Beta-glucuronidase as well as preparation method and application thereof

Abstract

The invention relates to beta-glucuronidase as well as a preparation method and application thereof, and belongs to the technical field of enzyme engineering. According to the recombinant beta-glucuronidase disclosed by the invention, a beta-glucuronidase coding gene from Thermobacillus composti is expressed in escherichia coli, so that the recombinant beta-glucuronidase with good hydrolysis specificity, thermal stability and pH stability can be prepared, and glycyrrhetinic acid can be prepared by efficiently degrading glycyrrhizic acid by utilizing the recombinant beta-glucuronidase; the problems that in the prior art, beta-glucuronidase is low in catalytic activity on glycyrrhizic acid, poor in hydrolysis specificity, poor in stability and the like are solved. In the production of glycyrrhetinic acid, glycyrrhizic acid or monoglucose glycyrrhetinic acid is taken as a substrate, methanol is added for assisting dissolution, the conversion efficiency is remarkably improved, the substrate conversion rate can reach 99% or above, and the highest yield can reach 95%. The biotransformation process established by the invention has the advantages of mild reaction conditions, high substrate utilization rate, good product purity and the like, and provides a new thought and method for industrial production of glycyrrhetinic acid.

Description

Technical Field

[0001] The present invention relates to the field of enzyme engineering technology, in particular to a beta-glucuronidase and a preparation method and application thereof. Background Art

[0002] β-glucuronidase (GUS) plays a role in regulating the glucuronidation of xenobiotics and endogenous compounds and participates in carbohydrate metabolism in various vertebrate tissues. Furthermore, as a glycosidase with hydrolytic activity, glucuronidase is widely used in the preparation of active pharmaceutical ingredients.

[0003] Licorice and Scutellaria baicalensis are traditional Chinese medicinal herbs. Glycyrrhizic acid, a derivative of the triterpenoid saponin glycyrrhizic acid, possesses anti-inflammatory, anti-tumor, antiviral, hepatoprotective, and lipid-lowering properties. Baicalein, a member of the flavonoid family, is primarily found in the traditional Chinese medicinal herb Scutellaria baicalensis. Numerous studies have demonstrated that baicalein possesses excellent free radical scavenging, anticancer, and antibacterial activities. Baicalein exhibits potent inhibitory effects against colorectal, lung, and breast cancers, and holds promise as a novel targeted cancer therapy. Currently, the main production routes for glycyrrhizic acid and baicalein involve direct extraction from the dried roots of licorice and Scutellaria baicalensis, or acidic cleavage, alkaline cleavage, and chemical synthesis using glycyrrhizic acid and baicalin (or their salts). However, traditional plant extraction and chemical synthesis methods fall far short of meeting market demand and suffer from drawbacks such as harsh reaction conditions, high energy consumption, and the use of extensive organic solvents, which can cause environmental pollution. Biosynthesis of glycyrrhetinic acid and baicalein offers advantages such as mild reaction conditions, good chemical bond selectivity, and high yields, meeting green manufacturing and environmental protection requirements. Inspired by the precise control enzymes provide over reaction outcomes, biocatalytic transformations are being used to supplement or replace traditional chemical routes. Consequently, the biosynthesis of glycyrrhetinic acid and baicalein has been extensively developed in recent years, representing an effective approach to addressing their resource shortages.

[0004] Glucuronidase, an important hydrolase that specifically hydrolyzes glycosidic bonds, has been widely used in the preparation of active pharmaceutical ingredients. Glucuronidase plays a key role in catalyzing the synthesis of glycyrrhizic acid and baicalein, catalyzing the hydrolysis of glycyrrhizic acid and baicalin to produce glycyrrhetinic acid and baicalein. However, currently discovered glucuronidases suffer from low hydrolytic activity, poor stability, and low specificity. Few enzymes are able to efficiently hydrolyze glycyrrhizic acid and baicalin to glycyrrhetinic acid and baicalein. Therefore, the search for a novel β-glucuronidase that can efficiently and specifically hydrolyze glycyrrhizic acid and baicalin to glycyrrhetinic acid and baicalein has great industrial application value. This would not only help address the environmental concerns of traditional processes but also improve product quality and yield, promoting the sustainable development of related industries. SUMMARY

[0005] To solve the above technical problems, the present application expresses β-glucuronidase from Thermobacillus composti KWC4 in Escherichia coli, and prepares recombinant β-glucuronidase with good hydrolysis specificity, thermal stability and pH stability, which can be used to efficiently degrade glycyrrhizin to prepare glycyrrhetic acid.

