Dual mutant of phenylalanine ammonialyase and application thereof

Abstract

The invention discloses a dual mutant of phenylalanine ammonialyase and application thereof, and relates to the technical field of biological medicine, the mutant has M222N and I149D mutations or M222N and N36S mutations on the basis of an amino acid sequence of the phenylalanine ammonialyase as shown in SEQ ID NO.1. The invention further discloses a preparation method of the dual mutant of the phenylalanine ammonialyase. The invention also discloses an application of the mutant in preparation of drugs for treating phenylketonuria. The enzyme activity of the phenylalanine ammonialyase is improved through a directional modification method, and the clinical application value of the phenylalanine ammonialyase is greatly increased.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a double mutant of phenylalanine ammonia lyase and application thereof. Background Art

[0002] Phenylketonuria (PKU) is a genetic disease caused by defective phenylalanine metabolism, which can cause intellectual development disorders in patients. Phenylalanine ammonia lyase (AvPAL) can degrade phenylalanine into trans-cinnamic acid and ammonia that can be excreted in urine. It has been used as a treatment for PKU, but it still has many problems such as poor activity, short half-life and poor compliance. More effective treatment drugs are urgently needed. Summary of the invention

[0003] In order to solve the above technical problems, the purpose of the present invention is to provide a double mutant of phenylalanine ammonia lyase. The present invention improves the enzymatic activity of phenylalanine ammonia lyase through a directed transformation method, thereby greatly increasing its clinical application value.

[0004] The technical solution of the present invention to solve the above technical problem is as follows: a double mutant of phenylalanine ammonia lyase is provided, which has M222N and I149D mutations, or M222N and N36S mutations based on the amino acid sequence of phenylalanine ammonia lyase as shown in SEQ ID NO.1.

[0005] The present invention also provides a gene encoding the double mutant of the phenylalanine ammonia lyase.

[0006] The present invention also provides a recombinant expression vector comprising the gene.

[0007] The present invention also provides a host cell of the above recombinant expression vector.

[0008] The present invention also provides the use of the double mutant, gene, recombinant expression vector and host cell of the phenylalanine ammonia lyase in preparing a drug for treating phenylketonuria.

[0009] The present invention has the following beneficial effects:

[0010] 1. The present invention, through directed transformation, designs and synthesizes the double mutants M222N / I149D and M222N / N36S of phenylalanine ammonia lyase, whose enzyme activity and catalytic efficiency are significantly improved compared with WT, and higher than the single point mutant M222N. Among them, the activity of mutant M222N / I149D on phenylalanine is increased to 2.36 times, and the catalytic activity of M222N / N36S is increased to 3.39 times, which has great application value in the treatment of phenylketonuria. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is the gel electrophoresis diagram of AvPAL;

[0012] Figure 2 The figure shows the results of enzyme activity assay of AvPAL and its mutants. DETAILED DESCRIPTION

[0013] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0014] Example 1

[0015] (1) Amplification of AvPAL and its mutants

[0016] According to the amino acid sequence of phenylalanine ammonia lyase (AvPAL) as shown in SEQ ID NO. 1, and according to the codon preference, the coding gene of the synthetic protein AvPAL was designed. According to the AvPAL sequence, PCR primers for introducing mutations were designed, and the AvPAL mutation primers are shown in Table 1. PCR amplification was performed respectively to obtain genes with amino acid mutations introduced.

[0017] Table 1 AvPAL mutation primers

[0018]

[0019]

[0020] The PCR amplification system was: ddH2O 21μL, template DNA 1μL, upstream and downstream primers 1.5μL each and KOD ONETM PCR Master mix 25μL. The PCR reaction conditions were: 98℃, 2min; 98℃ 10s, 60℃ 5s (30 cycles); 68℃ 50s, 68℃ 5min; stored at 4℃.

[0021] (2) Construction of expression plasmids for AvPAL and its mutants

[0022] After PCR amplification, 0.7% agarose gel electrophoresis was performed at a voltage of 120 volts. After cutting the gel, a gel recovery kit was used to recover the target DNA fragment of about 1800 bp, and then double-digested with Nde I and Xho I. The obtained DNA fragment was connected to the vector pET-21a(+), and the connection product was transformed into Escherichia coli DH5α competent cells and screened on LB plates containing ampicillin resistance.

