Protease immobilization method and application of protease immobilization method in active peptide preparation

Abstract

The invention belongs to the technical field of food and biological processing, and particularly relates to a protease immobilization method and application in active peptide preparation. According to the invention, ferrous salt and ferric salt modified superparamagnetic graphene oxide are adopted as a carrier, and a carboxylic acid functional group is introduced through a surfactant to functionalize the surface of the carrier, so that covalent binding of protease and the magnetic graphene oxide carrier is realized, and the complex is applied to hydrolysis of protein and controllable preparation of bioactive peptides. The protein hydrolysis peptide has the advantages of stable enzyme activity, easiness in recovery, reusability, good oxidation resistance, controllable molecular weight range and the like.

Description

Technical Field

[0001] The present invention belongs to the field of food and bioprocessing technology, and relates to a method for immobilizing a protease and its application in the preparation of active peptides, and specifically to a method for preparing a superparamagnetic graphene oxide nano-immobilized protease and its application, and especially to its application in preparing products related to bioactive peptides. Background Art

[0002] Bioactive peptides are short-chain peptides composed of amino acids with specific physiological functions. They have functions such as anti-oxidation, lowering blood pressure, inhibiting bacteria, lowering blood lipids and maintaining blood sugar balance. They can be developed into functional foods, drugs or auxiliary therapeutic products, so they are widely used in many fields such as food, biology and medicine. The use of proteases to hydrolyze complex, insoluble macromolecular proteins to prepare bioactive peptides can improve the bioavailability of parent proteins and reduce allergenicity, and have the advantages of good catalytic performance and safety and reliability. However, free proteases have problems such as unstable activity, difficulty in recycling and reuse, and high production costs during use. In addition, self-hydrolysis and self-aggregation caused by mutual recognition between protease molecules will occur, resulting in reduced protease catalytic effect or even inactivation. Therefore, it is necessary to immobilize the protease. At present, immobilization technologies include chemical cross-linking, physical adsorption and embedding, which can improve the stability of proteases to a certain extent, but there are still problems such as easy changes in enzyme structure or detachment from the carrier. Commonly used immobilization materials such as macroporous resins, silica gel and other carriers have poor mechanical strength, are not resistant to solvent corrosion, and are difficult to separate and recycle from high-viscosity protein solutions. In addition, due to the instability of protease activity and catalytic efficiency, the molecular weight of peptides prepared by enzymatic hydrolysis is uncontrollable. In summary, the selection of appropriate carrier materials and immobilization methods is the key to improving the catalytic activity, stability, recovery and reusability of proteases and achieving controllable and adjustable molecular weight preparation of bioactive peptides.

[0003] CN 108179143 A discloses a magnetic graphene oxide immobilized enzyme and its application in wet corn soaking. However, the preparation of the immobilized enzyme in this method is to combine the magnetic graphene oxide material with papain by physical adsorption, which has the problem of easy enzyme shedding. In addition, the patent cannot achieve complete hydrolysis of the protein in corn starch, and the molecular weight and distribution range of the hydrolyzed polypeptide are unclear, and the antioxidant activity of the polypeptide is also unknown. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a protease immobilization method and its application in the preparation of active peptides in view of the deficiencies in the above-mentioned prior art, specifically a preparation method and application of superparamagnetic graphene oxide nano-immobilized protease, which has the advantages of high and stable catalytic efficiency of immobilized protease, magnetic recovery, controllable and adjustable molecular weight of the prepared bioactive peptides and antioxidant activity, and has broad application prospects in the fields of food and biochemical engineering.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] The first technical purpose of the present invention is to provide a method for immobilizing proteases, which uses superparamagnetic graphene oxide modified by ferrous salts and ferric salts as a carrier, introduces carboxylic acid functional groups through surfactants to functionalize the carrier surface, and achieves covalent bonding of the protease and the magnetic graphene oxide carrier, which can effectively improve the catalytic stability of the immobilized protease.

[0007] Specifically, the protease immobilization method is operated as follows:

[0008] (1) Preparation of superparamagnetic graphene oxide nanocarrier (SGO)

[0009] 0.1-1g of graphene oxide is added to a 0.05-0.5M sodium hydroxide solution, and ultrasonically treated at room temperature for 10-60min, and the target solid is collected by centrifugation at 8000-12000g, and washed with deionized water until the pH value reaches neutral; the particles are dispersed in 10-20mL of degassed water by ultrasonic treatment, and 5mL of a liquid suspension containing 50-150mg / mL of iron salt and 10-100mg / mL of ferrous salt is added, and ultrasonic treatment is carried out for 30-180min, and then a 10%-20% ammonia solution is added to make the pH value of the suspension reach 9-12; after repeated washing and filtering, it is dried in an oven at 50-100°C overnight to obtain a superparamagnetic graphene oxide nanocarrier SGO;

[0010] (2) Surface functionalization of superparamagnetic graphene oxide nanocarriers

[0011] Add 0.1-1 g of the superparamagnetic graphene oxide nanocarrier SGO prepared in step (1) to 30-300 mL of 0.01-0.1 M phosphate buffer, treat in an ultrasonic water bath for 10-60 min, then add 15-200 mL of 1-5 M sodium hydroxide solution and treat in an ultrasonic water bath for 60-300 min, then add 5-12 M hydrochloric acid for neutralization, filter and rinse to obtain carboxylic acid-modified SGO;

[0012] The modified SGO was diluted with deionized water to 0.5-2 mg / mL, 2-10 mg of surfactant was added and ultrasonically treated for 1-10 min, 1-10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC was added and ultrasonically treated, and then EDC (15-30 mM) was added, and 100-500 uL β-mercaptoethanol was added after sufficient stirring to terminate the reaction, and the surface was washed with deionized water after centrifugation at 8000-12000 × g for 3-10 min, and vacuum dried at 20-60 ° C to obtain a surface functionalized magnetic graphene oxide nanocarrier;

[0013] (3) Preparation of superparamagnetic graphene oxide nanoparticles immobilized with protease

[0014] 0.01-0.1 g of the surface functionalized magnetic graphene oxide nanocarrier prepared in step (2) is dispersed in 0.01-0.1 M phosphate buffer to prepare a suspension, and a 20-60 mg / mL protease solution is prepared with the phosphate buffer; 0.1-2 g of the protease solution is added to the SGO-PEGA suspension, stirred sufficiently at room temperature, and the solid is collected by centrifugation, and further treated with phosphate buffer for at least 3 times to finally obtain a superparamagnetic graphene oxide nano-immobilized protease.

[0015] Preferably, the ferrous salt is selected from one or more combinations of ferrous chloride, ferrous sulfate, ferrous nitrate or their hydrates, the iron salt is selected from one or more combinations of ferric chloride, ferric sulfate, ferric nitrate or their hydrates, the surfactant is selected from one or more combinations of polyethylene glycol diamine, ethylenediamine, and o-phenylenediamine, and the protease is one or more combinations of papain, neutral protease, composite protease, alkaline protease, and flavor protease.

[0016] Furthermore, the iron salt and ferrous salt used in step (1) are respectively FeCl 3 6H 2 O and FeCl 2 ·4H 2 O aqueous solution, the concentration of the phosphate buffer in step (2) is 50 mmol / L, and the pH is 9.25; the modified SGO in step (2) is diluted to 1 mg / mL with deionized water, the concentration of the phosphate buffer in step (3) is 50 mg / mL, the pH of the protease solution is 6.5, and the pH of the SGO-PEGA suspension is 11.

[0017] Furthermore, the saturation magnetization intensity of the superparamagnetic graphene oxide nanocarrier is -60-60emu / g, and the immobilized protease can be recovered and enriched through the magnetic field.

[0018] Moreover, the ratio of the protease solution to the surface functionalized magnetic graphene oxide carrier is 1:1-1:10 (v / w).

[0019] Furthermore, the superparamagnetic graphene oxide nano-immobilized protease has a solid loading capacity of 500-1500 mg / h, a solid loading rate of 10-60%, a saturation magnetization intensity of -30-30emu / g, and can still maintain more than 50% of the initial activity after being reused 7 times.

[0020] The second technical purpose of the present invention is to provide a method for preparing a bioactive peptide, comprising:

[0021] The target protein sample is pretreated, and after the treatment, the superparamagnetic graphene oxide nano-immobilized protease or free enzyme is added, stirred or shaken at a certain temperature, hydrolyzed for a certain time, placed in boiling water for a certain time after the hydrolysis, centrifuged, and the supernatant is collected to obtain the bioactive peptide.

[0022] It should be noted that the more the amount of immobilized protease added, the longer the hydrolysis time, and the smaller the molecular weight of the active peptide; the specific molecular weight ratio and range are related to factors such as the amount of superparamagnetic graphene oxide nano-immobilized protease added, the reaction temperature and the reaction time.

[0023] Furthermore, the target protein samples include β-lactoglobulin, casein, bovine serum albumin, soy protein, rapeseed protein, peanut protein, sesame protein, walnut protein, flaxseed protein, perilla seed protein, hemp seed protein, almond protein and other animal and plant proteins.

[0024] Further, the target protein sample is pretreated specifically including: dissolving the target protein sample in a constant temperature water bath, treating at 20-100° C. for 10-100 min, preferably denaturing at 95° C. for 15 min, and adjusting the pH to 9-12 with 1M NaOH after the temperature drops to room temperature;

[0025] Alternatively, the target protein sample is dissolved in ultrapure water (1:10-1:40, w / v), stirred in a 20-30° C. water bath for 1-3 h, and the pH is adjusted to 9-12 with 1 M NaOH.

[0026] Furthermore, the dissolution temperature of the target protein sample is 25° C. and the stirring time is 1 h.

[0027] Furthermore, the ratio of the immobilized protease to the target protein sample is 1 mg: 1-10 mg.

[0028] Furthermore, the hydrolysis temperature of the immobilized protease is 20-90°C, and the hydrolysis time is 2-30h, preferably the hydrolysis temperature is 30-70°C, and the hydrolysis time is 4-24h.

[0029] Furthermore, after the hydrolysis is completed, the mixture is placed in boiling water for 1-20 minutes, centrifuged at 3000-12000×g for 5-20 minutes, and the supernatant is collected to obtain a crude product of the bioactive peptide.

[0030] Moreover, the molecular weight distribution of the prepared bioactive peptides is: <500Da is 30%-55%, 500-1500Da is 0-40%, 1500-3000Da is 2-45%, and above 3000Da is 6-70%; the prepared bioactive peptides have antioxidant properties, ABTS + The free radical scavenging rate is 50-70%, Fe 2+ The chelating activity is 20-90%.

[0031] In summary, the immobilized carrier described in the present invention is a superparamagnetic graphene oxide nanomaterial, which has the characteristics of high specific surface area, excellent magnetic recovery, good biocompatibility, etc.; the superparamagnetic graphene oxide nano-immobilized protease adopts magnetic graphene oxide modified by ferrous salt and ferric salt as a carrier, and by introducing carboxylic acid functional groups on the carrier surface, the carboxyl group and the amino group (PEGA) are promoted to form an imine bond under the action of EDC, and the amino functionalization of the graphene oxide surface is realized, and then the protease is combined with the magnetic graphene oxide carrier by electrostatic adsorption. Soy protein, sesame protein, flax protein, casein, bovine serum albumin, β-lactoglobulin, etc. are used as target protein samples for pretreatment, and immobilized protease is added after the treatment is completed, and hydrolysis is carried out under certain conditions to obtain bioactive peptides. The present invention realizes the functionalization of the surface of magnetic graphene oxide by introducing carboxylic acid functional groups, and constructs a method for fixing protease to hydrolyze protein, which has the advantages of stable enzyme activity, easy recovery, reusability, good antioxidant properties of proteolytic peptides, and controllable molecular weight range.

[0032] It can be seen from the above technical solutions that, compared with the prior art, the beneficial effects achieved by the present invention include at least:

[0033] (1) The present invention uses magnetic graphene oxide modified by ferrous salt and ferric salt as a carrier, introduces carboxylic acid functional groups through surfactants to functionalize the carrier surface, realizes covalent bonding of protease and magnetic graphene oxide carrier, and prepares a superparamagnetic graphene oxide nano-immobilized protease, which significantly improves the activity and stability of the protease and effectively reduces the problems of protease self-aggregation and self-hydrolysis.

[0034] (2) Compared with free proteases, immobilized proteases based on superparamagnetic graphene oxide are easy to recover magnetically and can be quickly separated from substrates and products. After being reused for 7 times, they can still maintain more than 50% of their relative activity, with significant effects on enzyme activity stability and reusability.

[0035] (3) The alkaline protease immobilized on superparamagnetic graphene oxide has wide applicability and can be used for the efficient hydrolysis of oil proteins such as soy protein, sesame protein, flaxseed protein, and different protein sources such as whey protein and bovine serum albumin. The molecular weight and range of the bioactive peptides prepared by hydrolysis are controllable and adjustable, and the antioxidant effect is significantly improved compared with the parent protein, which has good potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. The drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without creative work.

[0037] Figure 1 This is a schematic diagram of the preparation principle of superparamagnetic graphene oxide nano-immobilized protease in Example 1 of the present invention.

[0038] Figure 2 These are scanning electron micrographs of SGO, SGO-PEGA, and Alcalase@SGO-PEGA in Example 1.

[0039] Figure 3 Magnetic characteristic diagram of SGO-PEGA and Alcalase@SGO-PEGA based on vibrating sample magnetometer in Example 1.

[0040] Figure 4 This is a comparison chart of the thermal stability of the free enzyme and the superparamagnetic graphene oxide nanoimmobilized protease in Example 1.

[0041] Figure 5 This is a comparison chart of the pH tolerance of the free enzyme and the superparamagnetic graphene oxide nanoimmobilized protease in Example 1.

[0042] Figure 6 This is a diagram showing the reusability of the superparamagnetic graphene oxide nano-immobilized protease in Example 1 of the present invention.

[0043] Figure 7 This is a diagram showing the antioxidant effects of bioactive peptides prepared by hydrolyzing different proteins based on superparamagnetic graphene oxide nano-immobilized proteases in the present invention.

[0044] Figure 8 This is a confocal image of the superparamagnetic graphene oxide nanoimmobilized protease in the present invention.

[0045] Fig. 9 This is the infrared spectrum of the superparamagnetic graphene oxide nano-immobilized protease in the present invention. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] The word "embodiment" used here as an "exemplary" does not necessarily mean that any embodiment described is superior to or better than other embodiments. Unless otherwise specified, the performance index tests in the embodiments of this application are performed using conventional test methods in the art. It should be understood that the terms described in this application are only used to describe specific implementation methods and are not used to limit the content disclosed in this application.

[0048] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0049] In order to better illustrate the content of the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0050] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0051] The invention discloses a preparation method of superparamagnetic graphene oxide nano-immobilized protease and application of the superparamagnetic graphene oxide nano-immobilized protease in the preparation of active peptides.

[0052] It should be noted that the following experimental determination contents and methods are as follows:

[0053] Enzyme activity assay:

[0054] Alkaline protease activity is measured by determining the amount of tyrosine produced from casein:

[0055] 1 mL of free protease or magnetic graphene oxide immobilized protease was dispersed in 1 mL of PBS (50 mM, pH 11), incubated at 50 °C for 5 min, and then 1 mL of casein (2%, wt) was added to react for 10 min; then 2 mL of trichloroacetic acid (0.4 M) was added to terminate the reaction, and the supernatant was obtained by centrifugation for 1 min; 1.0 mL of the obtained supernatant was added to a 10 mL test tube, and 0.5 mL of Na 2 CO 3 The aqueous solution (0.4 M) and 1.0 mL of Folinol reagent were mixed and incubated at 40° C. for 20 min. The absorbance of the mixture at a wavelength of 680 nm was measured using a spectrophotometer, and the amount of released tyrosine was calculated using a standard casein calibration curve.

[0056] One unit of enzyme activity is defined as the amount of protease required to generate 1.0 μg of tyrosine in 1 min at pH 11 and 50°C.

[0057] The molecular weight of the bioactive peptides was detected by HPLC after dilution and membrane treatment:

[0058] HPLC was Shimadzu's LC 20AD, the liquid chromatography column was a TSK gel G2000 SWXL column (7.8×300 mm), the detector was a PDA detector, the wavelength was 220 nm, the column temperature was 30°C, the flow rate was 0.5 mL / min, the mobile phase was a mixture of acetonitrile, ultrapure water and trifluoroacetic acid (acetonitrile: ultrapure water: trifluoroacetic acid = 45:55:0.1, v / v), and the sample injection volume was 10 μL.

[0059] Antioxidant assay of bioactive peptides: by ABTS + The antioxidant capacity of bioactive peptides was determined by their free radical scavenging ability. Protein hydrolyzed samples (20 μL) with different concentrations and ABTS + The solution (180 μL) was added to a 96-well plate in sequence, reacted at room temperature for 6-8 minutes, and the absorbance was measured at 734 nm using an enzyme reader. + The free radical scavenging rate was calculated as follows:

[0060]

[0061] In the formula, A 0 Anhydrous ethanol and ABTS + Absorbance of solution mixture, A 1 For samples with ABTS + Absorbance of solution mixture, A 2 It is the absorbance of the sample mixed with distilled water.

[0062] Through Fe 2+The antioxidant capacity of bioactive peptides was determined by chelation activity: 0.1 mL of ferrous chloride solution (2 mm) and 0.2 mL of ferrous zinc solution (5 mm) were added to 5 mL of protein hydrolysate, incubated at room temperature for 10 minutes, and finally the absorbance at a wavelength of 562 nm was measured using a spectrophotometer. The iron ion chelation activity of different protein hydrolysates was calculated as follows:

[0063]

[0064] In the formula, B 0 is the absorbance of ultrapure water, B 1 is the absorbance of different protein hydrolysates.

[0065] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as a limitation of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0066] Embodiment 1:

[0067] A method for immobilizing a protease and its application in the preparation of active peptides, specifically comprising the following steps:

[0068] (1) Preparation of superparamagnetic graphene oxide nanocarrier (SGO):

[0069] 400 mg of graphene oxide was added to 70 mL of 0.1 M sodium hydroxide solution, and then the target solid was collected by centrifugation (10000 × rpm, 20 min) after ultrasonic treatment at room temperature for 45 min, and washed with deionized water to make its pH value reach about 7.1; the particles were dispersed in 20 mL of degassed water with 360 W ultrasonic treatment for 30 min, and 480 mg of FeCl was added. 3 6H 2 O and 176 mg FeCl 2 ·4H 2 5 mL of a liquid suspension of 200 μg of O was ultrasonically treated for 60 min, and then a 20% ammonia solution was added to adjust the pH of the suspension to 9.25; finally, the reaction contents were separated, washed with deionized water for multiple times, and dried in an oven at 60 ° C overnight to obtain a superparamagnetic graphene oxide nanocarrier (SGO) with a saturation magnetization of 51.5 emu / g.

[0070] (2) Surface functionalization of superparamagnetic graphene oxide nanocarriers (SGO-PEGA):

[0071] 15 mg of SGO was added to 5 mL of phosphate buffer (pH 9.25) and treated in an ultrasonic water bath for 30 min. 3 mL of NaOH (3 M) was added to the SGO solution. After ultrasonic treatment for 3 h, hydrochloric acid was added for neutralization. After filtration and washing, carboxylic acid-modified SGO was obtained. The modified SGO was diluted to 1 mg / mL with deionized water, and 4 mg of polyethylene glycol diamine (PEGA) was added and ultrasonically treated for 5 min. 5 mM of EDC was added to the solution and ultrasonically treated for 30 min. Sufficient EDC was added to 20 mM and stirred for 12 h. β-mercaptoethanol (200 μL) was added to terminate the reaction. The precipitate was collected by centrifugation (10000×g, 5 min), washed with deionized water, and vacuum dried for 8 h to obtain a surface functionalized superparamagnetic graphene oxide carrier (SGO-PEGA).

[0072] (3) Preparation of superparamagnetic graphene oxide nano-immobilized protease:

[0073] 5 mg SGO-PEGA was dispersed in 1.5 mL phosphate buffer (50 mM, pH 6.5) to prepare a SGO-PEGA suspension. At the same time, a 40 mg / mL Alcalase alkaline protease solution was prepared with phosphate buffer (50 mM, pH 11); 2 mL of the diluted Alcalase solution was added to the SGO-PEGA suspension, stirred at room temperature for 6 h, and then centrifuged (10000×g, 10 min) to collect the solid, which was further treated with phosphate buffer for at least 3 times to obtain an enzyme immobilized graphene oxide nano-immobilized protease (Alcalase@SGO-PEGA) with an enzyme immobilization capacity of 1249 mg / g, an immobilization rate of 41.7%, and a saturation magnetization intensity of 20.6 emu / g.

[0074] (4) Preparation of bioactive peptides based on superparamagnetic graphene oxide nanoimmobilized protease:

[0075] 1 mg of casein was dissolved in ultrapure water (1:20, w / v), the pH was adjusted to 11 with 1 M NaOH and 1 mg of Alcalase@SGO-PEGA was added. After reacting at 50°C for 14 hours, the reaction solution was treated in boiling water for 10-15 minutes to inactivate the enzyme, and the supernatant was collected by centrifugation at 8000 rpm for 10 minutes. The supernatant was passed through a membrane to prepare a sample, and about 35% of the polypeptides in the casein hydrolysis product had a molecular weight of <500 Da, about 45% of the polypeptides had a molecular weight of 500-1500 Da, and about 20% of the polypeptides had a molecular weight of about 1500-3000 Da.

[0076] The raw materials listed in the present invention, as well as the upper and lower limits and interval values ​​of the raw materials and enzyme amounts of the present invention, and the upper and lower limits and interval values ​​of process parameters (such as temperature, time, and homogenization speed) can all realize the present invention, and the embodiments are not listed one by one here.

[0077] Embodiment 2:

[0078] Immobilized protease Alcalase@SGO-PEGA catalyzes the hydrolysis of different proteins to prepare bioactive peptides. The specific steps are as follows:

[0079] 1 mg of soy protein isolate was dissolved in ultrapure water (1:10, w / v), stirred in a 25°C water bath for 1 h, adjusted to pH 11 with 1 M NaOH, and then added with 1 mg of immobilized protease. The enzymatic reaction was carried out at 30, 50, and 70°C for 4 h. Then, the reacted sample was placed in boiling water for 10-15 min to inactivate the biocatalyst, and the supernatant was collected by centrifugation at 8000 rpm for 10 min. The supernatant was passed through a membrane to prepare a sample, and the hydrolyzate of the soy protein isolate was detected by high performance liquid chromatography.

[0080] The results showed that 96.7% of small peptides were obtained by enzymatic hydrolysis of soy protein isolate using Alcalase@SGO-PEGA at 30°C, of ​​which 52.9% of small peptides were <500Da and 43.9% of small peptides were between 500-1500Da. Regardless of the reaction temperature, the DW distribution remained relatively constant, indicating the effectiveness of Alcalase@SGO-PEGA. However, the peptides with DW>1500Da decreased from 3.3% to 2.1% and 0, respectively, as the reaction temperature increased from 30°C to 50°C and 70°C. In other words, increasing the temperature can increase the degree of hydrolysis of soy protein isolate, so the immobilized protease Alcalase@SGO-PEGA exhibited higher catalytic activity at 70°C.

[0081] Embodiment 3:

[0082] The immobilized protease Alcalase@SGO-PEGA catalyzed the hydrolysis of sesame protein to prepare bioactive peptides, as in Example 2.

[0083] The results showed that 75.4% of short peptides were obtained by enzymatic hydrolysis of sesame protein with Alcalase@SGO-PEGA at 30°C, of ​​which 36.3% were <500Da and 39.1% were between 500-1500Da. As the reaction temperature increased from 30°C to 50°C and 70°C, the proportion of short peptides (<500Da) decreased to 29.6% and 25.4%, respectively.

[0084] Embodiment 4:

[0085] Immobilized protease Alcalase@SGO-PEGA catalyzed the hydrolysis of flaxseed protein to prepare bioactive peptides, as in Example 2.

[0086] The results showed that 45% of short peptides (<500Da) could be obtained by enzymatic hydrolysis of flaxseed protein with Alcalase@SGO-PEGA at 30°C. As the reaction temperature increased from 30°C to 50°C and 70°C, the short peptides (<500Da) decreased to 34.2% and 18.1%, respectively.

[0087] Embodiment 5:

[0088] Immobilized protease Alcalase@SGO-PEGA catalyzes the hydrolysis of β-lactoglobulin to prepare bioactive peptides. The specific steps are as follows:

[0089] 1 mg of β-lactoglobulin was dissolved in ultrapure water (1:10, w / v), denatured in a 95°C water bath for 15 min, and after cooling, 1 M NaOH was added to adjust the pH to 11, and then 1 mg of immobilized protease was added. The enzymatic reaction was carried out at 30, 50, and 70°C for 4 h. Then, the reacted sample was placed in boiling water for 10-15 min to inactivate the biocatalyst, and the supernatant was collected by centrifugation at 8000 rpm for 10 min. The sample was passed through a membrane to prepare the sample, and the hydrolysis product of β-lactoglobulin was detected by high performance liquid chromatography.

