A method for preparing a cell surface polyethyleneimine crosslinked silicone coating and applications thereof

Abstract

The application discloses a preparation method of a polyethylene imine cross-linked organic silicon coating on a cell surface. The method uses polyethylene imine as a cross-linking agent, adds the polyethylene imine and an organic silicon precursor into a bacterial suspension according to different coating methods, cross-links the bacterial body containing the organic silicon precursor through electrostatic adsorption by virtue of high cationic property of the polyethylene imine, and obtains the bacterial body coated with the polyethylene imine cross-linked organic silicon coating. The bacterial body and intracellular protein not only have significantly improved stability, but also have significantly improved catalytic activity. In the preparation process, only a small amount of polyethylene imine is added into the organic silicon cell coating, and the coating method, the molecular weight, the concentration, the reaction time and the reaction temperature of the polyethylene imine in the preparation process are changed, so that the cells with improved stability and catalytic activity are finally obtained. The application has low preparation cost, simple process operation and is suitable for large-scale production.

Description

Technical Field

[0001] This invention pertains to the preparation and application of cell coatings, and particularly relates to a method for preparing and applying a polyethyleneimine crosslinked silicone coating on the cell surface. Background Technology

[0002] Synthetic biology, a rapidly developing emerging discipline in the 21st century, utilizes advanced modular and system design techniques to engineer biological systems and organisms. It has been widely applied to the industrial production of amino acids, organic acids, aromatic compounds, sugars, and natural products. Cell modification is a crucial aspect of synthetic biology research. Coating cell surfaces can enhance cellular resistance to adverse conditions, control cell activity and metabolism, and significantly improve cellular productivity.

[0003] The method for preparing cell coatings based on organosilicon (application number 202110455041X) utilizes the bacterial cell surface as a template. Through the reaction of the organosilicon precursor 3-aminopropyltriethoxysilane (APTES) with the active groups on the bacterial cell surface and its own condensation, an organosilicon layer is formed on the bacterial cell surface. This organosilicon coating effectively protects the bacterial cell structure from damage and improves the stability of the bacterial cell itself and its intracellular proteins. Preferably, another organosilicon precursor, tetraethyl orthosilicate (TEOS), is added before the addition of 3-aminopropyltriethoxysilane. The silica particles formed by the hydrolysis of tetraethyl orthosilicate, combined with the modification effect of 3-aminopropyltriethoxysilane on the silica particles, increase the amount of silica particles in the organosilicon coating on the bacterial cell surface. This significantly enhances the organosilicon coating's ability to maintain the stability of the cells and their intracellular proteins.

[0004] Organosilicon coatings suffer from wear and degradation during recycling, leading to a loss of enzyme protection and a rapid decrease in enzyme activity. Modifying organosilicon coatings using crosslinking agents can overcome these shortcomings during recycling. The most common crosslinking agent is glutaraldehyde (GA), but glutaraldehyde has significant cytotoxicity. Long-chain polyamines are chemical components of diatom cell walls and, under physiological conditions, induce and regulate the rapid precipitation of silica, playing a key role in the biosilicification process. Based on the concept of biomimetic silicification, similar to long-chain polyamines in diatoms, polyethyleneimine (PEI) carries a high positive charge and can branch to rapidly form silica. Furthermore, compared to glutaraldehyde, polyethyleneimine has lower cytotoxicity, and when coated on the cell surface, it has a smaller impact on the activity of intracellular enzymes. Therefore, this invention constructs a coating with higher mechanical strength and more stable chemical properties based on organosilicon coatings. This invention introduces polyethyleneimine to enhance the mechanical strength and cellular stability of the organosilicon coating, while polyethyleneimine helps maintain the activity of intracellular enzymes, resulting in cells with higher catalytic activity. The raw materials used in this invention are inexpensive, the preparation process is simple, and it is suitable for large-scale production. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a polyethyleneimine cross-linked silicone coating on the cell surface. The method provided by this invention significantly improves the stability of cells and intracellular proteins by adding a small amount of polyethyleneimine, and also yields cells with higher catalytic activity. The raw materials used in this invention are inexpensive, the preparation process is simple, and it is suitable for large-scale production.

[0006] This invention provides a method for preparing a polyethyleneimine cross-linked organosilicon coating on the cell surface. Polyethyleneimine is used as a cross-linking agent, and an organosilicon precursor is added to a bacterial suspension using different coating methods. The organosilicon coating formed by the organosilicon precursor is an organosilicon layer containing silica particles. After the cross-linking reaction, a polyethyleneimine cross-linked organosilicon coating is obtained and coated on the cell surface. Furthermore, in the method for preparing the polyethyleneimine cross-linked organosilicon coating on the cell surface described in this invention, the bacterial suspension is a Gram-positive bacterial suspension. Bacillus subtilis has a higher application range and practical use in industrial production compared to other bacterial species. This invention preferably uses Bacillus subtilis as a representative Gram-positive bacterial species.

[0007] The specific steps of the method for preparing the polyethyleneimine crosslinked silicone coating on the cell surface are as follows:

[0008] Step 1: Polyethyleneimine and organosilicon precursors are added to the bacterial suspension using different coating methods to form a bacterial suspension containing polyethyleneimine and organosilicon precursors. The mass-volume concentration of polyethyleneimine is 0.05 g / L-1 g / L, and the molecular weight of polyethyleneimine is 600 Da-70000 Da. The bacterial suspension containing polyethyleneimine and organosilicon precursors is placed on a shaker at a temperature of 4℃-37℃ and shaken for 20 min-240 min to obtain a suspension with a polyethyleneimine cross-linked organosilicon coating on the cell surface.

