Phenylalanine ammonialyase mutant with high activity and stability and application of phenylalanine ammonialyase mutant

Abstract

The invention discloses a phenylalanine ammonialyase mutant with high activity and stability and application thereof, and relates to the technical field of biological medicine, the mutant has one of the following mutations on the basis of an amino acid sequence of phenylalanine ammonialyase as shown in SEQ ID NO.1: (1) one of H307N, A70G, I77V, V90L and Y304F mutations; (2) the gene has H307N and V90L mutations; (3) the gene has H307N and I77V mutations; (4) the gene has H307N and Y304F mutations; and (5) mutation of H307N, V90L and I77V is carried out. The invention also provides application of the mutant in preparation of drugs for treating phenylketonuria. According to the present invention, the AvPAL is subjected to semi-rational modification to obtain the mutant with significantly improved enzyme activity and stability, and the mutant has great application value in the preparation of phenylketonuria drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a highly active and stable phenylalanine ammonia-lyase mutant 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 highly active and stable phenylalanine ammonia lyase mutant and its application. The present invention obtains a mutant with significantly improved enzyme activity and stability by semi-rational transformation of AvPAL, which has great application value in the preparation of phenylketonuria drugs.

[0004] The technical solution of the present invention to solve the above technical problem is as follows: provide a highly active and stable phenylalanine ammonia lyase mutant, which has one of the following mutations based on the amino acid sequence of phenylalanine ammonia lyase as shown in SEQ ID NO.1:

[0005] (1) having one of the mutations H307N, A70G, I77V, V90L, and Y304F;

[0006] (2) with H307N and V90L mutations;

[0007] (3) with H307N and I77V mutations;

[0008] (4) with H307N and Y304F mutations;

[0009] (5) Has H307N, V90L and I77V mutations.

[0010] The present invention also provides a gene encoding the above-mentioned phenylalanine ammonia-lyase mutant with high activity and stability.

[0011] The present invention also provides a recombinant expression vector comprising the gene.

[0012] The present invention also provides a host cell comprising the above recombinant expression vector.

[0013] The present invention also provides the use of the highly active and stable phenylalanine ammonia-lyase mutant, the gene, the recombinant expression vector and the host cell in the preparation of a drug for treating phenylketonuria.

[0014] The present invention also provides a PEG-modified phenylalanine ammonia lyase, which is prepared by modifying the above-mentioned highly active and stable phenylalanine ammonia lyase mutant with a PEG reagent.

[0015] Furthermore, the PEG reagent is one of a mPEG-MAL reagent with a molecular weight of 10k, a mPEG-MAL reagent with a molecular weight of 20k, a MPEG-NHS reagent with a molecular weight of 20k, and a MPEG-NHS reagent with a molecular weight of 40k.

[0016] The present invention has the following beneficial effects:

[0017] 1. The present invention obtains the flexible region of AvPAL by comparing the RMSF of the AvPAL molecular dynamics simulation trajectory at 300K and 400K, and constructs a single-point saturation mutation library based on the above-mentioned sites, and further obtains mutants with improved activity through screening and iterative accumulation. Among them, the optimal mutant H307N / V90L has a k cat The value was increased by 134% compared with wild-type AvPAL, and k cat / K M The value is 2.16 times that of AvPAL.

[0018] 2. The present invention further improves the enzyme activity and plasma stability by PEG modification of the mutant H307N / V90L obtained by screening. Among them, after the mutant H307N / V90L was modified with MPEG-NHS-20K, the activity was increased by 65%, and k cat / K M It increased by 2.67 times, and after 216 hours of plasma incubation, the residual activity was 1.81 times that of the wild type. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the result of AvPAL molecular dynamics simulation;

[0020] Figure 2 This is a diagram showing the results of screening AvPAL mutants with improved activity;

[0021] Figure 3 It is a diagram showing the results of enzyme activity assay of AvPAL mutants;

[0022] Figure 4 It is the result diagram of the determination of kinetic parameters of AvPAL and its mutants;

[0023] Figure 5This is the result of non-denaturing gel analysis of H307N / V90L mutant after PEG modification;

[0024] Figure 6 The results of enzyme activity assay of H307N / V90L mutant after PEG modification are shown;

[0025] Figure 7 This is a graph showing the results of kinetic parameter determination of the H307N / V90L mutant after PEG modification;

[0026] Figure 8 This is a graph showing the plasma stability test results of the H307N / V90L mutant after PEG modification. DETAILED DESCRIPTION

[0027] 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.

