Method for preparing sol-gel immobilized enzyme by one-pot method in cosolvent-free system

Abstract

The invention relates to a method for preparing a sol-gel immobilized enzyme by a one-pot method in a cosolvent-free system. According to the method, a cosolvent is not used, and hydrolysis of the silane precursor is completed through simple heating and stirring; meanwhile, washing the aged gel with n-hexane, removing methanol and residual water formed by hydrolysis of a silane precursor, avoiding long-term contact between a polar solvent and the enzyme, reducing the surface tension of liquid in a system and preventing excessive shrinkage of a silicon network, and finally drying to obtain the sol-gel immobilized enzyme. The method is simple in process and easy and convenient to operate. No cosolvent is used in the preparation process, so that the risk of enzyme activity loss is reduced; the accessibility of active sites of the immobilized enzyme is improved by avoiding excessive shrinkage of a silicon skeleton, and the activity of the immobilized enzyme is further improved.

Description

Technical Field

[0001] The invention belongs to the field of enzyme catalytic reaction, in particular to a method for preparing immobilized enzyme in one pot in a co-solvent-free system based on a sol-gel method. Background Art

[0002] Enzymes have high catalytic activity and substrate specificity. Enzyme-catalyzed reactions conform to the principles of green chemistry and have the advantages of environmental friendliness and high selectivity. They have been widely used in pharmaceuticals, fine chemicals, and food. Immobilized enzyme technology can effectively improve the stability of enzymes and reduce the difficulty of enzyme recovery, further enhancing the economic value of enzymes in industrial applications. Immobilization methods usually include adsorption, covalent bonding, cross-linking, and embedding. Sol-gel immobilized enzymes, as a classic encapsulation method for immobilized enzymes, have broad application potential in the preparation of immobilized enzyme particles, powders, and coatings.

[0003] Most sol-gel enzyme immobilization processes inevitably use alcohol or acid to promote the hydrolysis of silane precursors. Ganonyan et al. (Entrapment of Enzymes in Silica Aerogels [J]. Materials Today, 2020, 33: 24-35.) used hydrogen chloride as a cosolvent to promote the hydrolysis of silane precursors, and used the sol-gel method to immobilize three different enzymes to achieve enzyme encapsulation in silica aerogels, but excess acid in the system would affect the activity of the immobilized enzymes. Mangiagalli et al. (Diverse Effects of Aqueous Polar Co-solvents on Candida Antarctica Lipase B [J]. International Journal of Biological Macromolecules, 2020, 150: 930-940.) studied the effects of three co-solvents (methanol, acetone, and dimethyl sulfoxide) on the structure and function of Candida Antarctica lipase B (CALB) immobilized by the sol-gel method. The study found that the binding between solvent molecules and proteins would lead to enzyme inactivation. Moreover, excessive methanol in the system will lead to aggregation or configuration change of CALB, resulting in CALB encapsulation failure or reduced activity yield of immobilized enzyme. In addition, in the drying stage of sol-gel, in order to protect the catalytic activity of the enzyme, it is also very important to avoid excessive shrinkage of the silicon network structure. The silicon network structure will shrink or even collapse due to capillary force during the drying process, which will not only squeeze the enzyme and destroy the spatial structure of the enzyme, but also increase the difficulty of contact between the enzyme active site and the substrate.

[0004] Therefore, maintaining high storage stability and thermal stability of sol-gel immobilized enzymes and improving the accessibility of active sites of immobilized enzymes are of great significance to the production and application of sol-gel immobilized enzymes. Summary of the invention

[0005] The purpose of the present invention is to provide a one-pot sol-gel immobilized enzyme preparation method in a cosolvent-free system in view of the shortcomings of the current technology. The method avoids the use of a cosolvent and completes the hydrolysis of a silane precursor by simple heating and stirring. At the same time, the aged gel is cleaned with n-hexane to remove methanol and residual water formed by the hydrolysis of the silane precursor, avoid long-term contact between the polar solvent and the enzyme, reduce the surface tension of the liquid in the system, prevent excessive shrinkage of the silicon network, and finally dry to obtain the sol-gel immobilized enzyme. The present invention has a simple process and is easy to operate. No cosolvent is used in the preparation process, which reduces the risk of enzyme activity loss. The accessibility of the active site of the immobilized enzyme is improved by avoiding excessive shrinkage of the silicon skeleton, and the activity of the immobilized enzyme is further improved.

