JP3152826B2 - Method for producing enzyme-containing composition - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing an enzyme-containing composition comprising an enzyme and a water-soluble polymer. The present invention particularly relates to a method for producing an enzyme-containing composition which can be advantageously used as a raw material for producing an enzyme-containing granule used in combination with a detergent.

[0002]

2. Description of the Related Art In recent years, it has been common to mix enzymes such as proteases and cellulases with powder detergents for washing clothes in order to improve the washing action by decomposing and removing stains on the clothes. It has become. However,
Enzymes may be inactivated by contact with other components of the detergent or may irritate the handler, so they are usually granulated with a water-soluble polymer such as polyethylene glycol and the granulated product. It is often added to detergents. On the other hand, an ordinary enzyme is obtained by culturing a microorganism (cells) that produces the enzyme and separating and removing the cells from the culture solution by a method such as filtration.

[0003]

SUMMARY OF THE INVENTION The present invention provides a novel method for producing an enzyme-containing composition comprising an enzyme and a water-soluble polymer. That is, the conventionally known filtration operation for separating an enzyme from a culture solution containing bacterial cells is not efficient, and the produced enzyme adheres to a filtration base material, so that the finally obtained enzyme is used. There is a problem that the yield of the compound decreases. Therefore, the present invention provides a high-efficiency separation method for separating an enzyme from a culture solution containing bacterial cells, and a method capable of recovering the produced enzyme in a high yield. With the goal. In particular, it is another object of the present invention to provide a method for producing an enzyme-containing composition which can be advantageously used as a raw material for producing an enzyme-containing granule used in combination with a detergent.

[0004]

SUMMARY OF THE INVENTION The present invention relates to a method for producing a two-phase partitioning system by combining an enzyme produced from cells with at least one kind of water-soluble polymer and the water-soluble polymer. An aqueous two-phase partitioning system formed from a substance and an aqueous two-phase partitioning system in the presence of a cationic surfactant, wherein an enzyme is prioritized over a phase to which the water-soluble polymer is distributed. And separating the enzyme together with the water-soluble polymer, followed by drying, to obtain an enzyme-containing composition.

The cationic surfactant used in the present invention is preferably a quaternary amine salt.

Examples of the enzyme used in the present invention include an enzyme having an action of decomposing dirt attached to clothes, a protease which is known to be used in combination with a detergent,
Enzymes such as esterase and carbohydrase can be mentioned. The following are specific examples of these enzymes. 1) Proteases pepsin, trypsin, chymotrypsin, collagenase, keratinase, elastase, splitisin, papain, aminopeptidase, carboxypeptidase. 2) Esterase Gastric lipase, pancreatic lipase, plant lipase, phospholipase, cholinesterase, phosphotase 3) Carbohydrase. Cellulase, maltase, saccharase, amylase,
Pectinase, α- and β-glycosinase. The enzymes used in the present invention are not limited to those described above, and any enzymes produced from bacterial cells that can be used in industrial products as granules with a water-soluble polymer. Any enzyme may be used.

[0007] The water-soluble polymer used in the present invention is an aqueous polymer utilizing at least one kind of water-soluble polymer and another substance which forms a two-phase distribution system in combination with the water-soluble polymer. It is a water-soluble polymer that can be used when performing a phase separation method. The aqueous two-layer separation method (also referred to as an aqueous two-phase partition method or simply a two-phase partition method) and a water-soluble polymer are already known.

That is, as a method for separating a target substance from a mixture of two or more kinds of substances, an aqueous solvent or an aqueous solution and an organic solvent are used, and the difference between the partition coefficients of each substance to those solvents is utilized. A method of separating (or extracting) is generally used. However, this method using an organic solvent often cannot be used particularly for separating biopolymers such as proteins. That is, many biopolymers are denatured by contact with an organic solvent. Therefore, a biopolymer such as a protein is prepared by using water alone as a partitioning solvent, and using an aqueous polymer that utilizes a water-soluble polymer and another substance that is combined with the water-soluble polymer to form a two-phase partitioning system. Phase separation methods have already been proposed and are in practical use at the laboratory level. Examples of the distribution system include a combination of two kinds of non-electrolyte polymers, a combination of a non-electrolyte polymer and an electrolytic polymer, a combination of two kinds of electrolytic polymers, and a combination of a polymer and a low-molecular substance. Combinations and the like are known, and various types are used depending on the target object to be separated. Specific examples of the combination of the partitioning systems used for the aqueous two-phase partitioning system in the present invention include a combination of polyethylene glycol and dextran, a combination of polyethylene glycol and phosphate (such as sodium salt and potassium salt), and a combination of polyethylene glycol and citric acid. Combinations with acid salts (sodium salts, potassium salts, etc.), combinations with polyethylene glycol and sulfates (sodium salts, potassium salts, etc.), combinations with polyethylene glycol and sodium carboxymethylcellulose, combinations with polyethylene glycol and dextran sulfate , A combination of polyethylene glycol and polyvinyl alcohol, and the like, depending on the purpose. As the salts, other alkali metal salts, alkaline earth metal salts, halides, thiocyanates and the like can also be used. The water-soluble polymer used in the present invention is preferably polyethylene glycol.

