JPH01168284A - Recovery of extracellular enzyme from full fermentation beer - Google Patents

Abstract

PURPOSE: To readily recover enzymes by adding a specific polymer and an inorganic salt to a fermented beer, and separating the obtained mixture to an enzyme-rich polymer phase and an enzyme-poor salt phase.

CONSTITUTION: (A) a polymer selected from polyethylene glycol, polypropylene glycol, an amine derivative thereof or a carboxylate derivative thereof, a poly (ethylene glycol) ester, polyethyleneimine, trimethylamino-polyethylene glycol, polyvinyl alcohol and polyvinylpyrrolidone, and (B) an inorganic salt (e.g. sodium chloride and sodium sulfonate) are added to the whole fermentation beer containing microorganism cells and extracellular enzyme. The obtained mixture is separated to an enzyme-rich polymer phase and an enzyme-poor salt phase and the enzyme is recovered from the former phase.

COPYRIGHT: (C)1989,JPO

Description

[Detailed description of the invention] Bacteria, fungi
The production of individual enzymes by culturing microorganisms such as and yeast in aqueous nutrient media is well known.

Depending on the nature of the particular microorganism, the enzymes produced can be either extracellular enzymes or intracellular enzymes.
traelIular) enzyme or a mixture thereof.

If the enzyme produced is an extracellular enzyme, the enzyme-containing supernatant is first separated from the microbial cells, and then the enzyme is recovered from the supernatant by well-known methods such as precipitation, ultrafiltration, and evaporation. generally obtained by If the enzymes produced are intracellular enzymes, they must first be released from the cell. This can be done chemically and/or mechanically. Once the enzymes are in solution, they can also be recovered by the well-known methods described above.

It is known that extracellular enzymes have different compositions and properties than intracellular enzymes. Extracellular enzymes generally have a much lower molecular weight than, for example, intracellular enzymes. A solution containing intracellular enzymes released from cells is a whole fermented beer (whole fermentation liquor) containing extracellular enzymes.
It also contains significant amounts of other intracellular substances not present in mention beer. These differences can lead to different recovery and purification methods even when they are both in aqueous solution. Therefore, recovery methods suitable for intracellular enzymes are not necessarily useful for extracellular enzymes.

To improve the efficiency and convenience of enzyme production and recovery, enzymes were purified by microbial fermentation in a biphasic nutrient medium containing a mixture of polyethylene glycol and dextran.
It is known that it is possible to m. At the end of fermentation, extracellular enzymes are concentrated in the upper polyethylene glycol phase and cells and other fermentation products are concentrated in the lower dextran phase. This is Enzyme and Micro Vial Technology, Volume f57, 7,333-
3 3 8 (1985) [EnzyIlle
and Microb, Technol, Vol.
, 7, 333-338 (1985)]. The partition coefficient of alpha amylase in that system was, for example, up to 4.

A variation of the above method is described in US Pat. No. 4,508,825. According to it, extracellular enzyme-containing (fl) fluid is separated from cells and cell-free supernatant is polyethylene glycol and cationic epihalohydrin/
Mixed with polyamine copolymer or dextran polymer to form two phases. This method can be used to separate extracellular proteases and amylases, where the protease is concentrated in the polyethylene glycol phase and the amylase is concentrated in the cationic copolymer or dextran phase.

The two-phase fermentation cord recovery method was also used for intracellular enzymes.

U.S. Pat. No. 4,144,130 discloses (1) a mixture of a high molecular weight unsubstituted or substituted polyalcohol, polyether, polyester, polyvinylpyrrolidone or polysaccharide with an inorganic salt; or (2) at least one of the above high molecular weight polymers. The use of a mixture of the two to recover intracellular enzymes from an aqueous solution in which they are released from cells is described.

For example, if a mixture of polyethylene glycol and inorganic salts is used, the desired intracellular enzymes will go to the upper polyethylene glycol layer and will be absorbed by the cell debris.
s) and other fermentation products go to the lower salt-containing layer. The partition coefficients of the various enzymes recovered in the glycol layer were approximately 0.3 when the whole fermented beer was treated.

The partition coefficient increased by about 3 when frozen cells were mixed with water and disrupted to release their enzymes.

The addition of polyethylene glycol to aid in the precipitation of enzymes from cell-free supernatants is described in US Pat. No. 4,016,039.

