Beta-glucuronidase mutant and application thereof

Abstract

The invention discloses a beta-glucuronidase mutant and application thereof, and belongs to the technical field of genetic engineering and protein engineering modification. The beta-glucuronidase mutant disclosed by the invention has the following mutation on the basis of an amino acid sequence as shown in SEQ ID NO.3: T512S or T512A. The catalytic efficiency of the beta-glucuronidase mutant T512S is improved by 13.92 times compared with that of AtGUS (-3t), and the beta-glucuronidase mutant T512S shows remarkable industrial application potential.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and protein engineering, and more specifically to β-glucuronidase mutants and their applications. Background Technology

[0002] Glycyrrhizic acid (GL) is the main active ingredient extracted from licorice root, possessing various pharmacological effects such as hepatocyte protection, anti-inflammation, antiviral activity, and anti-tumor cell activity. However, due to its high polarity, its bioavailability is low, and long-term use can cause side effects such as hypernatremia and hypokalemia. Glycyrrhizic acid can be hydrolyzed by β-glucuronidase (GUS, EC3.2.1.31) to produce glycyrrhetinic acid monoglucuronide (GAMG) and glycyrrhetinic acid (GA). Compared to glycyrrhizic acid, GAMG, with some sugars removed, has higher sweetness and emulsifying properties, while GA, with all sugars removed, has stronger pharmacological activity and higher absorption rate, showing broader application prospects in the pharmaceutical, food, and fine chemical industries.

[0003] β-Glucuronidases are widely distributed in microorganisms, plants, and animals, and can hydrolyze the glucuronic acid moiety from the non-reducing end of glycosides. To date, most identified β-glucuronidases belong to the GH2 family; they all possess a regular TIM(β / α)8 barrel structure and employ the same dual-retention mechanism. They originate from the fungus *Aspergillus terreus*. Aspergillus terreus Li-20 At GUS is a multi-domain homotetrameric protein. Each domain consists of 657 amino acid residues, including an N-terminal glycosyl-binding domain, a C-terminal TIM barrel domain, and a central immunoglobulin sandwich domain. Studies have shown that... At The mutant enzyme obtained by truncating the 65aa nonconserved C-terminal sequence of GUS At GUS(-3t) showed a 3.2-fold increase in enzyme activity. However, this enzyme still suffers from low specific activity and high production costs, requiring further optimization of its catalytic performance through molecular modification. Therefore, molecular modification of β-glucuronidase to improve its catalytic efficiency will provide a new technical method for the efficient enzymatic preparation of high-value glycyrrhizic acid derivatives GAMG and GA.

[0004] Therefore, providing β-glucuronidase mutants and their applications is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a β-glucuronidase mutant and its application. The catalytic efficiency of the mutant is significantly higher than that of the wild type, which is beneficial for its application in glycyrrhizic acid biotransformation.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The β-glucuronidase mutant has the following mutations based on the amino acid sequence shown in SEQ ID NO.3: T512S or T512A.

[0008] Furthermore, the DNA molecule encoding the β-glucuronidase mutant.

[0009] Furthermore, a recombinant expression plasmid containing the DNA molecule.

[0010] Furthermore, a recombinant expression host cell comprises the aforementioned recombinant expression plasmid; the host cell is... Escherichia coli BL21(DE3).

[0011] Furthermore, the β-glucuronidase mutant is used to improve the bioconversion efficiency of glycyrrhizic acid.

[0012] Furthermore, the application of the β-glucuronidase mutant in improving the specific activity of β-glucuronidase.

[0013] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a β-glucuronidase mutant and its application, using β-glucuronidase... At Based on GUS(-3t), a β-glucuronidase mutant containing a single-point mutation at T512S was provided. (This is in contrast to β-glucuronidase.) At Compared to GUS(-3t), the catalytic efficiency (k) of the β-glucuronidase mutant is significantly higher. cat / K m =152.33±3.50 mmol -1 ·L·s -1 Compared to Atgus(-3t) (k cat / K m =10.94±0.15 mmol -1 ·L·s -1 It increased by 13.92 times, demonstrating significant potential for industrial application.

