CN100427593C - Enzyme-immobilized silk fibroin nanoparticles and preparation method thereof - Google Patents

Abstract

The invention discloses a silk fibroin nanoparticle for producing immobilized enzymes by using silk fibroin and a preparation method thereof, belonging to the field of biotechnology enzyme engineering. The technical scheme is: after fully mixing the enzyme with the water-soluble regenerated silk fibroin solution, inject it into the excess water-soluble organic solvent stirred at a high speed, causing the denaturation and β-transformation of the silk fibroin to form milky white crystalline silk fibroin particles, and at the same time, the enzyme Entrapped and immobilized in microparticles. After filtering or centrifuging to remove the organic solvent, the crystalline immobilized enzyme silk fibroin nanoparticles are prepared. The average particle size of the immobilized enzyme is 35-125nm, the activity recovery rate is as high as 70%, the thermal stability is greatly improved, it is not easy to be decomposed by protease in vitro or in vivo, and the immunogenicity of the enzyme can be greatly reduced or eliminated. The immobilized enzyme silk fibroin nanoparticles have broad application prospects in drug sustained release systems, industrial enzyme reactors, food additives and advanced cosmetics.

Description

Enzyme-immobilized silk fibroin nanoparticles and preparation method thereof

technical field

The invention discloses a carrier for immobilized enzymes and a preparation method thereof, in particular to a silk fibroin nanoparticle for immobilized enzymes produced by silk fibroin and a preparation method thereof, which belongs to the field of biotechnology enzyme engineering and also belongs to the field of nanotechnology and polymer chemistry technology.

Background technique

Introducing enzymes or drugs into the body in the form of particles, microcapsules, liposomes, albumin, red blood cell carriers, etc. can prolong the half-life of enzymes or drugs, make them release slowly, and improve the effect of enzymes or drugs on hereditary enzyme deficiency and metabolic disorders. Therapeutic efficacy of diseases, tumors and cardiovascular diseases. There are many studies on the use of natural biological materials as enzyme immobilization carriers or drug slow-release carriers because of their safety, stability, biocompatibility and ability to metabolize in vivo. The silk fibroin produced by the silkworm is a natural polymer with a molecular weight of 370,000. Since this protein is non-toxic, harmless, and non-immune to the human body, it has long been used as a medical surgical suture. In recent years, the development and utilization of silk fibroin as a raw material for artificial skin, cosmetics and nutraceuticals or as a carrier for immobilized cells, enzymes and antibodies has attracted attention. In particular, the research on silk fibroin as an immobilized enzyme carrier has been particularly active in the past two decades, and many experiments have fully confirmed that silk fibroin is an excellent biomaterial for immobilized enzymes.

Using silk fibroin as an enzyme immobilization carrier has advantages that other polymer materials do not have. The principle of enzyme immobilization is based on the transformation of silk fibroin from a soluble random coil and α-helix structure into an insoluble antiparallel β -Folding structure, at the same time immobilization of the enzyme is also completed. There are various forms of silk fibroin as the immobilized enzyme carrier, such as silk film, silk fiber, silk powder or silk gel. Among them, the preparation and application research of silk fibroin powder is very active, and there have been a large number of patent reports, mainly through physical or chemical methods to induce silk fibroin protein aggregation, such as salting out, ultrasonic, aeration, high-speed stirring, isoelectric point aggregation, electrolysis, etc. Or mechanical pulverization after film formation or direct mechanical pulverization of silk fibers many times to make superfine silk fibroin powder ranging from 1 to 100 microns, which is used in cosmetics, nutritional foods, additives, super absorbent materials and coatings, etc. It has broad 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. Publication number is CN1560136 Chinese invention patent (international patent WO2005085327 A1) "manufacturing method of silk fibroin nanoparticles", discloses a water-soluble regenerated silk fibroin solution directly mixed with excess water-miscible organic solvent A method for preparing fibroin nanoparticles, but some organic solvents may cause enzyme inactivation and are not suitable for preparing immobilized enzymes.