That is, according to the present invention, by utilizing an aqueous two-phase separation method, an enzyme can be directly reacted with a water-soluble polymer from a culture of bacterial cells containing the enzyme without reducing its activity. By separating the mixture as a mixture and drying the mixture according to a known method, an enzyme-containing composition can be obtained at a high yield. Since this enzyme-containing composition is in the form of a composition with a polymer substance such as polyethylene glycol, it has high safety and stability, and can be used as it is in the production of enzyme granules to be added to detergents. Alternatively, it can be used for applications such as enzyme preparations for treating leather. Or,
After filtration of the cells from the culture of the cells containing the enzyme by a known method, the filtrate in the filtrate is subjected to an aqueous two-layer separation method to convert the colored substance in the filtrate into a water-soluble polymer phase to which the enzyme is transferred. Can be transferred to another phase, thus achieving purification of the enzyme.

In carrying out the aqueous two-phase separation method of the present invention, the presence of a cationic surfactant in the two-phase partitioning system facilitates the preferential transfer of the enzyme to the phase on the side of the water-soluble polymer. And the transfer of the coloring component to the other phase is promoted. Cationic surfactants that can be used in the two-phase distribution system used in the present invention include the following categories of cationic surfactants, and specific examples. Quaternary amine salts such as hexadecyltrimethylammonium salt, tetradecyltrimethylammonium salt, dodecyltrimethylammonium salt, hexadecylpyridinium salt, primary amine salts such as dodecylamine salt, octylamine salt, octadecylamine salt, and various other types Secondary and tertiary amine salts. In the present invention, it is preferable that the cationic surfactant is usually added in an amount of 0.001 to 5% by weight based on a component (a mixture containing water) forming an aqueous two-phase distribution system.

In the aqueous two-phase separation method of the present invention, generally, a water-soluble polymer such as polyethylene glycol and an enzyme preferentially gather in the upper layer to form a phase, and in the lower layer, an aqueous solution such as citric acid is formed. A compound and a coloring component corresponding to each other in the phase distribution system collect to form a phase. When the culture of the cells is directly subjected to the aqueous two-phase separation method, the cells move to the layer on the side opposite to the enzyme, that is, the lower layer. At this time, when a cationic surfactant coexists, migration and separation of these components are performed more quickly or more preferentially.

After the enzyme and the water-soluble polymer are transferred to one side (usually the upper layer) by the above-described aqueous two-layer separation method, the upper layer and the lower layer are separated, and the enzyme and the water-soluble polymer are separated. The target enzyme-containing composition can be obtained by once pulverizing the aqueous phase containing the compound by freeze-drying or the like.
In order to make the above-mentioned enzyme-containing composition into an enzyme granule to be used together with a detergent, for example, a granulation operation described in JP-A-62-257990 can be used. That is, a composition containing the enzyme and a water-soluble polymer (usually in a powder state) may be added with a hydrophilic polymer, if necessary, an enzyme stabilizer such as calcium chloride, sodium sulfate, or the like. After adding a powdering aid such as sodium chloride, this is subjected to a known granulation operation. Examples of granulation methods include:
Granulation methods such as stirring and rolling can be given.

[0013]

EXAMPLES [Example 1 (Reference Example) ] Bacillus sp. KSM-K1 producing extracellular protease
6 strains (described in JP-A-4-349882)
Was inoculated into a 3 liter medium (having the following medium composition) in a 5 liter jar fermenter, and cultured at 30 ° C. for 48 hours to obtain a culture solution containing the produced protease. This culture solution is passed through a microfiltration membrane (pore size: 0.4
The bacterial cells were removed by filtration using 5 μm (manufactured by Amicon Co., Ltd.), and the obtained protease solution was lyophilized to obtain an enzyme powder (colored dark brown).

Medium composition Glucose 2.0% by weight Polypeptone S 1.0% by weight Yeast extract (Difco) 0.05% by weight Monopotassium phosphate 0.1% by weight 0.02% by weight of magnesium sulfate 7H ₂ O 0.02% by weight of sodium carbonate 1.0 wt% 50 g of the above enzyme powder was dissolved in 1 liter of distilled water, and the enzyme activity was 2.05 P.L. by the casein method described below.
U. / ML of an enzyme aqueous solution was prepared.

