PHI29 DNA POLYMERASE MUTANT WITH INCREASED THERMOSTABILITY

Abstract

Provided are a phi29 DNA polymerase mutant with increased thermo stability, a method for preparing the mutant, the use of the mutant, and a method for increasing the stability of the phi29 DNA polymerase.

Claims

1. A protein comprising one or more amino acid mutations at positions 140, 171, 197, 215, 216, 217, 224, 239, 369, 323, 470, 474 and 526 relative to the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2.

2. The protein according to claim 1, wherein the protein comprises (1) to (16): (1) a protein obtained by mutating glutamic acid at position 239 into tryptophan and keeping remaining amino acids unchanged relative to the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2; (2) a protein obtained by mutating glycine at position 217 into glutamic acid and keeping remaining amino acids unchanged relative to the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2; (3) a protein obtained by mutating tyrosine at position 224 into lysine and keeping remaining amino acids unchanged relative to the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2; (4) a protein obtained by mutating phenylalanine at position 526 into lysine and keeping remaining amino acids unchanged relative to the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2; (5) a protein obtained by mutating glutamine at position 171 into glutamic acid and keeping remaining amino acids unchanged relative to the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2; (6) a protein obtained by mutating valine at position 470 into lysine and keeping remaining amino acids unchanged relative to the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2; (7) a protein obtained by mutating glycine at position 217 into aspartic acid and keeping remaining amino acids unchanged relative to the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2; (8) a protein obtained by mutating leucine at position 216 into lysine and keeping remaining amino acids unchanged relative to the amino acid sequence of phi29 DNA polymerase; (9) a protein obtained by mutating isoleucine at position 474 into lysine and keeping remaining amino acids unchanged relative to the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2; (10) a protein obtained by mutating threonine at position 140 into serine and keeping remaining amino acids unchanged relative to the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2; (11) a protein obtained by mutating glycine at position 197 into asparagine and keeping remaining amino acids unchanged relative to the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2; (12) a protein obtained by mutating threonine at position 140 into asparagine and keeping remaining amino acids unchanged relative to the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2; (13) a protein obtained by mutating serine at position 215 into aspartic acid and keeping remaining amino acids unchanged relative to the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2; (14) a protein obtained by mutating isoleucine at position 323 into asparagine and keeping remaining amino acids unchanged relative to the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2; (15) a protein obtained by mutating tyrosine at position 369 into arginine and keeping remaining amino acids unchanged relative to the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2; and (16) a protein obtained by mutating valine at position 470 into lysine, isoleucine at position 474 into lysine and keeping remaining amino acids unchanged relative to the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2.

3. The protein according to claim 1, wherein the protein is a fusion protein obtained by ligating a tag at the N-terminus or/and the C-terminus of the protein.

4. The protein according to claim 1, wherein the protein has increased stability compared to the phi29 DNA polymerase shown in SEQ ID NO: 2.

5. The protein according to claim 4, wherein the stability is thermal stability.

6. A nucleic acid molecule encoding the protein as defined in claim 1.

7. A method for improving stability of phi29 DNA polymerase comprising: mutating one or more amino acids at positions 140, 171, 197, 215, 216, 217, 224, 239, 369, 323, 470, 474 and 526 of the amino acid sequence of phi29 DNA polymerase shown in SEQ ID NO: 2.

8. The method according to claim 7, wherein the stability is thermal stability.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0078] FIG. 1 shows the DNA Nano ball (DNB) concentration produced in the presence of crude wild-type phi29 DNA polymerase and phi29 DNA polymerase mutant Y369R and remaining activity ratio thereof in the stability assay.

[0079] FIG. 2A and FIG. 2B show the detection results of pure wild-type phi29 DNA polymerase and phi29 DNA polymerase mutants V470K and I474K for stability, where FIG. 2A shows the comparison of enzyme activity of the pure wild-type phi29 DNA polymerase and phi29 DNA polymerase mutants V470K and I474K under different treatment conditions, and FIG. 2B shows the comparison of remaining activity ratio of the pure wild-type phi29 DNA polymerase and phi29 DNA polymerase mutants V470K and 1474K under different treatment conditions.

