WO2007112679A1

WO2007112679A1 - Silk fibroin nanoparticles fixed with enzyme and their production

Info

Publication number: WO2007112679A1
Application number: PCT/CN2007/001032
Authority: WO
Inventors: Yuqing Zhang
Original assignee: Yuqing Zhang
Priority date: 2006-03-30
Filing date: 2007-03-29
Publication date: 2007-10-11
Also published as: CN1834240A; CN100427593C

Abstract

Silk fibroin nanoparticles fixed with enzyme and their production method are disclosed. The method comprises the steps of: (1) completely mixing the enzyme and regenerated silk fibroin solution; (2) injecting into a water-soluble organic solvent under high speed stirring to obtain white crystalline silk fibroin nanoparticles fixed with the enzyme; and (3) centrifuging or filtrating to remove the organic solvent to obtain crystalline silk fibroin nanoparticles fixed with the enzyme. The average particle size of the nanoparticles is 35-125 nm, and the activity recovery is as high as 70%. The fixed enzyme has a high thermal stability, and is not easy to be decomposed by proteinase, therefore can largely reduce or even eliminate the immunogenicity of the enzyme. The nanoparticles fixed with the enzyme have important applications in sustained drug release, industrial enzyme reactor, food additives and cosmetics.

Description

Immobilized enzyme silk fibroin nanoparticle and preparation method thereof

Technical field

The invention discloses an immobilized enzyme carrier and a preparation method thereof, in particular to a silk fibroin nanoparticle prepared by using silk fibroin to produce an immobilized enzyme and a preparation method thereof, belonging to the field of biotechnology enzyme engineering, and also belonging to the field of nanotechnology. And the field of polymer chemistry technology. Background technique

Introducing the enzyme or drug into the body in the form of microparticles, microcapsules, liposomes, albumin, red blood cell carrier, etc., can prolong the half-life of the enzyme or drug, and release it slowly, extracting the enzyme or drug for hereditary enzyme deficiency, metabolism Therapeutic effects of disorders, tumors, and cardiovascular diseases. The use of natural biomaterials as an enzyme-immobilized carrier or as a sustained-release carrier for drugs has been studied because of its safety, stability, biocompatibility and metabolism in the body. The silk fibroin produced by silkworm is a natural terpene molecular polymer with a molecular weight of up to 370,000. Because this protein is non-toxic, harmless, and non-immunological to humans, it has long been used as a surgical suture. In recent years, development and utilization of silk fibroin in raw materials for artificial skin, cosmetics, and nutritious foods, or as carriers for immobilized cells, enzymes, and antibodies have been attracting attention. In particular, studies using silk fibroin as an immobilized enzyme carrier have been particularly active over the past two decades, and many experiments have fully confirmed that silk fibroin is an excellent immobilized enzyme biomaterial.

Silk fibroin as an enzyme immobilization carrier has advantages not found in other polymer materials. The principle of enzyme immobilization is based on the antiparallel of silk fibroin from soluble random coils and (X-helix structure to insoluble) The β-sheet structure, at the same time, completes the immobilization of the enzyme. Silk fibroin is used as an immobilized enzyme carrier in various forms such as silk fibroin film, silk fibroin fiber, silk fibroin powder or silk fibroin gel. The preparation and application research is very active, and there have been a large number of patent reports, mainly through the physical or chemical methods to induce silk fibroin condensation such as salting out, ultrasonic, aeration, high-speed stirring, isoelectric point condensation, electrolysis or prior After the film is mechanically pulverized or directly subjected to mechanical pulverization of the fiber, the ultrafine silk fibroin powder of 1 to 100 micrometers is prepared, and is widely used in cosmetics, nutraceuticals, additives, enamel water absorbing materials and coatings. Application prospects However, most of these methods or processing environments are not suitable for the preparation of immobilized enzymes with high activity and stable performance.

1

Fibre textbook Chinese Patent No. WO1560136 (International Patent WO 2005085327 A1), "Method for Producing Silk Fibro Nanoparticles", discloses a method of mixing a water-soluble regenerated silk fibroin solution directly with an excess of a water-miscible organic solvent. A preparation method of silk fibroin nanoparticles is prepared, but some organic solvents cause deactivation of the enzyme and are not suitable for preparing an immobilized enzyme.