[0015]Protease enzyme activity assay (casein
Law) Activity measurement method using casein substrate: containing 1% by weight of casein
50 mM boric acid-NaOH buffer (pH 10.0) 1
Mix with 0.1 mL of protease containing solution and mix
After reacting at 0 ° C for 10 minutes, add a reaction stop solution
(0.123M trichloroacetic acid-0.246M sodium acetate)
(Li-0.369M acetic acid) at 30 ° C.
Left for 0 minutes. The left reaction solution is then passed through a filter paper
No. 2), and the protein in the filtrate is filtered.
White degradation products are measured by an improved method of the Foreign Lorry method
did. In addition, 1P. U. Is 1 under the above reaction conditions.
The amount of enzyme that releases 1 mmol of tyrosine per minute
Was.

Separately, a separating funnel is prepared, and the above-mentioned enzyme aqueous solution is added thereto, and then a light liquid side phase forming substance (polyethylene glycol (PEG) 6000) for aqueous two-layer separation is prepared.
And a heavy liquid side phase forming substance (dipotassium phosphate) were added in powder form at 5% by weight and 15% by weight, respectively (based on the weight of the enzyme aqueous solution in the separating funnel). The mixture in the separating funnel was thoroughly stirred to dissolve the powder in the mixture, and then allowed to stand at 15 ° C. for 2 hours. It was confirmed that the degree of coloring was clearly higher than the degree of coloring. When only the lower layer having a high degree of coloring was discharged from the separating funnel, the liquid volume of the lower layer was 850 mL, and the liquid volume of the upper layer was 150 mL.
A large amount of polyethylene glycol was contained. Also,
The protease activity of the lower layer was 0.33P. U. And
The upper layer is 11.62P. U. Met. A powdery enzyme-containing composition containing protease and polyethylene glycol was obtained from the upper layer aqueous solution containing protease and polyethylene glycol by freeze-drying. 100 g of the above-mentioned enzyme-containing composition powder was added to sodium chloride (average particle size of 6
10 μm, 11% by weight of particles having a particle diameter of 500 μm or less,
9% by weight of particles having a particle diameter of 700 μm or more) 500
g, 350 g of sodium sulfate and 50 g of titanium oxide, and a high-speed mixer (Fukae Kogyo KK, F
(S-10 type) to obtain an enzyme-containing granulated product.

Example 2 (Reference Example) Extracellular cellulase-producing Bacillus sp. KSM-635
The strain (FERM P-8872) was inoculated into 2 liters of a medium (consisting of the following medium composition) in a 5 liter jar fermenter, and cultured at 30 ° C. for 72 hours.
A culture solution containing the produced cellulase was obtained. This culture solution is filtered through a microfiltration membrane (pore size: 0.45 μm, manufactured by Amicon Co., Ltd.) to remove the cells, and the resulting dark brown cellulase solution (activity value according to the cellulase activity measurement method described below) : About 3000 units / L).

Medium composition Meat extract 1.5 wt% Yeast extract 0.5 wt% Carboxymethylcellulose 1.0 wt% Monopotassium phosphate 0.1 wt% Sodium carbonate 0.75 wt%

Cellulase enzyme activity assay (CMC activity assay
Standard method) 2.5% carboxymethylcellulose (CMC) 0.2
mL, 0.5 M glycine buffer (pH 9.0) 0.1 m
0.1 mL of the enzyme solution is added to a substrate solution consisting of L and 0.1 mL of deionized water, and reacted at 40 ° C. for 20 minutes. After the reaction, the reducing sugar was quantified using the DNS (3,5-dinitrosalicylic acid) method. That is, 1 mL of the DNS reagent is added to 0.5 mL of the reaction solution, and
Heat at 00 ° C. to develop color. Then, after cooling,
Dilute by adding 4.5 mL of deionized water. Under these conditions, the amount of the enzyme that releases 1 μmol of reducing sugar in terms of glucose in one minute is defined as one unit.

Separately, a separating funnel is prepared, and the above-mentioned cellulase solution is added thereto, and then a light liquid side phase forming substance (polyethylene glycol (PEG) 6000) for aqueous two-layer separation is prepared.
And a heavy liquid side phase forming substance (dextran, molecular weight 100,000-
200,000) in powder form at 4% by weight and 14% by weight, respectively.
(Based on the weight of the cellulase solution in the separating funnel). Further, dipotassium phosphate was added in powder form as an upper layer transfer promoter of cellulase so as to be 8% by weight, and then the mixture in the separating funnel was thoroughly stirred to dissolve the powder in the mixture. When left at 15 ° C. for 2 hours, it was confirmed that the coloring components migrated preferentially to the lower layer, and the coloring of the lower layer was clearly higher than that of the upper layer. When only the lower layer having a high degree of coloring was discharged from the separating funnel, the liquid volume of the lower layer was 1800 mL, the liquid volume of the upper layer was 200 mL, and a large amount of polyethylene glycol was contained. The cellulase activity was 770 in the lower layer.
Unit / L, and the upper layer was 23000 Unit / L.
A powdery enzyme-containing composition containing cellulase and polyethylene glycol was obtained from the aqueous solution containing cellulase and polyethylene glycol in the upper layer by freeze-drying. To 100 g of the above-mentioned enzyme-containing composition powder, sodium chloride (average particle diameter: 610 μm, particles having a particle diameter of 500 μm or less are 11% by weight, and particles having a particle diameter of 700 μm or more are 9% by weight.
), 350 g of sodium sulfate and 50 g of titanium oxide were added and granulated using a high-speed mixer to obtain an enzyme-containing granulated product.