[0080] FIG. 3A and FIG. 3B show the detection results of pure wild-type phi29 DNA polymerase and phi29 DNA polymerase mutant V470K-I474K (Mut_1) for stability, where FIG. 3A shows the comparison of enzyme activity of the pure wild-type phi29 DNA polymerase and phi29 DNA polymerase mutant V470K-1474K under different treatment conditions, and FIG. 3B shows the comparison of remaining activity ratio of the pure wild-type phi29 DNA polymerase and phi29 DNA polymerase mutant V470K-1474K under different treatment conditions.

DETAILED DESCRIPTION

[0081] The experimental methods used in the examples as below are all conventional methods unless otherwise specified.

[0082] The materials, reagents and the like used in the examples are all commercially available unless otherwise specified.

[0083] The quantitative assays in the examples are all conducted in triplicate, and the results were averaged.

[0084] Lysis mixture used in examples (500 L per tube) is of a formulation comprising 440 L lysate (20 mM Tris-HCl. TB0194-500G; BBI), 500 mM NaCl (Analytial Reagent from Guangzhou Chemicalreagent Co., Lmt.), 0.5% Tween-20 (TB0560-500ML, BBI), 5% Glycerol (G5516-IL, SIGMA), 50 L 10 mg/ml lysozyme (1 mg/ml of final concentration, A610308-0005, BBI) and 10 L 0.1M Phenylmethanesulfonyl fluoride (PMSF, 10 mM of final concentration, 16A6063-25G, BBI). The lysis mixture is used right after it was ready.

[0085] Reaction Buffer 10phi29 used in examples (IL) is of a formulation comprising 60.57 g Tris (TB0194-5000 BBI), 6.17 g Dithiothreitol (DTT, A620058-0100, BBI), 13.2 g (NH.sub.4).sub.2SO.sub.4 (A610060-0500, BBI), 9.5 g MgCl.sub.2.6H.sub.2O (M0250-500, SIGMA) and 25 mM dNTP Solution Mix (1 ml, ENZYMATICS), pH 7.5.

[0086] 141 RCA Primer used in examples is TCTTAAGACCGCTTGGCCTCCGACT.

[0087] 141Ad ssDNA used in examples is made by BGI, which is a single-stranded loop library within a certain length range and has no fixed sequence.

Example 1 Obtaining Phi29 DNA Polymerase Mutants with a Single Mutation

[0088] 1.1 Construction of Expression Vectors

[0089] A recombinant vector WT was obtained by replacing the sequence between the BamHI and NdeI restriction sites of the pET28a vector (from Novagen) with the encoding gene of phi29 DNA polymerase shown in SEQ ID NO: 1, which can express a phi29 DNA polymerase having an amino acid sequence shown in SEQ ID NO: 2, i.e. the wide-type phi29 DNA polymerase.

[0090] A recombinant vector T140S was obtained by mutating C base at position 419 into G base relative to the encoding gene of phi29 DNA polymerase in the recombinant vector WT, which can express a phi29 DNA polymerase mutant having an amino acid sequence which includes a mutation at position 140 from threonine (Thr, T) to serine (Ser, S) and unchanged remaining amino acids compared to the amino acid sequence shown in SEQ ID NO: 2.

[0091] A recombinant vector T140N was obtained by mutating C base at position 419 into A base relative to the encoding gene of phi29 DNA polymerase in the recombinant vector WT, which can express a phi29 DNA polymerase mutant having an amino acid sequence which includes a mutation at position 140 from threonine (Thr, T) to asparagine (Asn, N) and unchanged remaining amino acids compared to the amino acid sequence shown in SEQ ID NO: 2.