The application of silk fibroin powder for the immobilization of enzymes and active substances originated in the early 1980s, mainly using salting out, high-speed agitation, protein freezing and thawing, adding a small amount of organic solvent or adjusting the isoelectric point to make silk fibroin. Precipitation, coagulation or denaturation, followed by drying and pulverization to prepare an immobilized enzyme. The water-soluble silk fibroin and the enzyme mixture are prepared by salting out an inorganic salt or an inorganic salt and an organic salt mixture to prepare an immobilized enzyme (Japanese Patent, JP-A-JP56-15687; JP-A-JP60-155125, International Patent, WO8503230; Special opening JP62-151180); or the silk fibroin and enzyme mixture first adjusted to acidic or alkaline, and then salted out, washed, dried to form a powdered immobilized enzyme (Japanese Patent, Special Open JP56-051983, JP-A-JP56-39783). In the silk fibroin and enzyme mixture, a small amount of organic solvent such as methanol is added to directly induce the aggregation of silk fibroin, or the silk fibroin mixture is first adjusted to the vicinity of the isoelectric point of silk fibroin, and a small amount of organic solvent is added to induce the aggregation of silk fibroin, and then the condensation is performed. After washing, drying and pulverizing, it is made into immobilized α-starch and sucrase (Japanese Patent, JP-A-JP1-313530), or made into an immobilized alkaline or neutral protease (Japanese Patent, JP-A-JP56-18590) ). The immobilized enzyme is produced by causing the silk fibroin and the enzyme mixture to generate a large amount of foam by agitation or inflating of the silk fibroin (Japanese Patent No. JP-A-60-227679, JP-A-61-187790). The mixture of silk fibroin and enzyme is first frozen and then thawed to denature the silk fibroin to prepare an immobilized alkaline phosphatase (Japanese Patent Laid-Open No. JP 01-120287). The silk fibroin powder is first prepared by salting out, and then the cross-linking agent is used to crosslink the silk fibroin powder with the saccharifying enzyme to prepare an immobilized enzyme powder [Sericulture Science, 25(2): 113-119, 1999 ]. In addition, there are no reports on immobilized enzymes using silk fibroin as a carrier at home and abroad. The reason for this is mainly because the silk fibroin powder prepared by the above methods has poor crystallinity, poor stability, small specific surface area, and low activity of immobilized enzyme. Moreover, the agglomerates or precipitates formed during the preparation process require drying and repeated pulverization and the like, and the silk fibroin powder thus prepared has a large particle size and a different shape, all of which are above the micron order. SUMMARY OF THE INVENTION

The object of the present invention is to overcome the deficiencies of the prior art and to provide a silk fibroin nanoparticle having an immobilized enzyme having good biocompatibility, high enzymatic activity recovery rate and simple production process, and a preparation method thereof.

The technical scheme adopted by the invention is: providing an immobilized enzyme silk fibroin nanoparticle, which has silk fibroin as a core, the enzyme is embedded and fixed in the surface layer of the microparticle, and the average particle size is 35~125ηιη, insoluble in water; The silk fibroin has a β-sheet structure and a crystallinity of 20% or more.

The method for preparing the above-mentioned immobilized enzyme silk fibroin nanoparticles, uniformly mixing the water-soluble silk fibroin solution and the enzyme, and injecting the silk fibroin mixture into the rapidly stirring water-soluble organic solvent, the silk fibroin solution and the organic solvent The mixing volume ratio is 1:2.3 or more, forming the milky silk fibroin as the core, and the spherical particles embedded in the surface layer and immobilized in the enzyme are dispersed in the organic solvent system to obtain the silk fibroin nanoparticle mixture or suspension of the immobilized enzyme, and then The organic solvent is removed to obtain a silk fibroin nanoparticle of the immobilized enzyme.

The water-soluble silk fibroin described in the above technical solution comprises purified from silk or wild silk, or silk fibroin produced by genetic engineering; the concentration of the silk fibroin solution is 0.1-20%. The enzyme mixed with the silk fibroin solution described in the above technical means is one or a mixture of two or more of an oxidoreductase, a hydrolase and an isomerase.