[0028] Example 1

[0029] The N-terminal sequence of AvPAL can affect the activity of AvPAL through changes in the secondary structure. Given that the flexible region of the protein may undergo conformational changes or violent movements, it is inferred that the modification of the flexible region in the AvPAL structure may improve the activity. First, molecular dynamics simulations were performed on AvPAL at 300K and 400K for 200ns. Because high temperatures will intensify the movement of proteins, the flexible region can be identified by comparing the movement trajectories at 300K and 400K. The results are as follows: Figure 1 As shown, A is the root mean square deviation (RMSD) analysis result, and B is the root mean square fluctuation (RMSF) analysis result.

[0030] Depend on Figure 1It can be seen that AvPAL tends to be stable at 20ns trajectory at both 300K and 400K, and the average RMSD value of AvPAL at 400K is much higher than the average RMSD value at 300K, indicating that the total motion amplitude of AvPAL increases when MD simulation is performed at elevated temperature. In order to facilitate subsequent analysis, the trajectory after 50-200ns was selected for analysis. Because the flexible structure region shows a higher value of root mean square fluctuation (RMSF) than the stable region, the flexible region can be identified by calculating the RMSF of each amino acid residue of AvPAL at 300K and 400K. The results show that compared with the RMSF value at 300K, the RMSF of AvPAL in the 5-24, 70-90 and 303-315 regions at 400K is significantly increased, and its RMSF is much higher than the value at 300K, so the amino acid sites in this region are selected for further study.

[0031] Example 2

[0032] AvPAL (NCBI: WP_011320679.1) from Anabaena variabilis was synthesized by BGI after codon optimization. Nde I and Xho I restriction sites were introduced at both ends of the gene, and the pET-21a(+)-AvPAL plasmid was constructed. Point saturation mutation primers were designed at each site of the 5-24, 70-90 and 303-315 regions of AvPAL, and the recombinant plasmid with introduced mutations was obtained by full plasmid PCR amplification, and then transformed into competent cells E.coli TOP10. The plasmid of the positive transformant was extracted and then transformed into the expression strain E.coli BL21 (DE3) to obtain a recombinant expression strain that can be used for AvPAL expression.

[0033] M9 medium was prepared for the screening of active amine enzymes in Escherichia coli cells. In M9 medium lacking nitrogen source, AvPAL can catalyze phenylalanine to produce NH 4+ and trans-cinnamic acid, E. coli utilizes NH 4 + Provide nitrogen source for growth, so the catalytic activity of AvPAL and its mutants can be related to the OD of bacterial growth. 600 The values ​​of were related, and mutants with improved activity could be obtained by screening with 96-well plates (e.g. Figure 2 As shown, the mutant of amino acid position 11 is taken as an example). 600 The value of the strain was significantly higher than that of the wild type, and the corresponding single colony was picked to contain ampicillin resistance (final concentration of 0.1 mg mL -1) overnight culture in LB medium, and then sequenced (Beijing Liuhe BGI Gene Technology Co., Ltd.), and the bacterial solution sent for sequencing was saved. After sequencing, mutants Q6S, A7S, K10N, T11N, F16S, T19C, T19S, S82T, A70G, I77V, V90L, Y304F, D306A, D306S and H307N were preliminarily determined to be potential beneficial mutations.

[0034] Example 3

[0035] The frozen bacterial solution of the mutant in the above Example 2 was taken out, thawed at room temperature, and then an inoculum containing ampicillin (0.1 mg mL -1 ) in LB medium at 37°C and 220 r·min -1 The bacteria were shaken overnight, and then expanded to obtain seed solution, which was then inoculated with 2% of the inoculum to contain ampicillin resistance (0.1 mg mL -1 ) in a culture medium at 37°C, 200 r·min -1 Cultivate until the OD of the bacteria 600 When the value reaches 0.6-0.8, add 0.2 mM IPTG and induce at low temperature for 15 h. Collect the bacteria by centrifugation and store them for later use.

[0036] After the frozen cells were thawed, they were resuspended in Lysis Buffer and a PMSF solution with a final concentration of 1 mM was added. When the cells were lysed using a high-pressure homogenizer, the broken cells were centrifuged, and the precipitates such as organelles and cell debris were discarded. The collected supernatant was further filtered through a 0.45 μm filter membrane and then filtered through Co 2+ The product was purified by affinity chromatography, and impurity proteins below 30 kDa were filtered out by ultrafiltration concentration tubes. Finally, ion exchange chromatography and molecular sieve chromatography were used for further purification to obtain the purified AvPAL mutant.

[0037] (1) Determination of enzyme activity

[0038] 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, and the reaction was continued at 12000 r·min -1The mixture was centrifuged at a speed of 1000 nm for 5 min, and the absorbance at 290 nm was measured on a UV spectrophotometer (blank adjustment was made to 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. The results are shown in Table 1. Figure 3 shown.