Example 3 Extracellular protease-producing Bacillus sp. Strain KSM-K16 (JP-A-4-3498)
No. 82) was inoculated into 50 mL of a liquid medium (consisting of the medium composition described in Example 1) in a volume of 500 mL = Sakaguchi flask, and cultured at 30 ° C. for 48 hours. Obtained. Separately, a separating funnel is prepared, and a light liquid side phase forming substance (polyethylene glycol (PEG) 4000) and a heavy liquid side phase forming substance (sodium citrate dihydrate) for aqueous two-phase separation are prepared. And 14% by weight in powder form. Then, a cationic surfactant (hexadecyltrimethylammonium bromide, HD
AB) was added in an amount of 0.5% by weight. The above culture solution was added to the separating funnel, and the mixture was stirred and mixed to dissolve the powder in the test tube to obtain a mixed solution of 500 mL. The separating funnel was allowed to stand at 15 ° C. for 1 hour to separate the cells into a lower layer, and then the PEG-protease solution in the upper layer was separated. 250mL of upper layer liquid taken out
And the volume of the lower layer liquid was 250 mL. The removed upper layer liquid contains about 35% by weight of PEG4000, 5% by weight of citrate, and 0.1% by weight of HDAB, and its coloring degree is clearly lower than that of the lower layer liquid. Was. The obtained aqueous solution containing the protease and polyethylene glycol in the upper layer was freeze-dried to obtain a powdery enzyme-containing composition. Sodium chloride (11% by weight of particles having an average particle diameter of 610 μm, particle diameter of 500 μm or less, and 9% of particles having a particle diameter of
% By weight), sodium sulfate and titanium oxide, and granulated using a high speed mixer to obtain an enzyme-containing granulated product.

Example 4 After culturing the strain of the genus Bacillus, the turbidity of the culture was adjusted to about 50 using an ultrafiltration membrane. Polyethylene glycol (PEG) 4000 and sodium phosphate were respectively added to the thus prepared cell turbidity-adjusted culture solution so as to be 14% by weight, and then hexadecyltrimethylammonium bromide was added to 0.5% by weight. Was added. Next, the sample solution was allowed to stand at 25 ° C. for 5 hours, and the turbidity of each of the separated upper and lower layers was measured. The turbidity of the upper layer was almost 0, and most of the cells migrated to the lower layer. It was confirmed that it was done. The coloring degree of the upper layer was clearly lower than that of the lower layer liquid. A powdery enzyme-containing composition containing protease and polyethylene glycol was obtained from the resulting aqueous solution containing protease and polyethylene glycol by freeze-drying. Sodium chloride (average particle diameter 610 μm, particle diameter 5
11% by weight of particles having a particle size of 00 μm or less and 9% by weight of particles having a particle size of 700 μm or more), sodium sulfate and titanium oxide, and granulate using a high-speed mixer to obtain an enzyme-containing granulated product. Obtained.

[0023]

The cationic surfactant of the present invention is present
By utilizing the method for producing an enzyme-containing composition by the aqueous two-phase separation method , the enzyme separated from the cells can be recovered in a high yield as a composition with a water-soluble polymer.
The enzyme-containing composition is particularly suitable for producing granules suitable for incorporation in detergents. Further, the enzyme-containing composition obtained by the present invention itself contains a water-soluble polymer substance, and is therefore useful as an enzyme preparation for treating leather.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kazuichi Kuroda 321-3 Nagashima, Sahara City, Chiba Prefecture (72) Inventor Shigeru Kawamura 1-8762-23, Doaihoncho, Hazaki-cho, Kashima-gun, Ibaraki Prefecture (56) References JP Hei 2-150280 (JP, A) JP-A-62-61922 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 9/96 C12N 9/00 BIOSIS (DIALOG) WPI ( DIALOG)

Claims (2)

(57) [Claims] Claims: 1. An enzyme produced from bacterial cells is mixed with at least one kind of water-soluble polymer and another substance which forms a two-phase partitioning system in combination with the water-soluble polymer. