An immobilization method and application for effectively improving the acid-resistant stability of zearalenone lactone hydrolase

Abstract

The present invention discloses an immobilization method and application for effectively improving the acid stability of zearalenone lactone hydrolase, belonging to the field of biotechnology. The present invention uses an injection gel method to encapsulate ZEN hydrolase in hydrogel beads based on potassium carrageenan, and the acid stability of the enzyme is improved by co-encapsulation with an alkaline buffer Mg(OH)2. The Mg(OH)2 buffer obtained by the present invention is insoluble under neutral conditions, but dissolves when hydrogen ions diffuse into the hydrogel beads, thereby ensuring a neutral internal pH value. The use of a hydrogel bead co-encapsulation system containing a Mg(OH)2 buffer improves the stability of ZEN hydrolase in an acidic environment, and is suitable for a food-grade delivery system of ZEN hydrolase for acid-sensitive enzymes, with potential for industrial application.

Description

Technical Field

[0001] The invention relates to an immobilization method and application for effectively improving the acid-resistant stability of zearalenone lactone hydrolase, belonging to the field of biotechnology. Background Art

[0002] Zearalenone (ZEN), also known as F-2 toxin, is primarily produced by Fusarium genus microorganisms during their secondary metabolism and has become one of the most widespread mycotoxins in recent years. ZEN is primarily found in moldy corn, wheat, and other moldy grains. Upon entering humans and animals through the food chain, ZEN, due to its phenolic dihydroxylactone structure similar to β-estrogen, competitively binds to β-estrogen receptors, triggering a range of reproductive toxicity, genotoxicity, carcinogenicity, and immunotoxicity. Numerous biological strategies have been developed to control this phenomenon. Among them, zearalenone lactone hydrolase (ZEN hydrolase) is a highly efficient biodegradation tool that shows great potential for ZEN biodegradation. However, the ZEN hydrolases identified so far exhibit poor acidic stability and are easily denatured and inactivated at low pH. In particular, ZEN hydrolases lose all their activity upon exposure to highly acidic gastric fluid. Therefore, the present invention is to develop an immobilization method to prepare hydrogel beads that can protect ZEN hydrolase from acid-induced inactivation when exposed to a highly acidic environment. Summary of the Invention

[0003] The present invention co-encapsulates the ZEN hydrolase derived from Gliocladium roseuma (GenBank accession number: ALI16790.1) with a sustained-release system consisting of carrageenan and magnesium hydroxide (Mg(OH)2), which significantly improves the acidic stability of the ZEN hydrolase and achieves efficient degradation of ZEN in a highly acidic environment.

[0004] The first object of the present invention is to provide a method for preparing immobilized ZEN hydrolase, which comprises:

[0005] Preparation of co-encapsulated sustained-release system of S1, carrageenan and Mg(OH)2:

[0006] Powdered carrageenan was dissolved in ultrapure water and stirred at 60°C for 1 hour. The temperature was then slowly lowered to room temperature under continuous stirring until the solution was completely dissolved to prepare a 2% (w / v) carrageenan aqueous solution. Mg(OH)2 powder was then added to the carrageenan aqueous solution and stirred for 30 minutes until the Mg(OH)2 was evenly distributed.

[0007] S2. Obtaining crude ZEN hydrolase solution:

[0008] The ZEN hydrolase derived from Gliocladium roseuma was expressed in Escherichia coli. The recombinant Escherichia coli was cultured, induced, and disrupted, and the cell supernatant was collected to obtain the crude ZEN hydrolase solution.

[0009] S3. Preparation of hydrogel beads:

[0010] 5 mL of crude zearalenone enzyme solution was added to 5 mL of the mixed solution prepared in S1, mixed in equal proportions, and stirred continuously for 30 minutes for co-encapsulation; the mixture was injected into a 10% potassium chloride (KCl) solution using a 1 mL syringe to prepare hydrogel beads; the formed hydrogel beads were kept in the KCl solution at 4°C for 30 minutes to promote the hardening of the hydrogel beads.

[0011] In one embodiment, Mg(OH)2 powder is added to a carrageenan aqueous solution to prepare solutions with concentrations of 0.1%, 0.2%, 0.3%, or 0.4%.

[0012] Preferably, the solution is prepared to have a concentration of 0.4%.

[0013] In one embodiment, the GenBank of the ZEN hydrolase is ALI16790.1, and the gene encoding the ZEN hydrolase is connected to PET-22b(+) to obtain a recombinant plasmid.

