CN116410958B - Preparation method of lysozyme inclusion particles and lysozyme inclusion particles - Google Patents
Preparation method of lysozyme inclusion particles and lysozyme inclusion particles Download PDFInfo
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- CN116410958B CN116410958B CN202310096142.1A CN202310096142A CN116410958B CN 116410958 B CN116410958 B CN 116410958B CN 202310096142 A CN202310096142 A CN 202310096142A CN 116410958 B CN116410958 B CN 116410958B
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- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 title claims abstract description 116
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- MNLRQHMNZILYPY-MDMHTWEWSA-N N-acetyl-alpha-D-muramic acid Chemical compound OC(=O)[C@@H](C)O[C@H]1[C@H](O)[C@@H](CO)O[C@H](O)[C@@H]1NC(C)=O MNLRQHMNZILYPY-MDMHTWEWSA-N 0.000 description 2
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 2
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 2
- 108010073771 Soybean Proteins Proteins 0.000 description 2
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- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
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- Chemical & Material Sciences (AREA)
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- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Animal Husbandry (AREA)
- Food Science & Technology (AREA)
- Enzymes And Modification Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention provides a preparation method of lysozyme inclusion particles and a product thereof, belonging to the technical field of immobilized enzyme preparation. The preparation method of the invention comprises the following steps: weighing AB-8 macroporous resin after vacuum drying, mixing with phosphate buffer solution for 20-40 min at 150-200 r/min, adding lysozyme after complete dissolution, mixing for 20-40 min at 150-200 r/min, adding glutaraldehyde solution with final mass concentration of 0.4-0.8 g/100mL, mixing, immobilizing for 4.3-4.8 h, filtering and washing to obtain lysozyme inclusion particles. After the lysozyme inclusion particles prepared by the invention are used for 4-6 times, the enzyme activity still remains 80-95%, the highest activity can be still kept at the temperature of 45-52 ℃, and the activity repeated availability and stability are obviously higher than those of free lysozyme.
Description
Technical Field
The invention belongs to the technical field of immobilized enzyme preparation, and particularly relates to a preparation method of lysozyme inclusion particles and a product thereof.
Background
Lysozyme (Lysozyme, EC 3.2.1.17) is called Lysozyme because of its bacteriolytic effect, i.e., the ability to precisely cleave the N-acetylglucosamine and N-acetylmuramic acid linked (. Beta. -1,4 glycosidic bond) on the bacterial cell wall, thus achieving the purpose of cell lysis. The lysozyme is applied to the food industry, such as anti-corrosion and fresh-keeping, health food additives, infant food additives and the like; medical applications such as inhibiting oral diseases, staphylococcal infections, etc.; in animal husbandry, such as preparing feed additive to achieve antibacterial, antiviral and immunity improving effects.
In the feed and food industry, lysozyme can selectively kill harmful bacteria in the intestinal tracts of livestock, such as escherichia coli and the like, can safely replace chemical additives harmful to animals so as to achieve a good antibacterial treatment effect, is an excellent feed additive without toxic or side effects, however, the activity is reduced and unstable due to the fact that free enzyme is easily influenced by temperature, pH, strong acid, strong alkali, storage and other environments in the process of functioning. Therefore, improving lysozyme activity and stability is a technical problem that human beings need to solve.
Disclosure of Invention
The invention provides a preparation method of lysozyme inclusion particles and a product thereof, and the active repeated availability and stability of the lysozyme inclusion particles prepared by the method are obviously higher than those of free lysozyme.
In order to solve the technical problems, the invention provides the following technical scheme:
The invention provides a preparation method of lysozyme inclusion particles, which comprises the following steps: weighing AB-8 macroporous resin after vacuum drying, mixing with phosphate buffer solution for 20-40 min at 150-200 r/min, slowly adding lysozyme after complete dissolution, mixing for 20-40 min at 150-200 r/min, adding glutaraldehyde solution with the final concentration of 0.4-0.8 g/100mL, mixing, immobilizing for 4.3-4.8 h, filtering and washing to obtain lysozyme inclusion particles.
Preferably, the mass ratio of the lysozyme to the AB-8 macroporous resin is 1:150 to 250.
