Non-specific peroxidase mutant with improved enzymatic activity and application of non-specific peroxidase mutant

Abstract

The invention relates to the technical field of gene engineering, and discloses a non-specific peroxidase mutant with improved enzymatic activity and application thereof. The amino acid sequence of the non-specific peroxidase derived from agrocybe cylindracea is subjected to single-point or any-point combined mutation of the 192nd site, the 243rd site, the 244th site, the 257th site, the 272nd site and the 317th site, so that the enzyme activity of the non-specific peroxidase is improved, especially the combined mutation (V244I / S272A) of the 244th site and the 272nd site reaches 8.9 U / mL, and the non-specific peroxidase can be used for preparing the non-specific peroxidase. When the enzyme is applied to one-step preparation of 25-hydroxy-vitamin D3, the conversion rate is increased by 14 times compared with that of an original enzyme.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a non-specific peroxidase mutant with enhanced enzyme activity and application thereof. Background Art

[0002] Nonspecific peroxidase from Agrocybe aegerita Aae UPO) is a highly glycosylated sulfhemoglobinase. 2 O 2 It is an electron acceptor and does not require any cofactors, which has unique advantages over P450 enzymes. AaeUPO can catalyze the hydroxylation, epoxidation, and dealkylation of aliphatic and aromatic compounds. In addition, AaeUPO has also been successfully used in the medical industry. AaeUPO catalyzes cholecalciferol and ergocalciferol to produce 25-monohydroxyvitamin A, which is both a vitamin and a steroid hormone. It plays an important role in promoting calcium absorption and utilization, regulating hormone secretion and immune function, and is an important compound for human health.

[0003] The heme binding region of PaDa-I consists of two conserved regions in which the PCP supersecondary structure (Pro35-Cys36-Pro37) interacts with the Fe 3+ This structure allows heme to be stably present in the pocket and participate in the catalytic reaction. On the other hand, the EGD supersecondary structure (Glu122-Gly123-Asp124) is involved in binding the Mg near the heme residue. 2+ Arginine (Arg189) and the adjacent glutamic acid (Glu196) act as charge stabilizers and acid-base catalysts, respectively, while Cys278 and Cys319 form a disulfide bond. The formation of a disulfide bond helps stabilize the structure of the protein, allowing it to fold correctly and perform its function.

[0004] Vitamin D3 was used as a substrate and the nonspecific peroxygenase from Agrocybe oleracea was used to catalyze the reaction. 2 O 2 ) as an oxidant, the activation and hydroxylation of the CH bond was successfully achieved, thereby preparing 25-hydroxy-vitamin D3 in one step. However, the activity of nonspecific peroxygenases in catalyzing C-25 hydroxylation is currently low. Summary of the invention

[0005] In order to solve the technical problem of low activity of non-specific peroxygenase from Agrocybe aegerita, the present invention provides a non-specific peroxidase mutant with improved enzyme activity and its application. The present invention improves the enzyme activity of non-specific peroxidase by performing single-point mutations at positions 192, 243, 244, 257, 272, and 317 or any combination of the above multiple points on the amino acid sequence shown in SEQ ID NO.1, especially the combined mutations at positions 244 and 272 (V244I / S272A), the enzyme activity of which reaches 8.9U / mL, and when applied to the one-step preparation of 25-hydroxy-vitamin D3, the conversion rate is increased by 14 times compared with the original enzyme.

[0006] The specific technical scheme of the present invention is: In a first aspect, the present invention provides a non-specific peroxidase mutant with enhanced enzyme activity, which is obtained by single-point mutation or multi-point combined mutation of the amino acid sequence shown in SEQ ID NO.1 at the following sites: Threonine at position 192 was mutated to serine; The glycine at position 243 was mutated to glutamine; Position 244 mutated from valine to isoleucine; Position 257 mutated from arginine to lysine; The serine at position 272 was mutated to alanine; The alanine at position 317 was mutated to aspartic acid.

[0007] The present invention improves the enzymatic activity of non-specific peroxidase by performing single-point mutations at positions 192, 243, 244, 257, 272, and 317 or any combination of the above multiple points on the amino acid sequence shown in SEQ ID NO.1, especially the combined mutations at positions 244 and 272 (V244I / S272A), the enzymatic activity of which reaches 8.9U / mL, and when applied to the one-step preparation of 25-hydroxy-vitamin D3, the conversion rate is increased by 14 times compared with the original enzyme. The non-specific peroxidase with the amino acid sequence shown in SEQ ID NO.1 is derived from Agrocybe aegerita.

