JPS6025116B2 - How to isolate enzymes - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating enzymes from cell debris or cells.

Solid-liquid separation methods for separating cell debris or cells from oxygen solutions still present technical problems, especially when large volumes have to be processed.

The usual methods for carrying out this type of separation are centrifugation or filtration. Both of these methods result in viscosity in the resulting suspension, colloidal properties in each component, and a slight difference in density between the suspended solid and liquid. This has the disadvantage of leading to a reduction in the outflow rate. (Naeher & Thum j
n Spencer, Industrial $pec
bofBiochemistry, 1974).
For example, pullulanase (PulManase) is
When isolated from Bsiellap umoniae, in the first step the enzyme is separated from intact cells with a detergent. This step can be carried out by any means without difficulty. However, when attempting to isolate these cells, a relatively high g-number is required, which is difficult to achieve by technical means. Similarly, separation by filtration is also very difficult to carry out due to the presence of detergents (for the state of the art see DE 22 45 342). Biochem Shout Vol. 12 No. 2525-253
According to p. 0 (1973), Escherichia
Enzyme from coli (Phospolipase AI)
It is already known that can be obtained using the phase partition method.
In this case, the cells are homogenized, the cell fluid is separated by centrifugation, and the enzyme is thereby eluted from the cell membrane. For this phase distribution, polypropylene glycol, polyethylene glycol, trimethylaminopolyethylene glycol, polyethylene glycol sulfonate, Ficoll (obtained by polymerizing sucrose and having an average molecular weight of about 40,000
A polyphasic system is used comprising a synthetic polysaccharide of 0) and dextran (a polysaccharide of the empirical formula (C6day, o05)x, glycose). Apart from the fact that this document does not indicate the transfer of the proposed teaching to use for obtaining other enzymes, this separation of cell fluids cannot be carried out on a technical scale.
This means that Eur. J. Bj ratio hem. 4th Tiger Climb 7th
5-81 (1974) and Eur. J. Bioch
em. The same is true for the phase partition method that became known from Vol. It does not start from the yeast decomposition product. The object of this invention is to provide a method for obtaining enzymes from cells on a technical scale, which does not require the separation of cell fluid, cell fragments or cells as previously done. Rather, the aim is to obtain enzymes from isolated or intact cells by partitioning in a multiphasic system. According to this method, one can start from any cell in order to isolate any enzyme. Such examples include, for example, pullulanase (P-anase) (amylobectin-6-glucanohydrase) from K1e broadiella sp. and for example E
Examples include amino-acyl-mNA-synthase from S. scherichiacoli. In this invention, it has surprisingly been found that a satisfactory separation can be achieved with an aqueous multiphase system unless the cell fluid is first separated from the cell debris or cells. According to the invention, the task of the invention is to solubilize the enzyme and add the system produced after enzyme solubilization together with the enzyme, insoluble part and cell fluid to an aqueous multiphase system, and to add the enzyme together with the insoluble part and cell fluid to an aqueous multiphase system. Separation of enzymes from cell debris or cells, consisting of partitioning between different phases of an aqueous multiphase system, separating the phases from each other, separating macromolecular compounds and salts from the enzymes, and optionally isolating them. In the method of The problem is solved by a method characterized by using

For example, a multiphasic system containing polyethylene glycol can be used.

When using a multiphasic system containing optionally substituted polyalcohols or polyethers, the average molecular weight is
It is desirable that the number be less than 40,000, especially less than 10,000. Also, by way of example, polyphasic systems containing sulfates, eg alkali sulfates, and/or phosphates, eg alkali phosphates such as potassium phosphate, can be used.

Additionally, the multiphasic system may include, for example, polymeric compounds such as polyethylene glycol and salts such as potassium phosphate.

Polymorphic systems of this kind are, for example, Escherichia
Aminoacyl-snake NA synthase (AmiMa) from Oli
It is suitable for separating enzymes such as cyl-tRNA-S thetase. In this type of system, water, polymeric compounds, and salts can be used in amounts such that these components alone form a phase before the addition of the soluble enzyme, insoluble portion, and cell fluid. . That is, in the case of such a multiphase system, it is thought that soluble enzymes, cell debris, etc. act as additional phase-forming components. The polymer compound should have an average molecular weight clearly 4.0 mm lower than the molecular weight of the enzyme, preferably less than 40,000, especially less than 10,000, and more preferably in the range of, for example, 50,000 to 1,500. Polyalcohols or polyethers which are less than 6000 and optionally substituted are used.

the enzyme enters the polyalcohol phase or polyether phase;
These polymeric compounds can be easily separated from the enzyme by, for example, ultrafiltration.