[0023] (3) Obtaining monoclonal cell lines of AvPAL and its mutants

[0024] The recombinant plasmids of AvPAL and its mutants were extracted using a plasmid extraction kit, and then transformed into the expression host E. coli BL21 (DE3) according to conventional methods, so as to obtain the engineered E. coli bacteria expressing the genes of AvPAL and its mutants.

[0025] (4) Expression of AvPAL and its mutants

[0026] 200 μL of the above-mentioned E. coli engineering bacteria liquid was inoculated into LB liquid medium containing ampicillin resistance from the bacteria-preserving cryopreservation tube, and cultured overnight at 37°C and 220 rpm to obtain seed liquid. Then 20 mL of the seed liquid was inoculated into 1 L of LB liquid medium containing ampicillin resistance (inoculation amount 2%), and cultured at 37°C and 220 rpm under shaking conditions. Cultured until OD 600 At about 0.6 (reached in about 3 hours), add IPTG with a final concentration of 0.2mM, induce expression at 16℃ and 160rpm for 16 hours, then centrifuge the bacterial solution at 4℃ and 6000rpm for 5 minutes, discard the culture supernatant, resuspend the bacteria in Lysis buffer, centrifuge again, discard the supernatant, and store the bacteria at -20℃.

[0027] (5) Purification of AvPAL and its mutants

[0028] Take out the cells from -20℃ and thaw them on ice. Add Lysis buffer and PMSF with a final concentration of 1mM, resuspend the cells and mix well. Use a high-pressure homogenizer precooled with a 4℃ cold circulation pump to lyse the cells. Set the pressure to 1000bar and repeat the lysis 3 times. Centrifuge the cell lysate at 11000rpm for 50min at 4℃, discard the precipitate, and filter the lysate supernatant through a 0.45μm filter membrane for later use. Use Co 2+ Resin (TALON Metal Affinity Resin) was used to purify the target protein containing the His tag, and an appropriate amount of Co 2+ The resin was loaded into the chromatography column, and the filler was first washed with 5 column volumes of pure water, and then the equilibrated chromatography column was washed with 5 column volumes of Lysis buffer. 2+ The resin filler is added to the filtered lysate supernatant and placed in a 4°C chromatography cabinet on a roller mixer for incubation and binding for 2 hours. The bound filler is reloaded into the chromatography column, and after the flow-through is exhausted, the impurities are washed with 30 mL of Lysis Buffer containing 10 mM imidazole, and then the target protein is eluted and collected with 20 mL of Elution Buffer containing 250 mM imidazole. The target protein eluate is centrifuged at 3000g at 4°C using a 30kDa ultrafiltration tube, PBS buffer is added to replace the Elution buffer, and finally centrifuged and concentrated to 500 μL to preserve the protein sample.

[0029] The eluate was tested by SDS-PAGE. Figure 1 As shown, there is a specific band of the target protein at 60 kDa, indicating that AvPAL (wild type, WT) and its mutants are successfully expressed in a soluble form.

[0030] Test Example 1 Activity determination of AvPAL and its mutants

[0031] The enzyme catalytic reaction system was 450 μL, and the concentration was 1 mg mL -1 , 5 μL of protein (AvPAL or its mutant, protein amount 5 μg) was added to a mixture of 50 mM Tris-HCl (pH 7.5) containing phenylalanine (10 mM), reacted at 40 °C for 5 min, and 50 μL of trichloroacetic acid (TCA) was added to terminate the reaction. -1 The mixture was centrifuged at a speed of 1000 nm for 5 min, and the absorbance at 290 nm was measured by a UV spectrophotometer (blank adjustment was a mixture of the reaction solution without enzyme and the stop solution). The amount of cinnamic acid generated was determined by the standard curve, and the enzyme activity of AvPAL and its mutants was calculated. The amount of PAL required to catalyze the generation of 1 μmol of trans-cinnamic acid per minute was defined as 1 U. Figure 2 shown.

