Disclosed herein is a novel collagenase "discolysin". It is useful for biochemical researches and the treatment of many diseases caused by collagen, such as hernia of intervertebral disc. It can be produced by culturing a discolysin-producing bacterium belonging to genus of Streptomyces in a culture medium and then collecting discolysin from the culture medium.

This invention relates to a novel collagenase "discolysin" having an 
activity capable of dissolving insoluble collagens and a production method 
thereof. 
As a collagenase having an activity of dissolving insoluble collagens, the 
enzyme produced by Clostridium histolycum has been known most popularly. 
The collagenase from this bacterium has found wide-spread commercial 
utility as a laboratory reagent for various biochemical researches. 
Recently, it has also become possible to use it for the treatment of 
diseases such as low back pain and hernia of intervertebral disc since it 
can decompose the intervertebral disc collagen. However, this bacterium is 
an anaerobic pathogen known as a gas gangrene for many years and produces 
toxins. Accordingly, this bacterium is not suitable for the mass 
production of a collagenase because it is difficult to culture it in a 
large quantity as it is an anaerobic bacterium and in addition, it 
produces dangerous toxins. Thus, the present inventor has widely looked 
for a collagenase which has excellent characteristics, permits ready mass 
production and hence is economical, and can be produced by a bacterium. As 
a result, a novel collagenase has been found from a culture medium of a 
bacterium in the genus of Streptomyces, leading to completion of this 
invention. 
Namely, this invention provides a novel collagenase which is useful for 
biochemical researches and the treatment of many diseases caused by 
collagen, such as hernia of intervertebral disc, and can be produced by a 
microorganism. 
The above enzyme has been found to dissolve both non-denatured and 
denatured collagens. This enzyme will hereinafter be called "discolysin". 
In one aspect of this invention, there is thus provided a novel collagenase 
"discolysin" having the following physical and chemical properties: 
(1) Molecular weight: 60,000-70,000, (SDS-Gel electrophoresis); 
90,000-110,000 (SDS-Gel electrophoresis; presence of 10 mM EDTA); 
(2) Formation of two collagenase-active bands by the disc gel 
electrophoresis; 
(3) Isoelectric point: pH 4.8 and 4.9 (determined by the focal 
electrophoresis); 
(4) Elementary analysis data: C, about 43%; H, about 7%; N, about 13%; 
(5) U.V. spectrum: FIG. 1; 
(6) I.R. spectrum: FIG. 2; 
(7) Precipitated by 45-81% saturation with ammonium sulfate; 
(8) Adsorbed on an ion exchanger, DEAE cellulose; 
(9) Decomposition of insoluble collagens and denatured collagen but 
extremely inert to casein; 
(10) Optimum pH for decomposition of insoluble collagens: 7.6-8.0; and 
(11) Inhibited by EDTA. 
This invention relates also to a method for culturing a bacterium in the 
family of Actinomycetes and collecting discolysin from the cultured 
mixture, especially, for culturing a bacterium in the genus of 
Streptomyces and collecting discolysin from the cultured mixture. 
In another aspect of this invention, there is also provided a method for 
producing a novel collagenase "discolysin", which comprises culturing a 
discolysin-producing bacterium belonging to genus of Streptomyces in a 
culture medium and then collecting discolysin from the culture medium.

Microorganisms useful in the practice of this invention are 
discolysin-producing bacteria in the genus of Streptomyces. As an 
exemplary strain which has been recognized to be particularly effective 
for the present invention, may be mentioned Streptomyces sp. C-51 strain. 
This strain has been deposited under FRI deposition FERM BP-710 with the 
Fermentation Research Institute, Agency of Industrial Science and 
Technology, Ministry of International Trade and Industry. 
The above strain was isolated by the present inventor from soil which had 
been obtained at Ohira, Numazu. Its mycological characteristics are as 
follows: 
(1) Form: 
No fragmentation of its vegetative mycelia is observed. Its hyphae and 
spore chains have wavy to spiral structures and contain 20 or more spores 
therein. Namely, it has properties between the Retinaculum-Apertum (RA) 
type and the Spira type. A scanning electron microscopic observation has 
found that its spores are pillow-like and 0.6 - 0.8 x 1.0 -1.2 m.mu. large 
and its spore surfaces are smooth. Organs such as asci, sclerotia or 
zoospores are not observed. 