[0006] The technical solution of the present invention is as follows:

[0007] A method for preparing a sol-gel immobilized enzyme by a one-pot process in a co-solvent-free system, the method comprising the following steps:

[0008] (1) mixing methyl orthosilicate, methyltrimethoxysilane and deionized water in a water bath at 25 to 70° C. for 15 to 90 minutes to obtain a homogeneous solution;

[0009] Wherein, the mass ratio of methyl orthosilicate, methyltrimethoxysilane and deionized water is 1:3.65-3.75:3.10-3.15;

[0010] (2) placing the homogeneous solution obtained in step (1) in an ice bath, adding the crude enzyme solution and polyethylene glycol, and continuing to mix and stir for 15 to 20 minutes in an ice bath to obtain a sol solution;

[0011] The mass ratio of crude enzyme solution to polyethylene glycol is 1:0.045-0.075; the mass ratio of homogeneous solution to crude enzyme solution to polyethylene glycol is 1:0.4-0.6:0.025-0.035;

[0012] (3) adding sodium fluoride aqueous solution to the sol solution obtained in step (2), and continuing to mix and stir for 5 to 10 minutes in an ice bath to form a gel network;

[0013] Wherein, the mass ratio of the sol solution to the sodium fluoride aqueous solution is 1:0.080-0.086;

[0014] The enzyme in the crude enzyme solution of step (2) is Candida antarctica lipase B (CALB);

[0015] The protein concentration of CALB in the crude enzyme solution of step (2) is 2-10 wt %;

[0016] The polyethylene glycol described in step (2) is polyethylene glycol 200, polyethylene glycol 400 or polyethylene glycol 600;

[0017] The concentration of sodium fluoride in step (3) is 0.05-5 mol / L;

[0018] (4) After aging, the gel is crushed and washed with n-hexane to remove residual water and methanol produced by silane hydrolysis;

[0019] The amount of n-hexane used for each washing is 3-8 mL / g;

[0020] (5) drying the gel at 25 to 70° C. for 24 to 48 hours to obtain a sol-gel immobilized enzyme;

[0021] The sol-gel immobilized enzyme obtained by the method is used for enzyme-catalyzed reactive distillation, and the coupling of the immobilized enzyme and reactive distillation is used to synchronize the reaction and separation processes.

[0022] The essential features of the present invention are:

[0023] In current technology, most sol-gel processes for preparing immobilized enzymes inevitably use alcohol or acid to promote the hydrolysis of silane precursors to obtain sol-gel immobilized enzymes.

[0024] The present invention does not add a cosolvent during the synthesis of the immobilized enzyme, thereby reducing the risk of enzyme activity caused by the presence of high-concentration alcohol substances. This is because the silane precursor and water can be effectively hydrolyzed at the phase interface, and the methanol produced by the hydrolysis of the silane precursor can also be used as a cosolvent of the system; and n-hexane is used to wash the aged gel to remove the methanol and residual water produced by the hydrolysis of the silane precursor, shortening the contact time between CALB and alcohol, reducing the capillary force of the liquid in the silicon network structure, thereby avoiding the extrusion of the enzyme caused by the excessive contraction of the silicon network, and reducing the mass transfer resistance of the substrate from the main solution to the enzyme active site, thereby better protecting the catalytic activity of the enzyme.

