JP3510308B2 - Lysozyme with antibacterial compound - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a lysozyme having an original antibacterial activity of lysozyme enhanced and an antibacterial spectrum broadened by binding the antibacterial compound to lysozyme. It is used as an anti-inflammatory agent, antibacterial agent, etc. in the cosmetics, food, feed fields, etc.

[0002]

2. Description of the Related Art Lysozyme, also called muramidase or mucopeptide hydrolase, is an enzyme protein that hydrolyzes β-1,4 bond between N-acetylmuramic acid and N-acetylglucosamine existing in peptidoglycan layer of bacterial cell wall. Is. Lysozyme has an antibacterial activity because it has an action of lysing bacteria by cleaving the cell wall of bacteria by its enzymatic action.

Lysozyme is widely distributed in the animal and plant kingdoms, and mammalian tears, saliva, urine, milk, avian egg whites, body surface mucus of fishes, microorganisms, lysozyme derived from bacteriophage T4, and the like are known. Lysozyme derived from egg white can be mass-produced and its antibacterial activity is
In the feed field, it is used as an anti-inflammatory agent, an antiseptic agent, a freshness-keeping agent, an antibacterial agent, a bactericidal agent and the like.

The antibacterial activity of lysozyme is particularly strong against gram-positive bacteria, and against gram-negative bacteria,
It is known to have little antibacterial activity. Gram-positive bacteria are bacteria that are stained purple by Gram stain. Cocci (Micrococcus, Staphylococcus, Streptococcus, etc.), Spore-forming bacilli (Bacillus, Clostridium, etc.), Lactic acid bacteria (Lactobacillus, etc.) ),
Coryneform bacteria (Corynebacterium, Nocardia, etc.), actinomycetes (Streptomyces, etc.), yeast, etc. are known. The cell wall is 20-80 nm
It is composed of a thick peptidoglycan layer and is highly sensitive to lysozyme. That is, lysozyme exhibits an action of easily hydrolyzing the peptidoglycan layer and lysing Gram-positive bacteria.

On the other hand, Gram-negative bacteria are bacteria that are not stained by Gram stain, and include photosynthetic bacteria (Rhodopseudomonas, etc.), Pseudomonas bacteria, enterobacteriaceae (Escherichia, Salmonella, etc.), chemoinorganic bacteria (Nitrobacter). Genus, Thiobacillus, etc.), methanogenic bacteria, and some cocci (Niceria, etc.), etc. are known. The surface layer of the cell has a thin peptidoglycan layer of about 2 to 3 nm on the outer side of the cell membrane (inner membrane), and further has an outer membrane containing lipopolysaccharide on the outer side thereof. Therefore, lysozyme cannot easily cleave the peptidoglycan layer and is not susceptible to lysozyme unless the outer membrane is damaged by ethylenediaminetetraacetic acid or the like.

[0006] Several attempts have been reported to make the antibacterial activity of lysozyme act not only on Gram-positive bacteria but also on Gram-negative bacteria, that is, to expand the antibacterial spectrum of lysozyme. For example, it has been disclosed that lysozyme bound to a polysaccharide such as dextran using the Maillard reaction has antibacterial activity against Gram-negative bacteria at a heating temperature of 50 ° C (Agric. Biol. Chem.,
54, 3057-3059, 1990). In addition, palmitic acid (J. Ag
ric. Food Chem., 39, 2077-2082, 1991), stearic acid, myristic acid (J. Agric. Food Chem., 41, 1164
-1168, 1993) and other lysozymes bound to fatty acids, and lysozymes obtained by introducing a hydrophobic peptide at the C-terminal of lysozyme by genetic engineering technology (Biosci. Biot
ech. Biochem., 56, 1361 to 1363, 1992) is disclosed to have strong antibacterial activity against Gram-negative bacteria.

[0007]

[Problems to be Solved by the Invention] Lysozyme exhibits a strong antibacterial activity, especially against Gram-positive bacteria, and almost no antibacterial activity against Gram-negative bacteria. Therefore, its use is limited to food shelf life improvers, cosmetic preservatives, anti-inflammatory drugs, etc. for the purpose of suppressing the growth of Gram-positive bacteria. However, in these applications, it is said that it is more important to suppress the growth of Gram-negative bacteria such as Escherichia coli and Salmonella, which have a bad influence on the life and health of humans and animals, than the Gram-positive bacteria.