When the silk fibroin solution is injected into the organic solvent, the stirring speed is 50 rpm or more.

The water-soluble organic solvent is ethanol or acetone.

The method for producing the above-mentioned immobilized enzyme silk fibroin nanoparticles has a working environment temperature of 10 to 45 ° C, preferably 25 to 37 ° C.

The organic solvent mixture or suspension of the immobilized enzyme silk fibroin nanoparticles is subjected to repeated centrifugal dehydration treatment, or repeated filtration and washing treatment until the organic solvent is completely removed.

The obtained silk fibroin nanoparticles were added to pure water or an aqueous solution and then ultrasonicated for 1 to 10 minutes to prepare an immobilized enzyme nanosiltin solution.

The organic solvent mixture or suspension of the obtained immobilized enzyme silk fibroin nanoparticles is subjected to vacuum freeze-drying to prepare an immobilized enzyme nanosiltin powder.

Water-soluble organic solvents are well known as one of the most commonly used protein denaturants, which are deactivated when biologically active proteases encounter high concentrations of organic solvents. Silk fibroin is also a high molecular egg White, when silk fibroin is made into regenerated water-soluble silk fibroin, it is susceptible to physical or chemical factors and protein denaturation occurs, especially when organic solvents are encountered, and structural changes are more likely to occur and coagulation precipitates. In the past studies, a small amount of an organic solvent was added to a mixed solution of silk fibroin and an enzyme to cause the silk fibroin to coagulate or precipitate, the enzyme was embedded in the silk fibroin precipitate, and then dried and pulverized to prepare an immobilized enzyme. The invention utilizes the two characteristics that the silk fibroin structure is susceptible to change and the water-soluble organic solvent easily causes protein denaturation, and a small amount of the mixed solution of silk fibroin and the enzyme is injected into a high-speed agitation of a large amount of pure organic solvent to promote silk fibroin. Rapid dispersion and denaturation, water-soluble silk fibroin is directly converted from α-helix and random coil into β-sheet structure, and while crystallized silk fibroin particles are formed, the enzyme is embedded and immobilized to obtain nano-scale properties. Stable, water-insoluble immobilized silk fibroin nanoparticles. The present invention also finds that ethanol and acetone are more effective in immobilizing the enzyme. Therefore, a preferred technical solution is that the organic solvent is ethanol or acetone.

Compared with the prior art, the present invention has the following advantages:

Since silk fibroin is mixed with an excess of a water-soluble organic solvent, the soluble silk fibroin is instantaneously converted from an irregular coil and an α-helical structure into an insoluble antiparallel β-sheet structure. Therefore, the obtained immobilized enzyme silk fibroin particles have an average of about 80 nm, and the enzyme activity recovery rate is up to 60%. The electron microscope observation is spherical, the crystallinity is more than 20%, the performance is stable, and it is not easily decomposed by proteases, and has strong resistance to ultraviolet radiation. Function, in the body can greatly reduce or eliminate the immunogenicity of the enzyme, and extend the half-life of the enzyme.

The silk fibroin nanoparticle of the immobilized enzyme provided by the technical scheme of the present invention has good stability, and can be placed at room temperature for a long time in both the particle suspension and the lyophilized powder state, and the enzyme activity is not affected at all, and does not need to be pure. The enzyme must be stored at 4 Ό or 4 ;. The immobilized silk fibroin nanoparticles in the lyophilized powder state can be resistant to high temperatures, and the enzyme activity is not lost when dried at 90 to 100 ° C for 1 hour. According to the technical solution of the present invention, since the toxic chemical reagent is not used in the preparation process, the obtained nanofilament of the immobilized enzyme is non-toxic, harmless, and has good biocompatibility, and is a green color. Environmentally friendly products. The organic solvent such as ethanol or acetone is mixed with the silk fibroin solution to form a suspension of the milky white immobilized enzyme silk fibroin nanoparticle, which is filtered or centrifuged, and the discarded filtrate or supernatant can be recovered and re-steamed; The aqueous solution of the organic solvent sublimed by vacuum freeze-drying can also be recovered and re-steamed and recycled. It also has a broad market prospect due to its simple product preparation process, low cost and low efficiency. DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a fluorescence emission spectrum of immobilized glucose oxidase silk fibroin nanoparticles obtained by the method of the present invention;

Figure 2 is an infrared absorption spectrum of immobilized glucose oxidase silk fibroin nanoparticles obtained by the production method of Example 1 of the present invention;

Figure 3 is an X-ray diffraction pattern of immobilized glucose oxidase silk fibroin nanoparticles obtained according to the manufacturing method of Example 1 of the present invention;

Fig. 4 is a graph showing the particle size distribution of immobilized glucose oxidase silk fibroin nanoparticles obtained by the production method of Example 1 of the present invention. detailed description

The invention is further illustrated by the following examples.

