JPH0751064B2 - Novel hyaluronidase SD-678 and method for producing the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel enzyme hyaluronidase SD-678 and a method for producing the same.

[Prior art and its problems] Hyaluronidase is a general term for an enzyme capable of degrading hyaluronic acid, which is a type of acidic mucopolysaccharide, and its presence is known to exist in animal tissues and cultures of certain microorganisms. There is.

As the animal-derived hyaluronidase, for example, those extracted and separated from the testicles of cattle and sheep are commercially available as research reagents, and as the microbial-derived hyaluronidase, for example, Streptomyces hyalurolyticus Nobu SP (Streptomyces hyalurolyticus
NOV.SP.) isolated from the culture is commercially available as a research reagent.

On the other hand, certain hyaluronidases are used as absorption / diffusion promoters for drugs in subcutaneous injection, and their use as therapeutic agents for myocardial infarction in the field of internal medicine is under study.

Conventionally, as a hyaluronidase derived from a microorganism, hyaluronidases derived from Streptomyces, Staphylococcus, Streptococcus, Propionibacterium, and Peptostreptococcus are known.

As a result of various researches by the present inventors, a novel hyaluronidase SD having different properties from conventional hyaluronidase in various points.
−678 to Streptococcus disgalactier (Str
The present invention was completed as a result of intensive research found in a culture of eptococcus dysgalactiae).

[Structure of Invention] I. Properties of Hyaluronidase SD-678: The hyaluronidase SD-678 of the present invention has the following physicochemical properties.

a) Action: endo β-hexosaminidase b) Substrate specificity: Decomposes hyaluronic acid and chondroitin, and does not decompose chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, heparin, heparan sulfate and keratosulfate.

Hyaluronidase SD-678 has high specificity for hyaluronic acid, and when the activity value when hyaluronic acid is used as a substrate is 100%, the relative activity value when chondroitin is used as a substrate is about 3%.

c) Optimum pH and stable pH range: a) Optimum pH: pH 5.8 to 6.6 The enzyme activity of hyaluronidase SD-678 at various pHs is added to the acetate buffer of various pHs in the titer determination method described below. , Phosphate buffer or tris-maleate buffer was used instead. Optimal pH of hyaluronidase SD-678
Is 5.8 to 6.6.

B) Stable pH: pH 5.0 to 9.0 Hyaluronidase SD-678 was allowed to stand for 15 minutes at 37 ° C in ethylenediamine-acetic acid buffer of various pH values of pH 3.0 to 10.0, and then its residual activity was determined according to the titer method described below. Was measured. Hyaluronidase SD-678 is stable in the pH range of 5.0 to 9.0.

d) Titration method: When hyaluronidase SD-678 was allowed to act on hyaluronic acid at 37 ° C., 1 micromol (μmole) of 2-
The amount of enzyme (titer) for liberating acetamido-2-deoxy-3-O- (β-D-gluco-4-enepyranosyluronic acid) -D-glucose (hereinafter referred to as “Δdi-HA”) is It is 1 unit.

Specifically, this enzyme is a lyase (leaving enzyme) that decomposes the β-hexosaminide bond of hyaluronic acid or chondroitin.

When this enzyme acts on hyaluronic acid, the unsaturated disaccharide Δdi-HA is released from hyaluronic acid. therefore,
When hyaluronic acid and this enzyme were reacted at 37 ℃, Δ in the reaction product
The titer is measured by measuring di-HA by the Morgan-Elson method.

The detailed titer measurement method is described below.

[Method for measuring titer of hyaluronidase SD-678] (a) Preparation of hyaluronic acid solution 0.2 g of sodium hyaluronate was dissolved in water and the total amount was 100 m.
Let l.

(B) Preparation of disodium hydrogen phosphate phosphate buffer _{(Na 2 HPO 4 · 12H 2} O) 6.62g and sodium dihydrogen phosphate _{(NaH 2 PO 4 · 2H 2} O) 11.32g and bovine serum albumin 0.2g Is dissolved in water to make the total volume 1000 ml.

(C) Preparation of potassium borate potassium borate solution _{(K 2 B 4 O 7 ·} 4H 2 O) 5g dissolved in water, the pH was adjusted to 9.0 with aqueous potassium hydroxide 2M, the total amount of 1
Set to 00 ml.