[0006] The first object of the present application is to provide a recombinant Escherichia coli producing β-glucuronidase, which expresses the β-glucuronidase encoding gene shown in SEQ ID NO. 1.

[0007] Further, the β-glucuronidase is derived from Thermobacillus composti KWC4.

[0008] Further, the amino acid sequence of the β-glucuronidase is shown in SEQ ID NO. 2.

[0009] Further, the β-glucuronidase is expressed using a pET28a vector.

[0010] Further, the plasmid pET28a is digested using restriction enzymes EcoR I and Xho I.

[0011] Further, the recombinant Escherichia coli uses Escherichia coli BL21(DE3) as a chassis strain.

[0012] The second object of the present application is to provide a microbial inoculant comprising the above-mentioned recombinant Escherichia coli.

[0013] The third object of the present application is to provide the use of the above-mentioned recombinant Escherichia coli or the above-mentioned microbial inoculant in the preparation of β-glucuronidase.

[0014] The fourth object of the present application is to provide a preparation method of β-glucuronidase, characterized in that the above-mentioned recombinant Escherichia coli or the above-mentioned microbial inoculant is added to the fermentation system.

[0015] The fifth object of the present application is to provide the use of the above-mentioned recombinant Escherichia coli or the above-mentioned microbial inoculant in the production of glycyrrhetic acid.

[0016] The sixth object of the present application is to provide a method for producing glycyrrhetic acid, which uses glycyrrhizin or monoglucosyl glycyrrhetic acid as a substrate and adds the above-mentioned recombinant Escherichia coli or the above-mentioned microbial inoculant to the fermentation system.

[0017] Further, the pH value of the fermentation system is 4.5-9.

[0018] Preferably, the pH value of the fermentation system is 6.5.

[0019] Further, the temperature of the fermentation system is 20-40℃.

[0020] Further, methanol is added to the fermentation system as a cosolvent.

[0021] Further, the proportion of methanol added to the fermentation system is 2-20%.

[0022] Preferably, the proportion of methanol added to the fermentation system is 10%.

[0023] A seventh object of the present application is to provide the use of the above-mentioned recombinant E. coli or the above-mentioned microbial inoculant in the production of baicalein, characterized in that the use uses baicalin as a substrate.

[0024] The beneficial effects of the present application are:

[0025] The present application screens a β-glucuronidase with excellent solubility, high catalytic efficiency and mild reaction conditions from Thermobacillus composti KWC4, which exhibits significant selective catalytic capacity for glycyrrhizinic acid substrate. Based on this, a β-glucuronidase catalytic application system in glycyrrhetic acid biosynthesis is innovatively developed, and the conversion efficiency exceeds 95%. Its precise hydrolysis specificity breaks through the technical bottlenecks of low substrate conversion rate and multiple by-products in traditional chemical methods, opens up an innovative path for the industrialized large-scale production of glycyrrhetic acid, and has broad market application potential in many fields such as medicine, food and cosmetics. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which:

[0027] Figure 1 It is a physical map of the pET28a-TcGUS recombinant plasmid in the embodiments of the present application;

[0028] Figure 2 It is a protein electrophoresis map of the recombinant β-glucuronidase in the embodiments of the present application, in which lane M is Marker, and lanes 1 and 2 are supernatant and precipitate of the recombinant genetically engineered bacteria BL21(DE3) / pET28a-TcGUS after induction, respectively;

[0029] Figure 3 It is a liquid phase detection map of the β-glucuronidase catalyzing glycyrrhizinic acid in the embodiments of the present application. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0031] The method for determining the enzyme activity of β-glucuronidase used in the following examples is as follows:

[0032] The activity of β-glucuronidase against the substrate glycyrrhizic acid was measured. The assay system consisted of an appropriate amount of enzyme solution, 1 mg / mL glycyrrhizic acid, and PBS (100 mmol / L, pH 7.0) buffer. The reaction was shaken at 1000 rpm at 40°C for 10 min. After the reaction, a sample was collected for liquid chromatography (HPLC) analysis. HPLC analysis conditions included a C18 column (250 × 4.6 mm, 5 μm), a mobile phase of methanol:0.2% phosphoric acid (95:5), a flow rate of 1 mL / min, a column oven temperature of 35°C, a detection wavelength of 250 nm, and an injection volume of 10 μL.

[0033] The enzyme activity unit (U) is defined as the amount of enzyme required for β-glucuronidase to catalyze the substrate to produce 1 μmol of product at 40°C.

[0034] The activity of β-glucuronidase against the substrate baicalin was determined. The assay system consisted of an appropriate amount of enzyme solution, 0.2 mg / mL baicalin, and PBS (100 mmol / L, pH 7.0) buffer. The reaction was incubated at 35°C with shaking at 1000 rpm for 15 min. After the reaction, samples were collected for liquid chromatography (HPLC) analysis. HPLC analysis conditions included a C18 column (250 × 4.6 mm, 5 μm), a mobile phase of methanol:0.2% phosphoric acid (85:15), a flow rate of 0.8 mL / min, a column oven temperature of 28°C, a detection wavelength of 275 nm, and an injection volume of 10 μL.

[0035] The enzyme activity unit (U) is defined as the amount of enzyme required for β-glucuronidase to catalyze the substrate to produce 1 μmol of product at 35°C.

[0036] Example 1: Construction of recombinant Escherichia coli

[0037] A β-glucuronidase encoding gene sequence derived from Thermobacillus composti KWC4 was chemically synthesized as shown in SEQ ID NO.1 and named TcGUS. The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0038] The plasmid pET28a was digested with restriction endonucleases EcoR I and Xho I, and then ligated with the nucleotide fragment of the TcGUS gene using T4 DNA ligase to obtain the recombinant expression vector pET28a-TcGUS (plasmid map shown in Figure 1 The constructed recombinant expression vector pET28a-TcGUS was transformed into competent E. coli BL21 (DE3), and coated on a kanamycin-resistant LB solid plate. After overnight culture, colony PCR was performed to verify that the positive clone was the recombinant E. coli BL21 (DE3) / pET28a-TcGUS. Positive clones were picked and cultured overnight in LB medium. The next day, they were transferred to fresh LB medium at a transfer volume of 1 mL / 100 mL and cultured until the OD 600 When the p-value reached 0.6-0.8, 0.2 mmol / L IPTG was added and cultured at 16°C for 16-18 hours. The cells were then collected by centrifugation at 8000 rpm for 5 minutes at 4°C. The collected cells were suspended in solution A (25 mmol / L Tris, 500 mmol / L NaCl, 20 mmol / L imidazole, pH = 7.4), disrupted by ultrasound, and analyzed for protein expression by SDS-PAGE.

[0039] Protein expression Figure 2 As shown, the target protein was present in a large amount in the supernatant, indicating that the recombinant enzyme was successfully expressed in a soluble form in E. coli.

[0040] Example 2: Properties of β-glucuronidase

[0041] (1) Isolation and purification of β-glucuronidase

[0042] Recombinant cells were suspended in solution A (25 mmol / L Tris, 500 mmol / L NaCl, 40 mmol / L imidazole, pH = 7.4) and ultrasonically disrupted by centrifugation to obtain a crude enzyme solution. Purification was performed using a HisTrap FF crude (nickel column) affinity column, which utilizes the histidine tag on the recombinant protein for affinity binding. First, the nickel column was equilibrated with solution A, the crude enzyme solution was loaded, and contaminants were eluted using solution A. After equilibration, gradient elution was performed with solution B (25 mmol / L Tris, 500 mmol / L NaCl, 500 mmol / L imidazole, pH = 7.4) to elute the recombinant protein bound to the nickel column, obtaining recombinant β-glucuronidase. The purified protein was analyzed by SDS-PAGE.

[0043] Protein purification results are as follows Figure 2As shown in the figure, after nickel column purification, a single band at around 70 kDa was observed with few impurities, indicating good nickel column purification. The purified β-glucuronidase was then replaced with PBS (20 mmol / L-1, pH = 7.0) using an ultrafiltration tube for enzymatic property analysis.

[0044] (2) Substrate spectrum analysis

[0045] The enzyme activity of β-glucuronidase (TcGUS) catalyzed by different triterpenoid saponin substrates (glycyrrhizic acid and monoglucose glycyrrhetinic acid) and a flavonoid substrate (baicalin) was measured. The enzyme activity measured with glycyrrhizic acid as the substrate was used as the 100% control, and the enzyme activities measured with other substrates were calculated as a percentage of the two. The results are shown in Table 1.

[0046] Table 1 Substrate spectrum analysis of TcGUS

[0047] Substrate Relative activity / % 1 Glycyrrhizic acid 100 2 Monoglucuronoglycyrrhizinic acid <1 3 Baicalin 60

[0048] Table 1 shows that TcGUS can catalyze the conversion of glycyrrhizic acid and monoglucose glycyrrhetinic acid to glycyrrhizic acid. At the same time, TcGUS can catalyze the conversion of baicalin to baicalein.

[0049] (3) Optimal pH of enzyme

[0050] 50 mmol / L PBS buffers of different pH values ​​(pH = 4.5-9.0) were prepared, and glycyrrhizic acid was used as a substrate to determine the relative enzyme activity of TcGUS in the buffers of different pH values. The results are shown in Table 2.