[0039] The results showed that through in vitro enzyme activity assay, it was finally determined that the activities of mutants Q6S, A7S, K10N, T11N, F16S, A70G, I77V, V90L, Y304F, D306A and H307N were increased by 21%, 12%, 17%, 21%, 18%, 56%, 75%, 69%, 63%, 25% and 104%, respectively, while the activities of mutants T19C, S82T and D306S were not improved in the in vitro activity test experiment.

[0040] The optimal single mutant H307N was selected, and the mutation sites I77V, V90L and Y304F with greatly improved activity were superimposed on it. Activity assays showed that the activities of the double mutants H307N / I77V, H307N / V90L and H307N / Y304F were increased by 99%, 123% and 81% respectively; further accumulation of mutation sites was performed on the mutant H307N / V90L with the best double mutation activity, and activity assays showed that the activities of the triple mutants H307N / V90L / I77V and H307N / V90L / Y304F were increased by 97% and 60% respectively, which were lower than the activity of the double mutant H307N / V90L, so the optimal mutant H307N / V90L with an activity increase of 123% was finally obtained.

[0041] (2) Study on Enzymatic Properties

[0042] 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 k was calculated by measuring the protein concentration. cat .

[0043] The purified protein was diluted to 1 mg mL with a mixed solution containing 20 mM Tris, 150 mM NaCl, pH 8.0. -129 μL of the diluted protein solution was taken, and 1 μL of protein fluorescent dye was added to it. After mixing, the mixture was heated from 25°C to 90°C at a heating rate of 1.625°C / min in a CFX 96 real-time polymerase chain reaction (PCR) system (BioRad, Hercules, CA, USA). T was calculated by the change in fluorescence intensity. m value.

[0044] The results of the kinetic parameter determination of AvPAL and its mutants are shown in Figure 4 The results showed that the k cat The k values ​​of H307N / V90L / I77V and H307N / V90L / Y304F were 105%, 134% and 100% higher than those of the wild type, respectively. cat The values ​​were 20% and 13% higher than those of the wild type, respectively. cat The K values ​​of the double mutation H307N / V90L were the highest in terms of the number of substrate molecules converted per second. The K values ​​of the triple mutation H307N / V90L / I77V and H307N / V90L / Y304F were the highest. M The values ​​of A7S, T19C, A70G, I77V, V90L, Y304F and H307N were 55% and 51% lower than those of the wild type, respectively, indicating that the affinity between the enzyme and the substrate was significantly enhanced. cat / K M The values ​​were 1.20, 1.27, 1.40, 1.37, 1.38, 1.54 and 1.85 times that of the wild type; the k values ​​of the double mutants H307N / I77V, H307N / V90L and H307N / Y304F cat / K M The values ​​were 2.02, 2.16 and 1.70 times that of the wild type; the k values ​​of the triple mutations H307N / V90L / I77V and H307N / V90L / Y304F cat / K M The values ​​were 2.66 and 2.28 times that of the wild type, but the values ​​of mutants H307N / V90L / I77V and H307N / V90L / Y304F were 49% and 52% lower than those of H307N / V90L, respectively. Based on the enzyme activity and enzyme kinetic parameters of the mutants, the double mutant H307N / V90L was finally determined to be the optimal mutant.

[0045] Example 4

[0046] PEG modification is widely used in the pharmaceutical field, especially in the research and development of protein drugs. It can improve the pharmacodynamics and pharmacokinetic properties of protein drugs. In this experiment, the H307N / V90L mutant was modified with PEG to test its activity, enzyme kinetics and plasma stability.

[0047] Prepare 20mM potassium phosphate solution with a pH of 8.0, and 1mM MPEG-NHS (or mPEG-MAL reagent) with molecular weights of 5k, 10k, 20k, and 40k, a total of two PEG reagents with different molecular weights. In the total reaction system, the PEG reagent and the protein are modified at a concentration ratio of 5:1. The total reaction system is 100μL. Add 10μL of protein at a concentration of about 18mg / ml to the reaction solution with a final concentration of 0.15mM PEG reagent and 75μL of 20mM potassium phosphate solution, and react overnight for 12h. The most active mutant H307N / V90L was PEG-modified with mPEG-MAL reagent (molecular weights of 5k, 10k, and 20k, respectively) and MPEG-NHS reagent (molecular weights of 2k, 5k, 10k, 20k, and 40k, respectively) and then subjected to non-denaturing gel analysis. The results are as follows Figure 5 As shown, the molecular weight of AvPAL is about 280 kDa, and it is a tetramer in solution. As the molecular weight of mPEG-MAL and MPEG-NHS increases, the molecular weight of the modified AvPAL also increases. The molecular weights of H307N / V90L-MAL-10k, H307N / V90L-MAL-20k, H307N / V90L-NHS-20k and H307N / V90L-NHS-40k exceed 880 kDa. The results show that the mutant H307N / V90L was successfully coupled with these 8 PEG materials under pH 8.0.