The methods of this invention may begin with any cell containing the desired enzyme. For the separation of pullulanase and aminoacyl-tRNA synthetase, it is advantageous to use a multiphase system containing polymeric compounds and inorganic salts. Enzymes can be solubilized, for example, by comminution of the cells and/or by treatment with detergents.

As a cleaning agent, for example, Triton
, Cholat, Denxicholat (戊s
cholat) and dodecyl sulfate (Dod
ecyls mountain at) is suitable. For example, cell debris or unbroken cells are considered insoluble components in a multiphasic system. In the method of the invention, for example salts can be added which have a cleaning or buffering effect.

Consequently, all macromolecular compounds that have been used for the distribution of microorganisms or macromolecular microbiological compounds in multiphasic systems can be used.

Regarding this, especially AI Mr. r$son, Paniti
On of Cell! Paniclesand N is c
romolecuies, NYI 971 (and Kensen). Examples of polymer compounds that can be used in this invention include polypropylene glycol, polyethylene glycol, methoxypolyethylene glycol, trimethylaminopolyethylene glycol, polyethylene glycol sulfonate, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, ethylhydroxyethylcellulose, and DEAE-cellulose. (diethylamino-cellulose), alkali carboxymethyl cellulose, de, xytran, hydroxyf.

Examples include lopyl dextraso, DEAE-dextran, dextran sulfate, alkali carboxymethyl dextran and Ficell. A polyphasic system with total volume/cell volume (limasse)=2, preferably 5 may be used. Also, preferably a pH value of 6 to
9, especially 7 to 8 is recommended.

Technically, this phase can be separated on a large scale, for example in separators, such as disk separators or in decanters.

According to the invention, it has been found that such phases can be separated using commercially available separators even when cell debris or cells are present at the same time.

Moreover, highly developed separation techniques for such enzyme separations can be used, and as a result, very high efflux rates can be achieved, and the reduction in enzyme activity due to long-term operation can be reduced. Besides this, another advantage of the liquid-liquid separation proposed here is that it not only allows the separation of insoluble components from the desired enzyme solution by partitioning between the multiphase systems, but also allows for the separation of different proteins. K-value (
It is also possible to carry out additional purification based on K-Wer).

A polymer compound constituting the multiphase system according to the present invention,
Proteins produced by cells can be separated using methods commonly known in the art.

Examples of this include enzyme precipitation or phase partitioning, ultrafurnace heating, dialysis,
Examples include gel permation (wipenheation), adsorption, ion exchange, and electrolysis.

The method of the invention can be carried out at room temperature, especially if the polymeric compound used stabilizes the enzyme.

A preferred method for separating the enzyme pullulanase from macromolecular compounds and proteins is to precipitate it with a compound of the following general formula: NRIR2R3R4× where RI is an organic compound having at least 10 carbon atoms. R2, R3 and R4 may be the same or different and represent a hydrogen atom or an organic residue having 1 to 20 carbon atoms.

In this case, these organic residues 1 to R4 are selected from alkyl groups, substituted alkyl groups, alkene groups, allyl groups, substituted allyl groups, aralkyl groups, and saturated and unsaturated cyclic groups. , R3 and R4 may be taken together to form a cyclic group, and if there is an unsaturated cyclic group, R2 may be omitted. X represents an inorganic or organic anion such as a halide ion, phosphate ion, nitrate ion, sulfate ion or acetate ion. Examples of this ammonium cation include dodecylmethylammonium ion, dodecyltrimethylammonium ion, tetradecylammonium ion, cetylmethylammonium ion, cetyldimethylammonium ion, cetyltrimethylammonium ion, octadecyltrimethylammonium ion, benzyldodecyldimethylammonium ion, N
, N-diethylmorpholinium ion, cetylpyridinium ion, decenyltrihydroxyethylammonium ion, dioctadecyldiethylammonium ion, dioctadecylmorpholinium ion, dilauryldimethylammonium ion, distearyl-2-chloroethylbutylammonium ion, etc. There is.

Naturally, purinalase can also be precipitated only after the separation of macromolecular compounds and proteins.