(2) Characteristics of cell wall: 
Its cell wall contains LL-diaminopimelic acid (LL-DAP) and glycine. No 
characteristic features are found on the saccharide which makes up the 
bacterium body. 
(3) State of growth: 
The state of its growth on various agar culture media were as shown in 
Table 1. 
TABLE 1 
__________________________________________________________________________ 
Color of Soluble 
Culture medium 
Growth back surface 
Hyphae pigment 
__________________________________________________________________________ 
Tryptone-yeast 
Good. Dull yellow 
Abundant. 
-- 
agar culture medium 
Light yellowish 
orange (5YR7/2) 
Dull yellow 
brown (10YR8/3) orange (5YR7/2) 
Yeast-malt Good. Dark yellowish 
Abundant. 
-- 
agar culture medium 
Light yellowish 
brown (2.5Y4/2) 
Dark yellowish 
brown (2.5Y6/3) brown (5YR4/1) 
Oat meal Good. Bright brownish 
Abundant. 
-- 
agar culture medium 
Bright olive 
gray (5Y6/1) 
Grayish olive 
gray (5Y7/1) (5Y7/1) 
Starch-inorganic salt 
Good. Yellowish brown 
Abundant. 
-- 
agar culture medium 
Light yellowish 
(2.5Y6/2) 
Bright brownish 
brown (5Y7/1) gray (1Y6/2) 
Glycerin-asparagine 
Good. Light yellow 
Abundant. 
-- 
agar culture medium 
Light yellow 
(2.5Y7/2) 
Bright brownish 
(2.5Y7/3) gray (10YR5/1) 
Peptone-yeast-iron 
Good. Light yellowish 
Abundant. 
-- 
agar culture medium 
Yellowish white 
brown (2.5Y8/4) 
Yellowish gray 
(2.5Y9/2) (5YR8/1) 
Tyrosine Good. Yellowish brown 
Abundant. 
-- 
agar culture medium 
Light yellowish 
(2.5Y5/3) 
Dark yellowish 
brown (2.5Y5/1) brown (5Y5/1) 
Glycerin-calcium 
Good. Dull yellow 
Abundant. 
-- 
maleate Yellowish white 
orange (10YR6/1) 
Grayish yellow 
agar culture medium 
(10YR7/2) brown (10YR5/1) 
Nutrient Good. Yellowish brown 
Abundant. 
-- 
agar culture medium 
Light yellowish 
(2.5Y6/2) 
Bright brownish 
brown (2.5Y7/3) gray (10YR6/1) 
Sucrose-nitrate 
Weak. Bright brownish 
Medium. -- 
agar culture medium 
Colorless (N-9) 
gray (10YR6/1) 
Light brownish 
gray (10YR7/1) 
Glucose-nitrate 
Weak. Bright brownish 
Medium. -- 
agar culture medium 
Colorless (N-9) 
gray (10YR6/1) 
Light brownish 
gray (10YR7/1) 
Glucose-asparagine 
Weak. Light brownish 
Abundant. 
-- 
agar culture medium 
Colorless (N-9) 
gray (10YR7/1) 
Bright brownish 
gray (10YR6/1) 
__________________________________________________________________________ 
(4) Physiological properties: 
Its growth temperature ranged from 10 to 40.degree. C. with the optimum 
growth temperature being near 28.degree. C. The starch-decomposing 
capacity, peptonizing capacity and coagulating capacity of skim milk, and 
liquefying capacity of gelatin (on a glucose-peptone gelatin culture 
medium) were all positive. On the other hand, the producing capacity of a 
melanine-like pigment, nitric acid reducing capacity, hydrogen sulfide 
producing capacity and cellulose-decomposing capacity were not observed. 
(5) Anabolism of carbon sources: 
It metabolized D-glucose, L-arabinose, D-mannit and D-fructose very well. 
It metabolized D-xylose and inositol rather well. However, it did not 
metabolize or metabolized extremely little L-rhamnose, raffinose, sucrose 
and cellulose. 
Summarizing the above properties, it can be concluded that the above strain 
is in the genus of Streptomyces, is of the RA or RAS type of the Gray 
series, has smooth spore surfaces, and is of the melanine-negative 
(non-chromogenic) type. 