Under these circumstances, several attempts have been reported to expand the antibacterial activity of lysozyme so that it also acts on Gram-negative bacteria. However, these conventional methods are effective against Gram-negative bacteria. In order to exhibit effective antibacterial properties, there are problems that heating at 50 ° C. or higher is required, solubility of lysozyme is significantly deteriorated by binding of fatty acids, and very complicated gene manipulation technology is required. However, it was not practical. Therefore, the problem to be solved by the present invention is to solve the above problems and to provide a practical lysozyme having antibacterial activity against Gram-negative bacteria.

[0009]

Means for Solving the Problems As a result of diligent studies on lysozyme having antibacterial activity against Gram-negative bacteria, the present inventors have found that lysozyme having an antibacterial compound bound thereto has antibacterial activity against Gram-positive bacteria. The present invention was completed by discovering that it markedly enhances the antibacterial activity and exhibits a strong antibacterial activity against Gram-negative bacteria. That is, the present invention relates to lysozyme, which is characterized by binding an antibacterial compound.

The lysozyme of the present invention is an enzyme protein which hydrolyzes the β-1,4 bond between N-acetylmuramic acid and N-acetylglucosamine, and its origin is human, egg white or fish. Lysozyme prepared by gene manipulation technology using body surface mucus-derived, microbial-derived, bacteriophage-derived and their lysozyme genes may be used. Particularly preferably, it is desirable to use lysozyme derived from chicken egg white, which can be easily prepared in a large amount.

The antibacterial compound of the present invention means a compound having bactericidal activity or growth inhibitory activity against bacteria,
Examples include plant-derived compounds, synthetic antibacterial agents, and antibiotics. Examples of plant-derived antibacterial compounds include eugenol, thymol, cresol, carvacrol, vanillin, salicylaldehyde, perylaldehyde, cinnamaldehyde, anisaldehyde, benzaldehyde, acetaldehyde, pinene, limonene, catechin, epicatechin, gallocatechin, epigalo. Examples include catechin, epicatechin gallate, epigallocatechin gallate, and the like. Among these, compounds having a high aldehyde group in the molecule having a high binding property to lysozyme, that is, vanillin, salicylaldehyde, perylaldehyde, cinnamaldehyde, anisaldehyde, benzaldehyde,
The use of acetaldehyde is desirable, and vanillin, salicylaldehyde, perylaldehyde and cinnamaldehyde are desirable from the viewpoint of antibacterial activity.

Examples of the synthetic antibacterial agents include oxophosphoric acid and sulfur agents. Among these, it is preferable to use sulfa drugs such as sulfadimethoxine, sulfamonomethoxine and sulfamethoxazole. Antibiotics include penicillins and cephalosporins β-lactam antibiotics, aminoglycoside antibiotics, chloramphenicol, tetracycline antibiotics, macrolide antibiotics, lincomycin antibiotics, peptides System antibiotics, ansa macrolide antibiotics, depsipeptide antibiotics, fusidic acid, novobiocin, fosfomycin, polyene antibiotics, griseofulvin, actinomycins, mitomycins, anthracyclines, aureolic acid derivatives, bleomycins, etc. To be Among these, it is desirable to use cell wall-acting antibiotics such as penicillin.

In the present invention, the bond between the antibacterial compound and lysozyme is not particularly limited as long as it chemically and enzymatically bonds the functional group in the antibacterial compound and the functional group in the constituent amino acids of lysozyme. is not. As the functional group, for example, a carbonyl group in the antibacterial compound, a hydroxyl group,
Examples thereof include an amino group, a carboxyl group, a sulfhydryl group and an aldehyde group. The functional group of the amino acid residue constituting lysozyme is, for example, a carboxyl group of glutamic acid or aspartic acid, an amino group at the η (eta) position in lysine, a phenolic hydroxyl group in tyrosine,
Examples include imidazole group of histidine, guanidyl group of arginine, alkyl group of valine and leucine, sulfhydryl group of cysteine and the like. In the preparation of the antibacterial compound-bound lysozyme of the present invention, these functional groups may be used to bond chemically or enzymatically.