(D) Preparation of p-dimethylaminobenzaldehyde solution 16 g of p-dimethylaminobenzaldehyde is dissolved in a mixed solution of 95 ml of acetic acid and 5 ml of hydrochloric acid.

(E) Reaction and measurement 0.25 ml of the above phosphate buffer solution to 0.25 ml of the above hyaluronic acid solution
After adding and stirring, 0.1 ml of a sample solution of hyaluronidase SD-678 is added and stirred, and the mixture is reacted at 37 ° C for 10 minutes.
Immediately after the reaction, the mixture was heated at 100 ° C for 1 minute, then cooled in ice water, 0.5 ml of the above potassium borate solution was added, and the mixture was stirred.
Heat at 00 ° C for 7 minutes. After heating, the mixture is cooled in ice water, 5 ml of acetic acid and 2 ml of the above-mentioned p-dimethylaminobenzaldehyde solution are added, stirred and left at 37 ° C. for 20 minutes, and then the absorbance at 585 nm is measured using a colorimeter.

e) Optimum temperature range of action: Optimum temperature: around 37 ° C Action temperature: 20 to 50 ° C Hyaluronidase SD-678 is reacted for 10 minutes by changing the reaction temperature of the above titration method to a predetermined temperature and reacting at each temperature. The enzyme activity was measured. Hyaluronidase SD-678
The optimum temperature is around 37 ℃, and the optimum temperature range for action is 20-50
℃.

f) Conditions for inactivation due to pH, temperature, etc .: i) Hyaluronidase SD-678 is allowed to stand for about 15 minutes at 37 ° C in an ethylenediamine-acetic acid buffer of pH 4.5 for about 15 minutes.
80% deactivated.

ii) Hyaluronidase SD-678 is inactivated by about 100% by allowing it to stand at 80 ° C for 30 minutes in phosphate buffer of pH 7.2.

iii) Hyaluronidase SD-678 containing 50 mM sodium sulfite, cysteine or mercaptoethanol at pH 6.5.
In phosphate buffer solution at 37 ℃ for 10 minutes
100% deactivated.

g) Inhibition and Stabilization: i) Chloride salts of various metals were added to hyaluronidase SD-678, and the activity was measured by the above titration method.
Fe ²⁺ , C are the main inhibitory metal ions at various metal ion concentrations.
u ²⁺ , Pb ²⁺ and Hg ²⁺ inhibit these by 90% or more. In addition, Zn ²⁺ inhibits about 80% and Cd ²⁺ inhibits about 40%.

ii) Hyaluronidase SD-678 5 units dissolved in 1 ml of 0.05M phosphate buffer (pH 6.2) and hyaluronidase
When 5 units of SD-678 and 0.25 mg of bovine serum albumin or 0.25 mg of gelatin were dissolved in 1 ml of 0.05M phosphate buffer (pH 6.2), the solution was allowed to stand at 4 ° C for 5 days, and its residual activity was measured. The residual activity of the solution containing bovine serum albumin or gelatin is about 95%, but the residual activity of the solution without addition is about 25%.
And hyaluronidase SD-678 is stabilized by the addition of bovine serum albumin or gelatin.

h) Molecular weight The molecular weight of hyaluronidase SD-678 was measured by the gel filtration method using Sephaacrylic S-200.

The molecular weight of hyaluronidase SD-678 by gel filtration was 12
It is 5,000 ± 10,000.

i) Mobility by electrophoresis Crystal structure analysis and elemental analysis were performed using hyaluronidase SD.
-678 cannot be performed because it is not crystallized. Therefore, the mobility of hyaluronidase SD-678 by electrophoresis was measured.

Cellulose acetate membrane as a support, 0.05M Tris-maleic acid buffer solution (pH 6.0), 0.5mA / cm at 30
Electrophoresis of hyaluronidase SD-678 is performed by energizing for minutes, and the mobility of bovine serum albumin migrated to the anode side is +
When set to 1.0, the mobility of hyaluronidase SD-678 is +
It is 0.3.

The microorganisms used in the present invention include Streptococcus
Any strain may be used as long as it is a strain belonging to Disgalactier. Examples of such strains include those that have been deposited at the Institute of Medical Science, University of Tokyo as IID678.