[0032] Depend on Figure 2It can be seen that compared with the wild-type AvPAL (WT), the activities of mutants F18G, F18I, F18K, F18L, F18P, F18T and F18V increased by approximately 24%, 13%, 16%, 9%, 9%, 30% and 4%, respectively, and the activities of mutants W106C, C424S and D533E located on the polymer binding surface increased by approximately 8%, 29% and 30%, respectively. In addition, three mutants with increased activity were also presented at the W279 and C503 sites: W279E, W279H and W279I, as well as C503F, C503L and C503R. Compared with WT, the activities of these mutants increased by 35%, 4% and 25% (for W279E, W279H and W279I), and 25%, 20% and 12% (for C503F, C503L and C503R), respectively. At the M222 site, two mutants with improved activity were identified, among which the activity of M222N was 83% higher than that of WT, making it the single-point mutant with the highest activity enhancement. Mutants C64L, P200N, D253V, I285L and C424S also showed a trend of increased activity. M222N / D533E and M222N / F18T are the only two of the six double mutants whose activity is lower than that of the wild type. Compared with WT, the activities of M222N / L375I and M222N / W279E increased by 65% ​​and 19%, respectively. Fortunately, the two double mutants, M222N / N36S and M222N / I149D, had higher activities than all the single-point mutants, 2.0 and 2.4 times that of WT, respectively, showing a positive epistatic effect.

[0033] Test Example 2 Enzyme kinetics assay

[0034] In order to estimate the enzyme kinetic parameter K M and k cat 20-40000 μM phenylalanine dissolved in 50 mM Tris-HCl (pH 7.5) was used as a substrate to react with 5 μg of enzyme solution, respectively, to determine the enzyme activity, and the Michaelis-Menten nonlinear fitting method in GraphPad Prism 8.0 was used to calculate K m and V max The corresponding kcat was calculated by measuring the protein concentration. The results are shown in Table 2.

[0035] Table 2 Enzyme kinetic parameters

[0036]

[0037] As shown in Table 2, among the single-point mutants, the k catThe value increased the most, which was 1.94 times that of WT. The k values ​​of M222N / N36S, M222N / W279E, M222N / L375I, M222N / I149D / F18T, M222N / I149D / N36S, M222N / I149D / W279E, M222N / I149D / L375I and M222N / I149D / D533E cat The values ​​were approximately 1.84, 1.18, 1.6, 1.9, 2.1, 1.46, 1.43, and 1.95 times that of WT, respectively. cat The value of F18I, F18V, P200N, D253V, W279E, C424S, C503F, M222N / L375I, M222N / I149D / W279E and M222N / I149D / D533E also increased the most, which was about 2.45 times that of WT. M The catalytic efficiency (k cat / K M ) are approximately 1.73, 1.78, 1.66, 1.58, 1.55, 1.36 and 1.73 times that of WT, respectively. In addition, the k cat / K M The value is approximately 3.39 times that of WT.

[0038] In summary, the enzyme activity and catalytic efficiency of the double mutants M222N / I149D and M222N / N36S of phenylalanine ammonia lyase were significantly improved compared with WT, and higher than the single point mutant M222N. Among them, the activity of mutant M222N / I149D on phenylalanine increased to 2.36 times, and the catalytic activity of M222N / N36S increased to 3.39 times, which has great application value in the treatment of phenylketonuria.

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A double mutant of phenylalanine ammonia lyase, characterized in that Based on the amino acid sequence of phenylalanine ammonia lyase as shown in SEQ ID NO.1, it has M222N and I149D mutations, or has M222N and N36S mutations.

2. A gene encoding the double mutant of phenylalanine ammonia lyase according to claim 1.

3. A recombinant expression vector comprising the gene according to claim 2. A host cell comprising the recombinant expression vector according to claim 3.

5. Use of the double mutant of phenylalanine ammonia lyase according to claim 1, the gene according to claim 2, the recombinant expression vector according to claim 3 and the host cell according to claim 4 in the preparation of a drug for treating phenylketonuria.

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