JPH10127281A - Peg-modified alginic acid lyase and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new enzyme consisting of a PEG-modified alginic acid lyase, stable in an organism, maintaining a high activity, weak in antigenicity and useful as a bio-film dissolving agent and for the prevention and treatment of refractory infectious diseases caused by the bio-film. SOLUTION: This new polyethylene glycol(PEG)-modified alginic acid lyase having 20-80% modifying ratio with PEG, is stable in an organism, has a low antigenicity while maintaining a high activity and is useful as a dissolving agent of a bio-film caused by bacteria and for the prevention and treatment of refractory infectious diseases caused by the bio-film. The PEG-modified alginic acid lyase is obtained by mixing a 5.0mM potassium phosphate buffer solution (pH6.5) of the alginic acid lyase with 0.5M boric acid-sodium hydroxide buffer solution (pH8.5), adding a modified PEG such as a polyethylene glycol succinimidesuccinate, for reacting with the amino group of a lysine residue in the enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an alginate lyase modified with polyethylene glycol (hereinafter referred to as PEG) and a biofilm dissolving agent containing the PEG-modified alginate lyase as an active ingredient.

[0002] The present invention also relates to a therapeutic / prophylactic agent for intractable infectious diseases, comprising a biofilm dissolving agent containing PEG-modified alginate lyase as an active ingredient and an antibacterial agent.

[0003]

2. Description of the Related Art Biofilm formation by bacteria has attracted attention as one of the causes of intractable infections. A biofilm is a mucous membrane composed of exopolysaccharide (glycocalix) produced by bacteria and biological components (fibrin, platelets, bacterial binding proteins, etc.). This biofilm is considered to be a defense mechanism against bacteria from the outside world. In diseases associated with such biofilm formation, the host immune mechanism, which is established by obtaining information from the surface of the bacterial cells, is not activated, and bacteria show resistance to the host defense reaction and Antibacterial agents cannot reach bacteria sufficiently. Therefore, diseases involving biofilm formation are considered to be refractory. Among them, the mucoid type Pseudomonas aeruginsa ( Pseudomonas aerugin)
Pseudomonas aeruginosa biofilms formed in living tissues by osa (hereinafter, referred to as Pseudomonas aeruginosa) have become a serious problem from the viewpoint of frequency, intractability, and severity during acute exacerbation. Is being done. at present,
It has been clarified that the main component of Pseudomonas aeruginosa biofilm is alginic acid.

[0004] Infections involving Pseudomonas aeruginosa biofilm include respiratory infections associated with cystic fibrosis, chronic respiratory infections such as diffuse panbronchiolitis, plastics, and metals. BACKGROUND ART Infectious diseases caused by adhesion of Pseudomonas aeruginosa to artificial materials such as artificial bones or artificial joints, endocarditis, osteomyelitis and intractable wound infections are known. In addition, in the urological field, as a typical example, complicated indwelling catheter urinary tract infections, infected stones,
Complex urinary tract infections with scarring and necrosis of the urothelial mucosa and severe urinary dysfunction are known, and periodontal disease caused by plaque formation and colonization of bacteria in the dental area is known. Are known.

[0005] Cystic fibrosis is a disease in which a viscous substance containing alginic acid produced by Pseudomonas aeruginosa infected to the respiratory tract accumulates in the lungs, causing bronchial obstruction and death. JP-A-6-197760 and JP-A-6-31956
No. 9 discloses that alginic acid in the viscous substance is decomposed and reduced in molecular weight, thereby reducing the viscosity of the viscous substance and improving the symptoms of cystic fibrosis. Alginate lyase having high activity and a purification method thereof, and the latter discloses an alginate lyase expression gene and a method for producing alginate lyase.

[0006] JP-A-6-197760 and Journal of Fermentation and Bioengineering (JOURNAL OF FERME)
NTATION AND BIOENGINEERIN
G, Vol. 76, No. 5, pp. 427-437, 1993
Alginate lyase AL-II, which has high degrading activity against acetylated alginic acid produced by Pseudomonas aeruginosa
I (molecular weight 38,000) is disclosed.

Further, Tsutomu Ozaki et al. Applied alginate-degrading enzyme to Pseudomonas aeruginosa biofilm, and aimed at improving the symptoms of respiratory tract infections by degrading alginic acid, and the results of purification and characterization of the enzyme. (Abstracts of the 116th Annual Meeting of the Pharmaceutical Society of Japan, 1996, p. 223, upper left column, Abstracts of the 23rd Annual Meeting of the Japan Society of Antimicrobial and Fungicide 1996, page 95, and the Japanese Journal of Agricultural Chemistry 1995) July, 1997 extra edition (1995 abstract of the meeting), p. 253, upper right column).