[0092] A recombinant vector Q171E was obtained by mutating C base at position 511 into G base and G base at position 513 into A base relative to the encoding gene of phi29 DNA polymerase in the recombinant vector WT, which can express a phi29 DNA polymerase mutant having an amino acid sequence which includes a mutation at position 171 from glutamine (Gln, Q) to glutamic acid (Glu, E) and unchanged remaining amino acids compared to the amino acid sequence shown in SEQ ID NO: 2.

[0093] A recombinant vector G197N was obtained by mutating G base at position 589 into A base and G base at position 590 into A base relative to the encoding gene of phi29 DNA polymerase in the recombinant vector WT, which can express a phi29 DNA polymerase mutant having an amino acid sequence which includes a mutation at position 197 from glycine (Gly, G) to asparagine (Asn, N) and unchanged remaining amino acids compared to the amino acid sequence shown in SEQ ID NO: 2.

[0094] A recombinant vector S215D was obtained by mutating A base at position 643 into G base, G base at position 644 into A base, and C base at position 645 into T base relative to the encoding gene of phi29 DNA polymerase in the recombinant vector WT, which can express a phi29 DNA polymerase mutant having an amino acid sequence which includes a mutation at position 215 from serine (Ser, S) to aspartic acid (Asp, D) and unchanged remaining amino acids compared to the amino acid sequence shown in SEQ ID NO: 2.

[0095] A recombinant vector L216K was obtained by mutating C base at position 646 into A base, T base at position 647 into A base, and G base at position 648 into A base relative to the encoding gene of phi29 DNA polymerase in the recombinant vector WT, which can express a phi29 DNA polymerase mutant having an amino acid sequence which includes a mutation at position 216 from leucine (Leu, L) to lysine (Lys, K) and unchanged remaining amino acids compared to the amino acid sequence shown in SEQ ID NO: 2.

[0096] A recombinant vector G217D was obtained by mutating 0 base at position 650 into A base and C base at position 651 into T base relative to the encoding gene of phi29 DNA polymerase in the recombinant vector WT, which can express a phi29 DNA polymerase mutant having an amino acid sequence which includes a mutation at position 217 from glycine (Gly. G) to aspartic acid (Asp, D) and unchanged remaining amino acids compared to the amino acid sequence shown in SEQ ID NO: 2.

[0097] A recombinant vector G217E was obtained by mutating G base at position 650 into A base and C base at position 651 into A base relative to the encoding gene of phi29 DNA polymerase in the recombinant vector WT, which can express a phi29 DNA polymerase mutant having an amino acid sequence which includes a mutation at position 217 from glycine (Gly, G) to glutamic acid (Glu, E) and unchanged remaining amino acids compared to the amino acid sequence shown in SEQ ID NO: 2.

[0098] A recombinant vector Y224K was obtained by mutating T base at position 670 into A base and T base at position 672 into A base relative to the encoding gene of phi29 DNA polymerase in the recombinant vector WT, which can express a phi29 DNA polymerase mutant having an amino acid sequence which includes a mutation at position 224 from tyrosine (Tyr, Y) to lysine (Lys. K) and unchanged remaining amino acids compared to the amino acid sequence shown in SEQ ID NO: 2.

[0099] A recombinant vector E239W was obtained by mutating G base at position 715 into T base. A base at position 716 into 0 base and A base at position 717 into G base relative to the encoding gene of phi29 DNA polymerase in the recombinant vector WT, which can express a phi29 DNA polymerase mutant having an amino acid sequence which includes a mutation at position 239 from glutamic acid (Glu, E) to tryptophan (Trp, W) and unchanged remaining amino acids compared to the amino acid sequence shown in SEQ ID NO: 2.

[0100] A recombinant vector Y369R was obtained by mutating T base at position 1105 into C base and A base at position 1106 into G base relative to the encoding gene of phi29 DNA polymerase in the recombinant vector WT, which can express a phi29 DNA polymerase mutant having an amino acid sequence which includes a mutation at position 369 from tyrosine (Tyr, Y) to arginine (Arg, R) and unchanged remaining amino acids compared to the amino acid sequence shown in SEQ ID NO: 2.