[0009] Step 2: Centrifuge the suspension of cells coated with polyethyleneimine cross-linked organosilicon obtained in Step 1, remove the supernatant, and wash with deionized water to finally obtain a polyethyleneimine cross-linked organosilicon coating on the cell surface.

[0010] Furthermore, in step one of the preparation method described in this invention, polyethyleneimine cross-linked organosilicon cell coatings with different degrees of cross-linking are obtained by changing different coating methods.

[0011] In the preparation method of this invention, the organosilicon precursors used in the coating of the polyethyleneimine crosslinked organosilicon cell coating are preferably tetraethyl orthosilicate and 3-aminopropyltriethoxysilane, with tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, and polyethyleneimine referred to as reagent 1, reagent 2, and reagent 3, respectively. The coating methods include, but are not limited to, the following:

[0012] 1) Add reagents 1, 2, and 3 simultaneously to the bacterial suspension to prepare a polyethyleneimine cross-linked organosilicon cell coating. This coating method is denoted as coating method 1 / 2 / 3.

[0013] 2) Add reagent 1, reagent 2 and reagent 3 to the bacterial suspension in sequence to prepare a polyethyleneimine cross-linked organosilicon cell coating. This coating method is denoted as coating method 1+2+3.

[0014] 3) Add reagent 1 and reagent 2 to the bacterial suspension in sequence, centrifuge and resuspend, then add reagent 3 to prepare a polyethyleneimine cross-linked organosilicon cell coating. This coating method is denoted as coating method 1+2+centrifugation+3.

[0015] 4) Add reagent 1 to the bacterial suspension first, and then add reagent 2 and reagent 3 to the solution at the same time to prepare a polyethyleneimine cross-linked organosilicon cell coating. This coating method is denoted as coating method 1+2 / 3.

[0016] 5) Add reagent 1, reagent 3 and reagent 2 to the bacterial suspension in sequence to prepare a polyethyleneimine cross-linked organosilicon cell coating. This coating method is denoted as coating method 1+3+2.

[0017] Furthermore, the polyethyleneimine cross-linked silicone cell coating of the present invention exhibits a higher half-life and catalytic activity. Preferably, at a temperature not exceeding 80°C, the half-life is extended by at least 20 times compared to the silicone cell coating without polyethyleneimine cross-linking with bacterial cells; more preferably, at a temperature not exceeding 70°C, the half-life is extended by at least 30 times compared to the silicone cell coating without polyethyleneimine cross-linking with bacterial cells; even more preferably, at a temperature not exceeding 60°C, the half-life is extended by at least 40 times compared to the silicone cell coating without polyethyleneimine cross-linking with bacterial cells. At a temperature not lower than 80°C, the polyethyleneimine cross-linked silicone cell coating increases the yield by at least 1.3 times compared to the silicone cell coating cross-linked with glutaraldehyde. After 10 cycles, the enzyme activity of the silicone cell coating without polyethyleneimine cross-linking is 47.98% of the initial activity, and after 20 cycles, the enzyme activity of the silicone cell coating with polyethyleneimine cross-linking decreases by no more than 45%.

[0018] The advantages of the method for preparing the polyethyleneimine crosslinked organosilicon cell coating proposed in this invention are: low raw material cost, mild preparation conditions, and simple and easy preparation process. Only a small amount of polyethyleneimine is added to the organosilicon cell coating. By changing the coating method, molecular weight, concentration, reaction time, and reaction temperature of polyethyleneimine during the preparation process, the degree of crosslinking of the polyethyleneimine crosslinked organosilicon coating can be controlled, ultimately obtaining cells with higher stability and higher catalytic performance. Compared with existing organosilicon cell coatings that are not crosslinked with bacterial cells by polyethyleneimine, the polyethyleneimine crosslinked organosilicon cell coating prepared by the method of this invention is more effective in maintaining the activity of cells and their intracellular proteins under adverse external environments (such as high temperatures). Compared with existing organosilicon cell coatings crosslinked with glutaraldehyde, the catalytic performance of the polyethyleneimine crosslinked organosilicon cell coating prepared by the method of this invention is significantly improved. Attached Figure Description

[0019] Figure 1 The graph shows the remaining activity of the polyethyleneimine cross-linked silicone cell coatings prepared in Comparative Example 1 and Example 1 after recycling. Detailed Implementation

[0020] The present invention proposes a method for preparing a polyethyleneimine crosslinked organosilicon cell coating. The design idea is to add polyethyleneimine, a crosslinking agent with high adhesion and high cationicity, to cells with organosilicon coatings, so that the organosilicon is crosslinked, resulting in a coating with a denser structure and higher mechanical stability. This makes the polyethyleneimine crosslinked organosilicon coating more effective in maintaining the stability of cells and their intracellular proteins, and at the same time, it makes the polyethyleneimine crosslinked organosilicon cells have higher catalytic activity.

[0021] The basic scheme of the preparation method of the organosilicon cell coating of the present invention is as follows: First, polyethyleneimine is added to the bacterial suspension with organosilicon cell coating and the mixture is shaken to react; then, the obtained bacterial suspension is centrifuged, the supernatant is removed, and the suspension is washed with deionized water by centrifugation. The result is cell@organosilicon@polyethyleneimine.

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, comparative examples and specific embodiments. In this description, tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, polyethyleneimine and glutaraldehyde are referred to as reagent 1, reagent 2, reagent 3 and reagent 4, respectively. The specific embodiments described are only for explanation and illustration of the present invention and are not intended to limit the present invention.