The immobilization research of silk fibroin powder for enzymes and active substances originated in the early 1980s, mainly using methods such as salting out, high-speed stirring, freezing and thawing of protein, adding a small amount of organic solvent or adjusting the isoelectric point to make silk fibroin Precipitation, aggregation or denaturation, followed by drying and crushing to make immobilized enzymes. Water-soluble silk fibroin and enzyme mixed solution are salted out with inorganic salt or inorganic salt and organic salt mixture to make it precipitate to prepare immobilized enzyme (Japanese patent, JP56-15687; JP60-155125, international patent, WO8503230; JP62-151180 of JP62-151180); or the silk fibroin and enzyme mixed solution are first adjusted to acidity or alkaline, and then powdered immobilized enzyme is made after salting out, washing and drying (Japanese patent, JP62-151180). JP56-051983, JP56-39783). Add a small amount of organic solvents such as methanol to the silk fibroin and enzyme mixture to directly induce silk fibroin aggregation, or first adjust the silk fibroin enzyme mixture to the vicinity of the isoelectric point of silk fibroin, then add a small amount of organic solvent to induce silk fibroin aggregation, and then agglomerate Make immobilized α-amylase and sucrase (Japanese patent, JP1-313530 of Japanese Patent, JP1-313530) after washing, drying and crushing of the thing, or make immobilized alkaline or neutral protease (Japanese patent, JP56-18590 of JP56-18590 of JP) ). The silk fibroin is agglomerated or aerated by high-speed agitation to cause a large amount of foam to be produced from the silk fibroin and enzyme mixture (Japanese Patent, JP60-227679, JP61-187790). The mixture of silk fibroin and enzyme is first frozen and then melted to denature the silk fibroin to make immobilized alkaline phosphatase (Japanese patent, JP01-120287). There is also the use of salting-out method to make silk fibroin powder, and then use cross-linking agent to cross-link silk fibroin powder and glucoamylase to make immobilized enzyme powder ["Sericulture Science", 25 (2): 113-119, 1999 ]. In addition, there are no more reports at home and abroad on the study of immobilized enzymes using silk fibroin powder as a carrier. Trace it to its cause mainly because the silk fibroin powder prepared by above-mentioned these methods has poor crystallinity, poor stability, small specific surface area and low immobilized enzyme activity. Moreover, the aggregates or precipitates formed during the preparation process require drying and repeated crushing and other processing techniques. The silk fibroin powder thus prepared has a large particle size and various shapes, all of which are above the micron level.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art, and provide a silk fibroin nanoparticle with good biocompatibility, high recovery rate of enzyme activity and simple production process for immobilizing enzymes and a preparation method thereof.

The technical scheme adopted in the present invention is: provide a silk fibroin nanoparticle with immobilized enzyme, which takes silk fibroin as the core, the enzyme is embedded and fixed in the surface layer of the particle, the average particle size is 35-125nm, and it is insoluble in water; The silk fibroin has a β-sheet structure and a crystallinity of more than 20%.

The method for preparing the above-mentioned silk fibroin nanoparticles with immobilized enzymes is to mix the water-soluble silk fibroin solution and the enzyme evenly, and then inject the silk enzyme mixed solution into a rapidly stirred water-soluble organic solvent, and the silk fibroin enzyme liquid and the organic solvent The mixed volume ratio is more than 1: 2.3, the milky white silk fibroin is formed as the core, and the surface layer embeds and immobilizes the spherical particles of the enzyme 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 the silk fibroin nanoparticles with immobilized enzyme.

The water-soluble silk fibroin described in the above technical solution is purified from domestic silkworm silk or wild silkworm silk, or silk-like protein 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 solution refers to one or more mixed enzymes among oxidoreductase, hydrolase and isomerase.

When the above-mentioned silk fibroin enzyme liquid is injected into the organic solvent, the stirring speed is above 50 rpm.

Described water-miscible organic solvent is ethanol or acetone.

For the above-mentioned manufacturing method of silk fibroin nanoparticles with immobilized enzymes, the working environment temperature for the preparation is 10-45°C, preferably 25-37°C.

The organic solvent mixture or suspension of immobilized enzyme silk fibroin nanoparticles is repeatedly centrifuged and dehydrated, or repeatedly filtered and washed until the organic solvent is completely removed.

The obtained silk fibroin nanoparticles are added into pure water or an aqueous solution, and then ultrasonically treated for 1 to 10 minutes to prepare a nano silk fibroin solution with immobilized enzymes.

The obtained organic solvent mixture or suspension of silk fibroin nanoparticles with immobilized enzymes is subjected to vacuum freeze-drying to prepare nano silk fibroin powder with immobilized enzymes.