Preferably, the concentration of the lysozyme is 0.5-2.5 mg/mL
Preferably, the immobilization temperature is 20-30 ℃.
The invention provides lysozyme inclusion particles prepared by the preparation method.
Preferably, the tolerant temperature of the lysozyme inclusion particle is 45-52 ℃, and the enzyme activity is still 80-95% after the lysozyme inclusion particle is used for 4-6 times.
The invention provides application of lysozyme inclusion particles in preparing feed.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, macroporous resin AB-8 is used as a carrier, enzyme is adsorbed under the action forces of intermolecular Van der Waals force, hydrogen bond and the like, glutaraldehyde is used as a cross-linking agent, so that lysozyme inactive molecules are cross-linked, and the adsorption stability of the carrier to the enzyme is improved; according to the invention, the two low-rotation-speed mixing is set in the preparation process, so that the lysozyme and macroporous resin can be effectively combined, and the lysozyme can be more effectively embedded; in the preparation process, proper raw material dosage, curing time and curing temperature are set, so that the reusability and stability of lysozyme are further improved.
(2) After the lysozyme inclusion particles prepared by the invention are used for 4-6 times, the enzyme activity still remains 80-95%, the highest activity can be still maintained at the temperature of 45-52 ℃, the tolerance capability of 5 ℃ is improved compared with that of the non-inclusion lysozyme, and the activity repeated availability and stability are obviously higher than those of the free lysozyme.
(3) The preparation method is simple and easy to implement, has no requirement on operators, does not generate peculiar smell and nutrient loss in the process, and does not need other energy consumption such as heating.
Drawings
FIG. 1m enzyme: and the influence result of the m carrier proportion on the lysozyme enzyme activity.
FIG. 2 results of the effect of glutaraldehyde mass concentration on the relative enzyme activity of lysozyme inclusion particles.
FIG. 3 results of the effect of immobilization time on lysozyme inclusion particle activity.
FIG. 4 shows the effect of immobilization temperature on lysozyme inclusion particle activity.
FIG. 5 determination of (A) optimum temperature and (B) optimum pH for the enzymatic reaction of lysozyme inclusion particles.
FIG. 6 results of a thermal stability assay of lysozyme inclusion particles with free enzyme.
FIG. 7 results of a repeatability assay of lysozyme-coated particles.
FIG. 8 shows the results of the antibacterial effect of lysozyme inclusion particles on E.coli.
Detailed Description
The invention provides a preparation method of lysozyme inclusion particles, which comprises the following steps: weighing AB-8 macroporous resin after vacuum drying, mixing with phosphate buffer solution for 20-40 min at 150-200 r/min, slowly adding lysozyme after complete dissolution, mixing for 20-40 min at 150-200 r/min, adding glutaraldehyde solution with the final concentration of 0.4-0.8 g/100mL, mixing, immobilizing for 4.3-4.8 h, filtering and washing to obtain lysozyme inclusion particles. The pH of the phosphate buffer solution according to the invention was 7.2. The concentration of the lysozyme is 0.5-2.5 mg/mL, preferably 1mg/mL.
In the invention, the mass ratio of the lysozyme to the AB-8 macroporous resin is 1:150 to 250, preferably 1:200. after the AB-8 macroporous resin is combined with lysozyme to a certain extent, enzyme molecules are basically adsorbed on the surface of the AB-8 macroporous resin, and the failure to adsorb is probably due to the fact that the washing resistance is improved after the mass of the AB-8 macroporous resin is increased, and the immobilization cost is increased.
In the present invention, the final glutaraldehyde concentration is preferably 0.6g/100mL. The glutaraldehyde is prepared into 50g/100ml of concentrated glutaraldehyde solution, and then different volumes of concentrated glutaraldehyde solution are added to enable glutaraldehyde in the system to reach corresponding final concentration. When the glutaraldehyde concentration is higher, the protein hydrophobicity is not favored, and when the glutaraldehyde concentration is slightly higher, the color of the soybean protein is changed. The strong interactions between them disrupt the higher structure of the protein, causing a change in the enzyme conformation.