[0023] Comparative Example 1

[0024] Preparation of Bacillus subtilis with catalytically active surface-coated organosilicon (enzyme@Bacillus subtilis@organosilicon)

[0025] Step 1: Preparation of Bacillus subtilis suspension with organosilicon coating (Bacillus subtilis@organosilicon):

[0026] Add 671.7 μl of tetraethyl orthosilicate to a Bacillus subtilis suspension with a concentration of 100 OD600, bringing the final concentration of tetraethyl orthosilicate to 150 mM. Incubate at 120 rpm. -1 The mixture was reacted in a water bath at 37°C for 20 min; then 164.7 μl of 3-aminopropyltriethoxysilane was added to the bacterial suspension to bring the final concentration of 3-aminopropyltriethoxysilane to 35.15 mM. The Bacillus subtilis suspension containing 3-aminopropyltriethoxysilane was then placed in a water bath at 120 rpm for 20 min. -1 The reaction was carried out in a 37℃ water bath shaker for 160 min; centrifuged to remove the supernatant, and washed with deionized water to finally obtain Bacillus subtilis@organosilicon; the coating method was recorded as coating method 1+2.

[0027] Step 2: Place the Bacillus subtilis@organosilicon obtained in Step 1 at 120 rpm. -1 The reaction was carried out in a 70℃ water bath shaker for 30 minutes to obtain an enzyme with catalytic activity @ Bacillus subtilis @ organosilicon.

[0028] The catalytically active enzyme@Bacillus subtilis@organosilicon obtained in Comparative Example 1 was incubated at 55℃, 65℃, and 75℃, respectively. The incubated enzyme@Bacillus subtilis@organosilicon was then removed, and substrate was added to catalyze the reaction. The product content was detected by high-performance liquid chromatography (HPLC). The formula was used to... The half-life of the enzyme was calculated, where E t E0 represents the residual activity of the enzyme after incubation at a specific temperature, while K represents the initial activity of the enzyme before incubation at that specific temperature. d t represents the thermal inactivation constant at a specific temperature, and t represents the incubation time. Enzyme activity is calculated based on the activity of intracellular proteins catalyzing the production of products from the substrate. The half-life of the intracellular enzyme is the incubation time at which 50% of the intracellular enzyme activity remains. The catalytically active enzyme@Bacillus subtilis@organosilicon obtained in Comparative Example 1 was incubated at 70°C. The incubated enzyme@Bacillus subtilis@organosilicon was then removed, and substrate was added to catalyze the reaction. The product content was detected by high-performance liquid chromatography. Enzyme yield is calculated based on the content of products obtained by intracellular proteins catalyzing the substrate over 24 hours.

[0029] Calculations showed that the half-life of the enzyme@Bacillus subtilis@organosilicon coating prepared in Comparative Example 1 was 278.42 min at 55℃, 149.92 min at 65℃, and 32.78 min at 75℃. The product yield obtained from the catalytic reaction of the enzyme@Bacillus subtilis@organosilicon coating prepared in Comparative Example 1 at 70℃ was 64.65 g / L. Figure 1As shown, after 10 cycles, the enzyme activity was 47.98% of the initial activity.

[0030] Comparative Example 2

[0031] Preparation of catalytically active Bacillus subtilis with a surface-coated glutaraldehyde-crosslinked organosilicon coating (enzyme@Bacillus subtilis@organosilicon@glutaraldehyde)

[0032] Step 1: Preparation of Bacillus subtilis suspension with glutaraldehyde-crosslinked organosilicon coating (Bacillus subtilis@organosilicon@glutaraldehyde), including:

[0033] Step 1-1) Add 671.7 μl of tetraethyl orthosilicate to a Bacillus subtilis suspension with a concentration of 100 OD600, bringing the final concentration of tetraethyl orthosilicate to 150 mM. Incubate at 120 rpm. -1 React in a 37℃ water bath with a shaker for 20 minutes;

[0034] Steps 1-2) Add 164.7 μl of 3-aminopropyltriethoxysilane to the bacterial suspension to bring the final concentration of 3-aminopropyltriethoxysilane to 35.15 mM. Incubate the Bacillus subtilis suspension containing 3-aminopropyltriethoxysilane at 120 rpm. -1 The mixture was reacted in a water bath at 37°C for 160 min to obtain a bacterial suspension of Bacillus subtilis@organosilicon.

[0035] Steps 1-3) Add 100 μl of glutaraldehyde to the bacterial suspension obtained in step 1-2) to bring the final concentration of glutaraldehyde to 0.25%. Incubate the Bacillus subtilis suspension containing glutaraldehyde at 120 rpm. -1 The mixture was reacted in a 37℃ water bath shaker for 60 min; centrifuged to remove the supernatant, and washed with deionized water to finally obtain Bacillus subtilis@organosilicon@glutaraldehyde; the coating method was recorded as coating method 1+2+4.

[0036] Step 2: Place the above-mentioned Bacillus subtilis@organosilicon@glutaraldehyde at 120 rpm. -1 The reaction was carried out in a 70℃ water bath shaker for 30 minutes to obtain an enzyme with catalytic activity @ Bacillus subtilis @ organosilicon @ glutaraldehyde.

[0037] The catalytically active enzyme@Bacillus subtilis@organosilicon@glutaraldehyde obtained in Comparative Example 2 was incubated at 55℃, 65℃, and 75℃. The incubated enzyme@Bacillus subtilis@organosilicon@glutaraldehyde was then removed and added to the substrate for catalytic reaction. The product content was detected by high-performance liquid chromatography. (The formula is used to...) The half-life of the enzyme was calculated, where E tE0 represents the residual activity of the enzyme after incubation at a specific temperature, while K represents the initial activity of the enzyme before incubation at that specific temperature. d t represents the thermal inactivation constant at a specific temperature, and t represents the incubation time. Enzyme activity is calculated based on the activity of intracellular proteins catalyzing the production of products from the substrate. The half-life of the intracellular enzyme is the incubation time at which 50% of the intracellular enzyme activity remains. The catalytically active enzyme@Bacillus subtilis@organosilicon@glutaraldehyde obtained in Comparative Example 2 was incubated at 70°C. The incubated enzyme@Bacillus subtilis@organosilicon@glutaraldehyde was then removed, and substrate was added to catalyze the reaction. The product content was detected by high-performance liquid chromatography. Enzyme yield is calculated based on the content of products obtained by intracellular proteins catalyzing the substrate over 24 hours.