It has never been suggested in the prior art that polyethylene glycol and inorganic salts can be used in a two-phase process to recover extracellular enzymes from whole fermented beer with a partition coefficient of at least 50.

According to the present invention, there is provided a method for recovering extracellular enzymes from whole fermented beer, which comprises adding (a) polyethylene glycol, amine derivatives of polyethylene glycol, polyethylene glycol to whole fermented beer containing microbial cells and extracellular enzymes; the group consisting of carboxylate derivatives of polypropylene glycol, amine derivatives of polypropylene glycol, carboxylate derivatives of polypropylene glycol, poly(ethylene glycol) esters, polyethyleneimine, trinotylamino-polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and mixtures thereof and (b) a mixture of an inorganic salt and this whole fermented beer - polymer -.
A method is provided for separating a geothermal mixture into an enzyme-rich polymer phase and an enzyme-poor geothermal phase and recovering the enzyme-rich product therefrom.

Whole-fermented beers containing extracellular enzymes useful as raw materials in the process of the invention are well known in the art. For example, Bacillus licheniformis Bacillus protease, amylase and microbial rennet (mi
It is known to produce extracellular enzymes such as crobial rennet. The resulting mixture of cell debris, extracellular soluble enzyme and other fermentation products can be used in the method of the invention without further separation of the cell debris from the soluble enzyme. As used herein, “
The term "cell debris" refers to whole cells.
s) and cell fragments (cell fraB+ents).

The whole fermented beer is then mixed with the polymer and inorganic salts to form a two-phase system. The desired extracellular enzyme collects in the polymer phase;
Cell debris will collect in the geological formations.

Suitable polymers include polyethylene glyfur, amine derivatives of polyethylene glycol, carboxylate 1 conductors of polyethylene glycol, polypropylene glyfur, amine derivatives of polypropylene glycol, carboxylate derivatives of polypropylene glycol, poly(ethylene glycol) esters, polyethylene Included are imine, trimethylamino/polyethylene glycol, polyvinylafurfur, polyvinylpyrrolidone and mixtures thereof. A preferred polymer is polyethylene glycol. Any form of polyethylene glycol that is soluble in aqueous fully fermented beer is suitable. A particularly useful polyethylene glycol has a molecular weight of about 3350. It is solid at room temperature and can be conveniently handled on an industrial production scale.

Inorganic salts have cations such as sodium, potassium, magnesium, and ammonium, and anions such as sulfates, carbonates, and carbonates.
onates), citrate ions (citrates)
, chlorides, phosphates, and mixtures thereof. Preferred salts are sodium chloride and sodium sulfate.

The polymers and inorganic salts are total fermented beer containing 64-90%, 1-15% polymer and 8-35% inorganic salts, where the percentages are based on the total weight of the mixture. Added to whole fermented beer in amounts to form a mixture. Total mixture is 73-79% total fermented beer, polymer 3-4
% and 15-24% of inorganic salts.

\, \circle m-\ Two phases will be formed after adding the polymer and salt. The polymeric phase will normally overlie the geological phase. The two phases can be formed by settling out by allowing the mixture to stand for about 5 hours.

The enzyme-containing polymer phase is then subjected to e.g. siphoning and decanting.
The enzyme can be separated and recovered by standard liquid/liquid separation methods such as decanting. However, it is preferred to use centrifugation to separate the phases. A useful separation device is a continuous solid ball centrifuge93
(solid-bowl centrifug6)
It is. In order to achieve the desired concentration of #g molecules in the final separated polymer phase, the volume ratio of enzyme-rich polymer to enzyme-poor geophase is preferably between 0.12 and 0.15. .

For the most efficient recovery of extracellular enzymes, centrifugal force m must be applied to ensure that nothing of the polymer phase is entrained in the geological phase to be discarded.
You should operate a. The resulting collected polymer phase may have some entrained geological phase mixed therein. The mixture is then subjected to a second centrifugation.

In the secondary centrifugation, the centrifuge is operated such that none of the polymer phase is entrained in the geological phase and only a small amount of the geological phase is entrained with the polymeric phase.

The enzyme-rich polymer phase so collected can be used directly as an enzyme source. Alkaline protease recovered in a polyethylene glycol phase is suitable for example in enzymatic detergent formulations.

This glycol is useful as an enzyme stabilizer.