The bond in the present invention is not particularly limited, but a covalent bond, an ionic bond, a coordinate bond, a hydrogen bond and the like are considered, and a covalent bond having a high bonding force is preferable. That is, as the binding method, the following binding method can be used depending on the combination of the type of functional group of the antibacterial compound and the type of functional group of the constituent amino acids of lysozyme. In the case of an antibacterial compound having an aldehyde group or a ketone group, lysozyme and the antibacterial compound are mixed,
A primary amine in the constituent amino acids of lysozyme, for example, a Schiff base (Schiff base) produced by dehydration condensation of an amino group at the η (eta) position of a lysine residue and an aldehyde group or a ketone group in an antibacterial compound is replaced with hydrogen. , Reduce with a reducing agent such as hydrogen iodide, hydrogen sulfide, lithium aluminum hydroxide, sodium borohydride, etc.
In this case, usually 1 to 200 for 1 mol of lysozyme.
The antibacterial compound may be mixed in a molar amount, preferably 10 to 100 mol. The conditions for forming the Schiff base and the conditions for reducing the Schiff base are preferably conditions that do not inactivate the enzyme activity of lysozyme as much as possible, and usually 0 to 70
It is desirable to use a temperature of ℃, a reaction time of pH 2 to 9, 5 to 120 minutes, more preferably 0 to 20 ℃, a reaction time of pH 5 to 8, 5 to 60 minutes.

In the case of an antibacterial compound to which a saccharide is bound, an excess amount of the compound, for example, 10 to 100 in molar concentration is used.
Add twice the amount of periodic acid to remove the adjacent hydroxyl group or amino group bonded to the carbon atom of the sugar from 0 to 30 ° C.
An aldehyde group produced by selective oxidation (periodic acid oxidation) at a reaction temperature of 4 to 9 at pH 4 to 9,
After reacting a primary amine in a constituent amino acid of lysozyme, for example, an amino group at the η (eta) position of a lysine residue to form a Schiff base (Schiff base), it is treated with hydrogen or iodide as described above. A binding method of reducing with a reducing agent such as hydrogen, hydrogen sulfide, lithium aluminum hydroxide or sodium borohydride can be used. In the case of an antibacterial compound to which a reducing sugar, such as glucose, maltose, or lactose, is bound, heat treatment is performed on a mixed aqueous solution of it and lysozyme, and the aminocarbonyl reaction that occurs between the carbonyl group of the reducing sugar and the amino group in the constituent amino acids of lysozyme is carried out. The coupling method used can be used. In this case, regarding the heating conditions, it is necessary to select a temperature, pH and treatment time at which lysozyme is less deactivated. For example, it may be carried out at a temperature of 70 ° C. or lower at pH 4 to pH 10 for 30 minutes to 2 hours. it can. In the case of an antibacterial compound having an amino group or a carboxyl group, add a carbodiimide reagent such as carbamic acid nitrile to a mixed aqueous solution of lysozyme and the compound, and, for example, use a bonding method in which the amino group and the carboxyl group contained in each are condensed. You can