Of course, the precipitation of purinalase can also be carried out in a method according to the invention to produce and use purinalase. In this case, purinalase can surprisingly be precipitated in the form of a stoichiometric compound of the empirical purinalase ammonium cation or the salt of empirical purinalase R4NX.

These precipitates can be redissolved in a salt solution with high ionic strength, as will be seen in the Examples below. It is known from DE 22 51 855 to treat enzymes, in particular purinalase, with long-chain quaternary ammonium compounds in order to improve their storage stability.

As can be seen from the bottom of page 4 to the top of page 5 of this publication, the treated enzyme may be present in stabilized form (Example 3) or intracellularly (Example 1) or in an unsaturated form. (Examples 2 and 4) on an active carrier. Therefore, the state of the art does not suggest that purinalase can be precipitated stoichiometrically. The stoichiometric precipitation of enzymes by the most chain ammonium compounds was previously unknown. The production of stoichiometric compounds for purinalase is even more surprising than the fact that, for example, Q-amylase and protease remain in solution when purinalase is precipitated with cetyltrimethylammonium bromide. . Particular precipitation of purinalase presents the advantage that the activities of amylases, glycosidases and proteases can be blocked and separated.

The precipitate formed can be easily centrifuged at a g-value of 4.

Purinalase is used, for example, in the brewing industry, in baking technology and in analytical chemistry to cleave the glycose-Q-1-6 bond and to completely degrade starch and glycogen together with other enzymes.

Pure purinalase turns starch into a bran, resulting in linear oligo- or polyglycan Q-1-4. An example for purinalase is the formation of an aqueous multiphase system with a polymeric compound from the group formed by polyalcohols, polyethers, polyesters and polysaccharides, optionally substituted, and a salt-free organic compound; The following describes the case where the treatment is carried out, for example, in the presence of polyethylene glycol and ammonium sulfate and optionally phosphoric acid.

Maltase (N-light ltase) The situation in maltase is essentially that of pullulanase, but in this case the effectiveness is clearly slightly lower.

This applies to embodiments of the process of the invention with at least two polymeric compounds: in the case of treatment in the presence of phosphistion, more than 0.1, preferably more than 0.3, especially more than 0.5 A phosphate ion concentration of or greater than Aminoacyl-tRNA-synthase (
Aminoacyl NAichi Synthetasen
) Furthermore, the following examples concerning aminoacyl-tRNA-synthases include one polymeric compound in the group formed by optionally substituted polyalcohols, polyethers, polyesters and polysaccharides and other polymeric compounds. "Molecular compounds" forming aqueous multiphase systems with cells or cell debris, in which case the treatment is carried out, for example, in the presence of polyethylene glycol and preferably in the presence of potassium phosphate. be.

Next, the present invention will be explained in detail by way of examples. M indicates average molecular weight.

U represents the enzyme unit, ie, the amount of enzyme that converts 1 mole of substrate into product per minute.

Experimental example 1 [Determination of optimal CTAB (cetyltrimethylammonium furomide) concentration] Based on the preliminary test results, a large CTA
It was confirmed that a decrease in the yield of purinalase activity occurred in the precipitate resulting in CTAB-precipitate at B-concentration.

Regarding the relationship between the precipitate and the molar fraction CTAB/purinalase, the CTAB concentration in the precipitate is then expressed in the form of mmol/U purinalase. This test was conducted as follows.

Purinalase solution (purity 60-70%, PO reed-0.0
2 to 0.03 skin, pH 7.0, 38 U/above) 10% CTAB solution was added in increasing amounts to the ground, and
Bow at ℃ for 15 minutes.

The precipitate was centrifuged and dissolved in potassium phosphate 0.5 (pH 7.5).
) dissolves in The results obtained can be seen from Table 1 below. Table 1 Yamamol CTAB/U Purinalase Activity-Yield% 0.
12 700.14
720.16
820.18
800.24 7
20.32 60 Examples
2 (Relationship between CTAB precipitation and purinalase concentration) This test was conducted in the following manner.

Purinalase solution (purity 70%; 42 U/', PO?
0.02 chloride, pH 7.5) was diluted with increasing amounts of 0.02 potassium phosphate solution (pH 7.5). Mole fraction 0.
Precipitation occurred by addition of a 10% CTAB solution corresponding to 16 rmol/U. The lamp was lit for 15 minutes at 0°C.