[0025] The beneficial effects of the present invention are:

[0026] (1) The present invention prepares sol-gel immobilized enzymes in a one-pot process in a cosolvent-free system, avoiding the use of cosolvents, and washing the aged gel with n-hexane to remove methanol and residual water formed by hydrolysis of the silane precursor, thereby reducing the capillary force of the liquid in the silicon network structure, avoiding the squeezing of the enzyme space structure due to excessive contraction of the silicon network structure, and reducing the mass transfer resistance of the substrate from the main solution to the enzyme active site;

[0027] (2) The activity of the immobilized enzyme prepared by the present invention is increased by 365.83% compared with the activity of the conventional immobilized enzyme, providing a higher catalytic efficiency for the application of the sol-gel method to prepare the immobilized enzyme;

[0028] (3) The sol-gel immobilized enzyme obtained in the present invention has high storage stability (see Examples 3 to 8, the sol-gel immobilized enzyme still maintains relatively stable activity after being stored at 40° C. for 60 days). BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a scanning electron microscope image of the sol-gel immobilized enzyme obtained in Example 1. DETAILED DESCRIPTION

[0030] In order to more specifically illustrate the one-pot preparation of sol-gel immobilized enzyme in a co-solvent-free system of the present invention, the present invention will be further described below in conjunction with specific examples.

[0031] Example 1

[0032] (1) Weigh 0.26 g of methyl orthosilicate, 0.96 g of methyltrimethoxysilane and 0.81 g of deionized water.

[0033] (2) The methyl orthosilicate, methyltrimethoxysilane and deionized water prepared in step (1) were mixed and stirred in a 40° C. water bath for 30 minutes to obtain a homogeneous solution.

[0034] (3) All the homogeneous solutions obtained in step (2) were placed in an ice bath, and 0.06 g of polyethylene glycol 400 and 0.86 g of crude enzyme solution (CALB; protein concentration of 6 wt%) were added and continued to mix and stir for 15 minutes in an ice bath, wherein the mass of the crude enzyme solution accounted for 29 wt % of the total mass.

[0035] (4) Add 0.245 g of sodium fluoride aqueous solution (concentration: 1 mol / L) to the sol solution obtained in step (3), and continue mixing and stirring in an ice bath for 5 minutes to form a gel network.

[0036] (5) After aging, the gel was crushed and washed three times with n-hexane to remove residual water and methanol produced by silane hydrolysis.

[0037] (6) Drying the gel of step (5) in an oven at 40° C. for 48 hours to obtain a sol-gel immobilized enzyme.

[0038] The obtained immobilized enzyme was characterized by scanning electron microscopy. Figure 1 As shown, from Figure 1It can be seen that the pores and network structure of the sol-gel immobilized enzyme are large, because the residual water in the aged gel is replaced by n-hexane, which reduces the capillary force of the liquid in the silica network structure.

[0039] Example 2

[0040] The sol-gel immobilized enzyme prepared in Example 1 was subjected to a storage stability test at 40°C, and the catalytic activity of the immobilized enzyme was determined by the reaction of the immobilized enzyme catalyzing the hydrolysis of p-nitrophenyl palmitate (p-NPP) to generate p-nitrophenol (p-NP). The experimental method is as follows: weigh 0.03g of p-NPP, add 6mL of anhydrous ethanol, and oscillate and heat until the solid is completely dissolved, which is recorded as solution A; weigh 0.01g of sol-gel immobilized enzyme, and add 3mL of phosphate buffer solution (50mM, pH=7.5), which is recorded as solution B. After solution B was placed in a 37°C constant temperature water bath for incubation for 5 minutes, 200μL of solution A was added, vortexed for 30 seconds, and then placed in a constant temperature water bath to continue the reaction for 2 minutes and 30 seconds, the solution was placed in an ultraviolet spectrophotometer, and the substrate concentration was detected at 410nm. All data experiments were tested in parallel 3 times. Specific activity of an enzyme (U / g): The amount of enzyme required to catalyze the conversion of 1 micromole (μmol) of substrate into product per minute.

[0041]

[0042] Wherein, c is the concentration of p-nitrophenol (p-NP) (calculated by substituting the absorbance into the standard curve) (mmol / ml); v is the volume of the reaction system (ml); m is the amount of immobilized enzyme (g); and t is the reaction time (min).