[0038] Calculations showed that the half-life of the enzyme@Bacillus subtilis@organosilicon coating prepared in Comparative Example 2 was 57850.62 min at 55℃, which was 207.78 times longer than that of Comparative Example 1; the half-life at 75℃ was 1718.96 min, which was 52.44 times longer than that of Comparative Example 1. The product yield of the enzyme@Bacillus subtilis@organosilicon@glutaraldehyde coating prepared in Comparative Example 2 was 51.29 g / L in the catalytic reaction at 70℃.

[0039] Example 1

[0040] Preparation of Bacillus subtilis with catalytically active surface-coated polyethyleneimine crosslinked organosilicon (enzyme@Bacillus subtilis@organosilicon@polyethyleneimine)

[0041] Step 1: Preparation of Bacillus subtilis suspension with a polyethyleneimine cross-linked organosilicon coating (Bacillus subtilis@organosilicon@polyethyleneimine), including:

[0042] To a Bacillus subtilis suspension with a concentration of 100 OD600, 671 μl of tetraethyl orthosilicate was added to bring the final concentration of tetraethyl orthosilicate to 150 mM. Simultaneously, 164.7 μl of 3-aminopropyltriethoxysilane was added to the suspension to bring the final concentration of 3-aminopropyltriethoxysilane to 35.15 mM. Also, polyethyleneimine with a molecular weight of 600 Da was added to the suspension to bring the final concentration of polyethyleneimine to 0.1 g / L. The Bacillus subtilis suspension containing polyethyleneimine was then incubated at 120 rpm. -1 The mixture was reacted in a 37℃ water bath shaker for 160 min; centrifuged to remove the supernatant, and washed with deionized water to finally obtain Bacillus subtilis@organosilicon@polyethyleneimine; the coating method was recorded as coating method 1 / 2 / 3.

[0043] Step 2: Place the above-mentioned Bacillus subtilis@organosilicon@polyethyleneimine mixture at 120 rpm. -1The reaction was carried out in a 70℃ water bath shaker for 30 minutes to obtain an enzyme with catalytic activity @ Bacillus subtilis @ organosilicon @ polyethyleneimine.

[0044] The catalytically active enzyme@Bacillus subtilis@organosilicon@polyethyleneimine obtained in Example 1 was incubated at 55°C, 65°C, and 75°C. The incubated enzyme@Bacillus subtilis@organosilicon was then removed, and substrate was added to catalyze the reaction. The product content was detected by high-performance liquid chromatography. The formula was used to... The half-life of the enzyme was calculated, where E t E0 represents the residual activity of the enzyme after incubation at a specific temperature, while K represents the initial activity of the enzyme before incubation at that specific temperature. d t represents the thermal inactivation constant at a specific temperature, and t represents the incubation time. Enzyme activity is calculated based on the activity of intracellular proteins catalyzing the production of products from the substrate. The half-life of the intracellular enzyme is the incubation time at which 50% of the intracellular enzyme activity remains. The catalytically active enzyme@Bacillus subtilis@organosilicon@polyethyleneimine obtained in Example 1 was incubated at 70°C. The incubated enzyme@Bacillus subtilis@organosilicon@polyethyleneimine was then removed, and substrate was added to catalyze the reaction. The product content was detected by high-performance liquid chromatography. Enzyme yield is calculated based on the content of products obtained by intracellular proteins catalyzing the substrate over 24 hours.

[0045] Calculations showed that the half-life of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 1 was 25956.79 min at 55°C, which was 93.23 times longer than that of Comparative Example 1; the half-life at 75°C was 1109.88 min, which was 33.86 times longer than that of Comparative Example 1. The product yield obtained from the catalytic reaction of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 1 at 70°C was 80.65 g / L, which was 1.57 times higher than that of Comparative Example 2. Figure 1 As shown, after 21 cycles, the enzyme activity was 44.91% of the initial activity.

[0046] Example 2

[0047] Example 2 is basically the same as Example 1 in terms of steps, except that in step 1, the concentration of polyethyleneimine in the solution is changed from 0.1 g / L to 0.5 g / L, and the Bacillus subtilis suspension containing polyethyleneimine is placed at 120 rpm. -1 The reaction was carried out at 37°C in a water bath with a shaker for 160 min, which resulted in the Bacillus subtilis suspension containing polyethyleneimine being placed at 120 rpm. -1 The reaction was carried out in a 25°C water bath shaker for 160 minutes. The coating method in Example 2 is coating method 1 / 2 / 3.

[0048] Calculations showed that the half-life of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 2 was 46620.16 min at 55°C, which was 167.45 times longer than that of Comparative Example 1; the half-life at 65°C was 21228.62 min, which was 141.60 times longer than that of Comparative Example 1; and the half-life at 75°C was 1200.72 min, which was 36.63 times longer than that of Comparative Example 1. The product yield obtained from the catalytic reaction of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 2 at 70°C was 78.76 g / L, which was 1.54 times higher than that of Comparative Example 2.