[0008] In vivo, infection
And Immunity (INFECTION AND I
MMUNITY, Vol. 60, No. 10, 3979-39
85, 1992), when a combination of alginate lyase and an antibacterial agent (amikacin) was administered to a rabbit endocarditis model, the eradication effect of mucoid Pseudomonas aeruginosa and the removal of exopolysaccharide were observed. Is described. However, when alginate lyase is administered as an enzyme preparation, the enzyme becomes unstable in a living body, and the elimination half-life of the enzyme is shortened. Therefore, the enzyme preparation must be administered in large amounts and frequently, and the antigenicity of the enzyme is rather a problem.

As described above, many attempts have been made to apply the alginate lyase enzyme to biofilm infections. However, there is currently no enzyme preparation of alginate lyase that has solved problems such as instability of the enzyme in the body, loss of activity, and antigenicity.Therefore, development of an alginate lyase preparation useful for biofilm infections is desired. ing.

On the other hand, by modifying the enzyme with a polymer,
It has been reported that the stability of the enzyme can be improved, the elimination half-life can be increased, and the antigenicity can be reduced. Among them, many reports have been reported on the effectiveness of using PEG as a modifying agent, and there is already a track record of being sold as a PEG-modified enzyme preparation.
As such PEG-modified enzyme preparations, for example, PEG-
Adenosine deaminase (ADAGEN
N, (registered trademark) include Enzon (commercially available from Enzon) (USA) and PEG-L-asparaginase (oncaspar (registered trademark) is commercially available from Enzon).

However, in an enzyme preparation, conditions such as the selection of a modifying agent, the mode of binding of a modifying agent to a protein, the modification conditions, and the degree of modification greatly affect retention of enzyme activity, maintenance of stability, and antigenicity. Creating useful enzyme preparations to do so is generally difficult. At present, no alginate lyase modified with PEG is known.

[0012]

It is an object of the present invention to provide an alginate lyase which is stable in vivo, has a longer elimination half-life and is less antigenic.

Another object of the present invention is to provide a biofilm dissolving agent containing the alginate lyase as an active ingredient.

Another object of the present invention is to provide an agent for treating or preventing intractable infectious diseases caused by biofilm, comprising a biofilm dissolving agent containing PEG-modified alginate lyase as an active ingredient and an antibacterial agent. And

[0015]

The inventors of the present invention have conducted intensive studies and have found that excellent effects can be obtained by selecting PEG as a modifier for alginate lyase, thereby completing the present invention. Was.

The present invention provides a PEG-modified alginate lyase, a biofilm dissolving agent containing PEG-modified alginate lyase as an active ingredient, a biofilm dissolving agent containing PEG-modified alginate lyase as an active ingredient, and a biofilm-producing microorganism. The present invention relates to a therapeutic / prophylactic agent for intractable infectious diseases comprising an antibiotic.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the PEG that modifies alginate lyase is not particularly limited as long as it can be used in pharmaceutical technology, pharmaceutical production technology, oral hygiene technology and the like. Semi-solid to solid forms are preferred. As such PEG, for example, an average molecular weight of about 1,000 to about 10
About 000, preferably about 2,000 to about 2,000
About 000, most preferably about 5,000 to
About 12,000 can be mentioned. These PE
G may have a certain molecular weight, or may be a mixture of various molecular weights. Further, commercially available PEG can be suitably used. As non-limiting examples of such commercially available PEGs, for example, Macrogol 1,000, 1,500, 1,54
0, 4,000, 6,000, and 20,000 (all are trade names, manufactured by Sanyo Chemical Industries). These are of relatively low molecular weight, but commercial products of high molecular weight can be used without any problems.

As the alginate lyase modified with PEG, any enzyme having the ability to degrade alginate can be suitably used, and in particular, those which degrade alginic acid produced by a microorganism belonging to the genus Pseudomonas. preferable.

Specific examples of such alginate lyase include Gram-negative aerobic bacilli such as Pseudomonas sp.
Xanthomonas (Xanthomonas) sp., Flavobacterium (Flavobacterium) genus Vibrio (Vibrio) spp., Klebsiella (Klebs
iella) genus and Enterobacter (Entero
alginate lyase isolated from the genus Bacter ), and preferably an alginate lyase isolated from a strain belonging to the genus Flavobacterium. Particularly preferably, Flavobacterium species OTC-6 (FERM BP) isolated from soil is used.
Alginate lyase AL-I and AL- isolated from the National Institute of Advanced Industrial Science and Technology
III (JP-A-5-15387, JP-A-6-197)
760, WO94 / 19006 pamphlet). Such alginate lyase may be isolated not only from a wild-type strain but also from a mutant strain, and further isolated from a genetically modified mutant strain into which an alginate lyase gene has been introduced and transformed. It may be something. Specifically, alginate lyases AL-I and AL-III isolated from Escherichia coli transformed by introducing the alginate lyase-expressing gene of Flavobacterium species OTC-6 (see JP-A-6-319569) ).

The physicochemical properties of the alginic acid lyases AL-I and AL-III isolated from the above-mentioned Flavobacterium species OTC-6 isolated from the soil will be described below.

