shake culture at 37 ℃ and 200rpm for 12 hours. And (3) centrifuging 1.5mL of activated bacterial liquid at 12000rpm for 3min, taking out supernatant, and adding 1mL of 100mM phosphate buffer solution for resuspension to obtain corresponding PNP mutant strain liquid.

Weighing 0.001g of adenine and 0.0005g of 2 '-fluoro-2' -deoxyuridine, adding 0.5mL of TP enzyme solution, 2.5mL of 100mM phosphate buffer solution and 1mL of mutant strain solution, reacting at 48 ℃ and at the rotating speed of 800r/min for 3h, sampling, inactivating the sample in 100 ℃ water bath for 12s, and detecting the yield of 2 '-fluoro-2' -deoxyadenosine by using high performance liquid chromatography, thereby screening out the mutant strain with better performance.

Rescreening of (di) purine nucleoside phosphorylase mutant strains

And on the basis of the mutant strains which are screened out primarily and have better catalytic performance, determining the enzyme activity of the screened mutant strains, and further obtaining the optimal mutant strains.

Adenine (0.0025 g) and 2 '-fluoro-2' -deoxyuridine (0.0025 g) were weighed, 2mL of the TP enzyme solution of example 5 and 7.5mL of 100mM phosphate buffer were added, the reaction temperature was 48 ℃ and the rotational speed was 800r/min, after 4 hours of reaction, 0.5mL of the PNP mutant enzyme solution of example 6 was added, after 1 hour of reaction, sampling was performed, the samples were inactivated in 100 ℃ water bath for 12 seconds, the yield of 2 '-fluoro-2' -deoxyadenosine was detected by high performance liquid chromatography, and the mutant strain exhibiting the best expression was selected from the yield of 2 '-fluoro-2' -deoxyadenosine.

Finally, a mutant strain is screened, the nucleotide sequence of the mutant coding gene of the corresponding purine nucleoside phosphorylase is shown as SEQ ID NO.3, the amino acid sequence of the purine nucleoside phosphorylase mutant coded by the mutant is shown as SEQ ID NO.4, and the mutants involved in the subsequent experiments are all the screened mutant enzymes.

EXAMPLE 8 purification of purine nucleoside phosphorylase enzyme solution

The purine nucleoside phosphorylase mutant enzyme solution selected in example 7 was purified from the wild-type purine nucleoside phosphorylase enzyme of example 6.

(1) Add about 2g of Medium Proteinlso Ni-NTA Resin to the column

(2) Adding 20% ethanol to submerge the medium, and washing for three times;

(3) adding pure water to immerse the medium, and washing for three times;

(4) washing with 20mM and 40mM imidazole phosphate buffer solution for three times respectively;

(5) adding the prepared crude enzyme solution;

(6) washing with 20mM, 40mM and 400mM imidazole phosphate buffer solution for three times, 1mL each time, adding twice and catching the filtered liquid by a centrifuge tube;

(7) after sampling, washing with 400mM imidazole phosphate buffer solution for multiple times;

(8) washing with pure water and 20% ethanol for several times;

(9) adding 20% ethanol, sealing;

(10) adding the filtrate of the 400mM imidazole phosphate buffer solution caught in the step (6) into an ultrafiltration centrifugal tube, centrifuging the ultrafiltration centrifugal tube at 8000rpm until the volume of the solution is concentrated to be less than 250 μ l, and adding 4mL of 100mM phosphate buffer solution;

(11) then, the mixture was centrifuged at 8000rpm until the volume of the solution became 250. mu.l-500. mu.l, the solution was added to an EP tube and diluted 10-fold with 100mM phosphate buffer solution, 65. mu.l of the enzyme solution was subjected to SDS polyacrylamide gel electrophoresis, and the remainder was stored in a refrigerator at-20 ℃ as shown in FIG. 2.

EXAMPLE 9 measurement of purine nucleoside phosphorylase enzyme solution concentration

The Protein concentration of the purified purine nucleoside phosphorylase wild-type and mutant enzyme solutions of example 8 was measured using Easy II Protein Quantitative Kit (BCA) Protein quantification Kit, and the results are shown in Table 4.

TABLE 4 concentration of enzyme solution of purine nucleoside phosphorylase

	Purine nucleoside phosphorylase content mg/L
		Mutants	30.4
Wild type	33.1

Example 10 measurement of the Performance of purine nucleoside phosphorylase mutants.