The precipitate was centrifuged and diluted with 0.5 potassium phosphate (pH 7.5).
) dissolved in The results obtained can be seen from Table 2 shown below. Table 12 Example 3 (Relationship between CTAB precipitation and pH value) Purinalase solution with a purity of 60 to 70% (4
20/Potassium phosphate 0.02 to 0.03, pH
7.5) Add 0.1 hydrochloric acid or 0 potassium hydroxide to 5'.
．． 1 desk and the pH value is 6.0, 6.5, 7.0,
Adjusted to 7.5 and 8.0 and diluted to 10.

The precipitation conditions resulting from this were as follows; PO reed-(CI-) 0.02-0.03 m
; pH value fluctuation; 0.16〃MoICTAB/U purinalase; 20-22 U/'. Addition of 10% CTAB solution also caused precipitation.

The rope was bowed for 18 minutes at 0°C.

The precipitate was centrifuged, and potassium phosphate (pH 7.5) 0.5
It was dissolved in the. The results obtained can be seen from Table 3 below. Table 3 Example 4 (CTAB-Relationship between precipitation and buffer salt concentration and ionic species) This test was conducted as follows.

Purinalase solution with a purity of 60 to 70% (approximately 42 U/machine,
Salt concentration PO reeds - 0.02) above 5, respectively, 1
potassium phosphate solution or ammonium sulfate solution (P
H7.0), molar concentration 0.04, 0.06 and 0.0
A solution volume of 10 with a salt concentration equivalent to 8' was used.
The precipitation conditions resulting from this were: 0.16 remol CTAB/U pullulanase, 20
to 22 U/di, pH 7.0 and varying salt concentration. 1
Precipitation occurred by adding 0% CTAB-solution.

I lit a candle for one minute at 0°C. The precipitate was centrifuged and dissolved in 0.5 potassium phosphate (pH 7.0). The results obtained can be seen from Table 4 below. 4- Example 5 (Relationship between activity yield and specific activity and solubility of CTAB-precipitate in saline solutions with increasing salt concentration) This test
It was carried out by the following method.

Each dialyzed upper phase 10 secretion was adjusted to pH 7.0,
Diluted to 15%. The precipitation conditions were as follows: PO reed-0.02', pH 7.0 about 30 U/U, about liU/U, and 0.16 mmol CTAB/U. 1
Precipitation occurred by adding 0% CTAB-solution.

The light was lit for 196 hours at 0℃.

The precipitate was centrifuged and dissolved in increasing concentrations in potassium phosphate or ammonium sulfate solution for 15 minutes. The results obtained can be seen from Table 5 below. Table 5 Examples 6 to 9 (CTAB-precipitation tests with different starting solutions) In the following tests, purinalase was precipitated from different starting solutions under the optimal conditions found in Examples 1 to 5. .

Example 6: Starting solution: Dialyzed upper phase 70', P02-
Salt concentration equivalent to 0.02 to 0.03 skin, pH 7.
Antistatic activity 72U/secretion, specific activity 3.6U by gel electrophoresis
/Kite [(=9% pure purinalase).

1 corresponding to 0.18 mol CTAB/U brinalase
Precipitation occurred by adding 0% CTAB solution.

It was incubated at 0℃ for about 2 hours.

The precipitate was centrifuged and diluted with 0.5 potassium phosphate (pH 7.5).
) dissolved in The following analytical values were obtained: activity yield 85
U/[=80%, specific activity approximately 21 U/3 (=52%)
, purity factor 5.8 (purity factor=specific activity in dissolved precipitate/specific activity of starting material).

Example 7: Starting solution: above dialyzed upper phase 50, PO reed-
Salt concentration equivalent to 0.03m, pH 7.0, activity 42
U/', specific activity by gel electrophoresis 12 U/cloth (30
%).

1 corresponding to 0.16 mmol CTAB/U purinalase
Precipitation occurred by adding a drop of 0% CTAB.

Takaazusa was carried out at 0°C for about 1 hour.

The precipitate was centrifuged and diluted with 0.5 potassium phosphate (pH 7.5).
) was dissolved in 10 units. As a result, the following analytical values were obtained: activity yield of 147 U/g = 70%, specific activity by gel electrophoresis of 30 U/9 (total 75%), purity coefficient of 2.5.
A protease activity test showed traces in the starting material, but this was not evidenced by CTAB-precipitation followed by dissolution of this precipitate.
In the Q-amylase test (Phade rat Q-amylase test/Pha dish acia), 30U was added in the starting material.
A value of /so=0.03U/yellow was found, but CTAB.
This was not demonstrated when the precipitate was dissolved after precipitation.