As the microorganism, for example, Streptococcus dysgalactiae is subjected to irradiation treatment with X-rays, γ-rays, ultraviolet rays or the like, or drug treatment with ethyl methanesulfonate, nitrosoguanidine, etc., transformation, transduction, conjugation, genetic manipulation. The hyaluronidase SD-678 production ability may be increased by a commonly used strain mutation treatment method such as.

Comparison with conventional hyaluronidase: Hyaluronidase SD-678 has physicochemical properties as described above.

Hyaluronidase has been known to exist in animal tissues and cultures of certain microorganisms.

All of the animal-derived hyaluronidases are hydrolases and act on hyaluronic acid to produce tetrasaccharides or hexasaccharides, and thus differ from lyase hyaluronidase SD-678 in the action.

On the other hand, hyaluronidases derived from microorganisms, as hyaluronidases whose physicochemical properties have been investigated, include Streptomyces hyalurolyticus Novo Espy, Streptomyces coganeiens No. 8231, Staphylococcus aureus, There is hyaluronidase produced by Peptostreptococcus sp. 84H14S, Propionibacterium acnes or Streptococcus pyogenes.

Here, the properties of hyaluronidase and hyaluronidase SD-678 derived from these microorganisms are compared and shown in the table.

The conventionally known microbial-derived hyaluronidase is a lyase that decomposes the β-hexosaminide bond of hyaluronic acid, and has a common action.

However, the hyaluronidases derived from Streptomyces, Peptostreptococcus and Propionibacterium and hyaluronidase SD-678 have a substrate specificity, an optimal p
The properties differ in terms of H and molecular weight. In addition, the hyaluronidase derived from Staphylococcus differs in its properties in terms of optimum pH and molecular weight.

The molecular weights of hyaluronidase SD-678 and Streptococcus pyogenes hyaluronidase differ, and hyaluronidase SD-678 has a higher molecular weight. Further, hyaluronidase SD-678 has a wide optimum pH range, which is advantageous in use. Furthermore, although Streptococcus pyogenes hyaluronidase is unstable to oxidants,
Since hyaluronidase SD-678 is hardly affected by oxidizing agents, this enzyme is more suitable for degrading hyaluronic acid in the presence of oxidizing agents. On the other hand, it is known that the hyaluronidase SD-678 is inactivated by a reducing agent, but the hyaluronidase of Streptococcus pyogenes is stabilized by the reducing agent.

Next, hyaluronidase SD-678 and Streptococcus
An antiserum of Piogenes hyaluronidase was prepared and examined for serological properties.

[Preparation of antiserum] Hyaluronidase SD-678 or Hyaluronidase derived from Streptococcus pyogenes was dissolved in 0.5 ml each of 100 units of physiological saline solution and mixed with an equal amount of complete adjuvant to prepare a suspension, which was prepared in rabbits. Subcutaneously, 0.2 ml was subcutaneously injected at 5 sites. This operation was performed 3 times at 1-week intervals, and 2 weeks later, whole blood was collected from rabbits and serum was separated to obtain antisera of each hyaluronidase.

[Reaction with antiserum] 1 unit of hyaluronidase SD-678 or 1 unit of hyaluronidase derived from Streptococcus pyogenes was used.
Dissolve it in 0.5 ml of 05M phosphate buffer (pH 6.2), add 0.5 ml of each antiserum to this solution, stir, and leave it at room temperature for 15 minutes, then measure its titer by the above titration method. did. The same measurement was performed using normal rabbit serum as a control.

Hyaluronidase SD-678 antiserum is hyaluronidase S
The enzyme activity of D-678 was inhibited, but the antiserum of normal rabbit serum and hyaluronidase derived from Streptococcus pyogenes did not inhibit the enzyme activity at all. On the other hand, hyaluronidase antiserum derived from Streptococcus pyogenes inhibited the enzymatic activity of hyaluronidase derived from Streptococcus pyogenes.
SD-678 antiserum and normal rabbit serum did not inhibit the enzyme activity at all.

Thus, various properties of hyaluronidase SD-678, as a result of comparative examination with known hyaluronidase before this application, the hyaluronidase SD-678 of the present invention is different from conventionally known animal-derived hyaluronidase and microbial-derived hyaluronidase. It was found to be a novel hyaluronidase that differs qualitatively in terms.