[0101] A recombinant vector I323N was obtained by mutating T base at position 968 into A base relative to the encoding gene of phi29 DNA polymerase in the recombinant vector WT, which can express a phi29 DNA polymerase mutant having an amino acid sequence which includes a mutation at position 323 from isoleucine (Ile, I) to asparagine (Asn, N) and unchanged remaining amino acids compared to the amino acid sequence shown in SEQ ID NO: 2.

[0102] A recombinant vector V470K was obtained by mutating G base at position 1408 into A base, T base at position 1409 into A base and G base at position 1410 into A base relative to the encoding gene of phi29 DNA polymerase in the recombinant vector WT, which can express a phi29 DNA polymerase mutant having an amino acid sequence which includes a mutation at position 470 from valine (Val. V) to lysine (Lys, K) and unchanged remaining amino acids compared to the amino acid sequence shown in SEQ ID NO: 2.

[0103] A recombinant vector 1474K was obtained by mutating T base at position 1421 into A base and T base at position 1422 into A base relative to the encoding gene of phi29 DNA polymerase in the recombinant vector WT, which can express a phi29 DNA polymerase mutant having an amino acid sequence which includes a mutation at position 474 from isoleucine (Ile, I) to lysine (Lys, K) and unchanged remaining amino acids compared to the amino acid sequence shown in SEQ ID NO: 2.

[0104] A recombinant vector F526K was obtained by mutating T base at position 1576 into A base, T base at position 1577 into A base and C base at position 1578 into A base relative to the encoding gene of phi29 DNA polymerase in the recombinant vector WT, which can express a phi29 DNA polymerase mutant having an amino acid sequence which includes a mutation at position 526 from phenylalanine (Phe. F) to lysine (Lys, K) and unchanged remaining amino acids compared to the amino acid sequence shown in SEQ ID NO: 2.

[0105] 1.2 Construction and Induced Expression of Recombinant Bacteria

[0106] 1.2.1 Construction of Recombinant Bacteria

[0107] Recombinant bacteria were obtained by transforming the recombinant vector WT, the recombinant vector T140S, the recombinant vector T140N, the recombinant vector Q171E, the recombinant vector G197N, the recombinant vector S215D, the recombinant vector L216K, the recombinant vector G217D, the recombinant vector G217E, the recombinant vector Y224K, the recombinant vector E239W, the recombinant vector Y369R, the recombinant vector I323N, the recombinant vector V470K, the recombinant vector 1474K and the recombinant vector F526K constructed in 1.1 into Escherichia coli BL21 (DE3) (TIANGEN, CB 105-02) respectively.

[0108] 1.2.2 Induced Expression of Recombinant Bacteria

[0109] The recombinant bacteria obtained in 1.2.1 were individually induced to express corresponding phi29 DNA polymerase, and wild-type phi29 DNA polymerase (WT), phi29 DNA polymerase mutant T140S, phi29 DNA polymerase mutant T140N, phi29 DNA polymerase mutant Q171E, phi29 DNA polymerase mutation G197N, phi29 DNA polymerase mutant S215D, phi29 DNA polymerase mutant L216K, phi29 DNA polymerase mutant G217D, phi29 DNA polymerase mutant G217E, phi29 DNA polymerase mutant Y224K, phi29 DNA polymerase mutant E239W, phi29 DNA polymerase mutant Y369R, phi29 DNA polymerase mutant I323N, phi29 DNA polymerase mutant V470K, phi29 DNA polymerase mutant 1474K and phi29 DNA polymerase mutant F526K were respectively obtained. The induction was conducted according to the following specific steps.

[0110] Step 1. Activation of Culture

[0111] The glycerol stocks of Escherichia coli BL21 (DE3) transformed with wild-type phi29 DNA polymerase or phi29 DNA polymerase mutant were seeded into 3 ml of liquid LB medium containing kanamycin and cultured overnight.