[0043] Embodiments 3 to 8

[0044] The test method is the same as that of Example 2. Under 40°C, the enzyme activity of the sol-gel immobilized enzyme prepared on the 2nd day, the 4th day, the 9th day, the 19th day, the 35th day, and the 65th day was tested. The results are shown in Table 1.

[0045] Table 1 Storage stability test results of sol-gel immobilized enzyme at 40°C

[0046] Enzyme activity (U / g) Example 3 921.47 Example 4 974.44 Example 5 916.68 Example 6 999.86 Example 7 902.37 Example 8 1010.76

[0047] It can be seen from Table 1 that the sol-gel immobilized enzyme has good storage stability after being stored at 40°C for 60 days.

[0048] Examples 9 to 11

[0049] The difference from Example 1 is that the addition amounts of crude enzyme solution are 31wt%, 33wt% and 35wt% respectively, and the other conditions are exactly the same as those in Example 1.

[0050] Example 12

[0051] The same test method as in Example 2 was used to test the enzyme activity of the sol-gel immobilized enzymes prepared in Examples 9 to 11 at 40° C. to optimize the addition amount of the crude enzyme solution.

[0052] Table 2 Effect of enzyme addition amount on immobilized enzyme activity

[0053] Enzyme activity (U / g) Example 1 723.51 Example 9 921.47 Example 10 812.37 Embodiment 11 777.99

[0054] It can be seen from Table 2 that at 40°C, when the addition amount of the crude enzyme solution is 31 wt%, the maximum unit immobilized enzyme catalytic activity can be obtained.

[0055] Matters not covered by the present invention are known technologies.

Claims

1. A method for preparing sol-gel immobilized enzyme by a one-pot process in a co-solvent-free system, characterized in that the method comprises the following steps: (1) mixing methyl orthosilicate, methyltrimethoxysilane and deionized water in a water bath at 25 to 70° C. for 15 to 90 minutes to obtain a homogeneous solution; in, The mass ratio of methyl orthosilicate, methyltrimethoxysilane and deionized water is 1:3.65-3.75:3.10-3.15; (2) placing the homogeneous solution obtained in step (1) in an ice bath, adding the crude enzyme solution and polyethylene glycol, and continuing to mix and stir for 15 to 20 minutes in an ice bath to obtain a sol solution; The mass ratio of crude enzyme solution to polyethylene glycol is 1:0.045-0.075; the mass ratio of homogeneous solution to crude enzyme solution to polyethylene glycol is 1:0.4-0.6:0.025-0.035; (3) adding sodium fluoride aqueous solution to the sol solution obtained in step (2), and continuing to mix and stir for 5 to 10 minutes in an ice bath to form a gel network; Wherein, the mass ratio of the sol solution to the sodium fluoride aqueous solution is 1:0.080-0.086; (4) After aging, the gel is crushed and washed with n-hexane to remove residual water and methanol produced by silane hydrolysis; The amount of n-hexane used for each washing is 3-8 mL / g; (5) Drying the gel at 25-70°C for 24-48 hours to obtain a sol-gel immobilized enzyme.

2. The method for preparing sol-gel immobilized enzyme by one-pot process in a co-solvent-free system as claimed in claim 1, characterized in that the enzyme in the crude enzyme solution is Candida antarctica lipase B (CALB).

3. The method for preparing sol-gel immobilized enzyme in a one-pot process without a co-solvent system as claimed in claim 1, characterized in that The protein concentration of CALB in the crude enzyme solution of step (2) is 2-10 wt%.

4. The method for preparing sol-gel immobilized enzyme in a one-pot process without a co-solvent system as claimed in claim 1, characterized in that The concentration of sodium fluoride in step (3) is 0.05-5 mol / L.

5. The method for preparing sol-gel immobilized enzyme in a one-pot process without a co-solvent system as claimed in claim 1, characterized in that The polyethylene glycol in step (2) is polyethylene glycol 200, polyethylene glycol 400 or polyethylene glycol 600.

6. The use of the sol-gel immobilized enzyme obtained by the method of claim 1, characterized in that it is used for enzyme-catalyzed reaction distillation.

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