CN112159802A - Lysozyme modified by cinnamyl aldehyde coupling and modification method thereof - Google Patents
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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)
-
- 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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- Organic Chemistry (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
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- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
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Abstract
The invention discloses a lysozyme modified by cinnamaldehyde coupling and a modification method thereof. The invention has the advantages that: has the advantages of improving the antibacterial effect on gram-negative bacteria, widening the antibacterial spectrum of lysozyme, expanding the application range of lysozyme and the like.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to lysozyme modified by cinnamaldehyde coupling and a modification method thereof.
Background
Lysozyme is an effective antibacterial enzyme, is white or white crystal in pure product, has strong thermal stability, can be stored for a long time under dry conditions, and can specifically hydrolyze beta-1, 4 glycosidic bond sites between N-acetylglucosamine and N-acetylmuramic acid so as to break cells. According to the biological source and action mechanism of LZ, LZ can be divided into microorganism, animal, plant and bacteriophage T4 source LZ, wherein animal source lysozyme includes c type, g type and i type. The species and site of action of the substrate may vary from source to source of LZ. Compared with LZ from other sources, human lysozyme has unique superiority and various pharmacological effects, and has been widely applied to the fields of food industry, animal husbandry and medical treatment. The human lysozyme belongs to C-type lysozyme, has the molecular weight of 14.7KD, consists of 130 amino acids, has the active centers of Glu35 and Asp52, and can catalyze and hydrolyze the beta-1, 4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine through the combined action of two key residues, namely glutamic acid at the 35 th position and aspartic acid at the 52 th position. HLZ is widely distributed in human tissues and several studies have indicated that HLZ has antifungal and antiviral effects. Compared with egg white lysozyme, the HLZ has better thermal stability, the pH value is within the range of 4-7, the lysozyme can still keep almost proenzyme activity after being treated for 1min at the temperature of 100 ℃, and the sterilization capacity is far higher than that of egg white lysozyme. No. 194 bulletin of Ministry of agricultural rural areas, namely growth-promoting drug feed additives are completely prohibited from being used in 7-1.7.2020, LZ is considered as a potential substitute of feed antibiotics, and in recent years, some researches show that LZ can achieve the same growth-promoting effect as antibiotic feed when used in antibiotic-free livestock and poultry feed. The previous research shows that the exogenous LZ added into the feed can improve the growth performance of livestock and poultry, improve the intestinal form, improve the immunity and improve the intestinal microflora structure.
Although the LZ has obvious bacteriostatic effect, the antibacterial spectrum is narrow, the antibacterial effect is obvious on gram-positive bacteria of which peptidoglycan is positioned on the surface of a cell wall, and the bacteriostatic effect is not obvious on gram-negative bacteria of which the outer side of the cell wall is coated by lipopolysaccharide, so that the wide-range application of the antibacterial agent is limited. The chemical modification of lysozyme is to utilize organic micromolecules or polysaccharide and other molecular compounds to carry out chemical modification on the surface of an LZ molecule, the hydrophobic organic micromolecules can be used as a hydrophobic carrier, and the hydrophobic organic micromolecules are combined with the LZ to convey the hydrophobic organic micromolecules to a plasma membrane layer. Research shows that medium-short chain saturated fatty acids C6:0 caproic acid, C10:0 capric acid and C14:0 myristic acid are combined with LZ, so that the antibacterial activity on E.
The cinnamaldehyde is a micromolecular organic matter with bacteriostatic activity, can change the metabolic activity of bacteria by influencing the integrity and permeability of a thallus envelope, and has been widely applied to the feed industry as a bacteriostatic mildew-proof feed additive. Human lysozyme modified by cinnamaldehyde coupling is utilized to improve the bacteriostatic effect of the human lysozyme on gram-negative bacteria, broaden the antibacterial spectrum of the lysozyme and expand the application range of the lysozyme, particularly in the field of replacing antibiotics by feeds.
Disclosure of Invention
In order to solve the problems, the invention provides the lysozyme modified by the cinnamaldehyde coupling and the modification method thereof, which can improve the bacteriostatic effect on gram-negative bacteria, widen the antibacterial spectrum of the lysozyme and expand the application range of the lysozyme.
