JPH0323152B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The production of individual enzymes by culturing microorganisms such as bacteria, fungi and yeast in aqueous nutrient media is well known. Depending on the nature of the particular microorganism, the enzyme produced can be an extracellular enzyme, an intracellular 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. The solution containing intracellular enzymes released from cells also contains significant amounts of other intracellular substances not present in a whole fermentation beer containing extracellular enzymes.
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.

In order to improve the efficiency and convenience of enzyme production and recovery, it is known that enzymes can be produced by microbial fermentation in a biphasic nutrient medium containing a mixture of polyethylene glycol and dextran. 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 Microvial Technology, Volume 7, 7333-338
(1985) [Enzyme and Microb. Technol.
Vol.7333-338 (1985)]. The partition coefficient of alpha amylase in that system was, for example, 4 at most.

A variation of the above method is described in US Pat. No. 4,508,825. Accordingly, the extracellular enzyme-containing supernatant is separated from the cells and the cell-free supernatant is mixed with polyethylene glycol and a cationic epihalohydrin/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.

A two-phase enzyme recovery method was also used for intracellular enzymes. U.S. Pat. No. 4,144,130 discloses (1) high molecular weight unsubstituted or substituted polyalcohols, polyethers,
(2) a mixture of polyester, polyvinylpyrrolidone, or a polysaccharide with an inorganic salt; or (2) a mixture of at least two of the above-mentioned high molecular weight polymers to recover intracellular enzymes from an aqueous solution in which they are released from cells. It is listed. 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 cell debris and other fermentation products will go to the lower salt-containing layer. The partition coefficients of various enzymes recovered in the glycol layer were approximately 0.3 when 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, in a method for recovering extracellular enzymes from whole fermentation broth, whole fermentation broth containing microbial cells and extracellular enzymes is provided with: (a) polyethylene glycol, an amine derivative of polyethylene glycol, a carboxylate of polyethylene glycol; derivatives selected from the group consisting of polypropylene glycol, amine derivatives of polypropylene glycol, carboxylate derivatives of polypropylene glycol, poly(ethylene glycol) esters, polyethylene imine, trimethylamino-polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and mixtures thereof. adding a mixture of a polymer and (b) an inorganic salt, separating the total fermentation broth polymer monosalt mixture into an enzyme-rich polymer phase and an enzyme-poor salt phase, and producing a cross-enriched product. A method is provided, characterized in that:

Whole fermentation broths containing extracellular enzymes useful as starting materials for the methods of the invention are well known in the art. For example, Bacillus licheniformis (ATCC14580; DSM13; NCIB9375;
NCTC10341), Bacillus amyloliquefaciens (ATCC23350; DSM7), Bacillus subbutilis (ATCC6051; DSM10; NCIB3610;
NCTC3610) and Mukhormiehei (Kekabi)
are known to produce extracellular enzymes such as proteases, amylases and microbial rennets when grown in suitable nutrient media. 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 and cell fragments.

The entire fermentation broth is then mixed with the polymer and inorganic salts to form a two-phase system. The desired extracellular enzymes will collect in the polymer phase and cell debris will collect in the salt phase.

Suitable polymers include polyethylene glycol, amine derivatives of polyethylene glycol, carboxylate derivatives of polyethylene glycol, polypropylene glycol, amine derivatives of polypropylene glycol, carboxylate derivatives of polypropylene glycol, poly(ethylene glycol) esters, polyethylene imine, trimethyl amines, Included are minnow polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and mixtures thereof. A preferred polymer is polyethylene glycol. Any form of polyethylene glycol that is soluble in the aqueous whole fermentation broth 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, citrates, chlorides, and phosphates. ) and mixtures thereof. Preferred salts are sodium chloride and sodium sulfate.

