Myrosinase rmyr-s342n-h350n and its use in preparing sulforaphane

Abstract

This invention discloses a black myrosinase Rmyr-S342N-H350N and its application in the preparation of raphanin, belonging to the field of functional enzyme technology. The amino acid sequence of the black myrosinase Rmyr-S342N-H350N is shown in SEQ ID NO.7. The application of the black myrosinase Rmyr-S342N-H350N in the preparation of raphanin is also described. The black myrosinase Rmyr-S342N-H350N of this invention is obtained by mutating black myrosinase Rmyr, and its enzyme activity is 169.76 U / mg, which is 13 times that of black myrosinase Rmyr. The high enzyme activity of the black myrosinase Rmyr-S342N-H350N of this invention makes it suitable for the industrial production of raphanin and has broad application prospects.

Description

Technical Field

[0001] This invention relates to a black mycosesac enzyme Rmyr-S342N-H350N and its application in the preparation of raphanin, belonging to the field of functional enzyme technology. Background Technology

[0002] Sulforaphene is a naturally occurring bioactive substance found in cruciferous plants, possessing bioactivities including anti-inflammatory, anti-cancer, anti-obesity, and improvement of cardiovascular and neurological diseases. However, sulforaphene is present in very low concentrations in cruciferous plants, making direct extraction difficult. Utilizing myrosinase-catalyzed hydrolysis of glucosinolates is a crucial pathway for the industrial production of sulforaphene.

[0003] CN 113736763 A discloses black myrosinase Rmyr and its application in the preparation of sulforaphane and raphanin, which utilizes black myrosinase Rmyr to catalyze the hydrolysis of glucosinolates. However, the enzyme activity of black myrosinase Rmyr is still not ideal, and it is necessary to mutate it to obtain black myrosinase with higher enzyme activity. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides a black mycosesacin Rmyr-S342N-H350N and its application in the preparation of raphanin, which belongs to the field of functional enzyme technology.

[0005] This invention is achieved through the following technical solution: A black myrosinase Rmyr-S342N-H350N, with the amino acid sequence shown in SEQ ID NO.7.

[0006] The application of the black myrosinase Rmyr-S342N-H350N in the preparation of raphanin.

[0007] Furthermore, in specific applications, the crushed radish seeds are dissolved in water, and myrosinase Rmyr-S342N-H350N is added. Under the catalytic action of myrosinase Rmyr-S342N-H350N, the raphanin in the radish seeds is hydrolyzed to produce raphanin.

[0008] The black myrosinase Rmyr-S342N-H350N of this invention is obtained by mutating and modifying the black myrosinase Rmyr. Its enzyme activity is 169.76 U / mg, which is 13 times that of the original black myrosinase Rmyr. The high enzyme activity of Rmyr-S342N-H350N makes it suitable for the industrial production of raphanin and has broad application prospects.

[0009] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0010] Figure 1 Results of molecular docking between myrosinase Rmyr and myrosinin.

[0011] Figure 2 Molecular docking results of mutant S342N with sinigrin.

[0012] Figure 3 Molecular docking results of mutant S342N-H350 with sinigrin.

[0013] Figure 4 : Schematic diagram of SDS-PAGE electrophoresis results, where M is the standard protein marker, lane 1 is the crude enzyme solution, and lane 2 is the pure enzyme solution.

[0014] Figure 5 Results of relative enzyme activity determination under different temperature conditions.

[0015] Figure 6 Results of relative enzyme activity determination under different pH conditions.

[0016] Figure 7 Results of temperature stability measurements.

[0017] Figure 8 Results of pH stability measurements.

[0018] Figure 9 Mass spectrometry chromatogram of hydrolysis products. Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0020] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0021] Example 1: Mutation of black myrosinase Rmyr Rmyr is an enzyme screened in the inventor's laboratory during previous research. Its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2.