Needless to say, bacteria in the genus of Streptomyces which bacteria are 
other than the above-mentioned bacterium may also be used including their 
varieties and variant strains, so long as they show discolysin-producing 
capacity. 
Discolysin can be produced in a culture medium by culturing a 
discolysin-producing strain aerobically in accordance with any culturing 
method which is known as a culturing method for Actinomycetes. 
Discolysin-producing bacteria grow at 10.degree.-40.degree. C. A 
temperature range of 25.degree.-35.degree. C. is generally preferred for 
the production of discolysin. For culturing a discolysin-producing 
bacterium in the genus of Streptomyces, it is possible to use any nutrient 
source known for culturing Actinomycetes and other microorganisms. For 
example, glucose, starch, dextrin, glycerin, sucrose and the like may be 
used as carbon sources. Of these carbon sources, glucose and sucrose are 
carbon sources both suitable for the production of discolysin. 
Nitrogen sources known for the growth of Actinomycetes and other 
microorganisms can all be used for the production of discolysin. For 
example, peptone, meat extract, yeast, yeast extract, soybean powder, 
peanut powder, corn steep liquor, rice bran, gelatin, various fish meal, 
inorganic nitrogen, etc. may be used. 
When discolysin is produced by culturing a discolysin-producing bacterium, 
one or more inorganic salts and metal salts may be added if necessary. One 
or more heavy metals may also be added in trace amounts. 
Discolysin can be obtained by culturing a discolysin-producing bacterium 
aerobically. For this purpose, a routinely-employed aerobic culturing 
method, for example, the solid culturing method, shake culturing method or 
aerated-stirring culturing method may be employed. If defoaming is 
required during culturing or in the course of sterilization of a culture 
medium, a defoaming agent such as silicone oil, surfactant or the like may 
be used. The preferred culturing temperature may be within the range of 
25.degree.-35.degree. C. 
The activity of discolysin can be measured by the following method in which 
discolysin dissolves collagen. Namely, 5 ml of a 0.067 M phosphate buffer 
(pH 7.4; containing 0.45% of NaCl), 1 ml of a discolysin solution and 25 
mg of collagen obtained from calf Achilles tendons are reacted at 
37.degree. C. for 18 hours (For a control, no enzyme is added). After the 
reaction, the liquid reaction mixture is filtered and 2.0 ml of the 
ninhydrin reagent is added to 0.2 ml of the filtrate. The resultant 
mixture is heated for 20 minutes in boiling water. Thereafter, the mixture 
is cooled in running water. The volume of the resultant mixture is 
adjusted to 10 ml with water. Fifteen minutes later, its absorbance is 
measured at a wavelength of 600 nm. From a standard curve obtained using 
L-leucine as a reference, the amount of amino acids freed during the 
reaction is determined. From the thus-determined amount, the physiological 
activities of discolysin can be determined. 
The culturing is continued until discolysin accumulates substantially. The 
extraction and separation of this substance from the cultured mixture may 
be carried out by suitably combining various methods on the ground of its 
properties found out by the present inventor, as will be shown in Examples 
which will be given hereinafter. Namely, there are the salting-out method 
making use of ammonium sulfate or the like, the precipitation relying upon 
an organic solvent such as acetone or methanol, the chromatography 
employing various ion exchangers, the gel filtration making use of various 
carriers, a variety of electrophoreses, the ultrafiltration, the 
lyophilization method, the dialysis method, the chromatofocusing, and so 
on. Discolysin can be isolated from the cultured mixture by combining 
these methods or using them repeatedly. 
Physical and chemical properties and biological activities of discolysin 
will next be given. 
(1) Molecular weight: 60,000-70,000 (determined by the SDS polyacrylic 
amide electrophoresis); 90,000-110,000 (determined by the SDS polyacrylic 
amide electrophoresis; presence of 10 mM EDTA); 
(2) Formation of two collagenase-active bands by the disc gel 
electrophoresis; 
(3) Isoelectric point: pH 4.8 and 4.9 (determined by the focal 
electrophoresis); 
(4) Elementary analysis data: C, about 43%; H, about 7%; N, about 13%; 
(5) U.V. spectrum: FIG. 1; 
(6) I.R. spectrum: FIG. 2; 
(7) Precipitated by 45-81% saturation with ammonium sulfate; 
(8) Adsorbed on an ion exchanger, DEAE cellulose; 
(9) Decomposition of insoluble collagens and denatured collagen but 
extremely inert to casein; 
(10) Optimum pH for decomposition of insoluble collagens: 7.6-8.0; 
(11) Activity-inhibited by EDTA. 