In addition, a method of binding a carboxyl group to an amino group as an acid azide derivative, a transesterification reaction,
A known chemical bonding reaction such as the glutaraldehyde method may be appropriately selected and used. Alternatively, a binding method using an enzymatic reaction of transglutaminase may be used. It can be carried out by a general method for separating and purifying lysozyme as a protein, such as gel filtration, cation exchange chromatography, or salting out. Among them, the method using the salting-out method is simple, and in order to separate lysozyme as a salting-out product, sodium sulfate or ammonium sulfate may be added so that the degree of saturation is 50% or more. Among these conjugation methods, as shown in the following examples, the aldehyde group of the antibacterial compound and the amino group at the η (eta) position of the lysine residue of lysozyme are formed by utilizing a dehydration condensation reaction. A method of reducing the Schiff base (Schiff base) with a reducing agent such as hydrogen, hydrogen iodide, hydrogen sulfide, lithium aluminum hydroxide and sodium borohydride is preferable in terms of operability. Further, the method for separating the lysozyme to which the antibacterial compound is bound by this reaction and the unreacted antibacterial compound is not particularly limited, and a general method for separating and purifying lysozyme as a protein, for example, gel filtration, ion It can be performed by exchange chromatography or salting out. In gel filtration, the molecular weight of lysozyme is about 14,000, but since the antibacterial compound used in the present invention has a molecular weight of 1,000 or less, for example, Sephadex G15 or G25 is used, which does not affect the enzymatic activity of lysozyme. It can be carried out with a solution having a pH of 4 to 8 nearby. In ion exchange chromatography, the isoelectric point of lysozyme is around 11, and in consideration of this, a cation exchanger can be used to adsorb antibacterial compound-bound lysozyme and separate unreacted antibacterial compounds. It is possible. Further, in the salting-out method, usually, antibacterial compound-bound lysozyme can be obtained as a salting-out product by adding sodium sulfate or ammonium sulfate to the solution after the reaction so as to have a saturation level of 50% or more, and then centrifugation or the like. It is possible to separate the unreacted antibacterial compound by the general method described above. Of these, the salting-out method is the simplest, so that its use is preferred.

[0017]

【Example】

Example 1 Preparation of perillaldehyde-bound lysozyme A conjugate of perillaldehyde, which is an antibacterial compound of perilla, and lysozyme derived from chicken egg white was prepared as follows. Ten
Lysozyme (1.5 g) was dissolved in 50 mM phosphate buffer (pH 7.4) containing 0 ml of 2.5 M NaCl. In addition, 50 times the amount of perillaldehyde as lysozyme was dissolved in 20 ml of hexane in a molar ratio. Mix the lysozyme solution and the perillaldehyde solution,
The mixture was stirred for 10 minutes under ice cooling to form a Schiff base.
After that, 120 ml of 0.3 M phosphate buffer (pH 7.0, containing 0.3 M NaCl) containing sodium borohydride dissolved at a concentration of 1 mg / ml was added, and the Schiff base was reduced by stirring for 30 minutes under ice cooling. It was The reduction reaction by adding sodium borohydride was repeated 3 times in total. Saturated sodium sulfate solution was added to the reaction solution so that the degree of saturation would be 66%.
After standing at ℃ for 1 hour, the salted-out precipitate was recovered by centrifugation. The salted-out precipitate was dissolved in 300 ml of distilled water, dialyzed thoroughly against distilled water, and then freeze-dried to obtain 1.35 g of perillaldehyde-bound lysozyme. The number of lysine residues in the obtained perylaldehyde-bound lysozyme and untreated lysozyme was 2,
As a result of measuring and comparing the absorbance at 344 nm using 4,6-trinitrobenzene sulfonic acid (TNBS) reagent, perylaldehyde-bound lysozyme was
It was bound with 4 mol of perillaldehyde.

Example 2 Preparation of cinnamaldehyde-bound lysozyme A conjugate of cinnamaldehyde, which is an antibacterial compound of cinnamon, and lysozyme derived from chicken egg white was prepared as follows. Cinnamaldehyde in a molar concentration 50 times that of lysozyme (1.5 g) was reacted in the same manner as in Example 1, and 1.28 g of cinnamaldehyde-bound lysozyme was obtained by freeze-drying. The number of lysine residues in the obtained cinnamaldehyde-bound lysozyme and untreated lysozyme was measured by comparing the absorbance at 344 nm using TNBS reagent. As a result, the cinnamaldehyde-bound lysozyme was 4 mol cinnamaldehyde Was a combination of.

Example 3 Preparation of salicylaldehyde-bound lysozyme A conjugate of salicylaldehyde and lysozyme derived from chicken egg white was prepared as follows. Example 1: 50 times the amount of salicylaldehyde in molar concentration with respect to 1.5 g of lysozyme.
The reaction was performed in the same manner as in (1) and freeze-dried to obtain 1.31 g of salicylaldehyde-bound lysozyme. The number of lysine residues of the obtained salicylaldehyde-bound lysozyme and the untreated lysozyme was measured using TNBS reagent and the absorbance at 344 nm was measured and compared. As a result, salicylaldehyde-bound lysozyme was 4 mol salicylaldehyde against 1 mol lysozyme. Was a combination of.

