Dear Venkatesham,

Please read the following text:

Dissolve pepsin at a concentration of 0.5 mg/ml in 0.01 N HCl. The solution obtained is clear, colorless solution.

http://www4.mpbio.com/ecom/docs/proddata.nsf/(webtds2)/102598

Hoping this will be helpful,

Rafik

 Method for solubilization of collagen fibers with proteolytic enzymes

In order to compare the solubilization ability of the present enzymes with that of pepsin, the following experiment was made. The pepsin used was a pure substance having an activity of 15,000,000 units for milk casein, and, in that case, the pH of hide was adjusted to 2.5 (the optimum pH of pepsin for collagen). In the case of the present new proteolytic enzymes having an activity of 150,000 units for milk casein, the pH of hide was adjusted to 3.0 (the optimum pH of the present proteolytic enzyme for collagen). The amount of the enzyme added was 0.01% per substrate by weight in the case of pepsin and 1.0% in the case of the present enzyme, so that the enzymic treatments by each enzyme-were made under the same condition from the viewpoint of the activity for casein. In this examination, the amount of the collagen fibers solubilized in 24 hours agitation was 20% in the case of pepsin, and 100% in the case of the present enzyme. Accordingly, the enzyme produced from Aspergillus niger is superior in the ability to solubilize the collagen fibers of adult steer hide to the proteolytic enzyme hitherto known.

3 EXAMPLE 2 Adult steer bone was deashed with 1 N hydrochloric acid. The collagen of the deashed bone was then neutralized and washed with water to obtain a sample of 200 g. (water content, 75%), to which 1 l. of dilute acetic acid was added to adjust the pH to 3.0. Thereafter, in the same way as in Example 1, the aforesaid enzyme (produced from Aspergillus niger) solution was added in an amount of 0.5% per substrate by weight, and the mixture was agitated for 24 hours at 25 C. to'form a viscous solution in which the collagen had been almost dispersed. After filtration, this solution was neutralized to form a fibrous precipitate, the dry weight of which was about 45 g., showing that about 90% of the collagen fibers was solubilized without being destroyed, the ability of reconstitution.

As shown in Examples 1 and 2 it is now clear that when the enzyme described therein is used, the collagen fibers of adult animals can be solubilized as easily as in the case where those of young animals are solubilized with pepsin.

EXAMPLE 3 The procedure described in Example 1 was followed using the proteolytic enzyme produced from Aspergillus sairoi (ATCC No. 14,332) in place of the enzyme used in Example 1. In this procedure, the collagen fibers were almost solubilized in 48 hours at about 25 C. and at a pH of about 2.5 to form a very viscous solution. In this case, the above-mentioned enzyme having an activity of 150,000 units for casein was used in an amount of 1% per substrate by weight. The collagen solution obtained by the above treatment had the same characteristics as those of the collagen solution obtained by the procedure in Example 1.

The collagen fiber-solubilizing activity of this enzyme produced from Aspergillus suitor was tested through comparison with that of pepsin. Pure pepsin (having an activity of 15,000,000 units for casein) was used in an amount of 0.01% per substrate by weight; on the other hand, the present crude enzyme (having an activity of 150,000 units for casein) produced from Aspergillus saitoi was used in an amount of 1% per substrate by weight, at a pH of about 2.5 and a temperature of about 25 C. for 24 hours.

The collagen fibers used were solubilized about 20% with pepsin and about 50% with the present enzyme. Therefore, it can be said that the enzyme produced from Arpergillus s'ar'toi showed an excellent activity to solubilize the collagen fibers.

EXAMPLE4 .The procedure described in Example 2 was followed using the proteolytic enzyme produced from Aspergillus saitoi in place of the enzyme used in Example 2. In this procedure, the collagen fibers are almost solubilizcd in 48 hours at a temperature of about 25 C. and at a pH of about 2.3, forming a viscous solution. The amount of the enzyme (having an activity of 150,000 units for casein) used was 1% per substrate by weight. The collagen solution obtained had the same characteristics as those of the collagen solution obtained by the procedure in Example 1.

. 4 EXAMPLE 5 The procedure described in Example 1 was followed using the proteolytic enzyme produced from Aspergr'llus oryzae as described in The Journal of Biochemistry, vol. 46, pages 1263-1270 (1959), in place of the enzyme used in Example 1. In this procedure, the collagen fibers were almost completely solubilized in 36 hours at a temperature of about 25 C. and at a pH of 2.8, to form a viscous solution. The solution had the same characteristics as those of the collagen solution obtained by the procedure in Example 1. The amount of the present proteolytic enzyme (having an activity of 150,000 units for casein) used was 1% per substrate by weight.

The comparison of the solubilizing ability of the present enzyme with that of pepsin was made. Pure pepsin (having an activity of 15,000,000 units for casein) was used in an amount of 0.01% per substrate by weight and, on the other hand, the present proteolytic enzyme produced from Aspergrllus oryzae (having an activity of 150,000 units for casein) was used in an amount of 1% per substrate by weight, at a pH of 2.8 and a temperature of 25 C. for 24 hours. The amount of the aolubilized collagen fibers was about 20% in the case of pepsin and in the case of the proteolytic enzyme produced from Aspergillus oryzae, showing that the collagen fiber-solubilizing ability of the latter is excellent.

2 W EXAMPLE 6 The procedure described in Example 2 was followed using the proteolytic enzyme mentioned in Example 5 in place of the enzyme used in Example 2. In this procedure, the collagen fibers were almost completely solubilized in 36 hours at a temperature of about 25' C. and at a pH of about 2.8, to form a viscous solution. The amount of the enzyme (having an activity of 150,000 units for casein) used was 1% per substrate by weight. The collagen solution obtained had the same characteristics as those of the collagen obtained by the procedure in Example 1.

For more plz read at following link.

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https://www.google.com/patents/US3530037

In 10mM or 25mM acetate buffer at ph 3 or 4.