As we all know, water-soluble organic solvents are the most commonly used protein denaturants. When biologically active proteases encounter high concentrations of organic solvents, they will be denatured and inactivated. Silk fibroin is also a high-molecular protein. When the silk fibroin fiber is made into regenerated water-soluble silk fibroin, it is susceptible to protein denaturation due to physical or chemical factors, especially when encountering organic solvents, it is more likely to undergo structural changes and coagulate. precipitation. In previous studies, a small amount of organic solvent was added to the mixed solution of silk fibroin and enzyme to promote the aggregation or precipitation of silk fibroin, so that the enzyme was embedded in the silk fibroin precipitate, and then dried and pulverized to make immobilized enzyme. The present invention utilizes the two characteristics that the structure of silk protein is easy to be changed and water-soluble organic solvents are easy to cause protein denaturation, and a small amount of mixed solution of silk fibroin and enzyme is injected into a large amount of pure organic solvent stirred at high speed to promote silk fibroin Rapid dispersion and denaturation, water-soluble silk fibroin directly transforms from α-helix and random coil to β-sheet structure, while forming crystalline silk fibroin particles, enzymes are embedded and immobilized, thereby obtaining nanoscale, high performance Stable, water-insoluble immobilized enzyme silk fibroin nanoparticles. The present invention also finds that ethanol and acetone are better for the immobilized enzyme effect, therefore, the preferred technical solution is that the organic solvent is ethanol or acetone.

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

After the silk fibroin is mixed with an excess of water-soluble organic solvent, the soluble silk fibroin instantly transforms from the random coil and α-helix structure into the insoluble antiparallel β-sheet structure. Therefore, the average size of the obtained immobilized silk fibroin particles is about 80nm, the recovery rate of the enzyme activity is as high as 60%, the electron microscope observation shows a spherical shape, the crystallinity is over 20%, the performance is stable, it is not easy to be decomposed by protease, and it has a strong function of blocking ultraviolet radiation , can greatly reduce or eliminate the immunogenicity of enzymes in vivo, and prolong the half-life of enzymes.

The silk fibroin nanoparticles of the immobilized enzyme provided by the technical scheme of the present invention have good stability, whether it is in the state of particle suspension or freeze-dried powder, it can be placed at room temperature for a long time, and the enzyme activity will not be affected in any way. Enzymes must be stored at or below 4°C; the immobilized enzyme silk fibroin nanoparticles in the state of freeze-dried powder produced can resist high temperature, and the enzyme activity will not be lost after drying at 90-100°C for 1 hour.

According to the technical scheme of the present invention, since no toxic chemical reagents are used in the preparation process, the obtained nano-silk fibroin with immobilized enzymes is non-toxic and harmless to the human body, and has good biocompatibility. It is a green Environmentally friendly products. After the organic solvents such as ethanol and acetone used are mixed with the silk fibroin enzyme solution, a milky white immobilized enzyme silk fibroin nanoparticle suspension is generated, which is filtered or centrifuged, and the discarded filtrate or supernatant can be recycled and re-distilled; or The sublimated organic solvent aqueous solution after vacuum freeze-drying can also be recovered and re-distilled for recycling. Also because the preparation process of the product is simple, the cost is low, and the benefit is high, it has broad market prospects.

Description of drawings

Fig. 1 is the fluorescence emission spectrum of the immobilized glucose oxidase silk fibroin nanoparticle obtained by the manufacturing method of Example 1 of the present invention;

Fig. 2 is the infrared absorption spectrum of the immobilized glucose oxidase silk fibroin nanoparticle obtained by the manufacturing method of Example 1 of the present invention;

Fig. 3 is the X-ray diffraction pattern of the immobilized glucose oxidase silk fibroin nanoparticle obtained by the manufacturing method of Example 1 of the present invention;

Fig. 4 is a particle size distribution diagram of the immobilized glucose oxidase silk fibroin nanoparticles obtained according to the manufacturing method of Example 1 of the present invention.

Detailed ways

The present invention will be further elaborated below in conjunction with embodiment.

Embodiment one:

After cleaning the seed cocoons, cocoon clothes, waste silk or waste silk cloth in sericulture, seed cocoon production, silk reeling, and textile production, add 30 times the amount of 0.5% sodium carbonate aqueous solution or other alkaline solutions or add surface active Boil for 1 hour, change the liquid once more, and boil for 1 hour to ensure that all the sericin is removed. The silk fiber from which the sericin has been removed is washed with water repeatedly and then dried for use.

Take the above-mentioned silk fiber from which sericin has been removed and mix it with 5-20 times the amount (W/V) of 8-9M lithium bromide aqueous solution or lithium bromide-methanol-water ternary mixed solvent, and dissolve at 40°C for 2-40 hours; or Mix the silk fiber with 5-20 times the amount of calcium chloride/ethanol/water ternary mixed solvent (molar ratio 1:2:8), and dissolve it at 50°C for 1-5 hours; or mix the above-mentioned silk fiber Dissolved in other concentrated salt solutions or organic solvents. Dialyze, desalt, and purify the various silk fibroin solutions obtained above to prepare a water-soluble silk fibroin solution with a concentration of 0.5-15%, preferably concentrated or diluted to a water-soluble silk fibroin solution with a concentration of 1.5-3.5%.

Take the above water-soluble silk fibroin solution with a concentration of 1.5-3.5%, add glucose oxidase which accounts for 0.0001-1% of the total amount of silk fibroin, stir the silk fibroinase mixed solution through a stirring device, and under the environmental conditions of 10-45°C, It is best to quickly add excess acetone (more than 70% of the final volume) under the environmental conditions of 25-37°C, so that the silk fibroin can be rapidly denatured into ultrafine particles, suspended in an organic solvent, filtered through filter paper, and the filtered The immobilized enzyme silk fibroin wet powder is placed in a buffer solution, and after ultrasonic treatment, the silk fibroin nanoparticle suspension of immobilized glucose oxidase can be directly used for the determination of enzyme activity; or the filtered immobilized enzyme silk fibroin wet powder Freezing at -20°C, and finally vacuum freeze-drying to obtain powdered immobilized glucose oxidase silk fibroin nanoparticles.

Glucose oxidase activity was measured by a slightly modified method previously reported (Kawahara Y: Journal of Sericulture Science of Japan, 62(4), 272-275, 1993). The specific operation steps are as follows: Add 2.4ml of staining buffer, 0.5mL of 10% substrate glucose solution and 0.1ml of peroxidase solution to the measurement tube, standard tube and blank tube respectively, shake well, and keep the temperature at 37°C for 2-5min Finally, add free enzyme to start the reaction, take the blank tube as a contrast, measure the absorbance change value of the reaction within 5 minutes at a wavelength of 500nm, and then divide by 5 to obtain the oxidation of 1.0mmol/L β-D-glucose into D-gluconic acid and Absorbance value increased _{by H2O2} . When measuring the immobilized enzyme, since silk fibroin particles are suspended in the cuvette while reacting, the measurement will block the transmission of ultraviolet light and interfere with the measurement. Therefore, a self-made immobilized enzyme reactor is made, and after 5 minutes of reaction, it is immediately filtered out The absorbance value of the reaction solution was measured. One unit of glucose oxidase activity is defined as the change in absorbance produced by catalyzing the oxidation of 1.0mmol/L β-D-glucose to D-gluconic acid and H ₂ O ₂ per minute under the condition of 37°C and pH 5.5 sodium phosphate buffer value. The values in the table are the average value of 5 repeated determinations. It can be seen from the table that the recovery rate of the activity of the immobilized enzyme is between 24.5% and 72.9%.

Table 1: Recovery rate of immobilized glucose oxidase activity

Referring to accompanying drawing 1, the fluorescence emission spectrum of immobilized glucose oxidase silk fibroin nanoparticle suspension in water and water-soluble silk fibroin solution containing enzyme is measured on Hitachi fluorescence spectrophotometer (F-4500 FL Spectrophotometer). Measurement conditions: excitation wavelength 290nm, excitation slit 10.0nm, emission slit 5.0nm, scanning speed 240nm/min, sensitivity 0.5s. It can be seen from the figure that after the silk fibroin is nano-sized, the fluorescence emission spectrum has a blue shift of about 10nm, indicating that the silk fibroin molecule has transformed from a random coil and an α-slow helix to a β-sheet structure.