On the other hand, Streptococcus disgalactiere, a hyaluronidase SD-678-producing bacterium, is a bacterium isolated from the skin of animals and the like, and is known to have little pathogenicity to humans, and in the production of hyaluronidase SD-678. Can use common equipment. On the other hand, Streptococcus pyogenes has pathogenicity to humans, and therefore, it is necessary to pay sufficient attention when producing hyaluronidase.

In addition, Streptococcus disgalactier IID-6
78 or Streptococcus pyogenes IID-715 were inoculated on blood agar medium (Kyokuto Pharmaceutical Co., Ltd.) and incubated at 37 ° C for 1 hour.
After overnight culture, sterilized yeast extract 2%, peptone
0.5%, dipotassium hydrogen phosphate 0.4%, glucose 2%
Two platinum loops of a bacterium were inoculated into a liquid medium (pH 7.2) of 100 ml, shake culturing was carried out at 37 ° C. for 24 hours, and the obtained culture was centrifuged to obtain a culture supernatant. When the hyaluronidase activity of this culture supernatant was measured by the above-mentioned titration method, the total enzyme activity of the culture supernatant of Streptococcus disgalactier was 230 units, and the total culture supernatant of Streptococcus pyogenes IID-715. It was about 60 times the enzyme activity.

Thus, this hyaluronidase SD-678 is easier to produce than the hyaluronidase derived from Streptococcus pyogenes and has good productivity.

II. Method for producing hyaluronidase SD-678: The hyaluronidase SD-678 of the present invention is produced, for example, by culturing Streptococcus disgalactiere in a medium, and separating and purifying hyaluronidase SD-678 from the obtained culture. To be done.

The culture method may be any method as long as it is in accordance with a general microorganism culture method, but a shaking or agitation culture method using a liquid medium is usually advantageous.

The medium used for culturing may be any medium component that allows Streptococcus disgalactiere to be used as a nutrient source.

For example, as the carbon source, glucose, mannose, starch, molasses, liquefied starch, glycerin and the like can be used.

Further, as the nitrogen source, for example, meat extract, yeast extract,
Peptone, casein hydrolyzate, gelatin, corn meal, soybean powder, ammonium phosphate, ammonium sulfate,
Urea or the like can be used.

In addition, various inorganic salts such as disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, calcium carbonate, manganese chloride, magnesium chloride, and sodium sulfate can be added if necessary.

On the other hand, the productivity of hyaluronidase SD-678 can be increased by adding hyaluronic acid, chondroitin or partially desulfated chondroitin sulfate as a hyaluronidase SD-678 inducer to the medium.

However, since hyaluronidase SD-678 is inactivated by the reducing agent, it is not preferable to add the reducing agent to the medium.

The above-mentioned medium components may be appropriately combined or added during the culture.

Further, when foaming is remarkable during culture, vegetable oils such as soybean oil and flaxseed oil, higher alcohols such as octyl alcohol, and antifoaming agents such as silicon compounds may be appropriately added.

A culture temperature of about 37 ° C is suitable, and a culture time of 10 to 30 hours is suitable.

Good results can be obtained by appropriately performing seed culture as the culture volume increases.

The culture conditions described above are applied by selecting the optimum conditions according to the characteristics of the strain used.

By culturing in this manner, hyaluronidase SD-678 is separated and purified from the obtained culture.

Since hyaluronidase SD-678 is mainly contained outside the cells in the culture, the culture is separated into cells and a culture solution by means of filtration, centrifugation, etc., and hyaluronidase SD is purified by a general enzyme purification method. Purify −678.

That is, vacuum concentration, freeze-drying, dialysis, ammonium sulfate fractionation, ultrafiltration, centrifugation, chromatography using an ion exchanger, gel filtration, separation of enzymes such as fractional precipitation using an organic solvent, a purification means alone or Any combination,
In addition, hyaluronidase SD-678 is separated, purified and collected from the culture filtrate by repeatedly using it as necessary.

[Examples of the Invention] Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples do not limit the scope of the present invention.

Example 1 Streptococcus disgalactiae IID-678 strain was inoculated on horse blood agar medium (Kyokuto Pharmaceutical Co., Ltd.) and incubated at 37 ° C. for 1 hour.
Cultured overnight.