Embodiment 1:

Add 30 times the amount of 0.5% sodium carbonate aqueous solution or other alkaline solution or add surface activity after washing the sericulture, seedling production, silk, textile production, sputum, waste silk or waste silk cloth. The agent was boiled for 1 hour, then changed to 1 time, and boiled for 1 hour to ensure that the sericin was completely removed. The silk fibroin-removed silk fibroin fiber is repeatedly washed with water and then dried for use.

The above-mentioned silk fibroin-removed silk fibroin fiber is mixed with 5 to 20 times (W/V) of 8-9 M lithium bromide aqueous solution or lithium bromide methanol water ternary mixed solvent, and dissolved at 40 ° C or higher for 2 to 40 hours; or The silk fibroin is mixed with 5 to 20 times the calcium chloride/ethanol/water ternary mixed solvent (molar ratio 1: 2:8), and dissolved at 50 Ό or more for 1 to 5 hours; or the above silk fibroin The fibers are dissolved in other concentrated salt solutions or organic solvents. The various silk fibroin solutions obtained above are dialyzed, desalted, and purified to prepare a water-soluble silk fibroin solution having a concentration of 0.5 to 15%, preferably concentrated or diluted to a 1.5 to 3.5% water-soluble silk fibroin solution.

Taking the above-mentioned concentration of 1.5 - 3.5% water-soluble silk fibroin solution, adding glucose oxidase accounting for 0.0001 - 1% of the total silk fibroin, stirring the silk fibroin mixed solution by a stirring device, under the environmental condition of 10 〜 45 ° C Preferably, the acetone is rapidly denatured into ultrafine particles suspended in an organic solvent by rapidly adding an excess amount of acetone (final volume of 70% or more) under an environment of 25 to 37 °C. In the filter paper, the filtered immobilized enzyme silk fibroin wet powder is placed in a buffer solution, and after sonication, it becomes a suspension of immobilized glucose oxidase silk fibroin nanoparticles, which can be directly used for enzyme activity determination; or The filtered immobilized enzyme silk fibroin wet powder was frozen at -20 Torr, and finally, vacuum freeze-dried to obtain powdery immobilized glucose oxidase silk fibroin nanoparticles. The glucose oxidase activity was measured by a slightly improved method using a previously reported method (Kawahara Y: Journal of Sericulture Science of Japan, 62(4), 272-275, 1993). The specific steps are as follows: Add 2.4ml staining buffer, 0.5mL 10% substrate glucose solution and 0.1ml peroxidase solution to the measuring tube, standard tube and blank tube, shake well, set at 37 °C constant temperature 2~ After 5 minutes, the reaction was started by adding the free enzyme. The blank tube was used as the control. The absorbance change of the reaction was measured at the wavelength of 500iun for 5 minutes. Then, except for 5, 1.0 mmol/L β-D-glucose was oxidized to D-gluconic acid per minute. And the absorbance value added by H ₂ 0 ₂ . When the immobilized enzyme is measured, the measurement of the silk fibroin particles in the colorimetric suit will block the transmission of the ultraviolet light and interfere with the measurement. Therefore, the immobilized enzyme reactor is prepared, and after the reaction is 5 m i, the filter is immediately filtered. The reaction solution was measured to measure the absorbance value. One unit of glucose oxidase activity was defined as 37 ° C, pH5.5 phosphate catalyst per minute 1.0 mmol / L β-D- glucose oxidase is under buffer conditions D- gluconic acid and H _₂ 0 ₂ produced an absorbance Change value. The values in the table are the average of 5 replicates. As can be seen from the table, the activity recovery rate of the immobilized enzyme is between 24.5 and 72.9%.