[0022] According to CN 113736763 A, the enzyme activity of black mycosesin Rmyr is 12.73 U / mg, which is relatively high, but still not ideal. Therefore, this invention attempts to mutate and modify it in order to obtain black mycosesin with higher enzyme activity.

[0023] The three-dimensional structure of black myrosinase Rmyr was analyzed using PymoL software. Its catalytic active site is "SDW" (amino acids at positions 336, 337, and 338). Non-conserved amino acids near the catalytic active site were searched for mutation sites. The inventors hypothesized that if serine at position 342 and histidine at position 350 near the catalytic active site were both mutated to asparagine, the attack distance of aspartic acid at position 337 (a nucleophilic residue) would be shortened. Under these conditions, when this nucleophilic residue attacks the anomeric carbon of the substrate glycosidic bond, it is more conducive to the formation of a covalent glycosyl-enzyme intermediate. Simultaneously, asparagine is a neutral amino acid and will not introduce electrostatic interference (in contrast, aspartic acid, which has a similar structure to asparagine, carries a negative charge under physiological pH conditions, interfering with the precise electrostatic microenvironment of the catalytic active site, potentially hindering the binding of negatively charged substrates or charge relay of catalytic residues). Furthermore, the amide group possesses both hydrogen bond donor and acceptor capabilities, enabling the formation of a more stable hydrogen bond network, thereby rigidifying the conformation of the catalytic triplet. Therefore, in this invention, the serine at position 342 of black myrosinase Rmyr is virtually mutated to asparagine to obtain mutant S342N. Based on this, the histidine at position 350 is virtually mutated to asparagine to obtain mutant S342N-H350N.

[0024] Molecular docking was performed between black myrosinase Rmyr, mutant S342N, and mutant S342N-H350N and black myrosinase glycosides, respectively. The docking results at the nucleophilic residue-substrate binding sites were demonstrated, and the differences were compared. Results: The molecular docking results between black myrosinase Rmyr and black myrosinase glycosides are as follows: Figure 1 As shown, the molecular docking results of mutant S342N with sinigrin are as follows: Figure 2 As shown, the molecular docking results of mutant S342N-H350 with sinigrin are as follows: Figure 3As shown in the figure, the number of hydrogen bonds between the substrate molecule and the enzyme protein after docking are 6, 7, and 8, respectively. The direct distances between the nucleophilic amino acid residues attacking the substrate and the anomeric carbon are 6.6 Å, 6.0 Å, and 5.0 Å, respectively. This indicates that the interaction between the double mutant and the substrate molecule is stronger, and it is speculated that the closer the attack distance of the nucleophilic amino acid residues, the faster the enzyme protein breaks the glycosidic bond and releases the glycosidase complex from the substrate. Meanwhile, the docking binding energies of myrosinase Rmyr, mutant S342N, and mutant S342N-H350N with myrosinase are -5.7 kcal / mol, -6.0 kcal / mol, and -6.3 kcal / mol, respectively, which means that mutant S342N-H350N has a stronger affinity for myrosinase. Based on the changes in the number of hydrogen bonds, the attack distance of nucleophilic residues, and the binding energy, it is predicted that the enzyme activity of the mutant S342N-H350N will be significantly better than that of black mysinase Rmyr.

[0025] Example 2 Heterologous expression of black myrosinase Rmyr-S342N-H350N Based on the analysis and virtual mutation prediction results of Example 1, this example involves a directed mutation of serine at position 342 of the black myrosinase Rmyr to asparagine and a directed mutation of histidine at position 350 to asparagine, resulting in a mutant named black myrosinase Rmyr-S342N-H350N. The black myrosinase Rmyr-S342N-H350N was heterologously expressed, extracted, and purified, following these steps: (1) Cloning of genes Using pET28a-Rmyr plasmid as a template, a pair of specific primers were designed at the site of the single amino acid mutation using the software Snapgene. The primers were extended simultaneously in the forward and reverse directions, and the mutated circular plasmid, pET28a-Rmyr-S342N-H350N plasmid, was obtained by PCR amplification.