[0008] As the preferred non-specific peroxidase mutant, the amino acid sequence of the non-specific peroxidase mutant is any one sequence selected from SEQ ID NO.2 to SEQ ID NO.12.

[0009] In a second aspect, the present invention provides a gene encoding the above-mentioned non-specific peroxidase mutant.

[0010] In a third aspect, the present invention provides a recombinant expression vector of the above-mentioned encoding gene.

[0011] Preferably, the recombinant expression vector is a plasmid, a phage or a viral vector.

[0012] In a fourth aspect, the present invention provides a host cell carrying the above-mentioned encoding gene or the above-mentioned recombinant expression vector.

[0013] Preferably, the host cell is a Pichia pastoris GS115 cell.

[0014] In a fifth aspect, the present invention provides the use of the above-mentioned non-specific peroxidase mutant in the preparation of 25-hydroxy-vitamin D3.

[0015] The application method comprises the following steps: using vitamin D3 as a substrate and the non-specific peroxygenase mutant according to claim 1 as a catalyst to catalyze the production of 25-hydroxy-vitamin D3.

[0016] Compared with the prior art, the present invention has the following technical effects: The present invention improves the enzymatic activity of nonspecific peroxidase by performing single-point mutation at positions 192, 243, 244, 257, 272 and 317 or any combination of the above multiple points on the amino acid sequence shown in SEQ ID NO.1, especially the combined mutation at positions 244 and 272 (V244I / S272A), whose enzymatic activity reaches 8.9 U / mL. When applied to the one-step preparation of 25-hydroxy-vitamin D3, the conversion rate is increased by 14 times compared with the original enzyme.

[0017] The present invention is transformed by site-directed mutagenesis technology Aae The UPO molecular structure, the resulting single point mutation mutants T192S, G243Q, V244I, R257K, S272A, A317D, their enzyme activities compared to the original enzyme also improved to varying degrees. Among them, the mutant A317D had the highest enzyme activity, reaching 8.35U / mL, followed by the mutant S272A, reaching 7.99U / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The relative enzyme activity assay results of each mutant in Example 1 are shown; Figure 2 The relative enzyme activity assay result of the optimal combination mutant in Example 2 is shown; Figure 3 This is a graph showing the HPLC detection results of vitamin D3 and calcifediol after the mutant catalyzed the reaction for 12 hours in Example 3. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should belong to the scope of protection of the present invention.

[0020] Example 1 Construction and screening of single point mutants First, the MOE software was used to perform molecular docking between the substrate vitamin D3 and the downloaded crystal structure of nonspecific peroxygenase (PDBID: 5OXU). The heme channel and substrate binding pocket of nonspecific peroxygenase were compared with the predicted results, and the amino acids with relevant interaction with the substrate were subjected to site-directed mutation. The points with high scores were screened and virtual mutations were performed through the online website Hirespot Wizard. After successful sequencing, the points were introduced into the yeast expression system, and then the crude enzyme solution was obtained by shaking the flask for reaction verification. The construction and expression of single-point mutants were carried out according to the following steps: (1) Construction of mutants Snapgene was used to design mutation primers, and the whole plasmid PCR technology was used to introduce single point mutations into the amino acid sequence of nonspecific peroxygenase (the sequence is shown in SEQ ID NO.1) using the recombinant vector pPICZαA-PaDa-I-SPGma as a template. The primers used are shown in Table 1. The single point mutants are: T192S: Threonine at position 192 is mutated to serine, and the amino acid sequence is shown in SEQ ID NO.2; G243Q: glycine at position 243 is mutated to glutamine, and its amino acid sequence is shown in SEQ ID NO.3; V244I: the 244th position is mutated from valine to isoleucine, and its amino acid sequence is shown in SEQ ID NO.4; R257K: position 257 is mutated from arginine to lysine, and its amino acid sequence is shown in SEQ ID NO.5; S272A: The serine at position 272 is mutated to alanine, and its amino acid sequence is shown in SEQ ID NO.6; A317D: The alanine at position 317 is mutated to aspartic acid, and its amino acid sequence is shown in SEQ ID NO.7.

[0021] Table 1 The PCR reaction system is as follows: 2× phanta Max buffer 25 μL, forward / reverse primer 1 μL, template DNA 1 μL, ddH 2 0 to 50 μL. PCR amplification conditions are shown in Table 2.