In the present invention, the immobilization temperature is 20 to 30 ℃, preferably 25 ℃. When the immobilization temperature is lower, the adsorption capacity of the carrier to lysozyme is reduced due to the heat required in the adsorption process, the adsorption of protease is facilitated by increasing the temperature at low temperature, but the temperature is increased again, so that a part of enzyme is deactivated, and the activity is reduced due to the fact that the crosslinking agent glutaraldehyde is used for improving ineffective crosslinking of free enzyme.
The invention provides lysozyme inclusion particles prepared by the preparation method. The tolerance temperature of the lysozyme inclusion particles is 45-52 ℃, preferably 50 ℃, and the tolerance capability of the lysozyme inclusion particles is improved by 5 ℃ compared with that of the lysozyme inclusion particles without free enzyme inclusion. The lysozyme inclusion particle disclosed by the invention has the advantages that the enzyme activity is still kept 83.6% after being used for 5 times, the enzyme activity is kept 60.5% after being used for 8 times, the enzyme activity is kept 54.9% after being used for 10 times, and the reusability is excellent and meets the expected result.
The invention provides application of lysozyme inclusion particles in preparing feed. The invention can be used as a feed additive to prepare feed by combining with other components which do not influence the activity of the enzyme. The use amount of the lysozyme inclusion particles is 0.35-10%.
In the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 Process investigation of lysozyme inclusion particles
1. The activity of the lysozyme inclusion particles is determined as follows:
(1) Sample preparation: taking 1.2g of lysozyme inclusion particles, precisely weighing, diluting to 100ml by using a citric acid-sodium citrate buffer solution with pH=6.2, magnetically stirring and dissolving for about 4 hours, and filtering by using filter paper to obtain a liquid sample;
(2) Preparation of the reaction substrate: 30mg of Micrococcus wall-dissolving dry powder is weighed, put into a cell grinder for grinding, washed into a small beaker with phosphate buffer solution, added to a scale of about 70ml, gently shaken to suspend the solution, the solution is cultured in a water bath at 35 ℃ for 30 minutes, the absorption value is measured to be adjusted to about 0.70+/-0.05, and the solution is kept at 25 ℃ in the water bath.
(3) The analysis step: firstly, zeroing a spectrophotometer by adding distilled water into a blank cuvette at the wavelength of 450nm, accurately measuring 3.0ml of substrate suspension, placing the substrate suspension into a 1ml cuvette, and measuring a light absorption value A0; accurately measuring 0.10ml of the sample solution prepared in the step (1), adding the sample solution into the cuvette, rapidly mixing, timing by using a stopwatch, and recording a light absorption value reading A when the time reaches 180 seconds; simultaneously, 0.10ml of a citric acid-sodium citrate buffer solution is accurately measured, the same method is operated, as a blank test, an absorption value A '0 of zero seconds and an absorption value A' of 180 seconds are measured, each sample is repeatedly processed for at least 2 times to ensure the accuracy and precision of measurement, and the enzyme activity is calculated: expressed in enzyme activity units (u/mg), the enzyme activity of the sample was calculated according to the following formula:
Wherein: a0-absorbance at zero time of non-loading of substrate suspension;
A-absorbance of the substrate suspension 180s after loading;
a' 0-absorbance value when substrate suspension is unbuffered;
Absorption value 180s after addition of buffer to A' -substrate suspension;
m-mass in sample solution (ug).
0.001-The value of the decrease in absorbance per minute caused by the unit lysozyme.
2. Immobilized mzyme/m vector ratio determination
Weighing 0.2g, 0.4g, 0.6g, 0.8g, 1.0g, 1.2g and 1.4g of AB-8 macroporous resin subjected to vacuum drying, mixing with phosphate buffer solution of pH7.2, uniformly mixing for 30min at 180r/min, slowly adding 4mL of lysozyme solution with concentration of lmg/mL after complete dissolution, uniformly mixing for 30min at 180r/min, adding glutaraldehyde solution with final mass concentration of 0.4g/100mL, shaking uniformly, fixing for 4h at 25 ℃, filtering and washing to obtain lysozyme inclusion particles. Statistically different m (enzymes): the relative enzyme activity of the immobilized enzyme obtained under the m (carrier) condition was measured in the same manner as in step 1 of this example. The results are shown in FIG. 1.