[0014] The catalytic efficiency of the β-glucuronidase mutant is significantly improved, which is beneficial to reducing production costs and promoting its application in the synthesis of glycyrrhizic acid derivatives GAMG and GA. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0016] Figure 1 The SDS-PAGE results are for the Thr512 site mutant.

[0017] Figure 2 The specific activity of the mutant enzyme at position 512. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0020] Strains and reagents: Cloned strains E.coli DH5α competent cells and expression host strains Escherichia coli BL21(DE3) competent cells were purchased from Qingke Biotechnology Co., Ltd.

[0021] DNA high-fidelity polymerase Fast Pfu DNA polymerase, Taq mix DNA polymerase, nucleic acid markers, and protein markers were purchased from Beijing TransGen Biotech Co., Ltd.; non-prestained protein molecular weight marker (26610) was purchased from Thermo Fisher Scientific; plasmid extraction kit was purchased from Tiangen Biotech Co., Ltd.; Bradford protein concentration assay kit was purchased from Beijing Solarbio Science & Technology Co., Ltd.; enzyme expression inducer isopropyl-β-D-thiogalactoside (TPTG) and substrate glycyrrhizic acid were purchased from Beijing Innovent Biologics Co., Ltd.; 4-nitrophenyl-β-D-pyranoglobulin was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; FD Dpn I was purchased from ABclonal; primer synthesis and sequencing results were obtained from Kingwise.

[0022] The culture media and reagents involved in the examples are as follows: Preparation of LB liquid medium: Weigh 5 g yeast extract, 10 g peptone, and 10 g NaCl into a 1 L beaker, add 800 mL of deionized water, stir well, and bring the volume to 1 L. Dispense the mixture into conical flasks of different sizes and sterilize at 121 ℃ for 20 min.

[0023] 2 g / L glycyrrhizic acid: Accurately weigh 0.2 g of glycyrrhizic acid, dissolve it in 50 mM sodium acetate-acetic acid buffer (pH 5.0), and bring the volume to 100 mL. Mix thoroughly.

[0024] 50×TAE electrophoresis buffer: Weigh 242 g Tris and 18.612 g EDTA into a 1 L beaker. Add approximately 800 mL of deionized water to the beaker and stir well. Add 57.1 mL of glacial acetic acid and dissolve completely. Adjust the pH to 8.3 with NaOH, and then add deionized water to bring the volume to 1 L. Store at room temperature. Dilute 50 times before use.

[0025] 5×SDS-PAGE electrophoresis buffer: Weigh 15.1 g Tris, 94 g g glycine, and 5.0 g SDS into a 1 L beaker. Add approximately 800 mL of deionized water to the beaker and stir to dissolve. Make up the volume to 1 L with deionized water and store at room temperature.

[0026] The amino acid sequence of Atgus is shown in SEQ ID NO.1.

[0027] MLKPRQTPFRDLISLDGLWKFALDSGDNATAAPWTGPLTTDLECPVPASYNDIFVDRQIRDHVGWVYYQREAIVPRAWSQQQYLVRVDAATHQGRIYINDNLVAEHRGGYTPFEADITGLVSAGDSFRLTIAVNNELTHETIPPGRIEVEEYTGKRVQVYQHDFFNYAGLARSVWLYSVPQQHIQDIKVVTHVKGSAGLINYLVTVSNSTTGRVKIDVIDKDGTTVAEASGARGSVTIDSVKLWQPGEAYLYQFRASIVGLNDSVVDTYCVETGVRTVKVSGNRFLINDKPFYFTGFGKHEDSAVRGKGYDPAYMVHDFQLMDWMGANSFRTSHYPYAEEVMEFADRHGIVVIDETPAVGLAFSIGSGVSSEDSPQTFTPEGINNNTREAHKQAIRELIARDKNHASVVMWSIANEPASQEVGAREYFAPLVDLAHELDPSRPVCFANYGDATYEVDRISDMFDVLCLNRYFGWYSQTGEVEEAEAALEKELLGWEGKYGKPIVITEYGADTMAGLHSVLALPWSEEFQVQLLDMYHRVFDRIDSVVGEHVWNFADFQTAVGIIRVDGNKKGVFTRERKPKAAAHTLKTRWSAGFAAMCLEHREKNFGLALVKARRRRGAGGTRRSLFRPSELAGPTTPAGISYIRPRRPLYFLFGA; SEQ ID NO.1。

[0028] The Atgus(-3t) nucleotide sequence is shown in SEQ ID NO.2.