Enzymatic activity detection of (mono) purine nucleoside phosphorylase

Adenine (0.0025 g) and 2 '-fluoro-2' -deoxyuridine (0.0025 g) were weighed, 2mL of the TP enzyme solution of example 5 and 7.5mL of 100mM phosphate buffer were added, the reaction temperature was 48 ℃ and the rotation speed was 800r/min, 0.5mL of the wild-type PNP enzyme solution and the mutant enzyme solution of example 8 were added after 4 hours of reaction, samples were taken after 1 hour of reaction, the samples were inactivated in 100 ℃ water bath for 12 seconds, and the yield of 2 '-fluoro-2' -deoxyadenosine was measured by high performance liquid chromatography. PNP enzyme activity definition: under the above conditions, the amount of enzyme required to produce 2 '-fluoro-2' -deoxyadenosine at 1. mu. mol/min was defined as one unit of enzyme activity. The results of enzyme activity detection of purine nucleoside phosphorylase are shown in Table 5.

TABLE 5 detection results of specific Activity of purine nucleoside phosphorylase

Experimental group	Specific activity (U/g)
		Mutant enzymes	3.1
Wild-type enzyme	2.1

As can be seen from Table 5, the specific activity of the purine nucleoside phosphorylase mutant provided by the invention can reach 3.1U/g, which is increased by about 47.6% compared with the wild-type enzyme.

Detection of thermostability of (di) purine nucleoside phosphorylase mutant

Setting the temperature at 50 ℃, standing for 1h, 6h, 12h, 18h and 24h respectively, and determining the residual enzyme activity. The results of measuring the thermostability of purine nucleoside phosphorylase are shown in Table 6.

TABLE 6 purine nucleoside phosphorylase thermostability assay results

As can be seen from Table 6, the enzyme activity of the purine nucleoside phosphorylase mutant and the wild type is basically kept unchanged when the mutant and the wild type are placed at 50 ℃ for 6h, the enzyme activity of the purine nucleoside phosphorylase mutant is reduced by 13% when the placing time reaches 12h, and the enzyme activity of the wild type is reduced by 41%; when the standing time reaches 24h, the enzyme activity of the purine nucleoside phosphorylase mutant is reduced by 39 percent, and the wild type is reduced by 49 percent; in conclusion, the purine nucleoside phosphorylase mutant has better thermostability than the wild type.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

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<221>variation

<222>(11)..(16)

<223>n stands for A/T/C/G.

<220>

<221>misc_feature

<222>(11)..(11)

<223>n is a, c, g, t or u

<220>

<221>misc_feature

<222>(12)..(12)

<223>n is a, c, g, t or u

<220>

<221>misc_feature

<222>(13)..(13)

<223>n is a, c, g, t or u

<220>

<221>misc_feature

<222>(14)..(14)

<223>n is a, c, g, t or u

<220>

<221>misc_feature

<222>(15)..(15)

<223>n is a, c, g, t or u

<220>

<221>misc_feature

<222>(16)..(16)

<223>n is a, c, g, t or u

<400>6

ccatcacgtc nnnnnnttcg ccgtc 25

Claims (10)

1. A purine nucleoside phosphorylase mutant characterized by having an E.coli purine nucleoside phosphorylase in which the 167 th methionine is mutated to arginine and the 168 th phenylalanine is mutated to alanine.

2. The purine nucleoside phosphorylase mutant according to claim 1, wherein the amino acid sequence is represented by SEQ id No. 4.

3. A gene encoding the purine nucleoside phosphorylase mutant according to claim 2.

4. The gene of claim 3, wherein the nucleotide sequence is as shown in SEQ ID No. 3.

5. A recombinant expression vector comprising the gene of claim 3 or 4.

6. The recombinant expression vector of claim 5, wherein the gene is inserted into a PET-28a (+) plasmid.

7. A genetically engineered bacterium comprising the recombinant expression vector of claim 6.

8. The genetically engineered bacterium of claim 7, wherein the host cell is E.coli BL 21.

9. Use of the purine nucleoside phosphorylase mutant according to claim 1 or 2 for preparing 2 '-fluoro-2' -deoxyadenosine.

10. A method for producing 2 '-fluoro-2' -deoxyadenosine, which comprises adding 2 '-fluoro-2' -deoxyuridine, adenine, thymine phosphorylase and the purine nucleoside phosphorylase mutant according to claim 1 or 2 to a reaction system,

the uridine on the 2 '-fluoro-2' -deoxyuridine is replaced by phosphate under the catalysis of thymine phosphorylase to obtain 2 '-fluoro-2' -deoxyribose-1 '-phosphate, and then the phosphate of the 2' -fluoro-2 '-deoxyribose-1' -phosphate is replaced by adenine under the catalysis of purine nucleoside phosphorylase mutant to obtain the 2 '-fluoro-2' -deoxyadenosine.

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