[0026] A pair of specific primers for mutations at the S342 site: the nucleotide sequence of the forward primer S342N-F is shown in SEQ ID NO. 3, and the nucleotide sequence of the reverse primer S342N-R is shown in SEQ ID NO. 4. A pair of specific primers for mutations at the S350 site: the nucleotide sequence of the forward primer S342N-H350N-F is shown in SEQ ID NO. 5, and the nucleotide sequence of the reverse primer S342N / H350N-R is shown in SEQ ID NO. 6.

[0027] The amino acid sequence of black myrosinase Rmyr-S342N-H350N is shown in SEQ ID NO.7, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.8.

[0028] (2) Construction of recombinant engineered bacteria The pET28a-Rmyr-S342N-H350N plasmid was transformed into Escherichia coli BL21 competent cells. The single colony that grew on the kanamycin sulfate resistant plate was the constructed recombinant engineered bacteria.

[0029] (3) Fermentation of recombinant engineered bacteria Select the above single colonies and inoculate them into 5 ml of LB liquid medium containing kanamycin sulfate. Activate at 37°C and 220 rpm for 12 h. Inoculate 1% of the colonies into ZYP-5052 medium containing kanamycin sulfate and culture at 20°C and 200 rpm for 48 h to induce the expression of black mycosesin.

[0030] (4) Extraction and purification of black myrosinase Rmyr-S342N-H350N Take the culture medium, centrifuge at 8000 g for 10 min at 4℃, collect the bacterial cells, resuspend them in 50 mM, pH 7.0 Tirs-HCl buffer, sonicate for 30 min, centrifuge at 12000 g for 15 min, and the supernatant is the crude enzyme solution.

[0031] The crude enzyme solution was purified by affinity chromatography using a Ni-NTA column: the column was equilibrated with equilibration buffer (500 mM NaCl, 50 mM Tris-HCl); weakly binding proteins were eluted with 20 mM imidazole solution (20 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl); the target protein was eluted with 200 mM imidazole solution (200 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl) to obtain the eluent, which was the pure enzyme solution.

[0032] SDS-PAGE analysis was performed on the crude enzyme solution and the purified enzyme solution to verify that the bands were uniform and of accurate size. A schematic diagram of the SDS-PAGE electrophoresis results is shown below. Figure 4 As shown, a band of size 69 kDa was obtained, consistent with the prediction.

[0033] Example 3 Enzyme activity assay The enzyme activity was determined as follows: 10 μL of purified enzyme solution was added to 40 μL of glucosinolate solution, and the reaction was carried out at 35℃ for 15 min. After the reaction, the supernatant was collected, 300 μL of DNS reagent was added, and the mixture was boiled in a water bath for 5 min for color development. The absorbance value was measured at 540 nm. The concentration of glucosinolate in the glucosinolate solution was 5 mg / mL, prepared using phosphate buffer at pH 6.0.

[0034] Enzyme activity is defined as the amount of glucose (μmol) produced per milligram of enzyme per minute under standard conditions.

[0035] The enzyme activity of black mysinase Rmyr-S342N-H350N was 169.76 U / mg, which is 13 times that of black mysinase Rmyr (enzyme activity is 12.73 U / mg), indicating a significant increase in enzyme activity.

[0036] Example 4: Determination of optimal temperature and optimal pH (1) Determination of the optimal temperature 10 μL of purified enzyme solution was added to 40 μL of myrosinase solution (concentration 5 mg / mL, pH 6.0), and the reaction was carried out at 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃ for 15 min, respectively. Other steps were the same as in Example 3.

[0037] The relative enzyme activity at each reaction temperature was calculated, with the highest enzyme activity defined as 100%. The results of the relative enzyme activity measurements at different temperatures are shown below. Figure 5 As shown, the optimal temperature is 35℃, and the enzyme activity is relatively high in the range of 25~40℃, with a relative enzyme activity of over 60%.