[0029] atgctgaagccccgacaaacaccttacc atggccggcctccattctgtgcttgctcttccatggagtgaggaattccaggtccaactactcgatatgtatcatcgggtgtttgaccgcattgactcagtggtaggagagcacgtctggaactttgctgatttccagacggctgtagggattattagggtggatggaaacaagaagggtgtctttaccagggaaaggaaaccaaaggctgcagct catacattgaaaacacggtggagttaa ; SEQ ID NO.2.

[0030] The amino acid sequence of Atgus(-3t) is shown in SEQ ID NO.3.

[0031] MLKPRQTPFRDLISLDGLWKFALDSGDNATAAPWTGPLTTDLECPVPASYNDIFVDRQIRDHVGWVYYQREAIVPRAWSQQQYLVRVDAATHQGRIYINDNLVAEHRGGYTPFEADITGLVSAGDSFRLTIAVNNELTHETIPPGRIEVEEYTGKRVQVYQHDFFNYAGLARSVWLYSVPQQHIQDIKVVTHVKGSAGLINYLVTVSNSTTGRVKIDVIDKDGTTVAEASGARGSVTIDSVKLWQPGEAYLYQFRASIVGLNDSVVDTYCVETGVRTVKVSGNRFLINDKPFYFTGFGKHEDSAVRGKGYDPAYMVHDFQLMDWMGANSFRTSHYPYAEEVMEFADRHGIVVIDETPAVGLAFSIGSGVSSEDSPQTFTPEGINNNTREAHKQAIRELIARDKNHASVVMWSIANEPASQEVGAREYFAPLVDLAHELDPSRPVCFANYGDATYEVDRISDMFDVLCLNRYFGWYSQTGEVEEAEAALEKELLGWEGKYGKPIVITEYGAD T MAGLHSVLALPWSEEFQVQLLDMYHRVFDRIDSVVGEHVWNFADFQTAVGIIRVDGNKKGVFTRERKPKAAAHTLKTRWS; SEQ IDNO.3.

[0032] Example 1 Cloning of β-glucuronidase gene Aspergillus terreus Aspergillus terreus Using the β-glucuronidase gene Atgus (amino acid sequence shown in SEQ ID NO.1) of Li-20 as a template, PCR amplification was performed using Gatgus-BamHI and Gatgus-NotI as primers to obtain a 1.7 kb DNA fragment (nucleotide sequence shown in SEQ ID NO.2, amino acid sequence shown in SEQ ID NO.3).

[0033] The primer sequences used are as follows: Gatgus-BamHI: 5'-CGC GGATCC ATGCTGAAGCCCCGACAAACACCTT -3';SEQ ID NO.4.

[0034] Gatgus-NotI: 5'-CAT GCGGCCGC TTAACTCCACCGTGTTTTCAATGTATG -3';SEQ ID NO.5.

[0035] The PCR products were recovered by gel extraction. The recovered PCR products were ligated with the vector pET28a, which had been double-digested with BamHI and NotI, and then transformed into *E. coli* DH5α. Correct transformants were selected for sequencing. Sequencing results showed that the amplified gene fragment sequence was SEQ ID NO.2, and it had 100% sequence similarity to the β-glucuronidase of *Aspergillus terreus* (excluding the terminal 65 amino acids). This plasmid was named pET28a(+)-Atgus(-3t).

[0036] Example 2: Rational Modification of Highly Catalytically Active β-Glucuronidase Mutant Literature reports that loop structures participate in enzyme catalysis, particularly sites in the loop region near the active site, which play a crucial role in promoting enzyme-substrate binding and influencing substrate selectivity. Molecular docking and structural analysis revealed that Thr 512 on the 7th βα loop of the TIM bucket is adjacent to the substrate glycyrrhizic acid molecule and exhibits multiple interactions. It is speculated that this site may promote substrate entry into the active pocket and product exit by controlling loop movement. Based on this, this invention designs substitutions of different amino acid residues (including T512A, T512S, T512Y, T512I, and T512D) according to the properties of the Thr 512 amino acid, altering the conformation of the local region, enhancing the substrate recognition ability of the catalytic active site, and increasing the reaction rate.

[0037] Based on the above concept, using the recombinant plasmid pET28a(+)-Atgus(-3t) as a template, corresponding primers were designed according to the selected mutation sites (Table 1). Site-directed mutagenesis was performed using the whole plasmid amplification mutagenesis method to clone the entire plasmid, while introducing mutation sites on the target amino acids. After the PCR amplification reaction, the methylated or hemimethylated template in the PCR product was digested with Dpn I digestion enzyme. Then, the digested product was transformed into E. coli DH5α competent cells to enrich the mutant plasmids pET28a(+)-Atgus(-3t)-T512A, pET28a(+)-Atgus(-3t)-T512S, pET28a(+)-Atgus(-3t)-T512D, pET28a(+)-Atgus(-3t)-T512Y, and pET28a(+)-Atgus(-3t)-T512I.

[0038] Table 1 Primer sequences

[0039] Example 3: Expression and catalytic activity verification of β-glucuronidase mutant 10 μL of recombinant plasmid was transformed into competent Escherichia coli BL21(DE3) cells. A single colony of *E. coli* was picked and inoculated into 10 mL of LB medium (containing 50 μg / mL kanamycin) and cultured overnight at 37 ℃ and 200 r / min to obtain a seed culture. The seed culture was then transferred to 400 mL of LB medium, with an inoculum size of 1%, and cultured at 37 ℃ for 3 h. The absorbance was then measured. 600 The concentration was approximately 0.6. 0.2 mM IPTG was added as an inducer, and the culture temperature was controlled at 16 ℃. Culture was continued for 20 h, and preliminary experiments determined that 20 h was the optimal induction time. The fermentation broth was centrifuged at 12000 r / min to collect the cells. The cells were resuspended in a 20 mM Tris-HCl / 100 mM NaCl equilibration buffer (pH 7.4), and the cells were lysed under high pressure and low temperature. The supernatant was then collected after centrifugation at 12000 r / min for 20 min to obtain the crude enzyme solution.

[0040] Based on the His tag and protein characteristics of the recombinant β-glucuronidase, the target protein was purified using an AKTA protein purification system via Ni-NTA affinity chromatography column. The purification system was used to wash away contaminating proteins with 50 mM Tris-HCl and 100 mM NaCl protein buffer (pH 7.4). The target protein was then eluted with a gradient of 50 mM, 150 mM, 200 mM, and 250 mM imidazole solutions. SDS-PAGE analysis showed that the target protein was eluted in 250 mM imidazole elution buffer, and its molecular weight was approximately 66 kDa. Figure 1 The proteins (enzyme solutions) collected at this time are used for subsequent protein functional characterization.

[0041] Take 20 μL of enzyme solution diluted to a certain concentration and mix it with 80 μL of glycyrrhizic acid (2.5 mmol·L⁻¹). -1 The solution was placed in a 2 mL EP tube, with a final glycyrrhizic acid concentration of 2 mmol·L⁻¹. -1 The reaction was carried out under certain conditions for a certain time, and then the reaction was terminated by boiling for 5 minutes. The concentrations of the substrate glycyrrhizic acid and the products GAMG and GA were detected by HPLC.

[0042] Sample preparation and detection: Add 900 μL of methanol to the reaction solution to dilute the sample 10 times. After mixing, filter the sample through a 0.22 μm organic filter membrane and then perform detection. The contents of GL, GAMG, and GA were determined by external standard method using high-performance liquid chromatography (HPLC).

[0043] HPLC detection parameters: C18 column (5 μm, 250 mm × 4.6 mm), UV detector wavelength of 254 nm, mobile phase of methanol:0.6% acetic acid = 81:19, flow rate of 1 mL / min, column temperature of 30 ℃.

[0044] The standard curve for GL is: y = 8127.9x + 31.817 (R² - 1 / 2)². 2 =0.992).

