Method for mechanochemical remodeling of new function of plant SOD (superoxide dismutase) enzyme

Abstract

The invention discloses a method for improving the activity and stability of SOD (superoxide dismutase) of plants. The plant SOD enzyme is derived from copper-zinc superoxide dismutase of corn. Comprising the following steps: placing the plant SOD enzyme in a centrifugal tube at normal temperature, and then carrying out ball milling to obtain the SOD enzyme subjected to the action of the mechanical force. Compared with the unacted SOD enzyme, the activity of the SOD enzyme is improved by 57%. Compared with a traditional plant SOD enzyme modification method, chemical reagents or organic solvents are not needed, the problem of environmental pollution is avoided, and the method has the advantages of being green and environmentally friendly. The operation process is relatively simple, enzyme modification can be achieved only by adjusting the rotating speed and the ball milling time of the ball mill, and industrial production is easy to achieve. Meanwhile, the prepared plant SOD enzyme has the characteristics of high activity, high temperature resistance, solvent resistance and high stability.

Description

Technical Field

[0001] This invention relates to the technical field of mechanochemical modification, and specifically to a novel method for mechanochemical modification of plant SOD enzymes. Background Technology

[0002] Mechanochemistry, fundamentally, is an interdisciplinary field that studies the chemical or physical changes induced by the interaction between mechanical energy and matter. Its core lies in directly driving atomic or molecular-scale reconstruction through mechanical means such as shearing, compression, and friction. From a materials science perspective, this process breaks through the limitations of traditional thermodynamic pathways, providing a disruptive technological route for the preparation of novel functional materials (such as high-entropy alloys, nanocomposite materials, and amorphous substances). The mechanism and source of mechanical force are key to understanding changes in material structure and properties. When mechanical force acts on matter, it can induce significant changes in crystal structure, including the generation of lattice defects, lattice distortion, crystal form transformation, and a decrease in crystallinity, even causing the matter to transform from a crystalline state to an amorphous state. Simultaneously, the surface energy of the matter increases under mechanical force, and the number of surface active sites also increases accordingly, further generating activation and inducing chemical reactions.

[0003] Superoxide dismutase (SOD) is a class of antioxidant enzymes widely found in living organisms. It catalyzes the dismutation of superoxide anion free radicals into oxygen and hydrogen peroxide, thereby effectively eliminating reactive oxygen species and protecting cells from oxidative damage. SOD enzymes have shown great potential in the fields of antioxidation and anti-radiation, but they still face some challenges in practical applications. For example, the activity of SOD enzymes is sensitive to environmental conditions; high temperatures, solvents, and high-energy radiation can easily lead to structural damage and loss of activity.

[0004] Based on this, the present invention develops a method to simultaneously enhance the activity and stability of plant SOD enzymes, namely, by modifying plant SOD enzymes through mechanical force. Results show that this method increases plant SOD enzyme activity by approximately 57%, while significantly improving tolerance to nonpolar solvents and thermal stability. Summary of the Invention

[0005] The purpose of this invention is to provide a method for simultaneously improving the activity and stability of plant SOD enzymes. This method involves applying mechanical force to the plant SOD enzyme by vibrating a grinding ball together with the enzyme in a grinding jar, providing friction to modify the enzyme. This method is simple to implement, has a short preparation time, and produces plant SOD enzymes with high activity and good stability.

[0006] The technical solution adopted to achieve the purpose of this invention is: a method for improving the activity and stability of plant SOD enzyme, characterized in that: by solid-phase ball milling of plant SOD enzyme, the conformation of the enzyme is adjusted under the action of mechanical force, and the contact between the active center of plant SOD enzyme and the substrate is strengthened, thereby improving the activity and stability of plant SOD enzyme.

[0007] A method for preparing plant SOD enzyme with enhanced activity and stability, wherein the specific operation of the preparation method is as follows: plant SOD enzyme and ball milling beads are added to a centrifuge tube at room temperature, and then a ball milling reaction is carried out. After the reaction is completed, the mechanochemically modified plant SOD enzyme is obtained.