Example 4 Preparation of vanillin-bound lysozyme A conjugate of vanillin and hen egg albumen-derived lysozyme was prepared as follows. A 50-fold molar amount of vanillin was reacted with 1.5 g of lysozyme in the same manner as in Example 1, and 1.31 g of vanillin-bound lysozyme was obtained by freeze-drying. The number of lysine residues of the obtained vanillin-bound lysozyme and untreated lysozyme was measured using TNBS reagent and the absorbance at 344 nm was measured and compared. As a result, 4 mol of vanillin was bound to 1 mol of lysozyme. It was a thing.

Comparative Example 1 Preparation of Fatty Acid-Binding Lysozyme As a comparative example of Examples 1, 2, 3, and 4, fatty acid-binding lysozyme was prepared. Its preparation method is J. Agric. Food Che
m., 39, 2077-2082 (1991).
That is, palmitic acid is used as a fatty acid, and this is N-
It was esterified with hydroxysuccinamide and bound to lysozyme derived from chicken egg white by transesterification. 1
Fatty acid-bound lysozyme having 4 mol of palmitic acid bound to 4 mol of lysozyme was obtained.

Comparative Example 2 Preparation of Mixture of Antibacterial Compound and Lysozyme As a comparative example of Examples 1, 2, 3 and 4, a mixture obtained by simply mixing the antibacterial compound used in each Example and lysozyme derived from hen egg white. Was prepared. The preparation method is peryl aldehyde, cinnamaldehyde, to 1.5 g of lysozyme,
Salicylaldehyde or vanillin was mixed in a molar amount of 4 times that of lysozyme by the same operation method as in Example 1, and immediately lyophilized to give perylaldehyde mixed lysozyme, cinnamaldehyde mixed lysozyme and salicyl. Aldehyde mixed lysozyme and vanillin mixed lysozyme were obtained.

Test Example 1 Solubility of antibacterial compound-bound lysozyme The solubility of each antibacterial compound-bound lysozyme obtained in Examples 1, 2, 3, 4 and the fatty acid-bound lysozyme and untreated lysozyme of Comparative Example 1 A comparison was made. Each lysozyme was dissolved in 50 mM phosphate buffer (pH 7.0) to a concentration of 0.1%. Then, each lysozyme solution was pressure filtered with a filter having a pore size of 0.45 micron, and the protein concentration of the obtained filtrate was measured. The results are shown in Table 1.

[0024]

[Table 1]

The protein concentration of the filtrate of untreated lysozyme
Assuming 100%, 91% of filtrate for perylaldehyde-bound lysozyme, 88% for cinnamaldehyde-bound lysozyme, 93% for salicylaldehyde-bound lysozyme, and 88% for vanillin-bound lysozyme. It was On the other hand, the solubility of the fatty acid-bound lysozyme of Comparative Example 1 is
It was 43%. As a result, the solubility of antibacterial compound-bound lysozyme is about 10% compared to that of untreated lysozyme.
Although it decreased to some extent, it had a very high solubility in comparison with the conventional fatty acid-bound lysozyme.

Test Example 2 Enzymatic activity of antibacterial compound-bound lysozyme Each of the antibacterial compound-bound lysozyme obtained in Examples 1, 2, 3 and 4 and the fatty acid-bound lysozyme of Comparative Example 1 and the untreated lysozyme were tested for enzymatic activity. A comparison was made. The enzymatic activity of lysozyme was compared by the lytic activity of the body of Micrococcus lysodeikticus. Each lysozyme was added to the cell suspension (initial turbidity A600nm = 0.75 to 0.80) suspended in 50 mM phosphate buffer (pH 6.2) to a final concentration of 1 μg / ml, and the turbidity was measured at 25 ° C. The change was measured. The results are shown in Table 1. Assuming that the turbidity change of untreated lysozyme is 100%, that of perylaldehyde-bound lysozyme is 81%, that of cinnamaldehyde-bound lysozyme is 82%, that of salicylaldehyde-bound lysozyme is 75%, and that of vanillin-bound lysozyme is 76%. there were. On the other hand, that of the fatty acid-bound lysozyme of Comparative Example 1 was 58%. As a result, the enzyme activity of antibacterial compound-bound lysozyme was reduced by about 20% compared to that of untreated lysozyme, but in comparison with the enzyme activity of conventional fatty acid-bound lysozyme, it has a very high enzyme activity. It was