[0090] The results showed that 74.4% of short peptides could be obtained by enzymatic hydrolysis of β-lactoglobulin at 30°C, of ​​which 31.2% were small peptides <500Da, 43.2% were short peptides between 500-1500Da, and 25.6% were medium-long peptides (>1500Da). As the reaction temperature increased to 70°C, the proportion of short peptides (<500Da) dropped to 23.1%.

[0091] Embodiment 6:

[0092] Immobilized protease Alcalase@SGO-PEGA catalyzes the hydrolysis of bovine serum albumin to prepare bioactive peptides. The specific steps are as follows:

[0093] 1 mg of bovine serum albumin was dissolved in ultrapure water (1:10, w / v), 1 M NaOH was added to adjust the pH to 11, 1 mg of immobilized protease was added, and the enzymatic reaction was carried out at 30, 50, and 70°C for 4 hours. Then, the reacted sample was placed in boiling water for 10-15 minutes to inactivate the biocatalyst, and the supernatant was collected by centrifugation at 8000 rpm for 10 minutes. The supernatant was passed through a membrane to prepare a sample, and the hydrolysis product of bovine serum albumin was detected by high performance liquid chromatography.

[0094] The results showed that 97% of short peptides could be obtained after enzymatic hydrolysis at 30 °C, among which 37.6% were short peptides (<500 Da), 59.4% were short peptides with molecular weights between 500 - 1500 Da, and only 3% were medium and long peptides with molecular weights >1500 Da. When the reaction temperature was increased to 70 °C, the short peptides (<500 Da) increased to 67.4%, indicating that increasing the temperature was beneficial for the hydrolysis of bovine serum albumin by the immobilized protease.

[0095] Example 7:

[0096] The immobilized protease Alcalase@SGO-PEGA was used to catalyze the hydrolysis of casein to prepare bioactive peptides, the same as in Example 6.

[0097] The results showed that after enzymatic hydrolysis at 30 °C for 4 h, 84.5% of short peptides could be obtained, including 48.7% (<500 Da) and 35.8% (500 - 1500 Da). As the reaction temperature increased, the short peptides (<500 Da) gradually decreased to 17.23%.

[0098] Example 8:

[0099] The immobilized protease Alcalase@SGO-PEGA was used to catalyze the hydrolysis of different proteins to prepare bioactive peptides, and the specific steps were as follows:

[0100] Dissolve 1 mg of soy protein isolate in ultrapure water (1:10, w / v), stir for 1 h in a 25 °C water bath, adjust the pH to 11 with 1 M NaOH, then add an appropriate amount of immobilized protease, carry out an enzymatic reaction at a temperature of 50 °C for 4 - 24 h. Then, place the reacted sample in boiling water for 10 - 15 min to inactivate the biocatalyst, centrifuge at 8000 rpm for 10 min to collect the supernatant, filter the supernatant to prepare the sample, and detect the hydrolysis products of soy protein isolate by high performance liquid chromatography.

[0101] The results showed that after enzymatic hydrolysis by Alcalase@SGO-PEGA for 4 h, the hydrolysis products of soy protein isolate consisted of 28.9% long peptides (MW > 3000 Da), 8.87% medium peptides (1500 < MW < 3000 Da), 20.26% short peptides (500 < MW < 1500 Da), and 41.97% short peptides (MW < 500 Da), and extending the reaction time to 14 h and 24 h had little effect on the molecular weight distribution of the peptides.

[0102] Example 9:

[0103] The immobilized protease Alcalase@SGO-PEGA was used to catalyze the hydrolysis of sesame protein to prepare bioactive peptides, the same as in Example 8.

[0104] The results showed that after 4 h of enzymatic hydrolysis by Alcalase@SGO-PEGA, the hydrolysis products of sesame protein consisted of 20.04% long peptides (MW>3000 Da), 19.09% medium peptides (1500<MW<3000 Da), 33.83% short peptides (500<MW<1500 Da), and 27.04% small peptides (MW<500 Da). Subsequently, the hydrolysis time was extended to 24 h, and the content of small peptides (MW<500 Da) increased from 27.04% to 29.96%. It can be seen that the hydrolysis time had a limited effect on the enzymatic reaction.

[0105] Example 10:

[0106] The immobilized protease Alcalase@SGO-PEGA was used to catalyze the hydrolysis of flaxseed protein to prepare bioactive peptides, the same as in Example 8.

[0107] The results showed that after 4 h of enzymatic hydrolysis by Alcalase@SGO-PEGA, the hydrolysis products of flaxseed protein consisted of 29.52% large peptides (MW>3000 Da), 15.94% medium peptides (1500<MW<3000 Da), 22.72% small peptides (500<MW<1500 Da), and 31.82% small peptides (MW<500 Da). When the hydrolysis time was extended to 24 h, the content of small peptides (MW<500 Da) increased to 42.7%. This indicated that the reaction time promoted the hydrolysis of flaxseed protein.