Example 8: Starting solution: 20 volumes of dialyzed upper phase; salt concentration 0.03, pH 7.0, activity 28 U/upper, QI
-9 (21%) with a specific activity of 8.4 U/by nebulophoresis.

10% CT equivalent to 0.18 molar/U purinalase
Precipitation occurred by adding the AB-solution.

The rice was worshiped at 4°C for about 15 minutes.

The precipitate was centrifuged and diluted with 0.5 potassium phosphate (pH 7.5).
) dissolved in 20 min. As a result, the following analytical values were obtained: activity yield 22.5 U/I = 80%, specific activity by QI-electrophoresis 23 U/9 (58%), purity factor 2.
8 Example 9: Starting solution: Amicon
orp. )/×M300 [Extreme furnace membrane. The upper phase 15 was dialyzed through an exclusion value of approximately 300,000 molecular weight (Amicon Corp)]: salt concentration 0.02 mPO reed-, pH
7.5, activity 19 U/I, Snake 1 - specific activity 23 U/ by flea aerophoresis 9 (streak %), specific activity 25 U/ by mountain whi
9 (62%).

10% CT equivalent to 0.16 mmol/U purinalase
Precipitation occurred by adding the AB solution.

The light was held at 0℃ for about 15 minutes.

The precipitate was centrifuged and diluted with 0.5% potassium phosphate (pH 7.5).
)10 dissolved in [. As a result, the following analytical values were obtained: activity yield 22.8 U/'=80%, specific activity by GI bursting 36 U/o (190%), purity factor 1.
45Q-amylase activity and protease activity tests were negative.

Example 10 It was investigated whether pullulanase could be precipitated using various cations of nitrogenous bases.

Starting from a nitrogen solution with the following values: activity 20.8 U/
Above, P--, salt concentration 0.02, pH 7.5.

The precipitates were each dissolved in 0.4% sodium chloride.
A 10% aqueous solution was prepared in each case using a precipitant. The results obtained can be seen from the following Table 6. 6th
Table Example 11 In this example, the quality of pullulanase precipitation from cetyltrimethylammonium furomide, N-dodecylpyridinium chloride, and N-cetylpyridium chloride was compared.

As a starting solution, a solution concentrated by passing through an ultra-furnace through Amicon
5, pfi7.5) was used. 0.15 to 0.2
0.2% (weight/
Weight) Precipitation was produced by adding each precipitant.

This was left at about 0°C for 15 minutes. The precipitate was dissolved in 0.4 volumes of sodium chloride. The results obtained can be seen from the following Table 7.

Table 7 CTAB precipitation gave the best results in terms of purity factor.

Other tests also showed better results. Example 1Es
6001 kg of herichiacoli MER was suspended in 0.05 kg of potassium phosphate aqueous solution (pH 8.0), and
times, high pressure homogenizer (Manton Gaulin)
The material was crushed at a rate of 600 kg/land.

The protein concentration was 9/47.2. This crushed material was used in various tests. The details are shown in Table 8.
According to the phase diagram of the polyethylene glycol (M6000)/potassium phosphate system, no phase separation should occur under the conditions of Test No. 3.

However, it is known that cells or cell debris enter phase diagrams as polymers. A clear phase field developed and the cell debris migrated to the highly viscous lower phase. Table 8 Example 2 Escherichia coli
i) MRE6001k9 was suspended in aqueous potassium phosphate buffer (Book 8.0) and ground as shown in Example 46.

To this suspension, add 50% polyethylene couricol solution (
M6000) 656 ml and 525 ml of 50% dipotassium hydrogen phosphate solution (pH 7.8) were added. This phase was transferred to a disc separator (Q-Lava1-GM) on the outlet length 14.5 side.
o-Tester B) at a flow rate of 160'/min. The length of stay was 16 days. A good yield of enzyme was obtained, with a concomitant improvement in the specific activity by a factor of about 2 and a good separation of the cell debris.

The details can be found in Table 9 below. Table 9 IRS=isoleucil-tRNA-synthetase LRS=leucil-tRNA-synthetase PRS=phenylalanine-tRNA-synthetase Example 3 Escherichjacoli 500 min.
suspended in 700 molar aqueous potassium phosphate buffer,
3 times, glass ball mill (D
achofen) (glass beads 0.25-0.5 kites, 5 m/h, 300 m).

