JPS6054037B2 - Manufacturing method of elastase - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing elastase.

Elastase is a type of so-called serine protease present in the pancreas of mammals, and is an extremely unique protease that degrades elastin, which is an insoluble hard protein. In recent years, attention has been focused on the relationship between elastin degradation and aging, arteriosclerosis, emphysema, pneumonia, vasculitis, etc.
ssue6(1)1 (1974)), it is thought that the use of elastase as a pharmaceutical will increase in the future. From this point of view, it is desired to develop a method for efficiently producing elastase on an industrial scale. Many reports have been made regarding methods for producing elastase, but most of them are produced at the laboratory level.

The most representative elastase production method reported to date is the method of Lewis et al. (J, Bio
lchemZ4 705 (1956)). This method involves adding ammonium sulfate to a dilute acetic acid extract of pancreas and pancreatin to 45% saturation, dissolving the resulting precipitate in O, IM carbonate buffer, and then desalting the euglobulin fraction resulting from O, IM. Precipitate the elastase by dissolving it in carbonate buffer and adding ammonium sulfate to 35% saturation.

Thereafter, it is dissolved again in O, IM carbonate buffer and crystallized. The method disclosed in Japanese Patent Publication No. 50-21557 is characterized in that the precipitate obtained by saturation with ammonium sulfate at 45% according to the method of Rewis et al. is dissolved in an aqueous solution (pH 5 to 10 g), left at 5 to 50 C for 1 hour or more, and then salted out again. This is basically the same method as that of Rewis et al.

The method disclosed in JP-A-52-110892 is characterized in that a pancreatic extract is directly applied to a cation exchanger to adsorb elastase, and then eluted and fractionated.

Furthermore, in Japanese Patent Application Laid-open No. 52-61287, the pancreas contains -
This shows a method for producing elastase, which is characterized by adding denum or an extract thereof to activate an elastase precursor.

In all of the above-mentioned methods, pretreatment for obtaining a pancreatic extract is completely absent or extremely insufficient, so it is extremely difficult to obtain a clear pancreatic extract with a good yield.

In addition, the content of proteins and other biological components in the extract is high, making the subsequent purification process complicated, making it difficult to use as a method for producing high-purity elastase on an industrial scale in good yield. hard.

As a result of research paying particular attention to these points, the present invention for obtaining highly pure elastase has been completed.

The present invention uses the pancreas of chewing mammals with 20 to 6 Cf'C, preferably 4
After autolysis at 0 to 50°C for at least 1 hour, 1% or more of an alkaline earth metal is added to the digestive fluid in the presence of a 10 to 30% water-soluble organic solvent, the precipitate formed is removed, and elastase is added. It is characterized by obtaining an extract.

If necessary, this elastase extract is dialyzed as usual, and then ammonium sulfate is added to saturation of 25 to 45%, preferably 35%, and the resulting precipitate has a pH of 8 to 11.
After dissolving in water in step 5, elastase precipitates the complex as a crystal by desalting and neutralizing the pH, or after the above desalting, passes through a weakly basic anion exchanger to remove the unadsorbed fraction. Pure elastase crystals can also be precipitated by neutralizing after collection.

Elastase according to the present invention is contained in the pancreas of dairy animals such as cows, pigs, and sheep, but since it is present in particularly large amounts in pig pancreas, it is difficult to use pig pancreas as a raw material for industrial production. is advantageous.

Purified elastase is extremely stable at low temperatures in the pH range of 4 to 10.5, but is known to be unstable at temperatures above 20°C. However, the stability of elastase during pancreatic autolysis is extremely high.
It was discovered that even when autolysis was carried out for 3 hours, almost no elastase degradation was observed as compared with the known method (Japanese Patent Application Laid-Open No. 52-61287).
The autolytic fluid obtained under these conditions has extremely low viscosity;
Therefore, the filtration was quick and a large amount of clear liquid was able to be obtained.

Furthermore, by autolysis at relatively high temperatures, many of the connective tissue proteins and other unnecessary proteins were reduced to low molecular weight molecules, making it possible to obtain a digested fluid suitable for the subsequent elastase production process.

After adding alkaline earth metals to the autolysis solution to a concentration of 1% or more, protein precipitates are removed by adding water-soluble organic solvents such as ethanol, methanol, acetone, etc. to a concentration of 10 to 30%. , to obtain an elastase-containing solution.

As alkaline earth metal salts that can be used here, for example, water-soluble salts such as calcium chloride and magnesium chloride are effective. The elastase purification degrees of the above method according to the present invention and the method of Lewis et al. are shown in the table below for reference.

The degree of purification is expressed as a magnification, assuming that the degree of purification of the crude extract of the elastase-containing solution obtained by the method of Le Wis et al. is 1.0. However, the elastin decomposition ability of elastase obtained by the method of the present invention is measured by calculating the amount of soluble elastin decomposed product produced by the decomposition of insoluble elastin as the tyrosine value, and using RObbins, where 1 unit is the activity of releasing 1 μy of tyrosine per minute. et al.'s method (Ar
ch. sBiOchem,. BiOphys,. plate, 3
96, (1975)).

Proteins are prepared using the LOWry method (JIBiOlIchem●
Quantification was performed using ovalbumin as a standard according to M. Ryo, 265, (1951)).

The present invention will be explained in more detail below with reference to Examples.

After autolyzing 1k9 pieces of shredded pork pancreas at 50°C for 3 hours with strong stirring, 3e of water was added and filtrated.
．． A clear solution of 5e was obtained.

After adding 80 f of calcium acetate to the clarified liquid, 500 ml of acetone was added. The resulting precipitate was removed by centrifugation to obtain elastase-containing centrifugal supernatant 3.8e. The supernatant was dialyzed to remove salts and organic solvents, solid ammonium sulfate was dissolved in the dialysis solution to a saturation of 35%, and the resulting precipitate was collected by centrifugation and suspended in 100 ml of water.
The precipitate was dissolved by adjusting the pH to 10 with aqueous ammonia.

After desalting by dialysis while maintaining the pH of dissolution at 8 to 11.5, the elastase complex was crystallized by adjusting the pH of the solution to 7 with acetic acid.

Claims (1)

[Claims] 1. After autolyzing the mammalian pancreas, 1.
A method for producing elastase, which comprises adding an alkaline earth metal salt to a concentration of 1% or more in the presence of a 0 to 30% water-soluble organic solvent, and removing the resulting precipitate to obtain an elastase extract. 2. The method according to claim 1, wherein autolysis of the pancreas is carried out at 40 to 50°C for at least 1 hour.