Referring to accompanying drawing 2, immobilized glucose oxidase silk fibroin nanoparticle lyophilized powder and enzyme-containing water-soluble silk fibroin solution lyophilized powder are prepared with a little KBr tablet, on Magna 550 infrared spectrophotometer (NicoletInstrument Corp.USA) For measurement, the scanning range is 4000-200 cm ^-1 . The infrared absorption spectrum of enzyme-containing water-soluble silk fibroin in the figure shows the characteristics of random coil and α-slow helix or crank-shaped structure (Silk I). When the silk fibroin is nano-sized, its absorption band shifts, and antiparallel β appears. - Construction of the fold (Silk II).

Referring to accompanying drawing 3, carry out silk fibroin sample analysis in MERCURY CCD-AFC8 type CCD single crystal X-ray diffractometer (Rigaku Electric Co., Ltd.), tube voltage is 4.0kV, tube current is 35mA, scanning speed 2 °/min, Ni filter. Scan from 2θ=5° to 45°, and obtain the X-ray diffraction patterns of the freeze-dried powder of the immobilized glucose oxidase silk fibroin nanoparticles and the freeze-dried powder of the enzyme-containing water-soluble silk fibroin solution. The enzyme-containing water-soluble silk fibroin freeze-dried powder can be confirmed as a completely amorphous structure, and after the silk fibroin is nano-sized in acetone, the molecular conformation of silk fibroin transforms from Silk I to Silk II, and becomes crystalline immobilized enzyme silk fibroin nanoparticles .

Referring to accompanying drawing 4, the immobilized glucose oxidase silk fibroin nanoparticle is diluted with water, puts directly into the sample cup after sonication, and measures particle size distribution situation on Zetasizer 3000HSa laser particle size analyzer (Malvern Instruments Ltd, Malvern UK). It can be seen from the figure that the particle size distribution of silk fibroin nanoparticles immobilized with enzyme is between 35 and 125 nm.

Embodiment two:

The preparation method is the same as that in Example 1. After mixing 1.5-3.5% water-soluble silk fibroin solution with superoxide dismutase, add it to excess acetone (final volume above 70%) that is stirred rapidly, and the rest of the preparation steps are the same. The acetone treatment method was used to prepare immobilized superoxide dismutase silk fibroin nanoparticle suspension or freeze-dried powder. The activity determination method and the activity recovery rate of the immobilized enzyme are shown in Table 2. The values in the table are the average value of 5 repeated determinations.

In this experiment, the superoxide dismutase (SOD) assay kit (produced by Nanjing Jiancheng Bioengineering Institute Branch 1) was used to measure the activity of solution enzyme or immobilized enzyme. Its determination method is to generate O ₂ ^- through the reaction system of xanthine and xanthine oxidase, and the latter oxidizes hydroxylamine to form nitrite, which turns purple under the action of chromogen, and measures its absorbance with Hitachi U3000 UV-Vis spectrophotometer. When the tested sample contains SOD, it has a specific inhibitory effect on superoxide anion free radicals, reducing the formed nitrite, and the absorbance value of the measuring tube is lower than that of the control tube during colorimetry, through the formula Calculate the SOD activity of the tested sample (NU/ml, expressed in nitrite units). The recovery rate of immobilized enzyme activity in the table is calculated based on the activity of bovine blood superoxide dismutase (produced by Shanghai Dongfang Livzon Biochemical Products Co., Ltd.) as 100%. The results showed that the activity recovery rate of immobilized superoxide dismutase was between 25% and 79.4%.

Table 2. Activity recovery of immobilized superoxide dismutase

Embodiment three:

The preparation method is the same as that in Example 1. After mixing 1.5-3.5% water-soluble silk fibroin solution with L-asparaginase, add it to excess acetone (final volume above 70%) that is stirred rapidly, and the rest of the preparation steps are the same. The immobilized L-asparaginase silk fibroin nanoparticle suspension or freeze-dried powder were respectively prepared by this acetone treatment method, and the activity determination method and the activity recovery rate of the immobilized enzyme are shown in Table 3. The values in the table are the average value of 5 repeated determinations.

L-asparaginase activity assay method (Mashburn, L.T., Wriston, J.C., jr.: Tumorinhibitory effect of L-Asparaginase. Biochen. Biophys. Res. Comm. 12: 50, 1963): use pipette to several Add 0.2ml Tris-HCl buffer solution (pH8.6) and 1.7ml substrate solution dropwise into the centrifuge tube, place in a water bath and adjust the temperature to 37°C. Add 0.10ml of free enzyme solution or immobilized enzyme and then accurately incubate for 10min. Add 0.10ml trichloroacetic acid solution to terminate the reaction. After centrifugation, take out 0.50ml of supernatant and drop into 7.0ml of distilled water, then add 1.0ml of Nessler's reagent. After standing at room temperature for 10 min, the absorbance was measured with a spectrophotometer (at 480 nm) with a blank tube as a control. After a series of free enzymes with different concentrations are measured by the same method as above, the relationship between the free enzyme concentration and absorbance is calculated, and a calibration curve is made to obtain a linear equation. Then, according to this linear equation (y=0.4286x+0.0555), calculate the enzyme activity actually measured in the immobilized enzyme sample, finally divide the added total amount of enzyme in the sample by the actual enzyme activity in the sample to obtain the recovery rate of the immobilized enzyme activity , the results are shown in Table 3. It can be seen from the table that the recovery rate of immobilized L-asparaginase activity is above 13%, and the highest can reach 76%.

The active recovery rate of table 3 immobilized L-asparaginase

Embodiment four:

The preparation method is the same as that in Example 1. After mixing 1.5-3.5% water-soluble silk fibroin solution with penicillin acylase, add it to rapidly stirred excess acetone (final volume above 70%), and the rest of the preparation steps are the same. The acetone treatment method was used to prepare the immobilized penicillin acylase silk fibroin nanoparticle suspension or freeze-dried powder, and the activity determination method and the activity recovery rate of the immobilized enzyme are shown in Table 4. The values in the table are the average value of 5 repeated determinations.

The activity of penicillin acylase (penicillin Acylase E.C.3.5.11) was determined by PDAB (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 (1000U/mL) cleaves penicillin at 37°C to produce 6-APA under acidic conditions, and then forms Schiff base with PDAB, with maximum absorption at 415nm. The operation steps of enzyme activity determination are as follows: 0.5ml of enzyme solution or about 20mg of immobilized enzyme is added to 4.5ml of phosphate buffer (pH 7.8) for dilution. Draw 1.0ml into a test tube and equilibrate at 37°C for 5min. In addition, equilibrate 4% penicillin G solution at 37°C for 5 minutes, draw 1.0ml into the test tube containing the enzyme, shake well, react accurately for 5 minutes, and add 3ml of ethanol to stop the reaction. Draw 0.75ml and add 5.25ml PDAB chromogenic solution, let stand for 3min. Measure the optical density at 415nm. Control the standard curve to obtain the concentration of 6-APA. The amount of enzyme required to catalyze the hydrolysis of penicillin G potassium salt to produce 1 μmol of 6-APA per minute at pH 7.8 and temperature 37°C is defined as 1 enzyme activity unit (U).

Prepare a series of penicillin acylase with different concentrations, measure the enzyme activity according to the above method, calculate the standard curve of enzyme concentration and absorbance, obtain the linear equation y=0.0984x+0.2526, 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 recovery rate of the activity of the immobilized penicillin acylase was between 20% and 80%.

Table 4. Enzyme activity recovery rate of immobilized penicillin acylase silk fibroin nanoparticles

Claims (2)

1, a kind of silk nano granular of immobilized enzyme is characterized in that: it is core with the silk fibroin, and enzyme is by embedding and be fixed in the particulate top layer, and mean particle size is 35～125nm, and is water insoluble; Described silk fibroin is the beta sheet structure, and degree of crystallinity is more than 20%; Described enzyme comprises a kind of or mixed enzyme more than two kinds in the middle of oxydo-reductase, lytic enzyme and the isomerase.

2, the method for the silk nano granular of preparation immobilized enzyme, it is characterized in that: water-soluble fibroin solution and enzyme are mixed, again fibroin enzyme mixed solution is injected in the water-miscible organic solvent of quick stirring, volume of organic solvent accounts for more than 70% of final volume, described organic solvent is an acetone, forming the oyster white silk fibroin is core, the spherical particle of top layer embedding and immobilized enzyme is dispersed in the organic solvent system, the silk nano granular mixed solution or the suspension of being fixed enzyme, remove organic solvent wherein, the silk nano granular of being fixed enzyme; Described enzyme comprises a kind of or mixed enzyme more than two kinds that oxydo-reductase, lytic enzyme and isomerase are central.

Images