Next, 100 ml of a medium solution consisting of 2% yeast extract, 0.5% peptone, 0.4% dipotassium hydrogen phosphate and 2% glucose (pH 7.2) was placed in each of 500 ml Sakaguchi flasks.
It was autoclaved at 121 ° C for 15 minutes. Two platinum loops of each of the above-mentioned cultures were inoculated into this flask and cultured at 37 ° C for 10 hours.

Separately, the medium solution 20 having the same composition as described above was put into 30 jar fermenters, sterilized at 121 ° C. for 20 minutes, and then separately sterilized, 1% sodium hyaluronate solution 2 was added aseptically, Add flask culture at 37 ° C
It was cultured for 12 hours. On the way, the pH of the medium is kept neutral by adding 3N NaOH solution appropriately.

The culture solution thus obtained was filtered using Radiolite (manufactured by Showa Kagaku Kogyo) as a filter aid to obtain a filtrate. The filtrate was water-cooled to 10 ° C., ammonium sulfate was added so as to have a saturated ammonium sulfate concentration of 70%, the mixture was stirred, and then allowed to stand for 2 hours while maintaining the liquid temperature at 10 ° C.

The precipitate produced by this salting out was collected by centrifugation, dissolved in 50 ml of 0.025 M Tris-hydrochloric acid buffer (pH 7.8), and then dialyzed against the same buffer 15 for 20 hours. Next, add this solution to DEAE
-Pass through a column packed with cellulose and load the column to 0.
After washing with 025 M Tris-hydrochloric acid buffer, elution with 0.025 M Tris-hydrochloric acid buffer containing 0.2 M NaCl, the eluate was collected and concentrated, and then the solution was diluted with 0.05 M phosphate buffer (pH 5.8). hand
It was dialyzed for 20 hours. This solution is passed through a column packed with Phospho-cellulose, washed well with 0.05M phosphate buffer (pH 5.8), and then pH is adjusted using 0.05M pH 5.8 and pH 7.0 phosphate buffer. Gradient elution was performed, and the fractions containing the desired product were collected and concentrated to 20 ml. This liquid is Sepha acrylic S-
Gel filtration was performed using a column packed with 300 (Pharmacia), and the fractions of the desired product eluted were collected and freeze-dried to obtain 12 mg of powdered hyaluronidase SD-678.

The specific activity of the thus obtained hyaluronidase SD-678 was measured by a titration method and was 1,500 units / mg.

EFFECTS OF THE INVENTION According to the present invention, a novel hyaluronidase can be provided.

Claims (2)

[Claims] 1. The following physicochemical properties: Action: endo β-hexosaminidase Substrate specificity: Decomposes hyaluronic acid and chondroitin, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, heparin, heparan sulfate and kerat sulfate. Without disassembling. Optimum pH and stable pH range: Optimum pH pH 5.8 to 6.6 Stable pH pH 5.0 to 9.0 Optimum temperature range of action: Optimum temperature: around 37 ° C Optimal temperature range of action: 20 to 50 ° C Loss due to pH and temperature Conditions of activity: i) 1 at 37 ° C in ethylenediamine-acetic acid buffer of pH 4.5
It is inactivated by about 80% when left for 5 minutes. ii) About 100% inactivation by leaving it in phosphate buffer of pH 7.2 for 30 minutes at 80 ℃. iii) 1 mM at 37 ° C. in a phosphate buffer of pH 6.5 containing 50 mM sodium sulfite, cysteine or mercaptoethanol.
About 100% deactivate by leaving it for 0 minutes. Inhibition and Stabilization: i) Inhibited by Fe ²⁺ , Cu ²⁺ , Pb ²⁺ and Hg ²⁺ . ii) Stabilize by adding bovine serum albumin or gelatin. Molecular weight: 125,000 ± 10,000 (gel filtration method with Sephaacrylic S-200) Mobility by electrophoresis: 0.5mA / using a 0.05M tris-maleic acid buffer solution (pH 6.0) with a cellulose acetate membrane as a support. 30 in cm
The mobility is +1.0 when the mobility of bovine serum albumin to be electrophoresed on the anode side is +1.0 by applying electricity for minutes.
It is 0.3. The hyaluronidase SD-678 derived from Streptococcus dysgalactiae, which comprises: 2. Streptococcus dysgalactiae or a mutant strain thereof is cultured in a medium, and hyaluronidase SD-67 is obtained from the obtained culture.
Hyaluronidase S characterized by separating and collecting 8
Manufacturing method of D-678.