Table 1: Recovery of immobilized glucose oxidase activity

Enzyme concentration determination enzyme amount absorbance value enzyme activity activity recovery rate

1 5. 8 0. 05 0. 29 0. 100 0. 345

2 3. 5 0. 10 0. 35 0. 189 0. 540 72. 98

3 2. 1 0. 20 0. 42 0. 176 0. 419 56. 62

4 1. 2 0. 0 0. 48 0. 162 0. 338 45. 68

5 1. 1 1. 00 1. 10 0. 199 0. 181 24. 46 Standard GOD 25μ1 8. OU/ml 0. 20 0. 148 0. 740 100 See Figure 1, immobilized glucose oxidase silk fibroin nano Fluorescence emission spectra measured on a Hitachi Fluorescence Spectrophotometer (F-4500 FL Spectrophotometer) in a suspension of particulate water and an enzyme-containing water-soluble silk fibroin solution. Measurement conditions: excitation wavelength 290 nm, excitation slit 10.0 ηιη, emission slit 5.0 nm, scanning speed 240 nm/min, sensitivity 0.5 s. Silk fibroin can be found in the picture After nanocrystallization, the fluorescence emission spectrum undergoes a blue shift of lOrnn, indicating that the silk fibroin molecules are converted from a random coil and an α-slow helix to a β-sheet structure.

Referring to Figure 2, immobilized glucose oxidase silk fibroin nanoparticle lyophilized powder and enzyme-containing water-soluble silk fibroin solution lyophilized powder were sampled with a few KBr pellets on a Magna 550 infrared spectrophotometer ( Nicolet Instrument Corp. USA) The measurement was performed on a scan range of 4000 to 200 cm - ¹ . The infrared absorption spectrum of the enzyme-containing water-soluble silk fibroin shows the characteristics of random coil and α-slow spiral or crank-shaped structure (Silk l). When the silk fibroin is nanosized, its absorption band is displaced, and antiparallel β appears. - The construction of the fold (Silk ll).

Referring to Figure 3, a silk fibroin sample was analyzed by a MERCURY CCD-AFC8 CCD single crystal X-ray diffractometer (Nippon Rigaku Corporation) with a tube voltage of 4.0 kV, a tube current of 35 mA, and a scanning speed of 2 °/min. Ni filtering. The X-ray diffraction pattern of the immobilized glucose oxidase silk fibroin nanoparticle lyophilized powder and the enzyme-containing water-soluble silk fibroin solution lyophilized powder was obtained by scanning from 2 Θ = 5° to 45°. The enzyme-containing water-soluble silk fibroin lyophilized powder can be confirmed as a completely amorphous structure, and after the silk fibroin is nanosized in acetone, the silk fibroin molecular conformation is converted from Silk l to Silk ll to become a crystalline immobilized enzyme silk fibroin nanoparticle. .

Referring to Figure 4, the immobilized glucose oxidase silk fibroin nanoparticles were diluted with water, sonicated and placed directly in a sample cup, and the particle size distribution was measured on a Zetasizer 3000HSa laser particle size analyzer (Malvern Instruments Ltd, Malvern UK). As can be seen from the figure, the size of the silk fibroin nanoparticles of the immobilized enzyme is between 35 and 125 nm.

Embodiment 2:

In the same manner as in the first embodiment, 1.5 to 3.5% of the water-soluble silk fibroin solution was mixed with the superoxide dismutase, and then rapidly stirred a large amount of acetone (final volume of 70% or more) was added, and the remaining preparation steps were all the same. The immobilized superoxide dismutase silk fibroin nanoparticle suspension or lyophilized powder was prepared by the acetone treatment method, and the activity measurement method and the activity recovery rate of the immobilized enzyme were shown in Table 2. The values in the table are the average of 5 replicates.