[0108] Example 11:

[0109] The immobilized protease Alcalase@SGO-PEGA was used to catalyze the hydrolysis of β-lactoglobulin to prepare bioactive peptides. The specific steps were as follows:

[0110] Dissolve 1 mg of β-lactoglobulin in ultrapure water (1:10, w / v), denature it in a 95 °C water bath for 15 min. After cooling, adjust the pH to 11 with 1 M NaOH, then add 1 mg of immobilized protease, and carry out an enzymatic reaction at a temperature of 50 °C for 4 - 24 h. Then, place the reacted sample in boiling water for 10 - 15 min to inactivate the biocatalyst, centrifuge at 8000 rpm for 10 min to collect the supernatant, filter it to prepare the sample, and use high performance liquid chromatography to detect the hydrolysis products of β-lactoglobulin.

[0111] The results showed that after 4 h of enzymatic hydrolysis by Alcalase@SGO-PEGA, the hydrolysis products of β-lactoglobulin consisted of 15.04% large peptides (MW>3000 Da), 27.61% medium peptides (1500<MW<3000 Da), 24.85% short peptides (500<MW<1500 Da), and 32.5% small peptides (MW<500 Da). When the enzymatic hydrolysis time was extended to 24 h, the small peptides (MW<500 Da) increased to 60.15%. This indicates that the reaction time has a promoting effect on the enzymatic hydrolysis of β-lactoglobulin.

[0112] Example 12:

[0113] The preparation of bioactive peptides by the catalytic hydrolysis of bovine serum albumin with the immobilized protease Alcalase@SGO-PEGA is as follows:

[0114] Dissolve 1 mg of bovine serum albumin in ultrapure water (1:10, w / v), adjust the pH to 11 with 1 M NaOH, add 1 mg of the immobilized protease, and carry out the enzymatic reaction at 50 °C for 4 - 24 h. Then, place the reacted sample in boiling water for 10 - 15 min to inactivate the biocatalyst, centrifuge at 8000 rpm for 10 min to collect the supernatant, prepare the sample by passing it through a membrane, and detect the hydrolysis products of bovine serum albumin by high performance liquid chromatography.

[0115] The results showed that after 4 h of enzymatic hydrolysis, the hydrolysis products of bovine serum albumin consisted of 19.85% large peptides (MW>3000 Da), 35.95% medium peptides (1500<MW<3000 Da), 21.66% short peptides (500<MW<1500 Da), and 22.54% small peptides (MW<500 Da). When the enzymatic hydrolysis time was extended to 24 h, the small peptides (MW<500 Da) increased to 24.3%. This indicates that increasing the hydrolysis time has little effect on the enzymatic hydrolysis reaction.

[0116] Example 13:

[0117] The preparation of bioactive peptides by the catalytic hydrolysis of casein with the immobilized protease Alcalase@SGO-PEGA is the same as in Example 12.

[0118] The results showed that after 4 h of enzymatic hydrolysis, the hydrolysis products of casein consisted of 4.34% large peptides (MW>3000 Da), 21.45% medium peptides (1500<MW<3000 Da), 49.59% short peptides (500<MW<1500 Da), and 24.62% small peptides (MW<500 Da). When the hydrolysis time was extended to 24 h, the small peptides (MW<500 Da) increased to 37.28%. Thus, it can be seen that increasing the hydrolysis time is beneficial to the formation of small peptides.

[0119] Embodiment 14:

[0120] Immobilized protease Alcalase@SGO-PEGA catalyzes the hydrolysis of different proteins to prepare bioactive peptides for ABTS+ elimination and Fe 2+ Chelation.

[0121] The results showed that the solution without hydrolyzed protein had a significant effect on ABTS+ scavenging and Fe 2+ The chelating activity of ABTS+ obtained after enzymatic hydrolysis of protein is very low (<20%). 2+ The chelating activity of ABTS+ was significantly improved. Compared with pure protein solution, the scavenging activity of ABTS+ was increased by about 4-8 times. 2+ The chelating activity was increased by about 2-33 times.

[0122] Embodiment 15:

[0123] Weigh 0.2 mg of fluorescein isothiocyanate (FITC) and dissolve it in 100 μL of dimethyl sulfoxide (DMSO) solution, add 2 mL of Alcalase solution (4 mg / mL), and react in the dark for 4 hours at 25°C and 220 rpm on a shaker. After the reaction, add a certain amount of anhydrous acetone to precipitate the Alcalase labeled with FITC. After centrifugation at 10000 rpm for 10 min, remove the excess FITC in the solution. Add PBS buffer (50 mM, pH 11.0) to make the final concentration of FITC-Alcalase solution 2 mg / mL. Then place 10 μL of immobilized protease on a slide and take a confocal (CLSM, LSM 980cococal, Zeiss, Germany).

[0124] The results show that the position of the fluorescently labeled Alcalase can be observed by comparing the bright field and the dark field. Combined with the position of the fluorescence distribution in Figure C, it is proved that Alcalase is successfully fixed on the graphene oxide.

[0125] Embodiment 16:

[0126] The prepared immobilized protease Alcalase@SGO-PEGA was characterized by infrared spectroscopy.

[0127] The results showed that 3450cm -1 The peaks at 1638 and 1607 cm correspond to the stretching vibration of -OH. -1 The peaks at 1065 and 1091 cm are related to the stretching vibration of C=C. -1 The peak at 1607 cm is related to the stretching vibration of CO. -1 and 1091cm -1The peak area of ​​​​Alcalase increased, indicating that Alcalase was fixed on graphene oxide, and its amino acid structure increased the content of C=C and CO.