[0112] Step 2. Transfer of Bacterial Solution

[0113] The bacterial solution obtained in step 1 was transferred into 2 L of liquid LB medium containing kanamycin at a volume ratio of 1:100 and cultured in a shaker at 37 C. for 3 hours.

[0114] Step 3. Induction of Expression

[0115] Isopropyl-beta-D-thiogalactopyranoside (IPTG) with a final concentration of 0.5 mM was added for inducing expression and the mixture was cultured in a shake at 30 C. for another 2 to 2.5 hours.

[0116] Step 4. Collection of Bacterial Pellet

[0117] The bacterial solution was centrifuged at 12,000 rpm for 1 minute and bacterial pellets were collected.

[0118] 1.3 Stability Assay of Phi29 DNA Polymerase

[0119] 1.3.1 Treatment of Bacterial Pellet

[0120] The bacterial pellets obtained in step 1.2.2 were mixed with the lysis mixture, lysed in a water bath at 30 C. for 10 minutes and centrifuged at 12000 rpm and 4 C. for 10 minutes, and the supernatant was collected as crude phi29 DNA polymerase which was placed on ice for use.

[0121] 1.3.2 Detection of Crude Enzyme for Stability Via RCA Reaction

[0122] 20 L of crude enzyme solution of wild-type phi29 DNA polymerase (WT) or individual phi29 DNA polymerase mutants obtained in step 1.3.1 was subjected to heat treatment at 37 C. for 10 minutes. 1 L of the crude enzyme solution before and after heat treatment was respectively added into 40.4 L of denatured RCA reaction system (refer to Table 1), and subjected to RCA reaction at 30 C. for 30 minutes. After that, 2.5 L of 0.5M EDTA as a stop solution (AM9261, INVITROGEN) was added to terminate the RCA reaction. Besides, a group of denatured RCA reaction system containing the heat-treated crude enzyme was added with the stop solution before the initiation of RCA reaction, which was used as a negative control group. All samples obtained were detected by using Qubit ssDNA Assay Kit (Q10212, INVITROGEN) for DNA Nano ball (DNB) concentration produced.

[0123] The remaining activity ratio of wild-type phi29 DNA polymerase or individual phi29 DNA polymerase mutants was calculated according to the following formula:

Remaining activity ratio=(N.sub.2 N.sub.0)/(N.sub.1 N.sub.0)*100%

[0124] in which, N.sub.0 indicates the DNB concentration of negative control, N.sub.1 indicates the DNB concentration produced in the presence of crude enzyme (without heat treatment) after the RCA reaction, and N.sub.2 indicates the DNB concentration produced in the presence of heat-treated crude enzyme after the RCA reaction.

TABLE-US-00001 TABLE 1 RCA reaction system Reagent Volume (1) Volume (10) 10phi29 Reaction 4.0 L 40 L Buffer 5 M 141 RCA Primer 0.2 L 2.0 L 25 mM dNTPs Mix 1.0 L 10 L 141Ad ssDNA 3 ng 30 ng H.sub.2O 35.2-sample) L (35.2-sample) 10 L

[0125] The detection results are shown in Table 2 and FIG. 1, which indicate that the wild-type phi29 DNA polymerase (WT) and the phi29 DNA polymerase mutants Y369R and I323N respectively have a remaining activity ratio of 43.26%, 52.03% and 67.13%, showing the remaining activity ratio of the phi29 DNA polymerase mutants Y369R and I323N has increased by 9% and 24% respectively compared to the wild-type phi29 DNA polymerase. The results demonstrate that the phi29 DNA polymerase mutants have increased stability compared to the wild-type phi29 DNA polymerase.