[0022] Table 2 Add 1 μL of DpnI enzyme (restriction endonuclease) and 5 μL of buffer to the PCR product, and then treat at 37℃ for 1h and 70℃ for 10min. Take 10 μL of PCR product and add it to 100 μL of competent E. coli suspension in an ice bath. After standing on ice for 20min, immediately heat shock at 42℃ for 90s. After ice bathing for 3min, add 800 μL of LB medium to recover for 1h, centrifuge at 5000rpm for 2min, discard part of the supernatant, suspend the remaining bacterial liquid and apply it to the plate, and incubate it upside down at 37℃ for 12h.

[0023] (2) Induced expression of recombinant engineered bacteria From the plate after plating in step (1), a single clone was picked and placed in 10 mL LB medium (containing bleomycin, final concentration 25 μL / mL), and cultured at 37°C and 180 rpm for 12 hours. The plasmid was extracted and linearized, and then electroporated into Pichia pastoris GS115. 100 μL was taken out and spread on YPD solid medium containing 100 μL / mL bleomycin. After culturing at 30°C for 3 days, colony PCR of Pichia pastoris GS115 was performed. After successful verification, the bacteria were picked and purified and streaked. Cultured at 30°C for 3 days, and then the seed liquid was inoculated into 30 mL BMGY medium at a 2% inoculation volume (volume ratio). 2% (volume ratio) methanol was added for induction every 24 hours. The induced culture was obtained by culture at 30°C for 96 hours, and the culture was centrifuged at 4°C and 8000 rpm for 8 minutes, and the supernatant was collected to obtain the crude enzyme solution.

[0024] The mutant starting enzyme nonspecific peroxygenase (PaDa-I-SPGma constructed in Example 1 of the patent application publication number CN117947077A) was used as a control enzyme to prepare a crude enzyme solution of the control enzyme in the same manner as described above.

[0025] (3) Enzyme activity detection The crude enzyme solutions of the mutant and control enzymes obtained in step (2) were used to determine the enzyme activity using the ABTS method. The method was as follows: 158 μL of 100 mM phosphate buffer (pH 4.4), 20 μL of 3 mM ABTS, and 20 μL of crude enzyme solution were added to a 96-well plate in sequence. After thorough mixing, 0.2 M H 2 O 22μL, detect the change of its absorbance at 418nm wavelength. Substitute into the formula to calculate the enzyme activity, the calculation formula is as follows: where Vsample is the enzyme volume of the reaction solution to be measured (20 µL); ε is the extinction coefficient of ABTS at 420 nm (ε = 36000 M −1 × cm −1 ); d is the cuvette diameter (d = 1 cm); Vtotal is the total volume of the measurement system (200 µL); Ew is the sample absorbance.

[0026] Enzyme activity definition: At 25°C and pH 4.4, the amount of enzyme required to hydrolyze 1 μmol of substrate (ABTS) to generate the corresponding product (ABTS free radical) per minute is 1 enzyme activity unit (U).

[0027] Definition of relative enzyme activity: enzyme activity / enzyme activity of control enzyme*100%.

[0028] This step was tested, and the enzyme activity results of each single mutant and control enzyme are shown in Table 3.

[0029] Table 3 Enzymes Enzyme activity (U / mL) T192S 7.08 G243Q 5.58 V244I 7.50 R257K 7.19 S272A 7.99 A317D 8.35 Control enzyme 4.65 From the results of enzyme activity detection, it can be seen that mutant A317D has the highest enzyme activity, followed by mutant S272A.

[0030] The relative enzyme activity results of each mutant are shown in the attached Figure 1 shown.

[0031] Example 2 Construction and screening of two-point combination mutants According to the single mutant sequence constructed in Example 1, a combined mutation was designed. Using the whole plasmid PCR technology, the single point mutation constructed in Example 1 was used as a template to perform a two-by-two combined mutation on the amino acid sequence of the non-specific peroxidase to obtain mutants T192S / G243Q, V244I / R257K, V244I / S272A, T192S / R257K, and V244I / A317D. Among them, the amino acid sequence of mutant T192S / G243Q is shown in SEQ ID NO.8; the amino acid sequence of mutant V244I / R257K is shown in SEQ ID NO.9; the amino acid sequence of mutant V244I / S272A is shown in SEQ ID NO.10; the amino acid sequence of mutant T192S / R257K is shown in SEQ ID NO.11; and the amino acid sequence of mutant V244I / A317D is shown in SEQ ID NO.12.

[0032] The PCR reaction system and reaction conditions were the same as those in step (1) of Example 1.

[0033] The preparation of the crude enzyme solution is the same as step (2) of Example 1.

[0034] The enzyme activity detection method, enzyme activity and relative enzyme activity definitions are the same as those in step (3) of Example 1.

[0035] This step was tested, and the enzyme activity results of each double mutant and control enzyme are shown in Table 4.

[0036] Table 4 Enzymes Enzyme activity (U / mL) T192S / G243Q 5.41 V244I / R257K 7.58 V244I / S272A 8.90 T192S / R257K 7.02 V244I / A317D 7.59 Control enzyme 4.65 According to the test in this example, the enzyme activity of the mutant V244I / S272A was the highest, which was 8.90 U / mL. Figure 2 shown.

[0037] Example 3 Application of the mutant V244I / S272A in catalyzing the hydroxylation reaction of 1 mM vitamin D3 In order to identify the ability of the non-specific peroxygenase AaeUPO mutant V244I / S272A to catalyze vitamin D3, 4 mL of the crude enzyme solution prepared in Example 2 and the crude enzyme solution of the control enzyme prepared in Example 1 were used to catalyze 1 mM vitamin D3. The 10 mL catalytic system was: 40% acetone, a final concentration of 1 mM vitamin D3, 20% 100 mM phosphate buffer (pH 8), 40% crude enzyme solution, and 1 mM / h hydrogen peroxide. In order to fully react with vitamin D3, samples were taken after 12 h of reaction and detected by HPLC liquid chromatography analysis.

[0038] HPLC liquid chromatography analysis method is: take 500μL sample and 500μL n-hexane extraction, shake well and let stand for 6min, after stratification, suck out the upper layer, add 500μL methanol to dissolve after evaporation, and then filter with a 0.22μm organic phase filter. The product 25(OH)VD3 was quantitatively analyzed by HPLC analysis method. Configuration of mobile phase: The mobile phase is divided into two bottles, where mobile phase A is pure methanol and mobile phase B is pure water, and then membrane ultrasound is performed. Chromatographic column: Welchrom C18 column (4.6mm×250mm, 5μm); working temperature: 30℃. UV detection wavelength: 264nm; flow rate: 1mL / min. The mobile phase is: methanol: pure water = 90:10 (v / v).

[0039] The HPLC test results of this example show that the conversion rate of the crude enzyme solution of the control enzyme is 4.8%, and the conversion rate of vitamin D3 catalyzed by the crude enzyme solution of the mutant V244I / S272A12h is 72.0%, which is 14 times higher than the conversion rate of the crude enzyme solution of the control enzyme. Among them, the HPLC test results of vitamin D3 and calcifediol after 12 hours of catalytic reaction of the mutant V244I / S272A12h are as follows: Figure 3As shown, the retention time of vitamin D3 is 25.638 min, and the retention time of calcifediol is 10.016 min.

[0040] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the field; the methods used in the present invention, unless otherwise specified, are all conventional methods in the field; the experimental methods in the present invention, unless otherwise specified, are all conventional methods. For example, the gene cloning operation can be found in the "Molecular Cloning Experiment Guide" compiled by J. Sambrook et al.

[0041] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A non-specific peroxidase mutant with enhanced enzyme activity, characterized in that: The amino acid sequence shown in SEQ ID NO.1 is obtained by single point mutation or multi-point combined mutation at the following sites: Threonine at position 192 was mutated to serine; The glycine at position 243 was mutated to glutamine; Position 244 mutated from valine to isoleucine; Position 257 mutated from arginine to lysine; The serine at position 272 was mutated to alanine; The alanine at position 317 was mutated to aspartic acid.

2. The non-specific peroxidase mutant with enhanced enzyme activity according to claim 1, characterized in that: The amino acid sequence is any one selected from SEQ ID NO.2 to SEQ ID NO.

12.

3. The gene encoding the non-specific peroxidase mutant according to claim 1.

4. A recombinant expression vector carrying the coding gene according to claim 2.

5. The recombinant expression vector according to claim 4, characterized in that: The recombinant expression vector is a plasmid, a phage or a virus vector.

6. A host cell carrying the coding gene according to claim 3 or the recombinant expression vector according to claim 5.

7. The host cell according to claim 6, characterized in that: The cells are Pichia pastoris GS115.

8. Use of the non-specific peroxidase mutant according to claim 1 in the preparation of 25-hydroxy-vitamin D3.

9. The use according to claim 8, characterized in that: The application method comprises the following steps: using vitamin D3 as a substrate and the non-specific peroxygenase mutant according to claim 1 as a catalyst to catalyze the production of 25-hydroxy-vitamin D3.

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