In order to solve the technical problems, the technical scheme provided by the invention is as follows: the lysozyme is characterized in that the lysozyme is obtained by covalently combining cinnamaldehyde essential oil and human lysozyme, and an imine bond is formed between an aldehyde group in the cinnamaldehyde essential oil and an amino acid residue of the human lysozyme.
Preferably, the cinnamaldehyde is C9H80[ 3-phenyl-2-propenal]。
The invention also comprises a modification method of lysozyme by utilizing cinnamaldehyde coupling modification, which comprises the following steps:
the method comprises the following steps: dissolving active ester of cinnamaldehyde in DMSO, adding NaHCO3 aqueous solution of lysozyme, performing condensation reaction under the condition of stirring, and obtaining a reaction mixture after the reaction is finished;
step two: and D, dialyzing the buffer solution obtained in the step I by using deionized water and a phosphate buffer solution in sequence, and carrying out centrifugal separation on the obtained dialysate to obtain the modified lysozyme.
Preferably, the lysozyme is human lysozyme.
Preferably, the addition amount of the cinnamaldehyde is 5.0-10.0g/L, and the addition amount of the lysozyme is 1.5-2.0 g/L.
Preferably, the mixing temperature of the lysozyme and the cinnamaldehyde is 30-50 ℃.
Preferably, the modification method comprises the following specific steps of dissolving cinnamaldehyde in 5mL of mixed solution of sodium cinnamate and sodium cinnamate to form a solution with the final concentration of 0.7mM, dropwise adding 25mL of 0.2mM of 1% of NaHCO3 solution of egg white lysozyme under the condition of stirring, and continuously and slowly stirring for reaction at 30 ℃ for 6 hours; after the reaction, 25mL of 100mM glycine solution was added to the system, the temperature was maintained at 30 ℃ for 10min, the mixture was dialyzed against deionized water at room temperature, then against 20mM phosphate buffer, pH 7.0, and the mixture was maintained at 4 ℃ for about 1 day.
Compared with the prior art, the invention has the advantages that: the invention utilizes the organic bacteriostatic agent cinnamyl aldehyde essential oil to carry out effective chemical modification on human lysozyme, the enzyme has small enzyme activity loss in the modification process, the spatial structure of the enzyme activity center is adjusted, the bacteriostatic effect of the lysozyme is enhanced, the bacteriostatic ability of the lysozyme to gram-negative bacteria (such as escherichia coli) is greatly improved, a novel lysozyme modifier and a modification method are developed, the mode is simple and flexible, and the method is a biological engineering technology with practical value; compared with natural lysozyme, the method for expanding the antibacterial spectrum of lysozyme provided by the invention has the advantages that the antibacterial activity of lysozyme on gram-positive bacteria is improved, the antibacterial capability on gram-negative bacteria is greatly improved, and the antibacterial spectrum of lysozyme is effectively expanded.
Drawings
FIG. 1 is a schematic flow chart of lysozyme modified by cinnamaldehyde coupling and a modification method thereof.
FIG. 2 is a chemical reaction scheme of lysozyme modified by cinnamaldehyde coupling and a modification method thereof.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Example 1
Covalent modification of human lysozyme by cinnamaldehyde
The final concentration of the solution formed by dissolving cinnamaldehyde in 5mL of mixed MSO is 0.7mM, 25mL of 0.2mM egg white lysozyme solution of 1% NaHCO3 is added dropwise with stirring, and the reaction is continued for 6h at 30 ℃ with slow stirring. After the reaction, 25mL of 100mM glycine solution was added to the system, the temperature was maintained at 30 ℃ for 10min, the mixture was dialyzed against deionized water at room temperature, then against 20mM phosphate buffer, pH 7.0, and the mixture was maintained at 4 ℃ for about 1 day.
Example 2
Determination of lysozyme activity
Respectively placing the enzyme solution to be detected and the substrate suspension in a water bath at 25 ℃ for heat preservation for 20min, sucking 2.8mL of the substrate suspension, placing in a 1cm cuvette for colorimetric determination of OD value under 450nm, wherein the OD value is read when the OD value is zero, then adding 0.2mL of the enzyme solution, quickly shaking uniformly, and determining the OD value under 450nm every 1min after adding the enzyme solution, and determining for 3 times in total. The enzyme activity units for this experiment were defined as: the OD drop per minute was 0.001 to 1 activity unit (25 ℃, pH 6.2), i.e., (Δ OD45/min) × 103/mass of sample (mg) per mg activity unit (U/mg). The enzyme activity of the cinnamaldehyde modified lysozyme is 94.8 percent of the natural enzyme activity.