If desired, the enzyme can be separated from the polymer by well known methods such as precipitation, ultrafiltration, or evaporation to produce a substantially polymer-free enzyme product.

In another preferred embodiment of the invention, the whole fermented beer contains 1.5-5.0% calcium chloride dihydrate, 0.1-1.2% monobasic sodium phosphate or monobasic potassium phosphate and hydroxide. A mixture of 0-0.6% calcium is pretreated, the percentage being on a weight/volume basis, calculated from the weight of the additive to the volume of fermented beer. This pretreatment step helps to aggregate the cell debris and improves the separation of the enzyme from the cell debris when the polymer and inorganic salts are subsequently added. Preferably, if the protease is to be recovered, the pretreatment step includes 1.5-5% calcium chloride dihydrate.
．． 0% and monosodium phosphate or monopotassium phosphate 0.2-1.2% should be used. If amylase is recovered, the pretreatment step includes 1.5-5.0% calcium chloride dihydrate, 0.1-1.0% monobasic sodium phosphate or monobasic potassium phosphate, and hydroxide. Calcium 0.1-0.6% should be used.

Partition coefficients are known in the art as a measure of separation effectiveness. It is expressed as the concentration of enzyme activity in the upper or polymeric phase subtracted by the concentration of enzyme activity in the lower or inorganic salt phase. Enzyme activity in each phase can be measured by well-known methods. The method of the present invention is capable of achieving partition coefficients of at least 50, representing a significant advance over the known prior art.

The invention is further illustrated by the following examples.

Example 1 A nutrient medium suitable for the production of alkaline protease was prepared by adding the following ingredients to a 6000 gallon fermentor: wheat glutin.
1500 lb (681k(1) Sodium citrate 165 lb (74,9k(]) Calcium chloride dihydrate 165 lb (74,9kg
) corn starch 5000 lb (22
70kg) Soybean flour (Soy Meal) 250
01b (1135kg) Thermostable alpha amylase 5 lb (2,27kQ
) Monosodium phosphate 400 lb (No. 81,6 kg) Sodium diphosphate 400 l
b (Article 81,6k(1) Anti-foaming agent
16.5 gallons (62,51>total including water
This medium was then inoculated with live cells of Bacillus licheniformis and fermented for 36 hours at 36°C. 70% (W/V) calcium chloride dihydrate aqueous solution 21 to the resulting whole fermented beer
4 gallons (8111) and monobasic sodium phosphate 300
1b (136ko) was added. This resulted in a mixture containing 2.5% (W/v) of calcium chloride dihydrate and 0.6% (W/V) of monobasic sodium phosphate based on the weight of each additive and the volume of fermented beer. can get. The l)H of the mixture was maintained at 6.8-7.6 while mixing by addition of sodium hydroxide in the additive. After mixing is complete, add sodium hydroxide to reduce l)H to 7.4
-7.5 adjustment to achieve aggregation. I)H was then adjusted to 6.4-6.6 by addition of 50% by weight aqueous acetic acid solution. Sodium chloride 116001b (5260 klll) was added to the resulting mixture at 25-27°C and the mixture was stirred for 1 hour to dissolve all the sodium chloride.

Then polyethylene glycol 31001b (1400 kg) with a molecular weight of 3350 and sodium sulfate 4
2501b (1930kl) was added and the total beer temperature was raised to 30-30°C.

The pH of the whole beer was reduced to 6.0 by adding acetic acid solution. 〇-6,2
and the whole mixture was stirred for 2 hours.

The whole mixture contained 15% sodium chloride (W/Vl),
Polyethylene glycol 4% (w/w), sodium sulfate 5.5% (w/w) and whole fermented beer 75
5% (W/W). This percentage was calculated based on the weight of the additive relative to the total mixture of fermented beer and additive. After mixing, the entire mixture is divided into an upper polyethylene glycol phase and a lower sodium chloride-sodium sulfate phase.
The cells were separated into a debris phase. The phase ratio of upper phase capacitance to lower phase capacitance is 0.
．． It was 15. The concentration of protease activity in the upper phase divided by the concentration of protease activity in the lower phase resulted in a partition coefficient of 60-80. The two phases were then separated using a continuous solid ball centrifuge. The centrifuge was adjusted to provide a primary separation so that the polyethylene glycol phase was not entrained in the discarded geological phase. The glycol phase contained 90-92% of the total enzyme activity of the fermented beer. The glycol phase also contained about 5-20% by volume of phase phase entrainers.

The glycol phase collected as described above was then subjected to a second separation using a similar apparatus configured to allow less than 2% by volume of geological phase to be entrained in the glycol phase. Ditch the geological phase and it contained nothing of the glycolic phase. The glycol phase collected as above was then placed in a cooled tank at 10"C to remove traces of sodium sulfate. A small amount of sodium sulfate crystals was added to induce the growth of sodium sulfate crystals. After gentle stirring for 2 hours at , the sodium sulfate-free glycol phase was discharged. It was then mixed with activated carbon and filter aid and filtered using a filter press.

The resulting alkaline protease-enriched glycol solution can be used directly in liquid enzymatic detergent formulations.

The method of Example 1 was repeated through the steps of adding calcium chloride dihydrate and monobasic sodium phosphate. The resulting whole fermented beer-additive mixture was then mixed with polyethylene glycol 21301b having a molecular weight of 3350 (970
ko) and sodium 5A acid 106601b (4850
1C1) and raised the total beer temperature to 30-32°C. Then, polyethylene glycol 3% (W
/W), sodium sulfate 15% (W/W) and total fermented beer 82% (W/W), the entire mixture was stirred for 1 hour. After mixing, the entire mixture was separated into an upper polyethylene glycol phase and a lower sodium sulfate-cell debris phase. The phase ratio of upper phase volume to lower phase volume was 0.12. The concentration of protease activity in the upper phase divided by the concentration of protease activity in the lower phase resulted in a partition coefficient of 60-80. The alkaline protease in the glycol phase was in the form of an amorphous solid. The two phases were then separated using a centrifugal separator as described in Example 1 to produce a glycol phase containing amorphous solid protease and less than 2% by volume of lamellar phase entrainers. . The solids are then recovered by centrifugation and they can be used as granular protease in granular enzyme detergent products.

Example 3 A nutrient medium suitable for the production of thermostable alpha-amylase was prepared by adding the following ingredients to a 6000 gallon (22712+) fermenter: 22.5 lb (10,2 k(1) ) Monobasic potassium phosphate 300 lb < 136.2
kg) Potassium diphosphate 700 lb (
317,8ka) Ammonium sulfate 250
lb (113,5Jl) Sodium citrate
100 lb (45,4k(+) (corn soaking liquid) 1000 lb (454k(1) lactose 7000 lb (3178k)
(+) (cottonseed flour) 1500 lb
(881Jl) Tei Soybean Flour 200 lb (908k
g) Anti-foaming agent 165 gallons (625
1) Total volume including water: 6000 gallons (227
This medium was then inoculated with live cells of Bacillus licheniformis and fermented for 108-110 hours at 40°C, maintaining the DH at 7.05-7.15 by periodic addition of sodium hydroxide. 343 gallons (13001) of a 70% (W/■) calcium chloride dihydrate aqueous solution and 10% (W/V) were added to the resulting total fermented beer.
) Added 240 gallons of calcium hydroxide aqueous solution (9801) and monobasic potassium phosphate 601b (27ko).

Based on the weight of each additive and the volume of fermented beer, this results in 4% (w/v') of calcium chloride dihydrate, 0.4% (w/v) of calcium hydroxide, and 0.12% (w/v) of potassium monophosphate. % (W/V) is obtained. The pH of the mixture was maintained at 7.6-8.4 while mixing by addition of sodium hydroxide in the additive. After mixing is complete, add sodium hydroxide to reduce l)H to 8.4
Adjusted to -8.6. Polyethylene glycol 26001b (1 kg) with a molecular weight of 3350 and sodium chloride 74201b (3370 kg) were added to the resulting mixture.
ka> was added at 25-27°C and the mixture was stirred for at least 1 hour. 59401b (2700 kg) of sodium sulfate was then added and the fermented beer mixture was reduced to 30-32 kg.
heated to ℃. The pH was adjusted to 7 by addition of sodium hydroxide.
．． 9-8.1 and the entire mixture was stirred for at least 2 hours. The whole mixture was made up of 10% sodium chloride (W/
W ), polyethylene glycol 3.5% (w 7w
), 8% (W/W) of sodium chloride and 78.5% (W/W) of total fermented beer. After mixing, the entire mixture was separated into an upper polyethylene glycol phase and a lower sodium chloride-sodium sulfate-cell debris phase.