[0128] Table 1. Preparation of bioactive peptides by hydrolysis of different proteins at different reaction temperatures

[0129]

[0130] Table 2. Preparation of bioactive peptides by hydrolysis of different proteins at different reaction times

[0131]

[0132]

[0133] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for immobilizing a protease, characterized in that: Superparamagnetic graphene oxide modified by ferrous salt and ferric salt is used as a carrier, and the surface of the carrier is functionalized by introducing carboxylic acid functional groups through surfactants to achieve covalent bonding of protease and magnetic graphene oxide carrier.

2. The method for immobilizing a protease according to claim 1, characterized in that: The specific operations are as follows: (1) Preparation of superparamagnetic graphene oxide nanocarrier (SGO) 0.1-1g of graphene oxide is added to a 0.05-0.5M sodium hydroxide solution, and ultrasonically treated at room temperature for 10-60min, and the target solid is collected by centrifugation at 8000-12000g, and washed with deionized water until the pH value reaches neutral; the particles are dispersed in 10-20mL of degassed water by ultrasonic treatment, and 5mL of a liquid suspension containing 50-150mg / mL of iron salt and 10-100mg / mL of ferrous salt is added, and ultrasonic treatment is carried out for 30-180min, and then a 10%-20% ammonia solution is added to make the pH value of the suspension reach 9-12; after repeated washing and filtering, it is dried in an oven at 50-100°C overnight to obtain a superparamagnetic graphene oxide nanocarrier SGO; (2) Surface functionalization of superparamagnetic graphene oxide nanocarriers Add 0.1-1 g of the superparamagnetic graphene oxide nanocarrier SGO prepared in step (1) to 30-300 mL of 0.01-0.1 M phosphate buffer, treat in an ultrasonic water bath for 10-60 min, then add 15-200 mL of 1-5 M sodium hydroxide solution and treat in an ultrasonic water bath for 60-300 min, then add 5-12 M hydrochloric acid for neutralization, filter and rinse to obtain carboxylic acid-modified SGO; The modified SGO was diluted with deionized water to 0.5-2 mg / mL, 2-10 mg of surfactant was added and ultrasonically treated for 1-10 min, 1-10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC was added and ultrasonically treated, and then 15-30 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC was added, and 100-500 uL β-mercaptoethanol was added after sufficient stirring to terminate the reaction, and the surface was washed with deionized water after centrifugation at 8000-12000 × g for 3-10 min, and vacuum dried at 20-60 ° C to obtain a surface functionalized magnetic graphene oxide nanocarrier; (3) Preparation of superparamagnetic graphene oxide nanoparticles immobilized with protease 0.01-0.1 g of the surface functionalized magnetic graphene oxide nanocarrier prepared in step (2) is dispersed in 0.01-0.1 M phosphate buffer to prepare a suspension, and a 20-60 mg / mL protease solution is prepared with the phosphate buffer; 0.1-2 g of the protease solution is added to the SGO-PEGA suspension, stirred sufficiently at room temperature, and the solid is collected by centrifugation to finally obtain a superparamagnetic graphene oxide nano-immobilized protease.

3. The method for immobilizing a protease according to claim 1 or 2, characterized in that: The ferrous salt is selected from one or more combinations of ferrous chloride, ferrous sulfate, ferrous nitrate or their hydrates; the iron salt is selected from one or more combinations of ferric chloride, ferric sulfate, ferric nitrate or their hydrates; the surfactant is selected from one or more combinations of polyethylene glycol diamine, ethylenediamine, and o-phenylenediamine; and the protease is one or more combinations of papain, neutral protease, composite protease, alkaline protease, and flavor protease.

4. An application of an immobilized protease in the preparation of an active peptide, characterized in that: The immobilized protease is obtained by the method of claim 1, and the preparation operation of the active peptide is as follows: The target protein sample is pretreated, and after the treatment, the superparamagnetic graphene oxide nano-immobilized protease or free enzyme is added, stirred or shaken at a certain temperature, hydrolyzed for a certain time, placed in boiling water for a certain time after the hydrolysis, centrifuged, and the supernatant is collected to obtain the bioactive peptide.

5. The use according to claim 4, characterized in that: The target protein samples include beta-lactoglobulin, casein, bovine serum albumin, soy protein, rapeseed protein, peanut protein, sesame protein, walnut protein, flaxseed protein, perilla seed protein, hemp seed protein, and almond protein.

6. The use according to claim 4, characterized in that: The target protein sample pretreatment specifically includes: dissolving the target protein sample in a constant temperature water bath, denaturing at 95° C. for 15 min, cooling the temperature to room temperature, and adjusting the pH to 9-12 with 1M NaOH; Alternatively, the target protein sample is dissolved in ultrapure water, the ratio of the target protein sample to ultrapure water is 1:10-1:40, w / v, stirred in a 20-30° C. water bath for 1-3 h, and the pH is adjusted to 9-12 with 1 M NaOH.

7. The use according to claim 4, characterized in that: The ratio of the immobilized protease to the target protein sample is 1 mg:1-10 mg, the hydrolysis temperature of the immobilized protease is 20-90° C., and the hydrolysis time is 2-30 hours.

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