[0008] In some embodiments, the plant SOD enzyme is an extract of prickly pear, corn, etc., or copper-zinc superoxide dismutase (Cu / Zn-SOD).

[0009] In some embodiments, the grinding balls are zirconia grinding balls.

[0010] In some embodiments, the ball mill is a vibratory ball mill.

[0011] In some embodiments, the ball mill type is a vibratory ball mill.

[0012] In some embodiments, the ball milling speed is 1200-1800 rpm and the time is 10-90 minutes.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] 1. The activity of the plant SOD enzyme of the present invention is increased by 57% compared with that of unused SOD enzyme.

[0015] 2. The advantage of this invention is that no chemical reagents or organic solvents are needed in the process of modifying plant SOD enzymes, thus avoiding environmental pollution problems and having the characteristics of being green and environmentally friendly.

[0016] 3. The advantage of the invention is that the operation process is relatively simple. For example, the modification of enzymes can be achieved simply by adjusting the speed of the ball mill and the ball milling time, which makes it easy to realize industrial production.

[0017] 4. The advantages of this invention are that the prepared plant SOD enzyme has the characteristics of high activity, high temperature resistance, solvent resistance and high stability. Attached Figure Description

[0018] Figure 1 Images showing the activity of plant SOD enzymes at different ball milling speeds obtained in Example 1;

[0019] Figure 2Images showing the activity of plant SOD enzymes under different ball milling times obtained in Example 2;

[0020] Figure 3 Images showing the changes in the Michaelis constant of plant SOD enzyme before and after ball milling, obtained in Example 3;

[0021] Figure 4 The image shows the effect of ball milling on the temperature stability of plant SOD enzymes obtained in Example 3.

[0022] Figure 5 Images showing the tolerance of plant SOD enzymes in different organic solvents obtained in Example 3;

[0023] Figure 6 The image shows the changes in the Michaelis constant of plant SOD enzyme before and after ball milling, as obtained in Example 3. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0025] Example 1 (Speed ​​adjustment at fixed intervals)

[0026] A method for enhancing the activity and stability of plant SOD enzymes, comprising the following steps:

[0027] First, weigh 200mg of plant SOD enzyme and add it to a 2mL round-bottom plastic centrifuge tube. Then add 10 clean zirconia microspheres with a diameter of 5mm and 100mg of clean zirconia microspheres with a diameter of 2mm.

[0028] Then, place it in the mold of a vibratory ball mill, adjust the rotation speed to 200-1200 rpm, and the ball milling time to 10-90 minutes to carry out sufficient stress modification, and the modified plant SOD enzyme can be obtained.

[0029] The plant SOD enzyme prepared in Example 1 is characterized in that: the modification method of the plant SOD enzyme is to apply mechanical force to the plant SOD enzyme, and the milling beads vibrate together with the plant SOD enzyme in the milling jar to provide frictional force, thereby modifying the plant SOD enzyme. Figure 1 Images show the activity of plant SOD enzymes at different ball milling speeds, indicating that the activity is highest at 800 rpm.

[0030] Example 2 (Fixed speed adjustment time)

[0031] A method for enhancing the activity and stability of plant SOD enzymes, comprising the following steps:

[0032] First, weigh 200mg of plant SOD enzyme and add it to a 2mL round-bottom plastic centrifuge tube. Then add 10 clean zirconia microspheres with a diameter of 5mm and 100mg of clean zirconia microspheres with a diameter of 2mm.

[0033] Then, place it in the mold of a vibratory ball mill, adjust the rotation speed to 200-1200 rpm, and the ball milling time to 10-90 minutes to carry out sufficient stress modification, and the modified plant SOD enzyme can be obtained.

[0034] The plant SOD enzyme prepared in Example 2 is characterized in that: the modification method of the plant SOD enzyme is to apply mechanical force to the plant SOD enzyme, and the milling beads vibrate together with the plant SOD enzyme in the milling jar to provide frictional force, thereby modifying the plant SOD enzyme. Figure 2 Images were taken to explore the activity of plant SOD enzymes under different ball milling times. Figure 2 It can be seen that at 800 rpm and 30 min, the slope of UV absorbance versus time is the smallest, the auto-oxidation rate of pyrogallol is the lowest, and the activity of plant SOD enzyme is the highest.