The phase ratio of upper phase volume to lower phase volume was 0.12.

The concentration of amylase activity in the upper phase divided by the degree of amylase activity in the lower phase resulted in a partition coefficient of 50-70. Then continuous solid ball centrifugation Il! 111i in Example 1
The amylase-rich glycol phase was recovered, which was then purified as described in Example 1 to produce an amylase-rich glycol product.

A nutrient medium suitable for the production of alpha-amylase was prepared II by adding the following ingredients to a 6000 gallon (22712+) fermenter: Calcium carbonate water.
530 lb (240,6k(1) fish meal
750 lb (3
40.5kg) (ground soybean flour) 2800
lb (1271kg) Corn soaking liquid 1
500 lb (681 kl lactose
7000 lb (3178k (1) Ammonium diphosphate 130 lb (59 kg > antifoam agent 40 gallons (151,41) total volume with water 6000 gallons (22712+) This medium was then injected with live Bacillus amyloliquefaciens. Cells were inoculated and I) maintained at 7.05-7.15 by periodic addition of sodium hydroxide.
Fermentation was performed at 4°C for 60 hours. 7 for the total fermented beer obtained.
257 gallons (9731) of 0% (W/V) calcium chloride dihydrate aqueous solution, 80 gallons (6811) of 10% (W/V) calcium hydroxide aqueous solution, and 3001b (136! J) of monobasic potassium phosphate aqueous solution. added. As a result, based on the weight of each additive and the volume of fermented beer, calcium chloride hydrate is 3% (W/V) and calcium hydroxide is 0.
．． 3% (W/V) and monopotassium phosphate 0.6% (W
/V) is obtained. In the additive the p+ of the mixture is increased while mixing by the addition of sodium hydroxide.
-+ was held at 6.8-7°6. After mixing was completed, the DH was adjusted to 7.4-7.6 by adding sodium hydroxide.

To the resulting mixture polyethylene glycol 25101b (11401L!J) having a molecular weight of 3350 and 1-IJ chloride 79601b (3600 kO) were added at 25-27°C and the mixture was heated to a molecular weight of at least 1911L!J.
1fl stirring. Next r1070011) (4870kg
) of sodium sulfate was added and the fermented beer mixture was heated to 30-32°C. l) H was adjusted to 7.4-7.6 by addition of sodium hydroxide and the whole mixture was stirred for at least 2 hours. The whole mixture contains 10% sodium chloride (W/W), 3% polyethylene glycol
．． 15% (w 7w ), sodium sulfate 13.5%
(*/W) and total fermented beer 73.35% (W/W
). After mixing, the entire mixture was mixed with 2 as described in Example 3 with a phase ratio of 0.12 and a partition coefficient of 50-70.
Separated into phases. The phase was treated as described in Example 3 to produce an amylase-enriched glycol product.

Claims (1)