In this experiment, the activity of a solution enzyme or an immobilized enzyme was measured using a superoxide dismutase (SOD) assay kit (manufactured by Nanjing National Institute of Bioengineering, Division 1). The method is determined by the xanthine and xanthine oxidase reaction system, the latter oxidizes hydroxylamine to form nitrite, which is purple-red under the action of a color developing agent, and the absorbance is measured by a Hitachi U3000 ultraviolet-visible spectrophotometer. When the sample to be tested contains SOD, it has a specific inhibitory effect on the superoxide anion radical, which reduces the formation of nitrite. The absorbance value of the tube in the colorimetric measurement is lower than the absorbance value of the control tube. The SOD activity (NU/mI, expressed in nitrite units) of the sample to be tested was calculated. The activity of the immobilized enzyme activity in the table was calculated based on the activity of bovine blood superoxide dismutase (manufactured by Shanghai Oriental Livzon Biochemical Co., Ltd.) of 100%. The results showed that the activity of immobilized superoxide dismutase was between 25 and 79.4%.

Table 2. Activity recovery of immobilized superoxide dismutase

Sample number Enzyme content Determination of enzyme amount Absorbance value Inhibition rate Enzyme activity Activity recovery rate

(U/mg) (U) (ABS550nm) (%) (NU/U) (%)

1 0. 1 0. 010 0. 536 2. 10 16. 04 79. 41

2 1. 0 0. 100 0. 451 17. 65 13. 77 68. 17

3 1. 7 0. 378 0. 266 51. 6 10. 64 52. 70

4 5. 0 0. 250 0. 417 23. 9 7. 49 37. 08

5 10. 0 0. 500 0. 369 32. 7 5. 11 25. 30 Standard SOD 2. lU/ml 0. 210 0. 250 54. 4 20. 20 100 Example 3:

In the same manner as in the first embodiment, after 1.5 to 3.5% of the water-soluble silk fibroin solution was mixed with L-asparaginase, a rapid agitation of excess acetone (final volume of 70% or more) was added, and the remaining preparation steps were all the same. The immobilized L-asparaginyl silk fibroin nanoparticle suspension or lyophilized powder was prepared by the acetone treatment method, and the activity measurement method and the activity recovery rate of the immobilized enzyme were shown in Table 3. The values in the table are the average of 5 replicates.

Method for determining L-asparaginase activity (Mashbu, LT, Wriston, JC, jr.: Tumor inhibitory effect of L-Asparaginase. Biochen. Biophys. Res. Comm. 12: 50, 1963): Pipette number A centrifuge tube was added dropwise 0.2 ml of Tris-HCl buffer solution (pH 8.6) and 1.7 ml of the substrate solution, and the mixture was placed in a water bath to adjust the temperature to 37 °C. After adding O.lOm free enzyme solution or immobilized enzyme, it was precisely kept for 10 minutes. The reaction was terminated by adding 0.10 ml of a solution of trichloroacetic acid. After centrifugation, 0.50 ml of the supernatant was taken out into 7.0 ml of distilled water, and 1.0 ml of Nessler's reagent was added. After standing at room temperature for 10 min, the absorbance was measured with a blank tube as a control using a spectrophotometer (at 480 nm). When a series of different concentrations of free enzyme were determined by the same method as above, the relationship between the concentration of free enzyme and the absorbance was calculated, and a calibration curve was obtained to obtain a linear equation. Then, according to this linear equation (y=0.4286x+0.0555) Calculate the enzyme activity measured in the immobilized enzyme sample. Finally, the total enzyme addition amount in the sample was divided by the measured enzyme activity in the sample to obtain the immobilized enzyme activity recovery rate. The results are shown in Table 3. As can be seen from the table, the recovery rate of immobilized L-asparaginase activity was above 13%, and finally reached 76%.

Immobilized L-asparaginase activity recovery

Coding sample enzyme concentration determination enzyme amount absorbance value measured enzyme activity activity recovery rate (rag) (U/mg) (U) (ABS450nm) (U) (%)

1 4.0 1.000 4.0 0.654 1.398 34.96

2 6.0 0.500 3.0 0.461 0.946 31.54

3 8.0 0.250 2.0 0.709 1.525 76.24

4 20.0 0.100 2.0 0.593 1.254 62.70

5 20.0 0.050 1.0 0.327 0.633 63.35

6 50.0 0.025 1.0 0.113 0.134 13.42 Example 4:

In the same manner as in the first embodiment, 1.5 to 3.5% of the water-soluble silk fibroin solution was mixed with the penicillin acylase, and then rapidly stirred a large amount of acetone (final volume of 70% or more) was added, and the remaining preparation steps were all the same. The immobilized penicillin acylase silk fibroin nanoparticle suspension or lyophilized powder was prepared by the acetone treatment method, and the activity measurement method and the activity recovery rate of the immobilized enzyme were shown in Table 4. The values in the table are the average of 5 replicates.