Physicochemical properties of alginic acid lyase AL-I; (1) action: decompose alginic acid into a sugar having a double bond between C _{4 and} C ₅ at the non-reducing end, and finally decompose 4-deoxy-5
-Decomposes to ketouronic acid (2) molecular weight: 60,000 (3) optimal pH: 8.0 (4) stable pH: 6.0-8.0 (5) optimal temperature: 70 ° C (6) substrate Specificity: Highly degrading activity against bacterial alginic acid.

Physicochemical properties of alginic acid lyase AL-III; (1) Action: Alginic acid is decomposed into a sugar having a double bond between C _{4 and} C ₅ at the non-reducing end, and finally 4-deoxy-5
-Decomposes to ketouronic acid (2) Molecular weight: 38,000 (3) Optimum pH: 8.0 (4) Stable pH: 6.0-8.0 (5) Optimum temperature: 70 ° C (6) Substrate Specificity: It has particularly high decomposition activity against alginic acid derived from bacteria.

The alginate lyase is of course preferably of high activity, but a slightly less active one can also be suitably used. For example, the non-reducing terminal C—C generated by the alginate lyase decomposing alginic acid
The change in the specific absorbance (235 nm) due to the double bond between the two is used as an indicator of the enzyme activity. When the amount of the enzyme that increases the absorbance by 1 per minute is defined as 1 unit (U), about 20 units per 1 mg of the enzyme is used. Can be suitably used, and more preferably about 30 units or more.

The PEG-modified alginate lyase of the present invention comprising the above-described alginate lyase and PEG is characterized in that a part of the amino group of the lysine residue of the alginate lyase is PEG-modified.
By combining this with, the reduction of antigenicity and maintenance of the enzymatic activity of alginate lyase are both achieved. In the present invention, the binding rate between the amino group of the lysine residue of the alginate lyase and PEG, that is, the modification rate, is such that the enzyme activity necessary for dissolving the biofilm is maintained and the antigenicity is reduced. Although not particularly limited, for example, about 20 to 80%, preferably about 25 to 7%
0%, more preferably about 35 to 60%.
This allows the antigenicity of unmodified alginate lyase to be 10
About 30% or less, preferably about 1
0% or less, particularly preferably about 1% or less;
In addition, the enzyme activity can maintain an activity of about 40% or more, preferably about 50% or more, particularly preferably 90% or more, as compared with the case where unmodified alginate lyase is set to 100.

The above-mentioned PEG-modified alginate lyase can be obtained from any PEG and any combination of alginate lyases. However, if the PEG is modified, a PEG having a high molecular weight has a lower antigenicity. From the viewpoint of maintaining the enzyme activity, for example, a molecular weight of 1
In the combination of 2,000 PEG and alginate lyase, the modification rate is about 25% and the enzyme activity is about 91%.
% And the antigenicity is only about 6%, and the same combination having a modification rate of about 50% maintains the enzyme activity of about 40% and the antigenicity of about 0.6%. Has disappeared. At first glance, the latter appears to have lower enzymatic activity, but the modified PEG does not necessarily have to maintain higher enzymatic activity compared to the unmodified one. That is, even if the enzyme activity is slightly reduced by PEG modification, the elimination half-life in vivo becomes longer than that of the unmodified product,
This is because, since the retention in blood is greatly improved, the activity can be maintained for a long time, and if the antigenicity is extremely low, there is a merit in use that the safety for the living body is extremely high.

The PEG-modified alginate lyase of the present invention can be produced by any method that does not cause a decrease in enzyme activity during the binding reaction between PEG and alginate lyase.

As such a method, for example, a condensing agent widely used in the field of peptide synthesis may be used.
A method called a so-called active ester method may be employed in which a modified PEG having increased reactivity by esterifying G is used.

In the case of using the active ester method, for example, succinimidyl succinate PEG (trade name: SS)
-PEG, manufactured by NOF Corporation) and alginate lyase in a solvent, whereby the desired product can be easily obtained.

Examples of the modified PEG include PEG activated with N-hydroxysuccinimide, trichloroformate and the like, and specifically, PEG succinimide succinate, PEG succinimide propionate, double-chain PEG succinimide and the like. Is raised. Further examples include a copolymer of maleic anhydride and PEG, and particularly preferred is PEG succinimide succinate.

The reaction suitably proceeds, for example, under cooling to heating, but from the viewpoint of maintaining the activity of the enzyme, it is preferably carried out at a low temperature and in a buffer solution, and particularly preferably at about 4 to 10 ° C. preferable. At the time of the reaction, it is preferable to appropriately use about 1 to 10 times the molar amount of the modified PEG with respect to the amino group of the lysine residue of the alginate lyase, and the buffer is neutral to weakly alkaline. Those can be suitably used, and for example, a boric acid-sodium hydroxide buffer is preferable.

Then, after adding a reaction terminator such as ε-aminocaproic acid, the desired product can be obtained by separating the desired product from the reaction solution.

Although the separation of the target substance can be carried out by a conventional method, it is preferable to carry out gel filtration or ultrafiltration in an appropriate combination from the viewpoint of preventing a decrease in enzyme activity.