TABLE-US-00002 TABLE 2 Detection results of crude phi29 DNA polymerase mutants Y369R and I323N for stability DNB concentration after RCA Polymerase Heat treatment reaction (ng/L) Remaining type temperature 0 min 10 min activity ratio WT 37 C. 12.9 5.58 43.26% Y369R 37 C. 14.3 7.44 52.03% I323N 37 C. 5.75 3.86 67.13%

[0126] Other heat-treated crude phi29 DNA polymerase mutants also have an increased remaining activity ratio compared to the wild-type phi29 DNA polymerase, for example, the phi29 DNA polymerase mutants T140S, T140N, Q171E, G197N, S215D, L216K, G217E, G217D, Y224K, E239W, V470K, V474K and F526K respectively have a remaining activity ratio of 52%, 73%, 51%, 47.7%, 63%, 47.27%, 65.24%, 59%, 74.76%, 69.83%, 63.98%, 85.54% and 60.5%, etc.

[0127] 1.3.3 Detection of Pure Enzyme for Stability Via RCA Reaction

[0128] 1 L of bacterial pellets containing wild-type phi29 DNA polymerase (WT) or individual phi29 DNA polymerase mutants subjected to IPTG-induction and obtained in 1.2.2 were respectively purified through the affinity chromatography and ion exchange by using the AKTA pure system, thereby obtaining pure wild-type phi29 DNA polymerase (WT) and individual phi29 DNA polymerase mutants respectively. 20 L of obtained pure enzyme solution was individually subjected to heat treatment at 20 C., 34 C. and 37 C. respectively for 10 minutes. 1 L of the pure enzyme solution after heat treatment at different temperatures was respectively added into 80.8 L of denatured RCA reaction system (with twice volume of that listed in Table 1) and subjected to RCA reaction at 30 C. for 30 minutes. After that, 5 L of 0.5M EDTA as a stop solution was added to terminate the RCA reaction. All samples obtained were detected by using Qubit ssDNA Assay Kit (Q10212, INVITROGEN) for DNB concentration produced in the presence of wild-type phi29 DNA polymerase or individual phi29 DNA polymerase mutants, and the remaining activity ratio of enzyme was calculated.

[0129] The results are shown in FIG. 2A and FIG. 2B and Table 3. After treatment at 34 C. and 37 C. for 10 minutes, the wild-type phi29 DNA polymerase respectively has a remaining activity ratio of 25.3% and 0%, the phi29 DNA polymerase mutant V470K respectively has a remaining activity ratio of 39.27% and 5.6%, and the phi29 DNA polymerase mutant 1474K respectively has a remaining activity ratio of 23.16% and 4.68%. It can be seen that the phi29 DNA polymerase mutants V470K and I474K each have increased thermal stability compared to the wild-type of phi29 DNA polymerase. In addition, the commercial enzyme Enzymatics after treatment at 37 C. for 10 minutes and 5 minutes has a remaining activity ratio of 5.02% and 17.06% respectively, which may slightly higher than some mutants with single-point mutation, but has no significant advantages.

TABLE-US-00003 TABLE 3 Detection results of pure phi29 DNA polymerase mutants V470K and I474K and commercial enzyme Enzymatics for stability Heat Heat Polymerase treatment treatment Enzyme activity Remaining type temperature time (U/L) activity ratio WT 20 C. N/A 9.29 N/A 34 C. 10 min 2.35 25.30% 37 C. 10 min 0.09 0.00% V470K 20 C. N/A 27.68 N/A 34 C. 10 min 10.87 39.27% 37 C. 10 min 1.55 5.60% I474K 20 C. N/A 15.37 N/A 34 C. 10 min 3.56 23.16% 37 C. 10 min 0.72 4.68% Enzymatics 20 C. N/A 88.85 N/A 37 C. 5 min 15.16 17.06% 10 min 4.46 5.02%

[0130] Other pure phi29 DNA polymerase mutants after treatment at 37 C. for 10 minutes also has an increased remaining activity ratio compared to the wild-type phi29 DNA polymerase, for example, the phi29 DNA polymerase mutants T140S, T140N, Q171E, G197N, S215D, L216K, G217E, E239W, G217D, Y224K, F526K, I323N and Y369R respectively have a remaining activity ratio of 3.81%, 1.51%, 6.06%, 3.18%, 1.40%, 5.04%, 14.76%, 15.27%, 5.21%, 12.84%, 10%, 1.25% and 1.1%, etc.