[0038] (2) Determination of optimal pH Sinapisin solutions with different pH values ​​(5 mg / mL) were prepared using buffer solutions ranging from pH 4.0 to 10.0. The buffer solutions used included: citrate buffers at pH 4.0, 5.0, and 6.0; phosphate buffers at pH 6.0, 7.0, and 8.0; Tris-HCl buffers at pH 8.0 and 9.0; and glycine-NaOH buffers at pH 9.0 and 10.0.

[0039] 10 μL of pure enzyme solution was added to 40 μL of myrosinase solution (concentration 5 mg / mL) at different pH values, and the reaction was carried out at 35°C for 15 min. Other steps were the same as in Example 3.

[0040] The relative enzyme activity at each pH was calculated with the highest enzyme activity defined as 100%. The results of the relative enzyme activity measurements under different pH conditions are as follows: Figure 6 As shown, the optimal pH is 6.0.

[0041] Example 5: Determination of Temperature Stability The pure enzyme solution was incubated at 25℃, 30℃, and 35℃ for 22 hours respectively. Samples were taken at different time points (2, 4, 6, 8, 10, 12, 14, 16, 19, and 22 hours). The residual enzyme activity was measured under the optimal conditions (temperature 35℃, pH 6.0), using the same method as in Example 3.

[0042] Using the enzyme activity before incubation as 100%, the relative enzyme activity after incubation at different temperatures for different times was calculated. The results of the temperature stability determination are as follows: Figure 7 As shown, the enzyme can maintain a relative enzyme activity of over 60% after incubation at 35℃ for 8 hours, demonstrating good temperature stability.

[0043] Example 6: Determination of pH stability 10 μL of pure enzyme solution was added to 40 μL of phosphate buffer at pH 6.0, 7.0 and 8.0 respectively, and incubated for 40 days. Samples were taken at different time points, and myrosin was added to make the concentration 5 mg / mL. The residual enzyme activity was measured at 4℃, using the same method as in Example 3.

[0044] Using the unincubated enzyme activity as 100%, the relative enzyme activity after incubation at different pH conditions for different times was calculated. The results of pH stability measurements are as follows: Figure 8 As shown in the figure, the enzyme activity remained above 60% of its original value after being stored for 40 days, indicating that the enzyme has good stability.

[0045] Example 7 Identification of Hydrolysis Products Rhamnoside was hydrolyzed using the myrosinase Rmyr-S342N-H350N, and the hydrolysis products were identified.

[0046] Radish seeds were treated at 120℃ for 12 h to inactivate the enzyme, and then pulverized. Using the pulverized radish seeds as a substrate, a radish seed solution was obtained by dissolving the seeds in water at a ratio of 1:10 (m:v, g:ml). 60 U of pure enzyme solution was added to 3 mL of the radish seed solution, and the reaction was carried out at 35℃ and 150 rpm for 15 min. After the reaction, the solution was extracted with twice the volume of ethyl acetate, evaporated to dryness, and reconstituted with an equal volume of acetonitrile. The peak time was detected by liquid chromatography, and further identification was performed by mass spectrometry.

[0047] Mass spectrometry identification of hydrolysis products as shown in the figure. Figure 9 As shown, under the catalysis of myrosinase Rmyr-S342N-H350N, raphanin in radish seeds is hydrolyzed to produce raphanin (Mr=176).

[0048] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A myrosinase Rmyr-S342N-H350N, characterized in that: The amino acid sequence is shown in SEQ ID NO.

7.

2. The application of the black myrosinase Rmyr-S342N-H350N according to claim 1 in the preparation of raphanin.

3. Use of the myrosinase Rmyr-S342N-H350N according to claim 2 for the preparation of sulforaphane, characterized in that: In practical applications, crushed radish seeds are dissolved in water, and myrosinase Rmyr-S342N-H350N is added. Under the catalytic action of myrosinase Rmyr-S342N-H350N, raphanin in the radish seeds is hydrolyzed to produce raphanin.

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