[0049] Example 3

[0050] Example 3 is basically the same as Example 1 in terms of steps, except that in step 1, tetraethyl orthosilicate, 3-aminopropyltriethoxysilane and polyethyleneimine are added to the bacterial suspension simultaneously, and the Bacillus subtilis suspension containing polyethyleneimine is placed at 120 rpm. -1 The reaction was carried out in a 37°C water bath with a shaker for 160 minutes. The coating method was changed from 1 / 2 / 3 to adding tetraethyl orthosilicate to the Bacillus subtilis suspension. The suspension containing tetraethyl orthosilicate was then placed in a 120 rpm water bath. -1 The mixture was reacted in a water bath at 25°C for 20 minutes, followed by the addition of 3-aminopropyltriethoxysilane to the bacterial suspension. The bacterial suspension containing 3-aminopropyltriethoxysilane was then placed at 120 rpm for 20 minutes. -1 The mixture was reacted in a water bath at 25°C for 160 min. Finally, polyethyleneimine was added to the bacterial suspension containing an organosilicon coating. The Bacillus subtilis suspension containing polyethyleneimine was then placed in a water bath at 120 rpm for 160 min. -1 The reaction was carried out in a water bath shaker at 25℃ for 30 minutes, i.e., the coating method was 1+2+3, and the concentration of polyethyleneimine in the solution was changed from 0.1g / L to 1g / L.

[0051] Calculations showed that the half-life of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 3 was 19047.35 min at 65°C, which was 127.05 times longer than that of Comparative Example 1; the half-life at 75°C was 1130.76 min, which was 34.50 times longer than that of Comparative Example 1. The product yield obtained from the catalytic reaction of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 3 at 70°C was 72.54 g / L, which was 1.41 times higher than that of Comparative Example 2.

[0052] Example 4

[0053] Example 4 is basically the same as Example 1 in terms of steps, except that in step 1, tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, and polyethyleneimine are added to the bacterial suspension simultaneously, and the Bacillus subtilis suspension containing polyethyleneimine is placed at 120 rpm. -1 The reaction was carried out in a 37°C water bath with a shaker for 160 minutes. The coating method was changed from 1 / 2 / 3 to adding tetraethyl orthosilicate to the Bacillus subtilis suspension. The suspension containing tetraethyl orthosilicate was then placed in a 120 rpm water bath. -1 The mixture was reacted in a water bath at 4°C for 20 minutes, followed by the addition of 3-aminopropyltriethoxysilane to the bacterial suspension. The bacterial suspension containing 3-aminopropyltriethoxysilane was then placed at 120 rpm for 20 minutes. -1 The mixture was reacted in a water bath at 4°C for 160 min. Finally, polyethyleneimine was added to the bacterial suspension containing an organosilicon coating. The Bacillus subtilis suspension containing polyethyleneimine was then placed in a water bath at 120 rpm for 160 min. -1 The reaction was carried out in a 4℃ water bath shaker for 30 minutes, i.e., the coating method was 1+2+3, and the concentration of polyethyleneimine in the solution was changed from 0.1g / L to 0.25g / L.

[0054] Calculations showed that the half-life of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 4 was 24093.79 min at 55°C, which was 86.54 times longer than that of Comparative Example 1; the half-life at 65°C was 12235.58 min, which was 81.61 times longer than that of Comparative Example 1; and the half-life at 75°C was 1029.77 min, which was 31.41 times longer than that of Comparative Example 1. The product yield obtained from the catalytic reaction of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 4 at 70°C was 77.33 g / L, which was 1.51 times higher than that of Comparative Example 2.

[0055] Example 5

[0056] Example 5 is basically the same as Example 1 in terms of steps, except that in step 1, tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, and polyethyleneimine are added to the bacterial suspension simultaneously, and the Bacillus subtilis suspension containing polyethyleneimine is placed at 120 rpm. -1 The reaction was carried out in a 37°C water bath with a shaker for 160 minutes. The coating method was changed from 1 / 2 / 3 to adding tetraethyl orthosilicate to the Bacillus subtilis suspension. The suspension containing tetraethyl orthosilicate was then placed in a 120 rpm water bath. -1 The mixture was reacted in a water bath at 18°C ​​for 20 minutes, followed by the addition of 3-aminopropyltriethoxysilane to the bacterial suspension. The bacterial suspension containing 3-aminopropyltriethoxysilane was then placed at 120 rpm for 20 minutes. -1The bacterial suspension was reacted in a water bath at 18°C ​​for 160 min. The supernatant was removed by centrifugation, and the suspension was washed with deionized water. Finally, polyethyleneimine was added, and the Bacillus subtilis suspension containing polyethyleneimine was incubated at 120 rpm. -1 The reaction was carried out in a water bath at 18°C ​​for 30 minutes, i.e., the coating method was 1+2+centrifugation+3, and the concentration of polyethyleneimine in the solution was changed from 0.1 g / L to 0.05 g / L.

[0057] Calculations showed that the half-life of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 5 was 10467.89 min at 55°C, which was 37.60 times longer than that of Comparative Example 1; the half-life at 75°C was 963.32 min, which was 29.39 times longer than that of Comparative Example 1. The product yield obtained from the catalytic reaction of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 5 at 70°C was 76.27 g / L, which was 1.49 times higher than that of Comparative Example 2.

[0058] Example 6

[0059] Example 6 is basically the same as Example 1 in terms of steps, except that in step 1, tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, and polyethyleneimine are added to the bacterial suspension simultaneously, and the Bacillus subtilis suspension containing polyethyleneimine is placed at 120 rpm. -1 The reaction was carried out in a 37°C water bath with a shaker for 160 minutes. The coating method was changed from 1 / 2 / 3 to adding tetraethyl orthosilicate to the Bacillus subtilis suspension. The suspension containing tetraethyl orthosilicate was then placed in a 120 rpm water bath. -1 The mixture was reacted in a water bath at 37°C for 20 minutes, followed by the addition of 3-aminopropyltriethoxysilane to the bacterial suspension. The bacterial suspension containing 3-aminopropyltriethoxysilane was then placed at 120 rpm for 20 minutes. -1 The bacterial suspension was reacted in a 37°C water bath with a shaker for 160 min. The suspension was then centrifuged to remove the supernatant, washed with deionized water, and finally polyethyleneimine was added. The Bacillus subtilis suspension containing polyethyleneimine was then incubated at 120 rpm. -1 The reaction was carried out in a 37℃ water bath shaker for 30 minutes, i.e., the coating method was 1+2+centrifugation+3, and the concentration of polyethyleneimine in the solution was changed from 0.1g / L to 1g / L.