The total mixture of polymers and inorganic salts contains 64-90% total fermentation broth, 1-15% polymer and 8-35% inorganic salts, where the percentages are by weight based on the total weight of the mixture. is added to the whole fermentation broth in an amount to form . The whole mixture is the whole fermentation broth73
-79%, 3-4% polymer and 15-24% inorganic salts.

Two phases will form after adding the polymer and salt. The polymer phase will normally be on top of the salt 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 can then be separated to recover the enzyme by standard liquid/liquid separation methods, such as siphoning and decanting. However, it is preferred to use centrifugation to separate the phases. A useful separation device is a continuous solid-bowl centrifuge. To achieve the desired concentration of enzyme in the final separated polymer phase, the enzyme-rich polymer to enzyme-poor salt phase volume ratio is preferably between 0.12 and 0.15.

For most efficient recovery of extracellular enzymes, the centrifuge should be operated such that nothing of the polymer phase is entrained in the salt phase, which should be discarded. The resulting collected polymer phase may have some entrained salt 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 with the salt phase and only a small amount of the salt phase is entrained with the polymer 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 total fermentation broth contains 1.5-5.0% calcium chloride dihydrate, 0.1-5% monobasic sodium phosphate or monobasic potassium phosphate.
a mixture of 1.2% and 0-0.6% calcium hydroxide,
However, the percentages are preconditioned on a weight/volume basis, calculated by the weight of the additive relative 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 ml of calcium chloride dihydrate.
-5.0% and monobasic sodium phosphate or monobasic potassium 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 calcium hydroxide 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 divided 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 alkaline protease production was prepared by feeding a 6000 gallon (22712 l) fermenter with the following ingredients: Wheat Gluten 1500 lb (681 Kg) Sodium Citrate 165 lb (74.9 Kg) Calcium Chloride Dihydrate Salt 165lb (74.9Kg) Corn Starch 5000lb (2270Kg) Soy Meal 2500lb (1135Kg) Thermostable Alpha-Amylase 5lb (2.27Kg) Monobasic Sodium Phosphate 400lb (181.6Kg) Dibasic Sodium Phosphate 400lb (181.6Kg) Antifoam 16.5 gallons (62.5l) Total with water 6000 gallons (22712l) The medium was then inoculated with live cells of Bacillus licheniformis and fermented for 36 hours at 36°C. 70% (w/v) in the total fermentation broth obtained
Calcium chloride dihydrate solution 214 gallons (811l)
and 300 lb (136 Kg) of monobasic sodium phosphate were added. This results in 2.5% calcium chloride dihydrate based on the weight of each additive and the volume of fermented beer.
(w/v) and monosodium phosphate 0.6% (w/v)
A mixture containing v) is obtained. The PH of the mixture was maintained at 6.8-7.6 while mixing by adding sodium hydroxide in the additive. After the mixing was completed, the pH was adjusted to 7.4-7.5 by adding sodium hydroxide to achieve flocculation. The pH was then adjusted to 6.4-6.6 by addition of 50% by weight aqueous acetic acid solution. Add 11,600 lb of sodium chloride to the resulting mixture at 25-27°C.
(5260Kg) was added and the mixture was stirred for 1 hour to dissolve all the sodium chloride. Then 3100lb of polyethylene glycol with a molecular weight of 3350
(1400 Kg) and 4250 lb (1930 Kg) of sodium sulfate were added and the total broth temperature was raised to 30-30°C. The pH of all beer was reduced to 6.0− by addition of acetic acid solution.
6.2 and the whole mixture was stirred for 2 hours.
The total mixture contains 15% (w/w) sodium chloride, 4% (w/w) polyethylene glycol, 5.5% (w/w) sodium sulfate and 75.5% total fermentation broth.
(w/w). This percentage was calculated by the weight of the additive relative to the total weight of the fermented beer and additive mixture. 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 capacity to lower phase capacity was 0.15. 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 salt phase. The glycol phase contained 90-92% of the total enzyme activity of the fermentation broth. The glycol phase also contained about 5-20% by volume of salt phase congeners. The glycol phase collected as above is then added to the glycol phase at a concentration of 2% by volume.
A second separation was carried out using a similar apparatus configured to allow entrainment of less salt phase.
The salt phase was discarded and it contained no glycol phase. The glycol phase collected as described 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 2 hours of gentle stirring at 10° C., 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.

Example 2 The method of Example 1 was repeated through the steps of adding calcium chloride dihydrate and dibasic sodium phosphate. The resulting total fermentation broth-additive mixture was then mixed with 2130 lb (970 Kg) of polyethylene glycol having a molecular weight of 3350 and 10660 lb of sodium sulfate.
(4850Kg) and bring the total broth temperature to 30−32
The temperature was raised to ℃. Then polyethylene glycol 3% (w/w), sodium sulfate 15% (w/w)
and the total mixture containing 82% (w/w) of total fermented beer 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 capacity to lower phase capacity was 0.12. The concentration of protease activity in the upper phase divided by the concentration of protease activity in the lower phase is
It yielded 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 centrifuge as described in Example 1 to produce a glycol phase containing amorphous solid protease and less than 2% by volume of salt phase entrainment. 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 l) fermenter: Calcium chloride dihydrate 22.5 lb (10.2 Kg) 1st Potassium phosphate 300lb (136.2Kg) Dibasic potassium phosphate 700lb (317.8Kg) Ammonium sulfate 250lb (113.5Kg) Sodium citrate 100lb (45.4Kg) (Corn soaking liquid) 1000lb (454Kg) Lactose 7000lb (3178Kg) (Cotton seed flour ) 1500 lb (681 Kg) Soy flour 2000 lb (908 Kg) Antifoam 165 gallons (625 l) Total volume with water 6000 gallons (22712 l) This medium was then inoculated with live cells of Bacillus licheniformis and periodically treated with sodium hydroxide. Fermentation was carried out at 40° C. for 108-110 hours while maintaining the pH at 7.05-7.15 by addition. 343 gallons (1300 l) of a 70% (w/v) calcium chloride dihydrate solution in the resulting total fermentation broth, 10% (w/v)
240 gallons (980 l) of aqueous calcium hydroxide solution and 60 lb (27 Kg) of monobasic potassium phosphate were added. This resulted in 4% (w/v) calcium chloride dihydrate, based on the weight of each additive and the volume of fermentation broth;
A mixture is obtained containing 0.4% (w/v) of calcium hydroxide and 0.12% (w/v) of monobasic potassium phosphate. The PH of the mixture was maintained at 7.6-8.4 while mixing by adding sodium hydroxide in the additive. After mixing was completed, the pH was adjusted to 8.4-8.6 by adding sodium hydroxide. The resulting mixture contains 2600lb (1180Kg) of polyethylene glycol with a molecular weight of 3350 and 7420lb of sodium chloride.
(3370Kg) was added at 25-27°C and the mixture was stirred for at least 1 hour. An amount of 5940 lb (2700 Kg) of sodium sulfate was then added and the fermentation broth mixture was
Heated to 32°C. The PH was adjusted to 7.9-8.1 by addition of sodium hydroxide and the whole mixture was stirred for at least 2 hours. The whole mixture is sodium chloride 10
% (w/w), polyethylene glycol 3.5%
(w/w), sodium sulfate 8% (w/w) and total fermented beer 78.5% (w/w). After mixing, the entire mixture was separated into an upper polyethylene glycol phase and a lower sodium chloride monosulfate-cell debris phase. The phase ratio of upper phase capacity to lower phase capacity is
It was 0.12. The concentration of amylase activity in the upper phase divided by the concentration of amylase activity in the lower phase resulted in a partition coefficient of 50-70. A continuous solid ball centrifuge is then used as described in Example 1 to recover the amylase-rich glycol phase, which is then purified as described in Example 1 to produce amylase-rich glycol. produced something.