[0051] Table 2 Enzyme activity of TcGUS at different pH

[0052]

[0053]

[0054] Table 2 shows that the optimal reaction pH for TcGUS is 6.5, and the enzyme activity is 25.23 U / mg. In PBS buffer with a pH of 8.0-9.0, the enzyme activity drops below 30%.

[0055] (4) pH stability of enzyme

[0056] Using glycyrrhizic acid as a substrate, the residual activity of TcGUS was determined after 5 hours of incubation in 50 mmol / L PBS buffer at different pH values ​​(pH = 4.5-9.0). The residual activity of the enzyme after incubation was calculated as a percentage of the residual activity at each pH value, with the unincubated enzyme activity at each pH value being considered 100%. The results are shown in Table 3.

[0057] Table 3 pH stability of TcGUS

[0058] pH Enzyme activity (U / mg) Residual enzyme activity (%) 4.5 1.32 98.94 5.0 1.34 101 5.5 1.34 98.22 6.0 15.39 96.43 6.5 23.26 92.20 7.0 18.11 76.73 7.5 6.60 47.15 8.0 1.98 37.07 8.5 1.11 41.44 9.0 1.15 30.68

[0059] Table 3 shows that TcGUS has good stability under acidic conditions. In PBS buffer with a pH of 8.0-9.0, the enzyme activity drops below 45%.

[0060] (5) Optimal temperature of enzyme

[0061] Using glycyrrhizic acid as a substrate, the enzyme activity of TcGUS was measured at different temperatures (25-60°C) for 10 minutes. The highest enzyme activity measured was defined as 100%, and the enzyme activities measured at other temperatures were calculated as a percentage of the highest activity. The results are shown in Table 4.

[0062] Table 4 Enzyme activity of TcGUS at different temperatures

[0063] Reaction temperature (°C) Enzyme activity (U / mg) Enzyme activity percentage (%) 25℃ 30.29 91 30℃ 32.62 98 35℃ 33.41 100 40℃ 29.68 90 45℃ 18.81 56 50℃ 5.58 17 55℃ 2.88 9 60℃ 4.01 12

[0064] Table 4 shows that the optimal reaction temperature of TcGUS is 35°C and the enzyme activity is 33.34 U / mg.

[0065] (6) Enzyme thermal stability

[0066] Using glycyrrhizic acid as a substrate, the thermal stability of TcGUS was measured at 50°C, 60°C, and 70°C. The initial enzyme activity at 0 hours was defined as 100%, and the enzyme activities measured at other time points were calculated as percentages relative to the initial enzyme activity at 0 hours. The results are shown in Table 5.

[0067] Table 5 Thermal stability of TcGUS

[0068]

[0069]

[0070] Table 5 shows that the enzyme activity of TcGUS can be maintained above 80% after being kept at 50℃ for 7 days; the enzyme activity can be maintained above 80% after being kept at 60℃ for 5 days, and the activity decreases to less than 50% of the initial activity after 7 days.

[0071] (7) Kinetic parameter analysis

[0072] The kinetic parameters of TcGUS activity against the substrate glycyrrhizic acid were determined. The enzyme activity assay system was as follows: PBS buffer (100 mmol / L, pH = 6.5), substrate glycyrrhizic acid (0.15-1.5 mmol / L). The reaction rate was characterized by calculating the specific enzyme activity, and thus the kinetic parameters were calculated. The results are shown in Table 6.

[0073] Table 6 TcGUS kinetic parameters

[0074] Enzyme Km (mM) <![CDATA[kcat(s -1 )]]> kcat / Km (mM -1 ·s -1 )]]> TcGUS 2.82 59.14 20.97

[0075] Table 6 shows that the kinetic parameters of TcGUS for glycyrrhizic acid are Km 2.82 mmol / L, V max It is 102.83μmol / mg / min.

[0076] (8) Effects of metal ions on enzyme activity

[0077] Metal ions were added to the reaction system at a final concentration of 0.2 mmol / L, and residual enzyme activity was measured in PBS buffer (100 mmol / L, pH 6.5) using glycyrrhizic acid as a substrate. Under the same conditions, the enzyme activity measured without the addition of any metal ions was set as 100%, and the enzyme activity measured with the addition of metal ions was calculated as a percentage of the control. The results are shown in Table 7.

[0078] Table 7 Effects of metal ions on TcGUS activity

[0079]

[0080]

[0081] Table 7 shows that the activity of TcGUS was not inhibited when EDTA was added, but was greatly inhibited when metal ions were added to the reaction system. Therefore, the enzyme is a metal ion-independent enzyme.

[0082] (9) Effects of cosolvent and addition ratio on enzyme activity

[0083] Methanol and dimethyl sulfoxide were added to the reaction system as substrate cosolvents to a final concentration of 5% (v / v). Relative enzyme activity was measured in PBS buffer (100 mmol / L, pH = 6.5) using glycyrrhizic acid as the substrate, with the highest measured activity defined as 100%. Relative enzyme activity was measured by adding methanol to a final concentration of 2%-20% (v / v), with the highest measured activity defined as 100%. Enzyme activities measured at other concentrations were calculated as a percentage of the highest activity. The results are shown in Tables 8 and 9, respectively.

[0084] Table 8 Effect of cosolvent types on TcGUS enzyme activity

[0085] Co-solvent Relative activity Methanol 100％±1.6 Dimethyl sulfoxide 14.59％±0.2

[0086] Table 8 shows that the TcGUS enzyme activity is highest when the cosolvent is methanol.

[0087] Table 9 Effect of methanol ratio on TcGUS enzyme activity

[0088] Methanol ratio (%) Relative activity 2％ 55.64％±1.2 5％ 94.06％±4.0 10％ 100％±6.4 15％ 49.77％±1.6 20％ 37.89％±2.8

[0089] Table 9 shows that the optimal addition amount of methanol is 10%, at which point the reaction effect is the best and the enzyme activity reaches 12.1 U / mg.

[0090] Example 3: Application of β-glucuronidase in the preparation of glycyrrhetinic acid

[0091] 0.01-0.1 g of the obtained recombinant β-glucuronidase and 0.1-0.5 g of glycyrrhizic acid were dissolved in PBS buffer (pH = 6.5, 100 mmol / L) and 10% methanol (Table 10) was added to a total reaction volume of 25 mL. The reaction was maintained at 35°C and samples were taken during the reaction. The reaction was terminated when no further product increase was detected.

[0092] The detection conditions were as follows: C18 column (250×4.6 mm, 5 μm), detection wavelength was 254 nm, mobile phase was methanol:0.2% phosphoric acid (95:5), and the flow rate was 1 mL / min.

[0093] The results are shown in Table 10. β-glucuronidase TcGUS can maintain good catalytic performance and hydrolysis specificity at high substrate concentrations. The substrate conversion rate can be maintained above 99% in 10 h, and the product formation rate reaches 95%.

[0094] Table 10 Preparation of glycyrrhetinic acid by TcGUS

[0095]

[0096] Comparative Example

[0097] Referring to the methods of Examples 1 and 2, the gene sequence encoding β-glucuronidase from Aspergillus parasiticus was chemically synthesized and named ApGUS. The gene was then induced to express in Escherichia coli BL21 (DE3) using plasmid pET28a. After isolation and purification, a recombinant ApGUS enzyme solution was obtained. Referring to the method of Example 3, glycyrrhizic acid was used as a substrate, and the recombinant ApGUS enzyme solution was added to construct a reaction system. The product was detected, and the conversion rate and yield were calculated. The results are shown in Table 11. It can be seen that compared with TcGUS, the conversion rate and yield of glycyrrhizic acid converted to glycyrrhetinic acid using ApGUS were significantly reduced.

[0098] Table 11 Preparation of glycyrrhetinic acid by ApGUS

[0099]

[0100]

[0101] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A recombinant Escherichia coli producing β-glucuronidase, characterized in that: The recombinant Escherichia coli expresses the β-glucuronidase encoding gene shown in SEQ ID NO.

1.

2. A microbial agent comprising the recombinant Escherichia coli according to claim 1.

3. Use of the recombinant Escherichia coli according to claim 1 or the microbial agent according to claim 2 in the preparation of β-glucuronidase.

4. A method for preparing β-glucuronidase, characterized in that: The recombinant Escherichia coli according to claim 1 or the microbial agent according to claim 2 is added to the fermentation system.

5. Use of the recombinant Escherichia coli according to claim 1 or the microbial agent according to claim 2 in the production of glycyrrhetinic acid.

6. A method for producing glycyrrhetinic acid, characterized in that: With glycyrrhizic acid or monoglucose glycyrrhetinic acid as a substrate, the recombinant Escherichia coli according to claim 1 or the microbial agent according to claim 2 is added to the fermentation system.

7. The method according to claim 6, characterized in that: Methanol is added into the fermentation system as a cosolvent.

8. The method according to claim 7, wherein: The proportion of methanol added in the fermentation system is 2-20%.

9. Use of the recombinant Escherichia coli according to claim 1 or the microbial agent according to claim 2 in the production of baicalein, characterized in that: The application uses baicalin as a substrate.

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