Test Example 3 Antibacterial activity of antibacterial compound-bound lysozyme against Gram-negative bacteria Each antibacterial compound-bound lysozyme obtained in Examples 1, 2, 3 and 4, antibacterial compound mixture obtained in Test Example 2 The antibacterial activity of lysozyme and untreated lysozyme against gram-negative bacteria was compared. E. coli (Escherichia coli K-12 strain) was used as a Gram-negative bacterium. Escherichia coli in the logarithmic growth phase was suspended in 10 mM phosphate buffer (pH 7.0) at 1 × 10 ⁶ cells / ml. Each lysozyme was added to the E. coli suspension to a final concentration of 100 μg / ml and incubated at 37 ° C for 1 hour, then a fixed amount of the suspension was inoculated on MacConkey agar medium and incubated at 35 ° C for 24 hours. The number of viable bacteria after culturing was measured. The results are shown in Table 1. The addition of 0.2% dimethylformamide (DMF) to the E. coli suspension had no effect on the growth of E. coli. As a result of comparing the viable cell counts of the untreated lysozyme with 100% as the viable cell count, perilaldehyde-bound lysozyme was 20%, cinnamaldehyde-bound lysozyme was 15%, salicylaldehyde-bound lysozyme was 16%, and vanillin-bound. That for lysozyme was 12%. Moreover, in the case of each mere mixture of lysozyme and the antibacterial biocompound, the viable cell count was 100% or more in any of the mixtures, and no enhancement of their antibacterial activity and expansion of the antibacterial spectrum were observed. This revealed that the antibacterial compound-bound lysozyme exhibited antibacterial activity against gram-negative bacteria Escherichia coli, and the antibacterial activity was about 5 to 10 times that of untreated lysozyme. Further, while a simple mixture of lysozyme and an antibacterial biocompound does not have antibacterial properties, the antibacterial activity against Escherichia coli of the present invention is caused by the chemical bond between lysozyme and the antibacterial biocompound.

Test Example 4 Antibacterial activity of antibacterial compound-bound lysozyme against Gram-positive bacteria Each antibacterial compound-bound lysozyme obtained in Examples 1, 2, 3 and 4, and antibacterial compound mixture obtained in Test Example 2 The antibacterial activity of lysozyme and untreated lysozyme against Gram-positive bacteria was compared. Staphylococcus aureus (Staphylococcus aureus IFO14462 strain) was used as a gram-positive bacterium. Bacteria in the logarithmic growth phase were suspended in 10 mM phosphate buffer (pH 7.0) at 1 × 10 ⁶ cells / ml. After adding each lysozyme to the final concentration of 100 μg / ml to the bacterial suspension and incubating at 37 ° C. for 1 hour,
Inoculate MacConkey agar with a fixed amount of the suspension,
The number of each viable bacterium was measured after culturing at 24 ° C. for 24 hours. The results are shown in Table 1. 0.2% DM for bacterial suspension
Addition of F had no effect on bacterial growth. As a result of comparing the viable cell count of each with the viable cell count of untreated lysozyme as 100%, that of perylaldehyde-bound lysozyme was 9% and that of cinnamaldehyde-bound lysozyme was 11%.
%, That of salicylaldehyde-bound lysozyme was 5%, that of vanillin-bound lysozyme was 5%. Further, in the case of a mere mixture of lysozyme and an antibacterial biocompound, the viable cell count was 100% or more in any of the mixtures, and no enhancement of their antibacterial activity and expansion of the antibacterial spectrum were observed. As a result, the antibacterial compound-bound lysozyme was able to enhance the antibacterial activity against Gram-positive bacteria by about 5 to 10 times as compared with that of untreated lysozyme. Further, each mere mixture of lysozyme and the antibacterial biocompound does not have an antibacterial effect enhancing effect, and the antibacterial activity enhancing effect against Gram-positive bacteria of the present invention is caused by the chemical binding of lysozyme and the antibacterial biocompound. It has been shown.