The biofilm dissolving agent of the present invention can be used in various dosage forms, such as tablets, capsules, buccal adhesives, spray preparations, injections, infusions, eye drops, ointments and the like. can give. Formulations of these various dosage forms may be used according to conventional methods, depending on the purpose of treatment, such as excipients, binders, solubilizers, solubilizers, emulsifiers, and suspending agents. Can be prepared.

Examples of such carriers include lactose, sucrose, glucose, starch, crystalline cellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, acacia, gelatin, magnesium metasilicate aluminate, anhydrous calcium phosphate, citric acid, trisodium citrate , Hydroxypropylcellulose, sorbitol, sorbitan fatty acid esters, polyvinylpyrrolidone, vegetable oils (peanut oil, olive oil, etc.), benzyl alcohol, propylene glycol, water and the like.

The administration route of the biofilm dissolving agent of the present invention is not particularly limited, and may be appropriately selected depending on the purpose of treatment. Oral administration, parenteral administration (for example, transdermal administration, transpulmonary administration, intravenous administration, Subcutaneous administration, intramuscular administration) is possible. For example, for pulmonary infections, application by pulmonary administration as a spray formulation, for urinary tract infections and endocarditis, etc., application by intravenous administration as an injection, and for wound infections May be applied as an ointment, applied to artificial medical materials by applying a liquid agent to the surface, and applied to periodontal disease as an oral adhesive.

The dose can be easily determined in consideration of the disease, weight, age, etc. of the patient according to the activity of the PEG-modified alginate lyase and the state of biofilm formation at the site of administration.

The biofilm dissolving agent of the present invention can be used for infectious diseases involving biofilms, for example, chronic respiratory infections such as cystic fibrosis and diffuse panbronchiolitis, plastics, metals, etc. It is useful for dissolving biofilms resulting from infections caused by P. aeruginosa adherence to materials such as catheters, artificial bones or artificial joints, endocarditis, osteomyelitis and intractable wound infections. It is also useful for complicated urinary tract infections, periodontal disease and the like.

Furthermore, when a biofilm dissolving agent containing the PEG-modified alginate lyase of the present invention as an active ingredient is administered in combination with an antibiotic, in addition to dissolving the biofilm, the bacteria are removed by the antibiotic, and the treatment is difficult. It is possible to treat existing biofilm infections, especially biofilm infections caused by Pseudomonas aeruginosa. In addition, when the biofilm is dissolved, there is no need to administer a large amount of the antibiotic over a long period of time, which is a conventional problem, and short-term and low dose of the antibiotic can be expected.

Specific examples of the antibiotics include macrolide antibiotics such as clarithromycin and erythromycin, combinations of the macrolide antibiotics and quinolone antibiotics, cefsulodin sodium, cefoperazone Third-generation cephem antibiotics such as sodium, ceftazim, secuprom sulfate, cefozopran hydrochloride, and aminoglycoside antibiotics such as amikacin sulfate.

In the present invention, when a preparation is prepared by blending PEG-modified alginate lyase and an antibiotic, the antibiotic is used in a dose according to its type, and the activity of the PEG-modified alginate lyase is determined by its activity and biofilm at the administration site. Can be easily determined by combining the amounts in consideration of the disease, weight, age, etc. of the patient according to the generation status of the patient. The dosage form is not particularly limited,
Various additives described in the biofilm dissolving agent can be used, and various dosage forms can be adopted. In addition to the formulation, in addition to a form in which both components are present in a single preparation, a form in which PEG-modified alginate lyase and an antibiotic are separately formulated and coexisted in a capsule or the like may be used. Alternatively, it may be in the form of simultaneous or sequential administration.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0042]

【Example】

Example 1 Modification with PEG having a molecular weight of 2,000 3.0 mg / ml alginate lyase AL-III (manufactured by Gunze Co., Ltd., having a high degrading activity against alginic acid derived from Pseudomonas aeruginosa) (5.0 mM) Potassium phosphate buffer, 0.15 M NaCl, pH 6.5) 0.4 ml
And 0.5M boric acid-sodium hydroxide buffer (pH
8.5) 0.4 ml was mixed to obtain a reaction solution. 5 times the molar amount of activated PEG modifying reagent to the amino group of lysine residue of alginate lyase (S
S-PEG, manufactured by NOF Corporation) at 30 ° C at 4 ° C.
The amino group was modified with PEG by stirring for minutes.
The reaction was stopped by adding a 20-fold amount of ε-aminocaproic acid (manufactured by Seikagaku Corporation) to the SS-PEG in a molar ratio of 20 times. The obtained reaction mixture was subjected to gel filtration chromatography (FPLC system, column: Superdex (trademark) 200HR10 /
30, manufactured by Pharmacia), and the PEG-modified product eluted in the high molecular fraction was collected. Furthermore, ultrafiltration (ultrafiltration membrane: UP-20, molecular weight cut-off 20,000, Advantech (Advante)
ch)) to obtain a modified PEG.