[0045] The standard curve for GAMG is: y = 9701.9x - 77.776 (R²). 2 =0.998).

[0046] The standard curve for GA is: y = 17469x - 91.122 (R²) 2 =0.9984).

[0047] Where x is the sample concentration in mg / mL and y is the peak area.

[0048] β-glucuronidase activity is defined as the amount of β-glucuronidase required per minute to catalyze the conversion of 1 μmol of glycyrrhizic acid into the product under optimal reaction conditions for each enzyme; that is, 1 U = 1 μmol·min. -1 The specific activity of the enzyme is calculated based on the protein concentration. The specific activity of the enzyme is expressed in units of enzyme activity per mg of protein, which is U / mg.

[0049] Under substrate concentration saturation, the specific activities of each mutant and Atgus(-3t) are as follows: Figure 2 As shown, the specific activities of mutant enzymes T512A and T512S for substrates GL and GAMG were both increased compared to Atgus(-3t), with mutant T512S exhibiting the best activity, 1.78 times that of Atgus(-3t). The specific activities of mutant enzymes T512Y, T512I, and T512D all decreased significantly. The specific activities of each mutant enzyme were highly dependent on the nature of the side chain at position 512, with polar uncharged amino acids (Ser) exhibiting greater activity than nonpolar amino acids (Ala), aromatic amino acids (Tyr), and charged amino acids (Asp).

[0050] Glycyrrhizic acid can be hydrolyzed sequentially by β-glucuronidase to produce glycyrrhetinic acid monoglucuronide (GAMG) and glycyrrhetinic acid (GA).

[0051] Example 4: Determination of the enzyme kinetic constant of the β-glucuronidase mutant T512S Atgus(-3t) and the mutant T512S were reacted with a final concentration of 2.5 mmol·L⁻¹ under optimal reaction conditions (pH 5.0, 45℃). -1 2 mmol·L -1 1.5 mmol·L -1 1 mmol·L -1 0.75 mmol·L -1 0.5 mmol·L -1 The enzyme was reacted with glycyrrhizic acid solution for 10 min, and the enzyme activity was measured. Based on the transformation of the Michaelis-Menten equation, the enzyme-catalyzed reaction kinetic parameters Vmax, Km, and kcat were calculated using the Lineweaver-Burk double reciprocal method and the Michaelis-Menten equation. The results are shown in Table 2.

[0052] Table 2 Kinetic parameters of Atgus(-3t) and mutant enzymes

[0053] Table 2 shows that the catalytic efficiency of the mutant enzyme T512S was significantly improved. The results of Atgus(-3t) and the mutant enzyme T512S... K m They are 1.549 mmol·L -1 and 0.136 mmol·L -1 It can be seen that the mutant enzyme T512S has an increased affinity for the substrate glycyrrhizic acid, and the number of transitions of the mutant T512S is increased. k cat It was also improved compared to Atgus(-3t), likely due to the mutated loop structure facilitating substrate entry into the enzyme's active site. Ultimately, the catalytic efficiency of the mutant enzyme T512S was... k cat / K m The value was 13.92 times higher than that of Atgus(-3t). This result indicates that the main reason for the improved catalytic efficiency of the enzyme is that the change in hydrophilicity or hydrophobicity at the mutation site promotes substrate entry into the active site. The change from threonine to serine at position 512 on the TIM barrel loop structure may have affected the binding of the enzyme active site to the substrate, leading to a significant increase in enzyme catalytic efficiency.

[0054] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A β-glucuronidase mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.3, the following mutations exist: T512S or T512A.

2. A DNA molecule encoding the β-glucuronidase mutant of claim 1.

3. A recombinant expression plasmid comprising the DNA molecule of claim 2.

4. A recombinant expression host cell, characterized in that, It comprises the recombinant expression plasmid as described in claim 3; the host cell is Escherichia coli BL21(DE3).

5. The application of the β-glucuronidase mutant according to claim 1 in improving the biotransformation efficiency of glycyrrhizic acid.

6. The application of the β-glucuronidase mutant according to claim 1 in improving the specific activity of β-glucuronidase.

Images