The following can be considered as embodiments of the present invention. (1) Lysozyme characterized by binding an antibacterial compound (2) The lysozyme according to (1) above, wherein the antibacterial compound is plant-derived (3) The above (1), wherein the antibacterial compound is perillaldehyde Lysozyme (4) The lysozyme according to (1) above, wherein the antibacterial compound is cinnamaldehyde (5) The lysozyme according to (1) above, wherein the antibacterial compound is salicylaldehyde (6) The above antibacterial compound is anisaldehyde ( 1) Lysozyme (7) The antibacterial compound is benzaldehyde Lysozyme (8) The antibacterial compound is acetaldehyde The lysozyme (9) Antibacterial compound is vanillin Lysozyme (10) The lysozyme (12) antibiotic according to (1), wherein the antibacterial compound is an antibiotic. The penicillin (10) lysozyme described (13) said antibiotic is a cephalosporin (1
The lysozyme (14) antibiotic according to 0) is an aminoglycoside, and the lysozyme (15) antibiotic according to (10) above is tetracycline.
The lysozyme (16) antibiotic according to 0) is a macrolide, and the lysozyme (17) antibiotic according to (10) is actinomycin.
(10) The lysozyme (18) antibiotic described in 0) above is mitomycin.
(10) wherein the lysozyme (19) antibiotic described is bleomycin.
The lysozyme (20) according to (20), wherein the antibacterial compound is a synthetic antibacterial agent.
The lysozyme (22) synthetic antibacterial agent according to (20) above, wherein the lysozyme (23) synthetic antibacterial agent according to (20) above is sulfamonomethoxine, and the lysozyme (24) synthetic antibacterial agent according to (20) above is sulfa. Lysozyme according to (20) above, which is methoxazole (25) Lysozyme according to (20) above, wherein the synthetic antibacterial agent is chloramphenicol.

(26) A Schiff base produced by mixing lysozyme and an antibacterial compound, and dehydrating and condensing the amino group at the η (eta) position of the lysine residue in the constituent amino acids of lysozyme and the aldehyde group in the antibacterial compound. (Schiff base) is reduced with a reducing agent such as hydrogen, hydrogen iodide, hydrogen sulfide, lithium aluminum hydroxide, sodium borohydride, and the like to prepare an antibacterial compound-bound lysozyme (27) lysozyme and an antibacterial compound. After mixing, the Schiff base (Schiff base) generated by dehydration condensation of the amino group at the η (eta) position of the lysine residue in the constituent amino acids of lysozyme and the ketone group in the antibacterial compound is converted into hydrogen and hydrogen iodide. Obtained by reduction with a reducing agent such as hydrogen sulfide, lithium aluminum hydroxide or sodium borohydride. Periodic acid is added to an antibacterial compound having an antibacterial compound-bonded lysozyme (28) saccharide to selectively oxidize an adjacent hydroxyl group or amino group bonded to a carbon atom of the saccharide (periodic acid oxidation). ) Generated by the reaction with the amino group at the η (eta) position of the lysine residue in the constituent amino acids of lysozyme to form a Schiff base, and then
Antibacterial compound-bound lysozyme (29) obtained by reducing with a reducing agent such as hydrogen, hydrogen iodide, hydrogen sulfide, lithium aluminum hydroxide, sodium borohydride, etc. A mixed aqueous solution of an antibacterial compound having a reducing sugar and lysozyme Lysozyme bound to an antibacterial compound obtained by heat treatment and utilizing the aminocarbonyl reaction caused by the carbonyl group of the reducing sugar and the amino group in the constituent amino acids of lysozyme