Example 2 Modification by PEG having a molecular weight of 5,000 Instead of the molecular weight of 2,000 used in Example 1, an SS-PEG modifying reagent having a molecular weight of 5,000 (manufactured by NOF Corporation)
Was used. Reaction, fractionation and concentration were carried out in the same manner as in Example 1 to obtain a modified PEG.

Example 3 Modification with PEG having a molecular weight of 12,000 Instead of the molecular weight of 2,000 used in Example 1, an SS-PEG modifying reagent having a molecular weight of 12,000 (manufactured by NOF Corporation)
PEG modified product was prepared at a molar ratio of 1 to the amino group of the lysine residue of alginate lyase. Separation was performed in the same manner as in Example 1, and the ultrafiltration membrane was concentrated using UK-50 (molecular weight cut-off 20,000, manufactured by Advantech) instead of UP-20 to obtain a PEG-modified product. Was.

Example 4 Modification with PEG having a molecular weight of 12,000 PE used at 2.5 times instead of 1 molar ratio to the amino group of lysine residue of alginate lyase used in Example 3
The G modification was made. Separation and concentration were performed in the same manner as in Example 3 to obtain a modified PEG.

Example 5 Modification with PEG having a molecular weight of 12,000 A modified PEG was prepared at a 5-fold molar ratio to the amino group of the lysine residue of alginate lyase used in Example 3, instead of using a molar ratio of 1-fold. Separation and concentration in the same manner as in Example 3 to PE
The G modification was obtained.

Example 6 Modification with PEG having a molecular weight of 5,000 3.0 mg / ml alginate lyase AL-III (manufactured by Gunze Co., Ltd., having a high decomposition activity against alginic acid derived from Pseudomonas aeruginosa) 15 ml of 5.0 mM potassium phosphate buffer, 0.15 M NaCl, pH 6.5 and 0.5 M boric acid-sodium hydroxide buffer (pH 8.
5) After mixing 15 ml, SS-PEG was used as a reaction solution.
The reaction was performed with the molar ratio of (molecular weight: 5,000) being 10 times. After stopping the reaction in the same manner as in Example 1, ultrafiltration (ultrafiltration membrane: UP-20, molecular weight cut-off 20,000)
0, manufactured by Advantech) to remove excess SS-PEG. Further, the column was loaded with HiLoad 16/60, Superdex 200 pg (Pharmacia (P
fractionated by the same method as in Example 1 and concentrated to obtain a modified PEG having a modification ratio of 56% and an activity remaining ratio of 29%.

Example 7 Modification with PEG having a molecular weight of 2,000 3.0 mg / ml alginate lyase AL-III (manufactured by Gunze Co., Ltd., having a high decomposition activity against alginic acid derived from Pseudomonas aeruginosa) 5.5 mM potassium phosphate buffer, 0.15 M NaCl, pH 6.5) 5.5 ml
And 0.5M boric acid-sodium hydroxide buffer (pH
8.5) 5.5 ml was mixed to obtain a reaction solution. SS-
The reaction was performed with the molar ratio of PEG (molecular weight: 2,000) being five times. After terminating the reaction in the same manner as in Example 1, fractionation was carried out in the same manner as in Example 6, and concentration was performed to obtain a modified PEG having a modification rate of 57% and an activity remaining rate of 43%.

Example 8 Modification with PEG having a molecular weight of 5,000 In place of the molecular weight of 2,000 in Example 7, a molecular weight of 5,000 was used.
Using SS-PEG of 00, a modified PEG having a modification rate of 43% and an activity remaining rate of 64% was obtained.

Example 9 Modification with PEG having a molecular weight of 12,000 In place of the molecular weight of 2,000 in Example 7, a molecular weight of 12,
By using 000 SS-PEG, a modified PEG having a modification rate of 54% and a residual activity rate of 51% was obtained.

With respect to the modified PEG produced in the above example, the enzyme concentration, modification rate, residual activity rate and immunoreactivity were evaluated by the following methods.

Enzyme concentration The absorbance at 280 nm was measured using a spectrophotometer UV-365 (manufactured by Shimadzu Corporation), and alginate lyase A was measured.
The enzyme concentration was calculated from a calibration curve prepared using L-III (hereinafter, referred to as an unmodified product).

Modification rate 0.5 ml of a solution containing 0 to 25 μg of a modified or unmodified PEG, 50 mM potassium phosphate buffer (pH 8.
0) 1.0 ml and 0.3 mg / ml fluorescamine (manufactured by Polyscience)
By mixing 0.5 ml of a 1,4-dioxane solution, free amino groups and fluorescamine can be removed at room temperature by 10%.
Allowed to react for minutes. Fluorometer F-4010 (manufactured by Hitachi, Ltd., fluorescence wavelength 390 nm, excitation wavelength 480 nm)
The free amino group is quantified by measuring the fluorescence intensity using, and the fluorescence intensity,
That is, the modification rate was calculated from the decrease in amino groups.