[0035] Example 3

[0036] The plant SOD enzymes obtained in Examples 1 and 2 were compared in terms of Michaelis constant, temperature tolerance, solvent tolerance, and CD spectrum. Figure 3 Images showing the changes in the Michaelis constant of plant SOD enzyme before and after ball milling. Figure 3 It can be seen that the Michaelis constant of plant SOD enzyme before ball milling was 60.3, and the Michaelis constant of plant SOD enzyme before ball milling was 39.8. The Michaelis constant value decreased significantly, indicating that after solid-phase ball milling, plant SOD enzyme is more likely to bind to the substrate superoxide anion, thus improving its catalytic performance.

[0037] Figure 4 Images showing the effect of ball milling on the temperature stability of plant SOD enzymes. Figure 4 It can be seen that the temperature tolerance of plant SOD enzymes increased after ball milling. After ball milling, the activity of plant SOD enzymes at 60℃ for 24 hours approached 100%, while the activity of plant SOD enzymes before ball milling approached 80%. After 24 hours at 80℃, the activity of plant SOD enzymes after ball milling approached 60%, while the activity of plant SOD enzymes before ball milling approached 40%.

[0038] Figure 5 Images showing the tolerance of plant SOD enzymes in different organic solvents, from... Figure 5 It can be seen that ball milling improved the solvent tolerance of plant SOD enzymes. In aqueous solution, ball milling increased the solvent tolerance of plant SOD enzymes by 40%. In methanol solution, the solvent tolerance of plant SOD enzymes increased by 30%.

[0039] Figure 6 The images show the circular dichroism spectrum and two-dimensional structural changes of plant SOD enzymes before and after ball milling. Figure 6 It can be seen that the proportion of β-sheets increased after ball milling, indicating that ball milling can improve the stability of plant SOD enzymes.

[0040] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for enhancing the activity and stability of plant SOD enzymes, characterized in that: By solid-phase ball milling of plant SOD enzymes, the conformation of the enzymes is adjusted under mechanical force, enhancing the contact between the active site of plant SOD enzymes and the substrate, thereby improving the activity and stability of plant SOD enzymes.

2. The method for enhancing the activity and stability of plant SOD enzyme according to claim 1, characterized in that: The method for modifying plant SOD enzyme involves applying mechanical force to the plant SOD enzyme, with the milling beads vibrating together with the plant SOD enzyme in a milling jar to provide friction and thus modify the plant SOD enzyme. The method of the present invention has good versatility and is applicable to a variety of different plant SOD enzymes.

3. The method for enhancing the activity and stability of plant SOD enzyme according to claim 1, characterized in that: The plant SOD enzyme prepared by this invention has the advantages of high activity, high temperature resistance, solvent resistance, and high stability. At the same time, the preparation process is simple, the time cycle is short, and it has the potential for large-scale commercialization.

4. The method for enhancing the activity and stability of plant SOD enzyme according to claim 1, characterized in that: This invention not only modifies plant SOD enzymes through solid-phase ball milling, but also introduces other functional small molecules to modify plant SOD enzymes, thereby expanding their functional applications.

5. The method for enhancing the activity and stability of plant SOD enzyme according to claim 1, characterized in that: The selected ball mill is a vibratory ball mill.

6. The method for enhancing the activity and stability of plant SOD enzyme according to claim 1, characterized in that: The ball milling speed is 200-1800 rpm, and the time is 10-90 minutes.

7. The method for enhancing the activity and stability of plant SOD enzyme according to claim 1, characterized in that: The highly active and stable plant SOD enzyme prepared by this invention can be applied to the prevention and subsequent treatment of radiation dermatitis. It also has significant commercial value and can be used in related fields such as cosmetics, healthcare, and food.

Images