[Claims] 1. A method for recovering extracellular enzymes from whole fermented beer, which comprises adding (a) polyethylene glycol, amine derivatives of polyethylene glycol, polyethylene to whole fermented beer containing microbial cells and extracellular enzymes; The group consisting of carboxylate derivatives of glycols, polypropylene glycol, amine derivatives of polypropylene glycol, carboxylate derivatives of polypropylene glycol, poly(ethylene glycol) esters, polyethyleneimine, trimethylamino-polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and mixtures thereof. (b) separating the whole fermented beer-polymer-salt mixture into an enzyme-rich polymer phase and an enzyme-poor salt phase; A method characterized in that an enzyme-rich product is recovered therefrom. 2. The inorganic salt has a cation of sodium, potassium,
Claim 1: Magnesium and ammonium, the anions of which are selected from the group of compounds selected from the group of sulfate, carbonate, citrate, chloride, phosphate and mixtures thereof. the method of. 3. The total mixture of total fermented beer, polymer and inorganic salt mixture contains 64-90% total fermented beer, 1-15% polymer and 8-35% inorganic salt, provided that the percentages are based on the total weight of the mixture 2. A method according to claim 1, wherein the method is by weight based on . 4. The total mixture of total fermented beer, polymer and inorganic salt mixture contains 73-79% of total fermented beer, 3-4% of polymer and 15-24% of inorganic salts, provided that the percentages are based on the total weight of the mixture 2. A method according to claim 1, wherein the method is by weight based on . 5. The method of claim 4, wherein said polymer is polyethylene glycol and said inorganic salt is a mixture of sodium chloride and sodium sulfate. 6. The method of claim 4, wherein said polymer is polyethylene glycol and said salt is sodium sulfate. 7. The method of claim 4, wherein said polymer is polyethylene glycol having a molecular weight of about 3350. 8. Calcium chloride dihydrate 1.5-5.0%, monobasic sodium phosphate or monopotassium phosphate 0.1-1.2% and calcium hydroxide 0 before adding the polymer and inorganic salt mixture. 5. The method of claim 4, wherein the percentage is on a weight/volume basis calculated on the weight of the additive to the volume of fermented beer. 9. Whole fermented beer with calcium chloride dihydrate 1.5-5.
0% and monobasic sodium phosphate or monobasic potassium phosphate 0
．． 9. The method of claim 8, wherein the mixture is contacted with a 2-1.2% mixture. 10. Whole fermented beer with calcium chloride dihydrate 1.5-5
．． 0%, calcium hydroxide 0.1-0.6% and monobasic sodium phosphate or monobasic potassium phosphate 0.1-1.0
9. The method according to claim 8, wherein the mixture is contacted with a mixture of % and %. 11. The method of claim 4, wherein the volume ratio of enzyme-rich polymer to enzyme-poor salt phase is 0.12-0.15. 12. The method according to claim 4, wherein the resulting phases are separated by continuous solid ball centrifugation. 13. A method for recovering extracellular alkaline protease, which comprises fermenting an appropriate strain of Bacillus licheniformis in a suitable nutrient medium to obtain a total fermentation containing Bacillus licheniformis cells and extracellular alkaline protease. producing beer and adding 2.5% of calcium chloride dihydrate and 0.6% of monobasic sodium phosphate or monobasic potassium phosphate to the total fermented beer; on a weight/volume basis, calculated on a weight basis, and adding to the above mixture 3% polyethylene glycol and 15% sodium sulfate, provided that the percentages are based on the weight of the total mixture of fermented beer and additives. and forming an alkaline protease-rich polyethylene glycol phase and an alkaline protease-poor sodium sulfate phase and separating the two phases to recover an alkaline protease-rich product therefrom. A method characterized by: 14. After adding calcium chloride dihydrate and monobasic sodium phosphate or monobasic potassium phosphate, the mixture is divided into 15% sodium chloride, 4% polyethylene glycol and 5.5% sodium sulfate, provided that the percentages are different from fermented beer and 14. The method of claim 13, wherein the contacting is by weight based on the total mixture weight of the additive. 15. A method for recovering extracellular thermostable alpha-amylase, which comprises fermenting an appropriate strain of Bacillus licheniformis in a suitable nutrient medium to produce a mixture containing Bacillus licheniformis cells and extracellular alpha-amylase. producing a fully fermented beer containing 0.4% calcium hydroxide and 0.12% monobasic sodium phosphate or monobasic potassium phosphate, provided that the percentages are additives relative to the volume of the fermented beer; on a weight/volume basis, calculated based on the weight of the fermented beer and additives. by weight based on the total mixture weight of , forming an amylase-rich polyethylene glycol phase and an amylase-poor sodium chloride-sodium sulfate phase and separating the two phases to form an amylase-rich polyethylene glycol phase and an amylase-poor sodium chloride-sodium sulfate phase. A method characterized in that a product is recovered therefrom. 16. A method for collecting extracellular alpha-amylase, which involves fermenting an appropriate strain of Bacillus or Amyloliquefaciens in an appropriate nutrient medium.
amyloliquefaciens cells and extracellular alpha-amylase, and adding to the whole fermented beer 3% calcium chloride dihydrate, 0.3% calcium hydroxide and monobasic sodium phosphate or Adding 0.6% potassium monophosphate, however, the percentage is based on a weight/volume basis calculated based on the weight of the additive to the volume of fermented beer, and polyethylene glycol 3.15% to the above mixture. %, sodium chloride 10%
and sodium sulfate 13.5%, provided that the percentages are by weight based on the total mixture weight of fermented beer and additives, and an amylase-rich polyethylene glycol phase and an amylase-poor sodium chloride-sulfate phase. A process characterized in that a sodium phase is formed and the two phases are separated to produce an amylase-enriched product therefrom.