(30) An antibacterial compound-bonded lysozyme obtained by adding a carbodiimide reagent to a mixed aqueous solution of an antibacterial compound having an amino group and lysozyme to condense the amino group of the antibacterial compound and the carboxyl group of the constituent amino acids of lysozyme (31) An antibacterial compound-bound lysozyme obtained by adding a carbodiimide reagent to a mixed aqueous solution of an antibacterial compound having a carboxyl group and lysozyme to condense the carboxyl group of the antibacterial compound and the amino group in the constituent amino acids of lysozyme (32) Shampoo containing the antibacterial compound-bound lysozyme obtained in the above (1) to (31) (33) Characterizing addition of the antibacterial compound-bound lysozyme obtained in the above (1) to (31) Rinse (34) obtained in (1) to (31) above Mouthwash characterized by blending antibacterial compound-bound lysozyme (35) Dentifrice characterized by blending antibacterial compound-bound lysozyme obtained in the above (1) to (31) (36) Above ( 1)-(31) The antibacterial-compound-bonded lysozyme is blended, and the kamaboko (37) is characterized by being blended with the antibacterial-compound-bound lysozyme obtained in (1)-(31) above Custard cream (38) Pickles containing the antibacterial compound-bound lysozyme obtained in the above (1) to (31) (39) The antibacterial compound-bound lysozyme obtained in the above (1) to (31) Sausage characterized by being compounded (40) Ham characterized by compounding the antibacterial compound-bound lysozyme obtained in the above (1) to (31) (41) (1) Cooked rice characterized by blending the antibacterial compound-bound lysozyme obtained in (31) (42) Chicken egg maker characterized by blending the antibacterial compound-bound lysozyme obtained in (1) to (31) above Product (43) A feed additive characterized by blending the antibacterial compound-bound lysozyme obtained in any one of (1) to (31) above (44) An antibacterial compound-bound lysozyme obtained in any one of (1) to (31) above. (45) A pharmaceutical product containing the antibacterial compound-bound lysozyme obtained in any of (1) to (31) above

[0032]

The antibacterial activity of lysozyme has hitherto been limited to that against gram-positive bacteria, and its purpose of use has been limited. Conventional antibacterial compounds are known to exhibit antibacterial activity against Gram-positive bacteria or Gram-negative bacteria, or both depending on the type, but their use and amount used depend on their taste, odor, toxicity, etc. Was limited. The antibacterial compound-bound lysozyme of the present invention has the effect of enhancing the antibacterial activity of conventional lysozyme against Gram-positive bacteria. Further, although the antibacterial spectrum of conventional lysozyme was limited to Gram-positive bacteria, the antibacterial compound-bound lysozyme of the present invention has the effect of expanding its antibacterial spectrum and exhibits antibacterial activity against Gram-negative bacteria. Show. Furthermore, the antibacterial compound-bound lysozyme of the present invention in which it is bound to lysozyme exhibits excellent antibacterial activity due to its synergistic action with lysozyme, even if the antibacterial compound is used alone at a concentration that does not show antibacterial activity. That is, the antibacterial compound-bound lysozyme of the present invention has the effect of reducing the effective use concentration of conventional antibacterial compounds.
The antibacterial compound-bound lysozyme of the present invention has the above-mentioned effects, as a drug for the purpose of anti-inflammation or antibacterial, cosmetics such as shampoo and conditioner for the purpose of suppressing the bacterium causing dandruff and the bacteria causing tooth decay or As quasi-drugs such as mouthwash and toothpaste, livestock and fishery products for improving the shelf life of foods such as kamaboko, custard cream, pickles, sausage, ham, cooked rice and egg egg products for the purpose of preventing rot and sterilization. It can be used as a feed additive or veterinary drug for the purpose of preventing animal diseases.

Continuation of front page (56) References Chem. Pharm. Bull. , 1983, Vol. 31, No. 9, pages 3277-83 Agric. Biol. Chem. , 1989, Vol. 53, No. 12, pages 3173-7J. Agric. Food Ch em. , 1992, Vol. 40, No. 11, pages 2328-2330 Z. Lebensm. Unter s. Forsch. , 1987, Vol. 185, No. 1, pages 10-3 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 9/14-9/46 CA (STN) JSTPlus (JOIS) REGISTRY (STN) BIOSIS / WPI (DIALOG)

Claims (2)

(57) [Claims] 1. Lysozyme, which is bound with one or more antibacterial compounds selected from perillaldehyde, cinnamaldehyde, salicylaldehyde, anisaldehyde, benzaldehyde and vanillin. 2. The lysozyme according to claim 1, wherein the bond is formed by a dehydration condensation reaction.