[0060] Calculations showed that the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 6 had a half-life of 33454.62 min at 55°C, which was 120.16 times longer than that of Comparative Example 1; a half-life of 11984.28 min at 65°C, which was 79.94 times longer than that of Comparative Example 1; and a half-life of 1095.49 min at 75°C, which was 33.42 times longer than that of Comparative Example 1. The product yield obtained from the catalytic reaction of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 6 at 70°C was 72.89 g / L, which was 1.42 times higher than that of Comparative Example 2.

[0061] Example 7

[0062] Example 7 is basically the same as Example 1 in terms of steps, except that in step 1, tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, and polyethyleneimine are added to the bacterial suspension simultaneously, and the Bacillus subtilis suspension containing polyethyleneimine is placed at 120 rpm. -1 The reaction was carried out in a 37°C water bath with a shaker for 160 minutes. The coating method was changed from 1 / 2 / 3 to adding tetraethyl orthosilicate to the Bacillus subtilis suspension. The suspension containing tetraethyl orthosilicate was then placed in a 120 rpm water bath. -1 The mixture was reacted in a water bath at 25°C for 20 minutes. Then, 3-aminopropyltriethoxysilane and polyethyleneimine were added simultaneously to the bacterial suspension. The Bacillus subtilis suspension containing 3-aminopropyltriethoxysilane and polyethyleneimine was then placed at 120 rpm for 20 minutes. -1 The reaction was carried out in a water bath at 25°C for 160 minutes, i.e., the coating method was 1+2 / 3, and the concentration of polyethyleneimine in the solution was changed from 0.1 g / L to 0.25 g / L.

[0063] Calculations showed that the half-life of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 7 was 17467.89 min at 55°C, which was 62.74 times longer than that of Comparative Example 1; the half-life at 65°C was 9656.83 min, which was 64.41 times longer than that of Comparative Example 1; and the half-life at 75°C was 1064.55 min, which was 32.48 times longer than that of Comparative Example 1. The product yield obtained from the catalytic reaction of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 7 at 70°C was 73.26 g / L, which was 1.43 times higher than that of Comparative Example 2.

[0064] Example 8

[0065] Example 8 is basically the same as Example 1 in terms of steps, except that in step 1, tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, and polyethyleneimine are added to the bacterial suspension simultaneously, and the Bacillus subtilis suspension containing polyethyleneimine is placed at 120 rpm. -1 The reaction was carried out in a 37°C water bath with a shaker for 160 minutes. The coating method was changed from 1 / 2 / 3 to adding tetraethyl orthosilicate to the Bacillus subtilis suspension. The suspension containing tetraethyl orthosilicate was then placed in a 120 rpm water bath. -1 The mixture was reacted in a water bath at 18°C ​​for 20 minutes. Then, 3-aminopropyltriethoxysilane and polyethyleneimine were added simultaneously to the bacterial suspension. The Bacillus subtilis suspension containing 3-aminopropyltriethoxysilane and polyethyleneimine was then placed at 120 rpm. -1 The reaction was carried out in a water bath shaker at 18℃ for 160 minutes, i.e., the coating method was 1+2 / 3, and the concentration of polyethyleneimine in the solution was changed from 0.1g / L to 0.05g / L.

[0066] Calculations showed that the half-life of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 8 was 6163.89 min at 65°C, which was 41.11 times longer than that of Comparative Example 1; the half-life at 75°C was 930.33 min, which was 28.38 times longer than that of Comparative Example 1. The product yield obtained from the catalytic reaction of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 8 at 70°C was 74.88 g / L, which was 1.46 times higher than that of Comparative Example 2.

[0067] Example 9

[0068] Example 9 is basically the same as Example 1 in terms of steps, except that in step 1, tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, and polyethyleneimine are added to the bacterial suspension simultaneously, and the Bacillus subtilis suspension containing polyethyleneimine is placed at 120 rpm. -1 The reaction was carried out in a 37°C water bath with a shaker for 160 minutes. The coating method was changed from 1 / 2 / 3 to adding tetraethyl orthosilicate to the Bacillus subtilis suspension. The suspension containing tetraethyl orthosilicate was then placed in a 120 rpm water bath. -1 The mixture was reacted in a water bath at 4°C for 20 minutes, followed by the addition of polyethyleneimine to the bacterial suspension. The suspension containing polyethyleneimine was then placed at 120 rpm. -1 The mixture was reacted in a water bath at 4°C for 30 minutes. Finally, 3-aminopropyltriethoxysilane was added to the bacterial suspension. The Bacillus subtilis suspension containing 3-aminopropyltriethoxysilane was then incubated at 120 rpm. -1The reaction was carried out in a 4℃ water bath shaker for 160 minutes, i.e., the coating method was 1+3+2, and the concentration of polyethyleneimine in the solution was changed from 0.1g / L to 0.5g / L.

[0069] Calculations showed that the half-life of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 9 was 39919.88 min at 55°C, which was 143.38 times longer than that of Comparative Example 1; the half-life at 65°C was 18513.92 min, which was 123.49 times longer than that of Comparative Example 1; and the half-life at 75°C was 1112.61 min, which was 33.94 times longer than that of Comparative Example 1. The product yield obtained from the catalytic reaction of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 9 at 70°C was 70.53 g / L, which was 1.38 times higher than that of Comparative Example 2.