As can be seen from fig. 1, the enzyme m: when the m carrier is changed from 1:50 to 1:150, the carrier quality is increased, the relative enzyme activity of the immobilized enzyme is greatly enhanced, and the relative enzyme activity is respectively increased by 40% and 79.7%; when the ratio is 1:200, the enzyme activity reaches the peak value, and when the carrier ratio is increased, the enzyme activity is basically consistent. That is, when the carrier is combined with lysozyme to a certain extent, enzyme molecules are basically adsorbed on the surface of the carrier, and the failure to adsorb the enzyme molecules is probably due to the fact that washing resistance is improved after the mass of the carrier is increased, and immobilization cost is increased. So the optimal mzyme is finally obtained: the m carrier ratio is 1:200.
3. Determination of mass concentration of immobilized glutaraldehyde
Slowly adding 4mL lmg/mL lysozyme solution into 1.0g AB-8 macroporous resin dissolved by phosphate buffer solution, uniformly mixing for 30min at 180r/min, respectively adding different volumes of concentrated glutaraldehyde solution (50 g/100 mL) to ensure that the mass concentration of glutaraldehyde is 0.2, 0.4, 0.6, 0.8 and 1.0g/100mL, slowly mixing at uniform speed in the dripping process, fixing for 4h at 25 ℃, filtering and washing to obtain lysozyme inclusion particles. The relative enzyme activity of the prepared lysozyme inclusion particles was measured, and the measurement method was the same as in step 1 of this example, except that the highest enzyme activity of the finally produced lysozyme by detecting glutaraldehyde at different concentrations was 100%, the other being the relative value. The results are shown in FIG. 2.
As can be seen from FIG. 2, when the glutaraldehyde mass concentration is lower than 0.6g/100mL, the relative enzyme activity of immobilized lysozyme increases with the increase of glutaraldehyde concentration, and when the glutaraldehyde mass concentration is higher than 0.6g/100mL, the glutaraldehyde concentration is increased to lower the enzyme activity of immobilized enzyme, because the higher glutaraldehyde concentration is unfavorable for protein hydrophobicity, and if the glutaraldehyde concentration is slightly higher, the color of soybean protein is changed. The strong interactions between them disrupt the higher structure of the protein; the excessive glutaraldehyde itself can be crosslinked intramolecularly, and solidified into irregular substances to be attached to the surface of a carrier, so that besides affecting enzyme immobilization, excessive aldehyde groups can be reacted with the enzyme to cause the change of enzyme conformation, so that the activity of the immobilized enzyme is reduced, and the optimal mass concentration of glutaraldehyde is selected when 0.6g/100mL is used for immobilization.
4. Determination of immobilization time
Adding 4mL of enzyme solution with the concentration of 1mg/mL into 1.0g of AB-8 macroporous resin, oscillating for 20min at 180r/min, adding glutaraldehyde solution with the final mass concentration of 0.6g/100mL, fully and uniformly mixing, respectively fixing at 25 ℃ for 2.0, 2.5, 3, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0h, and filtering and washing to obtain the lysozyme inclusion particles. The relative enzyme activity of the prepared lysozyme inclusion particles was measured, and the measurement method was the same as step 1 of this example, taking the highest enzyme activity as 100%. The results are shown in FIG. 3.
As can be seen from FIG. 3, the enzyme activity is continuously improved when the immobilization time is 2.0-4.5h, the enzyme activity reaches the maximum value in 4.5h, the enzyme activity is reduced when the immobilization time is prolonged, which means that the combination state of the enzyme and the carrier is saturated, the cross-linking agent glutaraldehyde forms a cross-linking structure between free enzymes to destroy the three-dimensional structure and the enzyme activity site of lysozyme, and when the enzyme immobilization amount on the carrier reaches the saturation state, the distance between protease molecules is too close, the combination efficiency of the substrate and the enzyme molecules is possibly blocked, and the enzyme activity measurement result of the enzymatic reaction is lower, so that 4.5h is selected as the optimal curing time.