Determination of the activity of penicillin acylase (EGicillin EG3.5.11) using the PABA (P-dimethylaminobenzaldehyde) method ( Balasshingham K, Warburton D, Dunnill P et al., The isolation and kinetics of penicillin amidase from Escherischia Coli., Biochim Biophys Acta , 1972, 276: 250-256). Penicillin acylase (100 O/mL) at 37. C cleavage of penicillin produces 6-APA under acidic conditions, which in turn forms Schiff base with PDAB and has maximum absorption at 415 nm. The enzyme activity assay procedure is as follows: 0.5 ml of the enzyme solution or about 20 mg of the immobilized enzyme is added to 4.5 ml of phosphate buffer (pH 7.8) to be diluted. Pipette 1.0 ml into a test tube and equilibrate 5 m i at 37 °C. Another 4% penicillin G solution was equilibrated at 37 V for 5 min, and 1.0 ml was pipetted into the above enzyme-containing tube, shaken, accurately reacted for 5 min, and reacted by adding 3 ml of ethanol. Pipette 0.75 ml and add 5.25 ml of PDAB chromogenic solution for 3 min. The optical density was measured at 415 nm. The 6-APA concentration was obtained against the standard curve. The amount of enzyme required to catalyze the hydrolysis of penicillin G potassium salt per minute to produce 6-ΑΡΑ of Ιμηιοΐ at pH 7.8, temperature 37 °C, is defined as one enzyme unit (U).

Prepare a series of different concentrations of penicillin acylase, determine the enzyme activity according to the above method, calculate the standard curve of enzyme concentration and absorbance, find the linear equation y = 0.0984x + 0.2526, and then calculate the enzyme activity measured in the actual sample, according to The amount of enzyme added during sample preparation was used to calculate the recovery rate of immobilized enzyme activity. The results showed that the activity of immobilized penicillin acylase was between 20 and 80%.

Table 4. Recovery of enzymatic activity of immobilized penicillin acylase silk fibroin nanoparticles

o

Preparation Sample Enzyme concentration Determination of enzyme amount Absorbance value Measured enzyme activity Activity recovery rate (mg) (U/mg) (U) (ABS415nm) (U) (%)

1 30.1 0.1 3.01 0.489 2.40 79.73

2 20.9 0.5 10.45 0.966 7.25 69.38

3 20.5 1.0 20.50 1.193 9.56 46.63

4 20.1 2.0 1.676 14.47 36.00

5 24.4 4.0 97.60 2.210 19.89 20.34

Claims

Claim

1. A silk fibroin nanoparticle immobilized with an enzyme, characterized in that: the silk fibroin is the core, the enzyme is embedded and fixed on the surface layer of the microparticle, and the average particle size is 35-125 nm, which is insoluble in water; The protein has a β-sheet structure and a crystallinity of 20% or more.

The enzyme according to claim 1, which comprises one or more of a mixed enzyme of an oxidoreductase, a hydrolase and an isomerase.

3. A method for preparing a silk fibroin nanoparticle immobilized with an enzyme, characterized in that: the water-soluble silk fibroin solution is uniformly mixed with an enzyme, and the silk fibroin mixture is injected into a rapidly stirring water-soluble organic solvent, and the silk fibroin The mixing volume ratio of the enzyme solution to the organic solvent is 1:2.3 or more, forming the milky white silk fibroin as the core, and the spherical microparticles embedded in the surface layer and immobilized in the enzyme are dispersed in the organic solvent system to obtain the silk fibroin nanoparticle mixture of the immobilized enzyme. Or a suspension, removing the organic solvent therein to obtain a silk fibroin nanoparticle of the immobilized enzyme.

The enzyme according to claim 3, which comprises one or more of a mixed enzyme of an oxidoreductase, a hydrolase and an isomerase.

The water-soluble organic solvent according to claim 3, which comprises ethanol or acetone.