[0070] Example 10

[0071] Example 10 is basically the same as Example 1 in terms of steps, except that in step 1, tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, and polyethyleneimine are added to the bacterial suspension simultaneously, and the Bacillus subtilis suspension containing polyethyleneimine is placed at 120 rpm. -1 The reaction was carried out in a 37°C water bath with a shaker for 160 minutes. The coating method was changed from 1 / 2 / 3 to adding tetraethyl orthosilicate to the Bacillus subtilis suspension. The suspension containing tetraethyl orthosilicate was then placed in a 120 rpm water bath. -1 The mixture was reacted in a water bath at 37°C for 20 minutes, followed by the addition of polyethyleneimine to the bacterial suspension. The suspension containing polyethyleneimine was then placed at 120 rpm. -1 The mixture was reacted in a water bath at 37°C for 30 minutes. Finally, 3-aminopropyltriethoxysilane was added to the bacterial suspension. The Bacillus subtilis suspension containing 3-aminopropyltriethoxysilane was then incubated at 120 rpm for 30 minutes. -1 The reaction was carried out in a 37℃ water bath shaker for 160 minutes, i.e., the coating method was 1+3+2.

[0072] Calculations showed that the half-life of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 10 was 18617.04 min at 55°C, which was 66.87 times longer than that of Comparative Example 1; the half-life at 75°C was 895.05 min, which was 27.30 times longer than that of Comparative Example 1. The product yield obtained from the catalytic reaction of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 10 at 70°C was 75.91 g / L, which was 1.48 times higher than that of Comparative Example 2.

[0073] Table 1 shows the thermal stability and catalytic activity of enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coatings prepared under different coating methods, concentrations, and reaction temperatures.

[0074] Table 1

[0075]

[0076] Example 11

[0077] Example 11 is basically the same as Example 1 in terms of steps, except that in step 1, the concentration of polyethyleneimine in the solution is changed from 0.1 g / L to 1 g / L, and the Bacillus subtilis suspension containing tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, and polyethyleneimine is placed at 120 rpm. -1 The reaction was carried out at 37°C in a water bath with a shaker for 160 min, which resulted in a suspension of Bacillus subtilis containing tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, and polyethyleneimine being placed at 120 rpm for 120 min. -1 React in a 37℃ water bath shaker for 240 minutes.

[0078] Calculations showed that the half-life of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 11 was 45956.79 min at 55°C, which was 165.06 times longer than that of Comparative Example 1; the half-life at 75°C was 2109.88 min, which was 64.36 times longer than that of Comparative Example 1. The product yield obtained from the catalytic reaction of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 11 at 70°C was 77.89 g / L, which was 1.52 times higher than that of Comparative Example 2.

[0079] Example 12

[0080] Example 12 is basically the same as Example 1 in terms of steps, except that in step 1, the molecular weight of polyethyleneimine in the solution is changed from 600 Da to 1800 Da, and the concentration of polyethyleneimine in the solution is changed from 0.1 g / L to 0.5 g / L. The Bacillus subtilis suspension containing tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, and polyethyleneimine is placed at 120 rpm. -1 The reaction was carried out at 37°C in a water bath with a shaker for 160 min, which resulted in a suspension of Bacillus subtilis containing tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, and polyethyleneimine being placed at 120 rpm for 120 min. -1 React in a 37℃ water bath shaker for 200 minutes.

[0081] Calculations showed that the half-life of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 12 was 7533.98 min at 65°C, which was 50.25 times longer than that of Comparative Example 1; the half-life at 75°C was 1465.17 min, which was 44.70 times longer than that of Comparative Example 1. The product yield obtained from the catalytic reaction of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 12 at 70°C was 80.65 g / L, which was 1.57 times higher than that of Comparative Example 2.

[0082] Example 13

[0083] Example 13 is basically the same as Example 1 in terms of steps, except that in step 1, the molecular weight of polyethyleneimine in the solution is changed from 600 Da to 10000 Da, and the concentration of polyethyleneimine in the solution is changed from 0.1 g / L to 0.25 g / L. The Bacillus subtilis suspension containing tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, and polyethyleneimine is placed at 120 rpm. -1 The reaction was carried out at 37°C in a water bath with a shaker for 160 min, which resulted in a suspension of Bacillus subtilis containing tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, and polyethyleneimine being placed at 120 rpm for 120 min. -1 React in a 37℃ water bath shaker for 120 minutes.

[0084] Calculations showed that the half-life of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 13 was 10634.10 min at 55°C, which was 38.19 times longer than that of Comparative Example 1; the half-life at 75°C was 1281.71 min, which was 39.10 times longer than that of Comparative Example 1. The product yield obtained from the catalytic reaction of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 13 at 70°C was 78.76 g / L, which was 1.54 times higher than that of Comparative Example 2.

[0085] Example 14

[0086] Example 14 is basically the same as Example 1 in terms of steps, except that in step 1, the molecular weight of polyethyleneimine in the solution is changed from 600 Da to 70000 Da, and the concentration of polyethyleneimine in the solution is changed from 0.1 g / L to 0.05 g / L.

[0087] Calculations showed that the half-life of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 14 was 10106.1 min at 65°C, which was 67.41 times longer than that of Comparative Example 1; the half-life at 75°C was 1621.10 min, which was 49.45 times longer than that of Comparative Example 1. The product yield obtained from the catalytic reaction of the enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coating prepared in Example 14 at 70°C was 81.54 g / L, which was 1.59 times higher than that of Comparative Example 2.

[0088] Table 2 shows the thermal stability and catalytic activity of enzyme@Bacillus subtilis@organosilicon@polyethyleneimine coatings prepared with different molecular weights, concentrations, and reaction times of polyethyleneimine.