Residual activity rate Eisenia bicyris
cli )) (Nacalai Tesque, Inc.) in 50 mM Tris-HCl buffer (pH
7.0) to give a substrate solution. After 0.1 ml of a solution containing 0 to 5 μg of the modified or unmodified PEG was mixed with 0.9 ml of the substrate solution, the increase in absorbance at 235 nm due to the decomposition of alginic acid was measured at 25 ° C. for 3 minutes. The residual activity rate is 1
The specific activity (ΔA 235 nm / min / mg / mg of enzyme) was obtained by dividing the increase in absorbance per minute by the amount of enzyme.
Protein) was determined, and the residual activity ratio (%) was calculated from the comparison with the unmodified product.

The binding ability (immunoreactivity) to the antigenic antibody was determined by ELISA (Enzy).
me-Linked Immunosorbent As
assay) and used as an index of antigenicity.

50 μg / ml of the PEG-modified or unmodified PEG was added to a 96-well microplate (manufactured by Nippon Intermed Co., Ltd.) in an amount of 50 μl to 9 wells.
The enzyme was adsorbed by leaving it at 4 ° C. overnight. After removing the supernatant, PBS containing 0.05% Tween 20 was used.
Washing was performed once with 250 μl (hereinafter referred to as T20 / PBS). 50% Block Ace (trade name, Snow Brand Milk Products Co., Ltd.)
Was fixed at room temperature for 2 hours, and washed three times with T20 / PBS.

An alginate lyase antiserum prepared in rabbits according to a conventional method using the unmodified product as an immunogen was
% Block ace using T20 / PBS
A 9-fold 4-fold dilution series of 0 to 6,553,600-fold was used. 100 μl of the diluted serum was sequentially added to each well,
The reaction was allowed to proceed at room temperature for 2 hours to bind the immobilized enzyme to the alginate lyase antibody. T20 / P for excess antibody
After washing and separation with BS, the stock solution was diluted 3,000-fold with T20 / PBS and horseradish peroxidase (hereinafter referred to as HRP) labeled anti-rabbit IgG.
50 μl (manufactured by Zymmet) was added to bind to the antibody bound to the enzyme. Remove excess labeled antibody from T20 / PBS
After washing and separation with HRP, a substrate solution for HRP (2,
2'-azinodi (3-ethylbenzodithiazoline)-
6'-sulfonic acid, manufactured by Wako Pure Chemical Industries, Ltd.) 100 μl
Was added and reacted at room temperature for 15 minutes. After stopping the reaction by adding 100 μl of 1.5% oxalic acid, the plate reader Model 3550 (Bio-Rad (Bio-Rad)) was added.
Rad) was measured at 405 nm.

An antigen-antibody reaction curve was prepared for each of the modified PEG and the unmodified PEG. The degree of decrease in immunoreactivity was calculated from the ratio of the dilution of the antiserum showing 50% binding as an index of the antigen titer.

The properties of the unmodified alginate lyase AL-III and the modified PEG obtained in Examples 1 to 5 are shown in Table 1.

[0060]

[Table 1]

Test Example 1 Transition of loss of activity of PEG-modified alginate lyase Unmodified form of Japanese white male rabbit and P obtained in Example 6
A physiological saline solution of the modified EG was administered intravenously via the ear vein at a dose of 5 mg enzyme / animal (5 mg / ml solution, 1 ml) (one case each). 30 minutes after administration, 1, 3, 5, 7, 2
At 4, 48, 72, and 96 hours, 0.5 ml of blood was collected from the ear vein. After leaving the blood at room temperature for 2 hours, centrifugation (1
(2,000 rpm, 10 minutes), and the serum was collected.

The alginate lyase activity in the serum was determined by using 0.1% sodium alginate (manufactured by Nacalai Tesque, Inc.) / 50 mM Tris-derived from Aisenia bicyclis.
Using a hydrochloric acid buffer (pH 7.0) as a substrate solution, the following thiobarbituric acid method (hereinafter referred to as TBA method)
Journal of Biological Chemistry (J
ownal of Biological Chem
Istry) Vol. 234, No. 4, pp. 705-709,
1959). That is, serum 10μ
was added to 1 ml of the substrate solution, and an enzyme reaction was carried out at 37 ° C. 30 minutes after the start of the reaction, 100 μl of 0.03 N periodic acid (manufactured by Wako Pure Chemical Industries, Ltd.) / 0.125 N sulfuric acid
Was added to stop the reaction. After standing at room temperature for 1 hour, 0.3% thiobarbituric acid (manufactured by Wako Pure Chemical Industries, Ltd.) / PH 2.0 hydrochloric acid (pH was determined by adding hydrochloric acid to water)
2.0 ml), and heat-treated at 100 ° C. for 10 minutes to obtain an enzymatic reaction product of alginic acid (β-formylpyruvate (β-formylpyrate).
rivate)). Centrifugation (2,000
rpm, 5 minutes).
Filter with a μm filter (KC Prep Omni, manufactured by Katayama Chemical Industry Co., Ltd.) and use a spectrophotometer U-2000.
The absorbance at 548 nm of the filtrate was measured using (manufactured by Hitachi, Ltd.).

The activity of alginate lyase in the TBA method was determined by measuring the amount of enzyme required to produce 1 μmol of β-formylpyruvate under the above conditions by 1 unit (uni).
t). In addition, 54 of β-formyl pyruvate
The absorbance at 8 nm is 0.01 μmol from the literature value.
(Journal of Biological Chemistry) Vol. 237, No. 2
No. 309-316, 1962).

FIG. 2 shows the disappearance of alginate lyase activity in each serum after intravenous administration of the unmodified product and the PEG-modified product obtained in Example 6 to rabbits. The serum activity of the unmodified product after intravenous administration shows a rapid biphasic elimination transition, with an elimination half-life of about 35 minutes in the α-phase (distribution phase) and about 5.6 in the β-phase (elimination phase). It was time. On the other hand, the modified PEG showed a tendency to maintain the activity in serum, and high activity was detected 96 hours after administration. Its half-life is about 1
It was 42 hours, indicating that the retention in blood was significantly improved by the PEG modification.

Test Example 2 Examples 7 to 9 were applied to biofilms formed in vitro.
A basic study was conducted on the effect of the PEG-modified product on biofilm by applying the PEG-modified product prepared in the above.

(1) Preparation of In Vitro Biofilm Model Mucoid Pseudomonas aeruginosa isolated from the pulmonary airway of a patient with cystic fibrosis (Dr. A. Prin
ce: Columbia University, Department of Microbiology, New York)
From a tryptic soy agar medium (hereinafter referred to as TS).
A medium, Eiken Chemical Co., Ltd.), and precultured at 37 ° C. for 40 hours. The colonies that appeared on the TSA medium were suspended in physiological saline, and the spectrophotometer SPECTRO was used.
NIC21 (Bosch and Rohm (BAUSH
& LOMB), and the bacterial concentration was adjusted to about 10 ⁸ CFU / ml while measuring the absorbance at 660 nm.
Next, a membrane filter (MF-Millipore, trade name, pore size 0.45 μm, diameter 25 mm, manufactured by Nippon Millipore Co., Ltd.) was used for Brain Heart Infusion Agar Medium (hereinafter referred to as BHI agar medium, Eiken Chemical Co., Ltd.).
100 μl of the bacterial solution was dropped on the membrane filter, and 1 μl of the membrane filter was used.
A 0 ⁷ CFU of P. aeruginosa were deposited. A biofilm was formed on the membrane filter by stationary culture at 37 ° C. for 4 to 10 days (Chemotherapy (Chemo).
therapy) Vol. 41, No. 10, 1064-10
70, 1993).

(2) Biofilm Evaluation Method Morphological Observation Method Using a Scanning Electron Microscope (hereinafter, referred to as SEM) (a) Pretreatment of Sample After the membrane filter with the biofilm attached thereto was washed with physiological saline, the sample was washed with physiological saline. 30% in 2.5 w / v% glutaraldehyde (Katayama Chemical Co., Ltd.) / PBS solution containing 15% ruthenium red (Katayama Chemical Co., Ltd.)
For 1 minute, followed by 1% osmium tetroxide (Merck (ME
RK) and immobilized for 30 minutes in a PBS solution. Next, the ascending ethanol series (50, 70 and 9
0 v / v%) for 30 minutes each and further with 1 ml of dehydrated ethanol.
It was dehydrated overnight. After performing the critical point drying with carbon dioxide gas using a critical point dryer CPD-750 (manufactured by Alliwa Shoji),
Sputter coating was performed with platinum-palladium using an ion sputter E-102 (manufactured by Hitachi, Ltd.) to obtain a sample for SEM observation.

(B) SEM observation conditions Using an SEM device S-2250N (manufactured by Hitachi, Ltd., acceleration voltage: 25 kV, observation magnification: 3,000 times) and an image processor EP-3000 (manufactured by Hitachi, Ltd.) Morphological observation was performed.

FIG. 3 shows the time course of biofilm formation on (a) immediately after the biofilm was attached to the membrane filter, (b) on the fourth day of culture, (c) on the seventh day and (d) on the tenth day. .

Method for quantifying alginic acid in biofilm (a) Method for recovering alginic acid The membrane filter on which the biofilm was formed was placed in 3 ml of physiological saline, and vigorously vortexed to peel the biofilm from the membrane filter. After adding 5 ml of 99.5% ethanol and vortexing again, centrifugation (3,000 rpm,
10 minutes) to precipitate the biofilm component. After washing once with 5 ml of 99.5% ethanol, 5 ml of physiological saline was added to dissolve alginic acid. The cells and insoluble components are centrifuged (12,000 rpm).
pm, 10 minutes), 50 μl of a 10% copper sulfate solution was added to 1 ml of the solution, and alginic acid was precipitated again.
After washing the precipitate twice with hydrochloric acid adjusted to pH 3.0,
It was dissolved in 1 ml of 1N aqueous ammonia. Alginic acid solution was obtained by removing insoluble components by centrifugation (3,000 rpm, 5 minutes).

(Ii) Determination of Alginic Acid 500 μl of an appropriately diluted alginic acid solution was mixed with 3 ml of a concentrated sulfuric acid test solution containing 0.1 M boric acid. 0.1% carbazole (manufactured by Tokyo Chemical Industry Co., Ltd.) / Ethanol solution 10
Alginic acid was colored by adding 0 μl and reacting at 55 ° C. for 30 minutes. Spectrophotometer UV-120
The absorbance at 530 nm derived from the colored substance was measured using (manufactured by Shimadzu Corporation), and the amount of alginic acid was calculated from the results of the sodium alginate standard solution (Analytical Biochemistry (Anal. Biochem)).
m. Vol. 24, pages 470-481, 1968).

FIG. 4 shows the change over time in the amount of alginate produced in the biofilm (day 4, 7 and 10 of culture).

As shown in FIGS. 3 and 4, Pseudomonas aeruginosa adhered to the membrane filter was placed on an agar medium for 4 to 10 minutes.
After culturing for days, fibrous and plate-like substances were produced, and formation of a biofilm was recognized. Alginate production in the biofilm increased over time.

(3) Investigation on the effect of the modified PEG on the biofilm PEG on the biofilm formed in the above (1)
The biofilm destruction effect of the modified product was examined from the change of alginic acid content. As the PEG modified product, those prepared in Examples 7 to 9 were used.

Applicable biofilm: Biofilm 7 days after the start of culture Applicable enzyme: (a) Physiological saline (control) (b) Unmodified product (c) PEG-modified product obtained in Example 7 (d) Implementation Modified PEG obtained in Example 8 (e) Modified PEG obtained in Example 9 Amount applied: 100 μl of enzyme solution (5 μg of enzyme) diluted to 50 μg / ml with physiological saline was used.

Application method: The biofilm prepared in the above (1) was transferred to a fresh BHI agar medium, and the enzyme solution was dropped directly on the biofilm (five examples in each group). 3
The culture was started again at 7 ° C., and the biofilm was taken out one day later. After washing with physiological saline, morphological observation by SEM and quantification of alginic acid were performed according to the method (2).

FIG. 5 shows a morphological observation by SEM on the first day after the enzyme solution was applied to the biofilm formed for 7 days.

FIG. 6 shows the change in the amount of alginic acid one day after the enzyme solution was applied to the biofilm formed for 7 days.

As shown in FIGS. 5 and 6, the effect of the modified PEG on the biofilm formed on the biofilm formed for 7 days was examined. The amount of residual alginic acid 1 day after the application of the enzyme was significantly reduced as compared with the control group (physiological saline) for both the unmodified product and the three modified products modified with PEG having different molecular weights.

[0080]

Industrial Applicability The PEG-modified alginate lyase of the present invention has improved stability, maintains high enzyme activity and has low antigenicity, and is a refractory infectious disease caused by a biofilm dissolving agent and a biofilm. It is useful for the development of therapeutic and prophylactic agents.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a reaction mode between SS-PEG and an amino group in a PEG-modified alginate lyase of the present invention.

2 is a graph showing changes in serum alginate lyase activity after intravenous administration of the modified PEG obtained in Example 6 to rabbits in Test Example 1. FIG.

FIG. 3 is an SEM photograph showing the time-dependent change in biofilm formation observed in Test Example 2 (2).

FIG. 4 is a graph showing the change over time in the amount of alginic acid production in a biofilm calculated in Test Example 2 (2).

FIG. 5 is an SEM photograph showing the morphology of a biofilm observed in Test Example 2 (3).

FIG. 6 is a graph showing the change in the amount of alginic acid on the first day after applying the enzyme solution to the biofilm formed for 7 days, calculated in Test Example 2 (3).

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Hiroyuki Sakakibara 1-19-19 Osaka, Otsu City, Shiga Prefecture (72) Inventor Shirou Abe 1 Ikura Shinmachi Ishiburo, Ayabe City, Kyoto Gunze Kyoto Research Institute ( 72) Inventor Tomohiro Kuno 1 Ikura Shinmachi Ishiburo, Ayabe-shi, Kyoto, Japan Gunze Co., Ltd.Kyoto Research Institute

Claims (4)

[Claims] 1. A polyethylene glycol-modified alginate lyase. 2. The polyethylene glycol-modified alginate lyase according to claim 1, wherein the degree of modification with polyethylene glycol is 20 to 80%. 3. A biofilm dissolving agent comprising polyethylene glycol-modified alginate lyase as an active ingredient. 4. A therapeutic or prophylactic agent for intractable infectious diseases, comprising a biofilm dissolving agent comprising polyethylene glycol-modified alginate lyase as an active ingredient and an antibiotic effective against biofilm-producing microorganisms.