The resulting suspension was mixed with 10% polyethylene glycol (M
6000) and 8% potassium phosphate (pH 7.8) and separated as in Example 2 in a separator.
The high yield and specific activity of the enzyme were improved to a factor of 2, and the cell debris could be well separated from the upper phase.

The details are clear from the following Table 1. 101 See Table 9 for IRS, LRS and PRS.

Examples 4 and 5 The relationship between the partition coefficient K of pullulanase and various parameters in an aqueous two-phase system containing polyethylene glycol and ammonium sulfate was investigated.

Example 4: To form a two-phase system, polyethylene glycol (M4000, 50% aqueous solution), solid ammonium sulfate and each enzyme solution (60% purity, pH 7.
A salt concentration of about 0.02 mPO poison-) was used.

Detailed values can be found from Table 11. Example 5:
Polyethylene glycol (M
400050% aqueous solution), solid ammonium sulfate, enzyme solution (purity 60%, pH 7.5, salt concentration P
0.02) and potassium phosphate buffer (pH 7.02).
5) was used. Individual numerical values can be found from Table 12. It can be seen from Tables 11 and 12 that pullulanase significantly disappears in the upper phase containing polyethylene with increasing concentrations of ammonium sulfate and polyethylene glycol.

Furthermore, a significant increase in the K-value is observed with increasing phosphate concentration. Table 11 Table 12

Claims (1)

[Claims] 1. Solubilizing oxygen and adding the system produced after oxygenation and solubilization to an aqueous multiphase system together with oxygen, insoluble parts, and cell fluid,
From partitioning oxygen together with insoluble parts and cell fluids between the different phases of an aqueous multiphasic system, separating the phases from each other, and separating and optionally isolating macromolecular compounds and salts from oxygen. A phase-forming polymeric compound selected from the group consisting of optionally substituted polyalcohols, polyethers, polyesters, polyvinylpyrrolidone and polysaccharides and phase-forming A method characterized in that a multiphase system containing an inorganic salt is used. 2 Polypropylene glycol, polyethylene glycol, methoxypolyethylene glycol, trimethylaminopolyethylene glycol, polyethylene glycol sulfonate, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, ethylhydroxyethylcellulose, DEAE-cellulose, alkali carboxymethylcellulose, dextran, hydroxypropyldextran, DEAE 2. Process according to claim 1, characterized in that a polymeric compound of the group formed by dextran, dextran sulfate, alkylcarboxymethyl dextran and Ficoll is used. 3. Process according to claim 1 or 2, characterized in that the aqueous multiphasic system containing polyethylene glycol is used. 4. Claims characterized in that the aqueous multiphasic system is used which contains optionally substituted polyalcohols or polyethers with an average molecular weight of less than 40,000 and in particular less than 10,000. The method described in any one of 1 to 3. 5. Any one of claims 1 to 4, characterized in that the aqueous multiphase system is used which contains a sulfate, such as an alkali metal sulfate, and/or a phosphate, such as an alkali metal phosphate. Method described. 6. Process according to any one of claims 1 to 5, characterized in that the aqueous multiphase system containing potassium phosphate is used. 7 Patent characterized in that said aqueous multiphasic system containing a polymeric compound, for example polyethylene glycol, and a salt, for example potassium phosphate, is used for the separation of an enzyme, for example aminoacyl-tRNA-synthase, for example from Escherichia coli. A method according to any one of claims 1 to 6. 8 Water: A patent characterized by the use of an aqueous multiphase system containing polymeric compounds and salts in such amounts that these components alone form one phase before the addition of soluble enzymes, insoluble parts, and cell fluids. The method according to claim 7. 9. A method according to any one of claims 1 to 8, characterized in that a polyphasic system is used in which the ratio of total volume/cell volume is ≧2, preferably ≧5. 10 Claims 1 to 9 characterized in that a polyphasic system is used with a pH value of 6 to 9, preferably 7 to 8.
The method described in any of the above. 11. The method according to any one of claims 1 to 10, characterized in that phases are separated using a separator. 12 by enzyme precipitation or by phase partitioning, ultrafiltration, dialysis, gel permeation.
12. The method according to any one of claims 1 to 11, characterized in that the polymer compound is separated from the enzyme by adsorption, ion exchange, or electrophoresis.