[0089] Table 2

[0090]

[0091] In summary, the preparation method described in this invention utilizes cells coated with an organosilicon coating. By adding polyethyleneimine, a cross-linking agent with high adhesion and cationicity, the bacterial cells containing organosilicon precursors are cross-linked through electrostatic adsorption. This results in bacterial cells that are superior to those with organosilicon coatings in protecting the bacterial structure from damage and improving the stability of the bacterial cells and intracellular proteins. The yield is also higher than that of bacterial cells with glutaraldehyde-crosslinked organosilicon coatings. This invention significantly enhances the ability of the organosilicon coating to maintain the stability of cells and their intracellular proteins by adding only a small amount of polyethyleneimine to the organosilicon cell coating, while simultaneously giving the cells coated with polyethyleneimine-crosslinked organosilicon coatings higher catalytic activity. This invention improves the stability of intracellular proteins and catalytic activity by changing the coating method, molecular weight, concentration, reaction temperature, and reaction time of polyethyleneimine during the preparation process. This invention has low preparation cost, simple operation, and is suitable for large-scale production.

[0092] Although the present invention’s polyethyleneimine crosslinked silicone cell coating and its application in improving intracellular protein stability have been described above with reference to the accompanying drawings, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many modifications under the guidance of the present invention without departing from the spirit of the present invention, and these modifications are all within the protection scope of the present invention.

Claims

1. A method for preparing a polyethyleneimine cross-linked organosilicon coating on a cell surface, characterized in that, Polyethyleneimine is used as a crosslinking agent and is added to a bacterial suspension along with an organosilicon precursor in different coating methods. The organosilicon coating formed by the organosilicon precursor is an organosilicon layer containing silica particles. After the crosslinking reaction, a polyethyleneimine crosslinked organosilicon coating is obtained and coated on the cell surface. The preparation steps are as follows: Step 1: Polyethyleneimine and organosilicon precursors are added to the bacterial suspension using different coating methods to form a bacterial suspension containing polyethyleneimine and organosilicon precursors. The mass-volume concentration of polyethyleneimine is 0.05 g / L-1 g / L, and the molecular weight of polyethyleneimine is 600 Da-70000 Da. The bacterial suspension containing polyethyleneimine and organosilicon precursors is placed on a shaker at a temperature of 4℃-37℃ and shaken for 20 min-240 min to obtain a suspension with a polyethyleneimine cross-linked organosilicon coating on the cell surface. The organosilicon precursor includes tetraethyl orthosilicate and 3-aminopropyltriethoxysilane, denoted as Reagent 1 and Reagent 2, respectively. The polyethyleneimine is denoted as Reagent 3. Using polyethyleneimine as a crosslinking agent, it is added to the bacterial suspension along with the organosilicon precursor according to one of five coating methods: A) Add reagents 1, 2, and 3 to the bacterial suspension simultaneously; B) Add reagent 1, reagent 2 and reagent 3 to the bacterial suspension in sequence; C) Add reagent 1 and reagent 2 to the bacterial suspension in sequence, centrifuge and resuspend, then add reagent 3; D) Add reagent 1 to the bacterial suspension first, and then add reagent 2 and reagent 3 to the bacterial suspension at the same time; E) Add reagent 1, reagent 3, and reagent 2 to the bacterial suspension in sequence; Step 2: Centrifuge the suspension of cells coated with polyethyleneimine cross-linked organosilicon obtained in Step 1, remove the supernatant, and wash with deionized water to finally obtain a polyethyleneimine cross-linked organosilicon coating on the cell surface. The degree to which the half-life of the polyethyleneimine cross-linked silicone coating on the cell surface is prolonged compared with that of the silicone cell coating without polyethyleneimine cross-linking with the bacterial cells under the following temperature conditions is as follows: At a temperature not exceeding 80°C, the half-life of the polyethyleneimine cross-linked silicone coating on the cell surface is prolonged by at least 20 times compared with that of the silicone cell coating without polyethyleneimine cross-linking with the bacterial cells; at a temperature not exceeding 70°C, the half-life of the polyethyleneimine cross-linked silicone coating on the cell surface is prolonged by at least 30 times compared with that of the silicone cell coating without polyethyleneimine cross-linking with the bacterial cells; at a temperature not exceeding 60°C, the half-life of the polyethyleneimine cross-linked silicone coating on the cell surface is prolonged by at least 40 times compared with that of the silicone cell coating without polyethyleneimine cross-linking with the bacterial cells. The polyethyleneimine cross-linked silicone coating on the cell surface, when the temperature is not higher than 80°C, has a yield that is at least 1.3 times higher than that of the glutaraldehyde cross-linked silicone cell coating. The activity of the enzyme in the polyethyleneimine cross-linked silicone coating on the cell surface decreases by no more than 45% after 20 cycles of use.

2. The method for preparing a polyethyleneimine cross-linked organosilicon coating on the cell surface according to claim 1, characterized in that, The bacterial suspension is a Gram-positive bacterial suspension.

3. The method for preparing a polyethyleneimine cross-linked organosilicon coating on the cell surface according to claim 2, characterized in that, The Gram-positive bacterial suspension mentioned above is Bacillus subtilis.

4. The method for preparing a polyethyleneimine cross-linked organosilicon coating on the cell surface according to claim 3, characterized in that, In step one, polyethyleneimine cross-linked silicone cell coatings with different degrees of cross-linking are obtained through different coating methods.

5. The application of a polyethyleneimine cross-linked silicone coating on the cell surface, characterized in that, The polyethyleneimine cross-linked organosilicon coating on the cell surface prepared by any one of claims 1 to 4 is used to maintain the activity of cells and their intracellular proteins under adverse external environments; during cell catalysis, it improves cell stability and catalytic activity, thereby improving cell recyclability.

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