Example 4 A nutrient medium suitable for the production of alpha amylase was prepared by adding the following ingredients to a 6000 gallon (22712 l) fermenter: Calcium carbonate 530 lb (240.6 Kg) Fish meal 750 lb (340.5 Kg) (ground) Soy flour) 2800lb (1271Kg) Corn soaking liquid 1500lb (681Kg) Lactose 7000lb (3178Kg) Ammonium diphosphate 130lb (59Kg) Antifoam 40 gallons (151.4L) Total volume with water 6000 gallons (22712L) Then this medium were inoculated with live cells of Bacillus amyloliquefaciens and fermented for 60 hours at 34°C, maintaining the pH at 7.05-7.15 by periodic addition of sodium hydroxide. To the resulting total fermentation broth was added 257 gallons (973 l) of a 70% (w/v) calcium chloride dihydrate aqueous solution, 180 gallons (681 l) of a 10% (w/v) aqueous calcium hydroxide solution, and 300 lb (136 kg) of monobasic potassium phosphate. ) was added. This resulted in 3% (w/v) calcium chloride dihydrate based on the weight of each additive and the volume of fermentation broth;
A mixture is obtained containing 0.3% (w/v) calcium hydroxide and 0.6% (w/v) monobasic potassium phosphate. The PH of the mixture was maintained at 6.8-7.6 while mixing by adding sodium hydroxide in the additive. After mixing was completed, the pH was adjusted to 7.4-7.6 by adding sodium hydroxide. Polyethylene glycol with a molecular weight of 3350 in the resulting mixture
2510lb (1140Kg) and sodium chloride 7960lb (3600
Kg) was added at 25-27°C and the mixture was stirred for at least 1 hour. An amount of 10,700 lb (4,870 Kg) of sodium sulfate was then added and the fermentation broth mixture was heated to 30-32°C. The PH 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% (w/w) sodium chloride, polyethylene glycol
3.15% (w/w), sodium sulfate 13.5% (w/w)
w) and 73.35% (w/w) of total fermented beer. After mixing, the entire mixture was separated into two phases as described in Example 3 with a phase ratio of 0.12 and a partition coefficient of 50-70. The phase was treated as described in Example 3 to produce an amylase-enriched glycol product.

Claims (1)