(12) Its decomposition activity for insoluble collagens is either equal to 
or slightly higher than the collagenase produced by Clostridium 
histolycum; and 
(13) Its acute toxicity (LD.sub.50): 1 g/Kg or more (determined by its oral 
administration to mice). 
Discolysin can be used widely in a variety of biochemical, physiological 
and pharmacological researches which require decomposition of collagens. 
In addition, it can also be used extensively for the prevention and 
treatment of various diseases caused by collagens, for examples, hernia of 
intervertebral disc, Peyronie disease (spongiositis), certain liver 
diseases, etc. For example, a dog under narcosis was subjected to 
abdominal incision. While confirming an intervertebral disc by palpation, 
a discolysin solution (0.05 ml) was administered by injection through back 
peritoneum to nucleus pulposus located in a central part of the 
intervertebral disc. One week after the administration, the dog was killed 
and the intervertebral disc was observed. As a result, the nucleus 
pulposus was found to have been significantly dissolved where 100 units 
(ABC units) or more of discolysin was administered per each intervertebral 
disc. By the way, no serious toxicity or side effect was observed. 
As mentioned above, it has been confirmed that discolysin is effective for 
diseases caused by certain disorder in collagens and can hence be used as 
medical drugs, non-medical drugs or food additives. 
Discolysin may be administered either orally or non-orally, for examples, 
in the form of capsules, tablets, injectable preparations or the like. Its 
dose may vary depending on age, symptom, body weight, etc. However, 1-100 
mg/day may usually be administered in 1-3 portions to an adult. It may 
however be administered in a larger dose or more often as needed. 
Examples of this invention will hereinafter be described. Since various 
properties of discolysin have been found as mentioned above by the present 
invention, it is however possible to make various modifications to the 
collection of discolysin from a cultured mixture or its related substances 
on the basis of these findings. The present invention is thus not 
necessarily limited to the following Examples but should include all 
methods readily inferable from findings and knowledge which have already 
been reported. Example 1: 
Fifteen liters of a culture medium containing 1% of sucrose, 1% of peptone, 
0.3% of gelatin, 0.2% of yeast extract, 0.2% of Na.sub.2 HPO.sub.4, 0.25% 
of Na.sub.2 CO.sub.3 and 0.04% of MgSO.sub.4 7H.sub.2 O were charged in a 
jar having an internal volume of 30 liters. After sterilization, 
Streptomyces C-51 strain was inoculated and cultured aerobically at 
30.degree. C. for 30 hours. After completion of the culturing, a filtrate 
(12 liters) was obtained. Ammonium sulfate was then added to the filtrate. 
Fractions which precipitated at 45-80% saturation were collected. They 
were dissolved and dialyzed against 10 mM tris-HCl buffer (pH 7.5)/4 mM 
CaCl.sub.2, followed by adsorption on a DEAE cellulose column 
(3.2.times.26 cm). The column was then developed with a gradient system 
which consisted of 1 liter of the above buffer and 1 liter of a solution 
obtained by adding 1 mole of NaCl to the same buffer, thereby collecting 
discolysin fractions. These fractions were then dialyzed against 10 mM 
citric acid (Na) buffer (pH 7.0)/4 mM CaCl.sub.2, followed by adsorption 
on a DEAE cellulose column (2.0.times.26 cm) maintained under the same 
buffer conditions. Thereafter, the column was developed with a gradient 
system which consisted of 1 liter of the above buffer and 1 liter of 10 mM 
citric acid (Na) buffer (pH 4.0)/4 mM CaCl.sub.2, thereby collecting 
principal active fractions. These fractions were then dialyzed against 0.2 
mM CaCl.sub.2 and lyophilized to obtain 51 mg (as proteins) of purified 
discolysin.