[0014] In one embodiment, the ZEN hydrolase encoding gene from Gliocladium roseuma is connected to the expression vector pet-22b(+) to obtain a recombinant plasmid, the recombinant plasmid is chemically transformed into Escherichia coli BL21(DE3), and the recombinant Escherichia coli is cultured overnight in 4 mL of liquid LB medium containing 50 mg / L ampicillin at 37°C to obtain a seed solution.

[0015] In one embodiment, the seed solution is added to 200 mL of LB liquid medium containing 50 mg / L ampicillin and cultured to an OD of 600 The pH was 0.6-0.8, and IPTG (final concentration 1 mM) was added to induce protein overexpression at 25°C for 6 h.

[0016] In one embodiment, the induced culture medium is centrifuged at 8000 rpm for 2 minutes to collect the bacterial cells for preparing a crude enzyme solution; 20 mL of pH 7.0 cell lysis buffer is added to the bacterial cells, and the cells are resuspended and ultrasonically disrupted in an ultrasonic cell disruptor for 20 minutes.

[0017] In one embodiment, after ultrasonic treatment, the cell disruption solution is centrifuged at 8000 r / min for 10 min, and the supernatant is finally filtered through a 0.45 μm microporous filter to obtain a crude enzyme solution.

[0018] Beneficial effects of the present invention:

[0019] The present invention uses an injection gel method to encapsulate ZEN hydrolase in potassium carrageenan-based hydrogel beads. The enzyme's acid stability is enhanced by co-encapsulation with the alkaline buffer Mg(OH)2. The resulting Mg(OH)2 buffer is insoluble under neutral conditions but dissolves when hydrogen ions diffuse into the hydrogel beads, thereby ensuring a neutral internal pH. This hydrogel bead co-encapsulation system containing the Mg(OH)2 buffer improves the stability of ZEN hydrolase in acidic environments, making it suitable for a food-grade ZEN hydrolase delivery system for acid-sensitive enzymes and showing potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The shapes and laser confocal microscopy images of the hydrogel beads prepared using a 1 mL syringe in Example 1 are shown.

[0021] Figure 2 This is the effect of Mg(OH)2 content in the co-encapsulated sustained-release system on the enzymatic activity of ZEN hydrolase in Example 3.

[0022] Figure 3 This is the effect of KOH content in the co-encapsulated sustained-release system on the enzymatic activity of ZEN hydrolase in Example 3.

[0023] Figure 4 Schematic diagram of the difference in acid stability between immobilized enzyme and free enzyme in the sustained-release system co-encapsulated with Mg(OH)2 in Example 4. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with the accompanying drawings and specific preferred embodiments, but this does not limit the technical solutions in the embodiments of the present invention to a clear and complete description.

[0025] Definition of enzyme activity and relative enzyme activity:

[0026] (1) Enzyme activity: The amount of enzyme required to degrade 1 μg of ZEN per unit time is 1 U.

[0027] (2) Relative enzyme activity: The initial enzyme activity under optimal conditions (35°C, pH 7.0) was defined as 100%, and the relative enzyme activity was the residual enzyme activity relative to the initial enzyme activity.

[0028] Example 1: Preparation of ZEN hydrolase hydrogel bead sustained-release system

[0029] Step 1: Preparation of a co-encapsulated sustained-release system of carrageenan and Mg(OH)2

[0030] Prepare a 2% (w / v) carrageenan aqueous solution by dissolving 0.1 g of powdered carrageenan in 5 mL of ultrapure water and stirring at 60°C for 1 hour. Then, slowly lower the temperature to room temperature while stirring until the solution is completely dissolved. Then, add different amounts of Mg(OH)2 powder to the carrageenan aqueous solution and continue stirring for 30 minutes until the Mg(OH)2 is evenly distributed.

[0031] Step 2: Obtaining crude and pure ZEN hydrolase enzyme solutions

[0032] (1) The ZEN hydrolase encoding gene from Gliocladium roseuma (GenBank accession number: ALI16790.1) was ligated to the expression vector pet-22b(+) to obtain a recombinant plasmid. The recombinant plasmid was chemically transformed into Escherichia coli BL21(DE3), and the recombinant E. coli was cultured overnight at 37°C in 4 mL of liquid LB medium containing 50 mg / L ampicillin (Amp). Subsequently, the seed solution was added to 200 mL of LB liquid medium containing 50 mg / L ampicillin and cultured to an OD of 600 At this time, IPTG (final concentration 1 mM) was added to induce protein overexpression at 25°C for 6 h.

[0033] (2) The cells were collected by centrifugation at 8000 rpm for 2 min to prepare a crude enzyme solution. 20 mL of cell lysis buffer (pH 7.0) was added to the cells, and the cells were resuspended and ultrasonically disrupted in an ultrasonic cell disruptor for 20 min. After ultrasonic treatment, the cell disruptor was centrifuged at 8000 rpm for 10 min, and the supernatant was filtered through a 0.45 μm microporous filter to obtain a crude enzyme solution.

[0034] (3) The crude enzyme solution was loaded onto a HisTrap HP Ni-NTA column at a flow rate of 0.5 mL / min. The column was flushed with buffer A (50 mM PBS, 500 mM NaCl, 50 mM imidazole, pH 7.0) to remove weakly bound proteins. Subsequently, elution was performed with buffer B (50 mM PBS, 500 mM NaCl, 500 mM imidazole, pH 7.0).

[0035] (4) The purified target enzyme was dialyzed twice in protein dialyzate A (total time of not less than 12 h) to remove impure metal ions, and then dialyzed twice in protein dialyzate B (total time of not less than 12 h), collected into a 10 mL EP tube and stored at 4 °C for later use.

[0036] Step 3: Preparation of hydrogel beads

[0037] 5 mL of ZEN hydrolase crude enzyme solution was added to 5 mL of the mixture prepared above and stirred continuously for 30 min for co-encapsulation. The mixture was injected into 10% KCl solution using a 1 mL syringe to prepare hydrogel beads ( Figure 1 The formed hydrogel beads were kept in KCl solution at 4°C for 30 min to promote the hardening of the hydrogel beads.

[0038] Example 2: Activity measurement of ZEN hydrolase hydrogel sustained-release system

[0039] The specific implementation steps are as follows:

[0040] (1) Prepare ZEN standard solution (4 mg / mL) by dissolving ZEN powder (98%) in pure acetonitrile (99.9%).

[0041] (2) The total reaction volume was 250 μL, which contained 10 hydrogel beads, 5 μL ZEN standard solution (4 mg / mL), and 245 μL sodium acetate buffer (50 mM, pH 4.0);

[0042] The control group system consisted of 20 μL crude enzyme solution (1 mg / mL), 5 μL ZEN standard solution (4 mg / mL) and 240 μL sodium acetate buffer (50 mM, pH 4.0).

[0043] After incubation in a thermostat shaker (200 rpm, 35° C.) for 2 h, 750 μL of methanol was added to terminate the reaction.

[0044] (3) To determine the ZEN content in the reaction system, the reaction system was centrifuged at 10,000 rpm for 2 min. The supernatant was collected and filtered using a 0.22 μm organic syringe filter. The supernatant was analyzed by high-performance liquid chromatography (HPLC) using a Hypersil ODS-C18 column (250 × 4.6 mm, 5 μm) and a UV detector. The mobile phase was acetonitrile:water (6:4); the flow rate was 0.6 mL / min; the injection volume was 10 μL; and the detection wavelength was 254 nm.

[0045] Example 3: Effects of different Mg(OH)2 and KOH contents on the hydrogel bead sustained-release system

[0046] Mg(OH)2 and KOH powders were added to 5 mL of a 2% (w / v) carrageenan aqueous solution to concentrations of 0.1%, 0.2%, 0.3%, and 0.4%, respectively. The mixtures were stirred on a magnetic stirrer for 30 minutes. Once the powders were evenly distributed, 5 mL of crude enzyme solution was added, stirred, and co-encapsulated. The mixtures were then injected into a 10% KCl solution using a 1 mL syringe to prepare hydrogel beads. Subsequently, enzyme activity was monitored under acidic conditions of pH 4.0 and 5.0. The relative enzyme activity after co-encapsulation with different Mg(OH)2 and KOH levels was compared, and the group with the highest activity was selected as the optimal immobilization condition.

[0047] The results are as follows Figure 2 The results showed that at pH 5.0, the crude enzyme solution without the co-encapsulated acid-base buffer Mg(OH)2 retained only approximately 15% activity. As the concentration of co-encapsulated Mg(OH)2 increased, the enzyme activity gradually increased. At a 0.4% Mg(OH)2 content, the enzyme activity within the hydrogel beads reached its highest level, maintaining over 70% activity after a 2-hour incubation. At pH 4.0, the crude enzyme solution without the co-encapsulated Mg(OH)2 had an activity of less than 5%. However, at a 0.4% Mg(OH)2 content, the enzyme activity within the hydrogel beads still retained approximately 40% activity after a 2-hour incubation. This indicates that the hydrogel sustained-release system effectively prevents acid denaturation of the ZEN hydrolase. This is because the Mg(OH)2 maintains a relatively high pH environment within the hydrogel beads, protecting the ZEN hydrolase from acid attack.