[0048] (1) The enzyme activity of the PEG-modified H307N / V90L mutant was tested with reference to Example 3. The results are as follows: Figure 6 shown.

[0049] Depend on Figure 6 It can be seen that the activity of the H307N / V90L mutant modified with mPEG-MAL reagents with molecular weights of 5k, 10k and 20k respectively increased by 42%, 61% and 65% compared with the wild-type AvPAL; the activity of the H307N / V90L mutant modified with MPEG-NHS reagents with molecular weights of 2k, 5k, 10k, 20k and 40k respectively increased by 58%, 41%, 57%, 65% and 71% compared with the wild-type AvPAL.

[0050] (2) The enzymatic properties of the PEG-modified H307N / V90L mutant were studied with reference to Example 3. The results are as follows: Figure 7 shown.

[0051] k of H307N / V90L-MAL-10k, H307N / V90L-MAL-20k, H307N / V90L-NHS-20k and H307N / V90L-NHS-40k cat The values ​​were 46%, 47%, 41% and 37% higher than those of wild-type AvPAL. M The values ​​of β-catenin and β-catenin decreased by 39%, 33%, 47% and 39% respectively compared with the wild type, indicating that the affinity of the enzyme to the substrate phenylalanine has been enhanced. cat / K M , the k of samples H307N / V90L-MAL-10k, H307N / V90L-MAL-20k, H307N / V90L-NHS-20k and H307N / V90L-NHS-40k cat / K M The values ​​are 2.40, 2.18, 2.67 and 2.24 times that of the wild type. According to the experimental results, the activity of sample H307N / V90L-NHS-20k after PEG modification is the best.

[0052] (3) Using the wild-type AvPAL without PEG modification as the blank control group, the above-mentioned superior PEG-modified mutants H307N / V90L-MAL-10k, H307N / V90L-MAL-20k, H307N / V90L-NHS-20k and H307N / V90L-NHS-40k were filtered through a 0.22 μm filter membrane, and 5 μL of each was added to an EP tube containing 45 μL of plasma in a 37°C metal bath. At intervals of 24 h, 400 μL of reaction solution (10 mM Phe, 50 mM Tris-HCl, pH 7.5) was added to the EP tube. The blank was adjusted to a mixture of plasma without enzyme, reaction solution and stop solution, and the remaining OD was measured. 290 , monitor plasma stability every 24 hours, and the results are as follows Figure 8 shown.

[0053] The results showed that after 216 hours of incubation, the plasma stability of PEG-modified H307N / V90L was improved to varying degrees compared with wild-type AvPAL, among which H307N / V90L-MAL-20k and H307N / V90L-NHS-20k had the highest residual activity, which were 1.86 times and 1.81 times that of AvPAL, respectively, laying the foundation for subsequent drug development.

[0054] In summary, AvPAL and its mutant H307N / V90L were modified with PEG. Through activity determination, enzyme kinetics evaluation and plasma stability determination, it was found that the activity of mutant H307N / V90L was increased by 65% ​​after modification with MPEG-NHS-20K, and the k cat / K M It increased by 2.67 times, and after 216 hours of plasma incubation, the residual activity was 1.81 times that of the wild type.

[0055] 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 highly active and stable phenylalanine ammonia lyase mutant, characterized in that: Based on the amino acid sequence of phenylalanine ammonia lyase as shown in SEQ ID NO.1, it has one of the following mutations: (1) having one of the mutations H307N, A70G, I77V, V90L, and Y304F; (2) with H307N and V90L mutations; (3) with H307N and I77V mutations; (4) with H307N and Y304F mutations; (5) Has H307N, V90L and I77V mutations.

2. A gene encoding the highly active and stable phenylalanine ammonia-lyase mutant 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 highly active and stable phenylalanine ammonia-lyase mutant 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.

6. A PEG-modified phenylalanine ammonia lyase, characterized in that: The phenylalanine ammonia lyase mutant with high activity and stability as claimed in claim 1 is modified by a PEG reagent to obtain the product.

7. The PEG-modified phenylalanine ammonia lyase according to claim 6, characterized in that The PEG reagent is one of a mPEG-MAL reagent with a molecular weight of 10k, a mPEG-MAL reagent with a molecular weight of 20k, a MPEG-NHS reagent with a molecular weight of 20k, and a MPEG-NHS reagent with a molecular weight of 40k.

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