5. Immobilization temperature determination
Adding 4mL of lysozyme solution with the concentration of 1mg/mL and 180r/min into 1.0gAB-8 macroporous resin, uniformly mixing for 30min, adding glutaraldehyde solution with the final mass concentration of 0.6g/100mL, fully mixing, fixing for 4.5h at the temperature of 4, 15, 25, 35, 45, 50 and 55 ℃ respectively, filtering and washing to obtain lysozyme inclusion particles. The relative enzyme activity of the prepared lysozyme inclusion particles was measured, and the measurement method was the same as that of step 1 in this example, and the enzyme activity was measured at the highest temperature condition and was taken as 100%. The results are shown in FIG. 4.
As can be seen from fig. 4, in the lower temperature range, the relative enzyme activity of lysozyme increases with increasing temperature, and the optimum temperature of lysozyme is immobilized at 25 ℃; after exceeding 25 ℃, the lysozyme enzyme activity gradually decreases with the increase of the temperature. The reason is that under the condition of lower temperature, the adsorption process of protein needs heat, the temperature is increased at low temperature to be favorable for the adsorption of protease, but at the temperature, the temperature is increased again to inactivate a part of enzymes, and the crosslinking agent glutaraldehyde can also increase the ineffective crosslinking of free enzymes to reduce the activity, so that the optimal temperature for the immobilization of lysozyme is selected at 25 ℃.
The best suitable parameters in the preparation of the lysozyme inclusion particles are as follows according to the exploration of the technological parameters: menzyme mCarrier=1: 200, glutaraldehyde final mass concentration 0.6g/100mL, immobilization time 4.5h, immobilization temperature 25 ℃.
Example 2 preparation of lysozyme inclusion particles
Weighing 1.0g of AB-8 macroporous resin subjected to vacuum drying, mixing with phosphate buffer solution with pH of 7.2, uniformly mixing for 30min at 180r/min, and after complete dissolution, mixing according to the mass ratio of lysozyme to AB-8 macroporous resin of 1:200 mg/mL of lysozyme solution is added, 180r/min is mixed for 10min, glutaraldehyde solution with the final mass concentration of 0.6g/100mL is added, the mixture is mixed uniformly, immobilized for 4.5h at 25 ℃, and lysozyme inclusion particles are obtained through filtration and washing.
Example 3 preparation of lysozyme inclusion particles
The mixing speed was 200r/min and the time was 40min as compared with example 1, and the other steps were the same as in example 1.
Example 4
Preparation of lysozyme inclusion particles
The final mass concentration of glutaraldehyde solution was 0.8g/100mL as compared to example 1, and the rest of the procedure was the same as in example 1.
Example 5 determination of optimal temperature and optimal pH for enzymatic reaction of lysozyme inclusion particles
Determining the optimal temperature for the enzymatic reaction by measuring the relative enzyme activities of the lysozyme inclusion particles prepared in example 2 at 20, 25, 30, 35, 40 and 45 ℃ according to the measurement method in step 1 of example 1, wherein the activity under the temperature condition with the highest activity is 100%; the relative enzyme activities were measured under the optimum temperature conditions at pH values of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0 according to the measurement method in step 1 of example 1, and the optimum pH of the enzymatic reaction was determined at the highest activity pH value of 100%, as shown in FIG. 5.
As can be seen from FIG. 5, the optimum temperature in the enzymatic reaction of the lysozyme inclusion particle prepared in example 2 of the present invention is 35℃and the optimum pH is 6.
Example 6 comparative experiments on the thermal stability of lysozyme inclusion particles and free lysozyme
The lysozyme inclusion particles and free lysozyme prepared in example 2 were stored at 25, 35, 40, 45, 55, 65 and 75℃for 0.5 hours, and then the relative enzyme activities of the lysozyme inclusion particles and free lysozyme were measured at 35℃and pH6.0, respectively, in the same manner as in step 1 of example 1. The residual enzyme activities at other temperatures were measured with the activity at the temperature at which the enzyme activity was highest being 100%. The effect of temperature on the enzyme activity of the free and immobilized enzymes is shown in FIG. 6.
As can be seen from fig. 6, the lysozyme inclusion particle has the optimal temperature for enzyme activity that is the highest in stability at 50 ℃ and 10 ℃ higher than the free enzyme; the relative enzyme activities of the lysozyme inclusion particles are higher than that of free lysozyme in the temperature range of 45-60 ℃, and the results prove that the enzymatic properties of the lysozyme are improved after the AB-8 macroporous resin is immobilized.
Example 7 determination of the recyclability of lysozyme inclusion particles
The lysozyme inclusion particles prepared in example 2 were subjected to degradation of chitosan acetate solution at 35 ℃ and pH6, and after the reaction, the chitosan acetate solution was washed with phosphate buffer solution at pH6.2, and immobilized microspheres were recovered, and the above operations were repeated, and the enzyme activity was measured each time, and the reusability of the immobilized enzyme was examined with the initial enzyme activity being 100% of the relative enzyme activity, and the better the repeatability was, the greater the industrial application potential was. The results are shown in FIG. 7.
As can be seen from FIG. 7, after the immobilized enzyme was coated with the AB-8 macroporous resin, the enzyme activity remained 83.6% after 5 uses, and at 8 uses, the enzyme activity remained 60.5% and at 10 times, 54.9%. The repeated usability is excellent, and the expected result is met.
Example 8 determination of antibacterial Effect of lysozyme inclusion particles
2.0G of immobilized lysozyme prepared in example 2 was weighed and dissolved in a sodium citrate buffer at pH6.0 for 4 hours to prepare a lysozyme solution. In a super clean bench, 50. Mu.L of lysozyme solution was spotted onto LB agar plates coated with E.coli, A, B, C, D, F as a control plus H 2 O group, G, E as a lysozyme group, and left to stand at 37℃for 24 hours. The results of the bacteriostasis experiment are shown in figure 7.
As can be seen from FIG. 8, G, E groups had a clear zone of inhibition on the E.coli plate, whereas none of the control groups had a clear effect. Lysozyme breaks the beta-1, 4 glycosidic bond of N-acetylglucosamine and N-acetylmuramic acid through specific recognition on bacterial cell walls, so that the cell walls are depolymerized, and bacteria are killed. The lysozyme can still achieve ideal antibacterial effect after being combined by AB-8 macroporous resin, and proves the feasibility of the technology.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (3)
1. The preparation method of the lysozyme inclusion particle is characterized by comprising the following steps: weighing AB-8 macroporous resin after vacuum drying, mixing with phosphate buffer solution for 20-40 min at 150-200 r/min, slowly adding lysozyme after complete dissolution, mixing for 20-40 min at 150-200 r/min, adding glutaraldehyde solution with the final concentration of 0.4-0.8 g/100mL, mixing, immobilizing for 4.3-4.8 h, filtering and washing to obtain lysozyme inclusion particles;
the mass ratio of the lysozyme to the AB-8 macroporous resin is 1:150-250;
the concentration of the lysozyme is 0.5-2.5 mg/mL;
the immobilization temperature is 20-30 ℃.
2. The lysozyme inclusion particle prepared by the method of claim 1.
3. Use of the lysozyme inclusion particle as claimed in claim 2 in the preparation of a feed.
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| CN107058278A (en) * | 2017-04-11 | 2017-08-18 | 熊科 | A kind of preparation method of Carboxypeptidase A immobilised enzymes |
| CN110656102A (en) * | 2019-10-22 | 2020-01-07 | 中北大学 | Preparation method of beta-glucosidase immobilized enzyme |
| CN110862981A (en) * | 2019-11-26 | 2020-03-06 | 南京工业大学 | Method for immobilizing lipase by using nano material |
| CN112680489A (en) * | 2021-01-15 | 2021-04-20 | 驻马店华中正大有限公司 | Method for improving monensin bioavailability |
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| CN107058278A (en) * | 2017-04-11 | 2017-08-18 | 熊科 | A kind of preparation method of Carboxypeptidase A immobilised enzymes |
| CN110656102A (en) * | 2019-10-22 | 2020-01-07 | 中北大学 | Preparation method of beta-glucosidase immobilized enzyme |
| CN110862981A (en) * | 2019-11-26 | 2020-03-06 | 南京工业大学 | Method for immobilizing lipase by using nano material |
| CN112680489A (en) * | 2021-01-15 | 2021-04-20 | 驻马店华中正大有限公司 | Method for improving monensin bioavailability |
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