[Scope of Claims] 1. A method for recovering extracellular enzymes from whole fermentation broth, wherein the whole fermentation broth containing microbial cells and extracellular enzymes contains (a) polyethylene glycol, an amine derivative of polyethylene glycol, a carboxylic acid derivative of polyethylene glycol; selected from the group consisting of polypropylene glycol, amine derivatives of polypropylene glycol, carboxylate derivatives of polypropylene glycol, poly(ethylene glycol) esters, polyethylene imine, trimethylamino-polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and mixtures thereof. and (b) an inorganic salt, separating the total fermentation broth polymer monosalt mixture into an enzyme-rich polymer phase and an enzyme-poor salt phase, and producing an enzyme-rich product. A method characterized by collecting things. 2. Inorganic salts are selected from the group of compounds whose cations are sodium, potassium, magnesium and ammonium and whose anions are sulphate, carbonate, citrate, chloride, phosphate and mixtures thereof. The method according to claim 1, wherein the method comprises: 3 The total mixture of total fermentation broth, polymer and inorganic salts contains 64-90% total fermentation broth, 1-15% polymer and 8-35% inorganic salts, where percentages are based on the total weight of the mixture. 2. The method according to claim 1, wherein the method is based on the weight. 4 The total mixture of total fermentation broth, polymer and inorganic salts contains 73-79% total fermentation broth, 3-4% polymer and 15-24% inorganic salts, where percentages are based on the total weight of the mixture. 2. The method according to claim 1, wherein the method is based on the weight. 5. The method of claim 4, wherein the polymer is polyethylene glycol and the inorganic salt is a mixture of sodium chloride and sodium sulfate. 6. The method of claim 4, wherein the polymer is polyethylene glycol and the salt is sodium sulfate. 7. The method of claim 4, wherein the polymer is polyethylene glycol having a molecular weight of about 3350. 8. A mixture of 1.5-5.0% calcium chloride dihydrate, 0.1-1.2% monobasic sodium phosphate or monopotassium phosphate and 0-0.6% calcium hydroxide before adding the mixture of polymer and inorganic salt, where 5. The method of claim 4, wherein the percentage is on a weight/volume basis calculated based on the weight of the additive relative to the volume of the fermentation broth. 9. Convert the whole fermentation broth to calcium chloride dihydrate 1.5−
9. The method of claim 8, wherein the mixture is contacted with a mixture of 5.0% and 0.2-1.2% of monosodium phosphate or monopotassium phosphate. 10 Add 1.5% of the whole fermentation broth to calcium chloride dihydrate
-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 Claim 4 in which the volume ratio of enzyme-rich polymer to enzyme-poor salt phase is 0.12-0.15.
The method described in section. 12. The method of claim 4, wherein the resulting phases are separated by continuous solid ball centrifugation. 13 To the whole fermentation broth containing Bacillus licheniformis cells and extracellular alkaline protease obtained by fermenting a suitable strain of Bacillus licheniformis in a suitable nutrient medium, 2.5% calcium chloride dihydrate and sodium monophosphate or Potassium monophosphate 0.6%, where the percentages are weight/volume % calculated based on the weight of additives relative to the volume of fermentation broth, is added, and then to the above mixture 3% polyethylene glycol and 15% sodium sulfate, where The percentages are by weight based on the total mixture weight of fermentation broth and additives, are added to form an alkaline protease-rich polyethylene glycol phase and an alkaline protease-poor sodium sulfate phase, and these two phases are 2. The method of claim 1, comprising separating the alkaline protease-enriched product. 14 After adding calcium chloride dihydrate and monobasic sodium phosphate or monobasic potassium phosphate to the whole fermentation broth, the mixture was reduced to 15% sodium chloride, 4% polyethylene glycol and 5.5% sodium sulfate.
14. The method of claim 13, wherein percentages are by weight based on the weight of the total mixture of fermentation broth and additives. 15 Calcium hydroxide 0.4 to the total fermentation broth containing Bacillus licheniformis cells and extracellular alpha amylase obtained by fermenting a suitable strain of Bacillus licheniformis in a suitable nutrient medium.
% and monosodium phosphate or monopotassium phosphate 0.12%, where the percentage is weight/volume % calculated based on the weight of the additive to the volume of fermentation broth, and then to the above mixture polyethylene glycol 3.5%, sodium chloride 10% and sodium sulfate 8%, where the percentages are by weight based on the total mixture weight of fermentation broth and additives, to the amylase-rich polyethylene glycol phase and amylase. forming a lean sodium sulfate-sodium sulfate phase;
2. The method of claim 1, further comprising separating the two phases to recover the alkaline protease-enriched product. 16 Total fermentation broth containing Bacillus amyloliquefaciens cells and extracellular alpha-amylase obtained by fermenting a suitable strain of Bacillus amyloliquefaciens in a suitable nutrient medium was supplemented with 3% calcium chloride dihydrate and water. calcium oxide
0.3% and monosodium phosphate or monopotassium phosphate 0.6%, where the percentages are weight/volume % calculated based on the weight of the additive with respect to the volume of the fermentation broth, and then to the above mixture polyethylene Glycol 3.15%, Sodium Chloride
10% and sodium sulfate 13.5%, where the percentages are by weight based on the total mixture weight of fermentation broth and additives, plus an amylase-rich polyethylene glycol phase and an amylase-poor sodium chloride-sodium sulfate phase. 2. The method of claim 1, comprising forming a phase and separating the two phases to recover the amylase-enriched product.