JPS60133887A - Production of elastase - Google Patents

Abstract

PURPOSE:To obtain industrially and advantageously the titled substance, useful for medicines, etc., by autolyzing the pancreas of a mammal, filtering the resultant autolyzate, adding an alkaline earth metal salt to the resultant autolyzate filtrate in the presence of a specific amount of a water-soluble organic solvent, removing the formed precipitates, and separating the resultant extract of elastase. CONSTITUTION:The pancreas of a mammal, e.g. a pig, are shredded, and autolyzed at 50 deg.C for 3hr with vigorous agitation. Water is added thereto to filter the resultant mixture. An alkaline earth metal salt, e.g. calcium acetate, is added to the resultant autolyzate filtrate in the presence of 10-30% water-soluble organic solvent, e.g. acetone, to give >=1% alkaline earth metal salt concentration. The formed precipitates are removed by centrifugation to separate an extract of elastase, which is then dialyzed to remove salts and organic solvents. Ammonium sulfate is then added to the dialyzate to saturation, and the formed precipitates are separated to afford the aimed substance.

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 mammalian animals, and is an extremely unique protease that degrades elastin, which is an insoluble hard protein. In recent years, the relationship between elastin degradation and aging, arteriosclerosis, emphysema, 1Ft inflammation, vasculitis, etc. has attracted attention.
ve Ti5sue 6 (1) 1 (1974))
It is thought that the use of elastase as a medicine will increase in the future. From this point of view, it is desired to develop a method for producing elastase efficiently (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 Ewis et al.
-Biol che+n 222705 (1956)
).

This method involves adding ammonium sulfate to dilute acetic acid extracts of pancreas and pancreatin to 45% saturation, dissolving the resulting precipitate in 0.1 M carbonate buffer, and then desalting the eicloprine fraction to 0.1 Dissolve ammonium sulfate in 3M carbonate buffer.
Obtain 5% saturation plus elastase precipitation. Thereafter, it is dissolved again in 0.1M carbonate buffer and crystallized.

The method according to Special Publication No. 50-21557 is Lewis
The precipitate obtained at 45% ammonium sulfate saturation in the method of et al.
The method is basically the same as the method of Lewis et al., which is characterized in that it is dissolved in an aqueous solution of ~1O, left at 5 to 50°C for 1 hour or more, and then salted out again.

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, Japanese Patent Application Laid-Open No. 52-61287 discloses a method for producing elastase, which is characterized in that the duodenum of a mammal or an extract thereof is added to the pancreas to activate an elastase precursor.

In all of the above methods, the pretreatment for obtaining the pancreatic extract is either absent or extremely insufficient, making it extremely difficult to obtain a clear pancreatic extract with a good yield. It is.

In addition, the extract contains a large amount of tannozuri and other biological components, and the subsequent purification process requires less effort and complexity, making it insufficient as a method to produce high-purity elastase on an industrial scale with good yield. It is difficult to say that.

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

In the present invention, the mammalian pancreas is heated at 20-60°C, preferably at 40-60°C.
After autolysis at 50°C for a short time (at least 1 hour), 1% or more of an alkaline earth metal was added to the digested P solution in the presence of a 10-30% water-soluble organic solvent, and the resulting precipitate was is removed to obtain an elastase 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 is 1. H8-11
After dissolving in water of 5, desalting and neutralizing H causes elastase to precipitate the complex as a crystal, or
After the above desalting, pure elastase crystals can be precipitated by passing through a weakly basic anion exchanger, collecting unadsorbed fractions, and then neutralizing them.

Elastase according to the present invention is contained in the pancreas of mammals 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 0.5, but is known to be unstable at temperatures below 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 at °C, no decomposition of elastase was observed compared to the known method (Japanese Unexamined Patent Publication No. 52-61287).

The autolytic fluid obtained under these conditions has extremely low viscosity;
Therefore, the filtration was rapid and a large amount of clear P liquid could be obtained.

Furthermore, autolysis at a relatively high temperature reduced the molecular weight of many of the connective tissue proteins and other unnecessary proteins, making it possible to obtain a digested P solution that was convenient for the subsequent elastase production process.

After adding alkaline earth metals to the autolyzed clarified rho solution to a concentration of 1% or more, a water-soluble organic solvent such as ethanol, methanol, acetone, etc. is added to a concentration of 0 to 30% to remove protein precipitates. 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 of 180, which is the degree of purification of the crude extract of the elastase-containing solution obtained by the method of Lewis et al.

Purification according to the method of LeWlS et al. Purification according to the present invention Elastase-containing solution 3. , O1,0 45% ammonium sulfate precipitate 28 Euglobulin precipitate 47 Ammonium sulfate 35% precipitate 91 57 Elastapi complex crystal 12,0 11.2 However, the elastin decomposition ability of elastase obtained by the method of the present invention is measured by decomposing insoluble elastin. The amount of soluble elastin decomposition product produced is calculated as the tyrosine value, and the activity of releasing 1 μg of 1,000 rosin per minute is defined as R
Obl) lnS et al.'s method (Arch, Biochem
, Biophys, 66.396, (, 1975)
) was measured using a modified method.

Proteins were obtained using the LOWry method (J,]3io1・che
193, 265, (1951)) using ovalbumin as a standard.

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

1 minced pork pancreas 1 K9 at 50℃ with strong stirring 3
After 31 hours of autolysis! of water was added and a clearness of 3.51 was obtained by ρ filtration. After adding 80 grams of calcium acetate to the clear liquid, 500 grams of acetone was added.

The resulting precipitate was removed by centrifugation to obtain elastase-containing centrifuge a3.31.

The supernatant was dialyzed to remove salts and organic solvents, and solid ammonium sulfate was dissolved in the dialysis solution to a saturation of 35%.
The resulting precipitate was collected by centrifugation, suspended in 100-g water, and the precipitate was dissolved by adjusting the pH to 10 with aqueous ammonia.

After desalting the solution by dialysis while maintaining J of 8 to 11.5, the elastase complex was crystallized by adjusting the H of the solution to 7 with acetic acid. The crystals were collected by ρ filtration and washed with cold distilled water to obtain elastase complex 617 of 150 units/my protein.

Applicant: Okura Pharmaceutical Co., Ltd.

Claims (1)

[Claims] ■ After autolyzing the pancreas of a feeder animal, 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 one hour.