Example 3
Determination of the inhibition zone: the cup and dish method is adopted. Taking 20mL of dissolved solid culture medium in a culture dish, after solidification, taking 0.2mL of each bacterial suspension with the concentration of 106-. Each sample solution experiment was repeated 3 times and the average was taken.
The present invention and its embodiments have been described above, and the description is not intended to be limiting, and the drawings are only one embodiment of the present invention, and the actual structure is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (7)
1. The lysozyme is characterized in that the lysozyme is obtained by covalently combining cinnamaldehyde essential oil and human lysozyme, and an imine bond is formed between an aldehyde group in the cinnamaldehyde essential oil and an amino acid residue of the human lysozyme.
2. The lysozyme with cinnamaldehyde coupling modification and the modification method thereof according to claim 1, wherein: the cinnamaldehyde is C9H80。
3. The lysozyme with cinnamaldehyde coupling modification and the modification method thereof according to claim 1, wherein: the modification method comprises the following steps:
the method comprises the following steps: dissolving active ester of cinnamaldehyde in DMSO, adding NaHCO3 aqueous solution of lysozyme, performing condensation reaction under the condition of stirring, and obtaining a reaction mixture after the reaction is finished;
step two: and D, dialyzing the buffer solution obtained in the step I by using deionized water and a phosphate buffer solution in sequence, and carrying out centrifugal separation on the obtained dialysate to obtain the modified lysozyme.
4. The lysozyme with cinnamaldehyde coupling modification and the modification method thereof according to claim 3, wherein: the lysozyme is human lysozyme.
5. The lysozyme with cinnamaldehyde coupling modification and the modification method thereof according to claim 3, wherein: the addition amount of the cinnamaldehyde is 5.0-10.0g/L, and the addition amount of the lysozyme is 1.5-2.0 g/L.
6. The lysozyme with cinnamaldehyde coupling modification and the modification method thereof according to claim 3, wherein: the mixing temperature of the lysozyme and the cinnamaldehyde is 30-50 ℃.
7. The lysozyme with cinnamaldehyde coupling modification and the modification method thereof according to claim 3, wherein: dissolving cinnamaldehyde in a 5mLDMSO solution to form a solution, wherein the final concentration of the solution is 0.7mM, dropwise adding 25mL of 0.2mM of 1% NaHCO3 egg white lysozyme solution under the condition of stirring, and continuously and slowly stirring for reacting for 6 hours at 30 ℃; after the reaction, 25mL of 100mM glycine solution was added to the system, the temperature was maintained at 30 ℃ for 10min, the mixture was dialyzed against deionized water at room temperature, then against 20mM phosphate buffer, pH 7.0, and the mixture was maintained at 4 ℃ for about 1 day.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113876934A (en) * | 2021-11-12 | 2022-01-04 | 浙江莱康生物工程有限公司 | Lysozyme composition with antibacterial effect |
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|---|---|---|---|---|
| JPH07236479A (en) * | 1994-02-24 | 1995-09-12 | Taiyo Kagaku Co Ltd | Lysozyme bonded with antibacterial compound |
| JPH0827027A (en) * | 1994-07-11 | 1996-01-30 | Taiyo Kagaku Co Ltd | Modified lysozyme-containing antimicrobial agent |
| CN103555705A (en) * | 2013-11-13 | 2014-02-05 | 江南大学 | Method for improving ability of lysozyme for inhibiting gram negative bacteria through cinnamic acid modified lysozyme |
| CN103589709A (en) * | 2013-11-08 | 2014-02-19 | 江南大学 | Method for improving capacity of ferulic acid modified lysozyme for restraining gram-negative bacteria |
| US20190127716A1 (en) * | 2017-10-27 | 2019-05-02 | Chengdu Beacon Bio-Technology Co., Ltd. | Lysozyme having improved enzymatic activity |
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2020
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07236479A (en) * | 1994-02-24 | 1995-09-12 | Taiyo Kagaku Co Ltd | Lysozyme bonded with antibacterial compound |
| JPH0827027A (en) * | 1994-07-11 | 1996-01-30 | Taiyo Kagaku Co Ltd | Modified lysozyme-containing antimicrobial agent |
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