[0048] The results are as follows Figure 3 The results showed that at pH 5.0 and 4.0, the activity of the crude enzyme solution without co-encapsulated KOH was only about 15% and 5% respectively. As the concentration of co-encapsulated KOH increased, the enzyme activity remained relatively low, with no significant change. This indicates that the KOH hydrogel sustained-release system has no significant effect on the acid denaturation of ZEN hydrolase. This is because KOH is readily soluble in water and cannot be encapsulated in solid form within the hydrogel beads. When placed in a strongly acidic environment, it cannot achieve a sustained-release effect, and high concentrations of hydrogen ions still cause acid denaturation of ZEN hydrolase.

[0049] Example 4: Determination of acid stability of the sustained-release system of ZEN hydrolase and Mg(OH)2 co-encapsulated hydrogel beads

[0050] (1) Using the optimized Mg(OH)2 content in Example 3, 0.4% Mg(OH)2 powder was added to a 2% (w / v) carrageenan aqueous solution, and the ZEN hydrolase pure enzyme solution was co-encapsulated to prepare hydrogel beads.

[0051] (2) The pure enzyme solution (unencapsulated) and hydrogel beads (encapsulated) were incubated at 35°C and pH values ​​of 7.0, 6.0, 5.0, and 4.0 for 20 min, and the residual enzyme activity was determined.

[0052] (3) Record the experimental data under different pH conditions and find out the difference in acid resistance stability between the two.

[0053] The results are as follows Figure 4 The results showed that at pH 5.0, the activity of the pure enzyme solution without co-encapsulation of Mg(OH)2 was less than 40%, while the activity of the enzyme in the hydrogel beads exceeded 80% when 0.4% Mg(OH)2 was co-encapsulated. At pH 4.0, the activity of the pure crude enzyme solution was less than 5%, but the hydrogel beads co-encapsulated with 0.4% Mg(OH)2 still retained approximately 40% of the activity.

[0054] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing immobilized zearalenone (ZEN) hydrolase, characterized in that: The method is: Preparation of co-encapsulated sustained-release system of S1, carrageenan and Mg(OH)2: Dissolving powdered carrageenan in ultrapure water to prepare a carrageenan aqueous solution with a concentration of 1 to 3% (w / v), then adding Mg(OH)2 powder to the carrageenan aqueous solution, stirring evenly to obtain a mixed solution, and preparing a solution with a concentration of 0.1% to 0.4%; S2. Obtaining crude ZEN hydrolase solution: Recombinant Escherichia coli expressing ZEN hydrolase from Gliocladium roseuma was fermented to produce enzyme to obtain crude enzyme solution; S3. Preparation of hydrogel beads: The obtained crude enzyme solution is added to the mixed solution obtained in S1 for co-encapsulation, and the co-encapsulated mixture is injected into a potassium chloride solution to prepare hydrogel beads, which are hardened at 3-5°C; the crude enzyme solution and the mixed solution prepared in S1 are mixed in equal proportions.

2. The method according to claim 1, characterized in that In the S1, powdered carrageenan is dissolved in ultrapure water, stirred at 60° C. for 1 hour, and a carrageenan aqueous solution with a concentration of 1 to 3% (w / v) is prepared.

3. The method according to claim 2, characterized in that The step S1 is to add Mg(OH)2 powder into the carrageenan aqueous solution and stir until the Mg(OH)2 is evenly distributed.

4. The method according to claim 3, characterized in that Mg(OH)2 powder was added into the carrageenan aqueous solution to prepare a solution with a concentration of 0.4%.

5. The method according to any one of claims 1 to 4, characterized in that: The GenBank of the ZEN hydrolase is ALI16790.

1. The gene encoding the ZEN hydrolase is connected to a plasmid to obtain a recombinant plasmid, which is then transformed into Escherichia coli to obtain a recombinant Escherichia coli.

6. The method according to claim 5, characterized in that The obtained recombinant E. coli was cultured to OD 600 The pH value was 0.6-0.8, and IPTG was added to a final concentration of 1-2 mM, and induced at 25-30°C for 6-12 h.

7. The method according to claim 6, characterized in that The induced cells were collected, cell lysis solution was added thereto, and the crude enzyme solution was obtained after ultrasonic disruption and filtration.

8. The method according to claim 7, characterized in that After continuous stirring for 25-30 min for co-encapsulation, the mixture was injected into 10% potassium chloride solution using a 1 mL syringe.

9. The method according to claim 8, characterized in that The formed hydrogel beads were kept in a potassium chloride solution at 4° C. for 25 to 30 minutes to promote the hardening of the hydrogel beads.

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