Example 2 Obtaining Phi29 DNA Polymerase Mutant with Multiple Mutations

[0131] 2.1 Construction of Recombinant Vector

[0132] A recombinant vector V470K-1474K was obtained by mutating G base at position 1408 into A base, T base at position 1409 into A base, G base at position 1410 into A base, T base at position 1421 into A base and T base at position 1422 into A base relative to the encoding gene of phi29 DNA polymerase in the recombinant vector WT, which can express phi29 DNA polymerase mutant V470K-1474K having an amino acid sequence which includes a mutation at position 470 from valine (Val, V) to lysine (Lys, K), a mutation at position 474 from isoleucine (Ile, I) to lysine (Lys, K) and unchanged remaining amino acids compared to the amino acid sequence shown in SEQ ID NO: 2.

[0133] 2.2 Construction and Induced Expression of Recombinant Bacteria

[0134] Recombinant bacteria were obtained by transforming the recombinant vector V470K-I474K constructed in 2.1 into Escherichia coli BL21 (DE3), which was then induced to express phi29 DNA polymerase mutant V470K-1474K according to the method in 1.2.2, and bacterial pellts were obtained.

[0135] 2.3 Detection of Pure Enzyme for Stability

[0136] The bacterial pellets subjected to induction and obtained in 2.2 were purified according to the method in 1.3, thus obtaining pure phi29 DNA polymerase mutant V470K-1474K. 20 L of obtained pure phi29 DNA polymerase mutant V470K-1474K was subjected to heat treatment at 37 C. for 5 minutes and 10 minutes respectively. 1 L of the pure enzyme solution after heat treatment at different treatment times was respectively added into 40.4 L of denatured RCA reaction system (refer to Table 1) and subjected to RCA reaction at 30 C. for 30 minutes. After that, 5 L of 0.5M EDTA as a stop solution was added to terminate the RCA reaction. All samples obtained were detected by using Qubit ssDNA Assay Kit (Q10212, INVITROGEN) for DNB concentration produced in the presence of the phi29 DNA polymerase mutant V470K-1474K, and the remaining activity ratio of enzyme was calculated.

[0137] The results are shown in Table 4 and FIG. 3A and FIG. 3B. The phi29 DNA polymerase mutant V470K-1474K (i.e. Mut_1) has a remaining activity ratio of 62.02% and 17.48% respectively, while the wild-type phi29 DNA polymerase has a remaining activity ratio of 37.56% and 4.55% respectively. The results demonstrate that the stability of phi29 DNA polymerase mutant V470K-1474K is significantly higher than that of the wild-type of phi29 DNA polymerase, which is also higher than that of phi29 DNA polymerase mutants V470K and 1474K with a single-point mutation.

TABLE-US-00004 TABLE 4 Detection results of pure phi29 DNA polymerase mutant V470K-I474K for stability Heat Heat Polymerase treatment treatment Enzyme Remaining type temperature time activity (U/L) activity ratio WT 37 C. 0 min 6.15 N/A 5 min 2.31 37.56% 10 min 0.28 4.55% V470K-I474K 37 C. 0 min 40.05 N/A 5 min 24.84 62.02% 10 min 7.00 17.48%

INDUSTRIAL APPLICATION

[0138] The present disclosure has improved the stability of wild-type phi29 DNA polymerase through amino acid mutating, and a group of phi29 DNA polymerase mutants with increased stability have been obtained after screening. The present disclosure realizes to prepare and store the obtained phi29 DNA polymerase mutants under a reduced condition by mutating the amino acid sequence of wild-type phi29 DNA polymerase, which has reduced the production cost and the phi29 DNA polymerase mutants produced have prolonged validity period and improved efficiency.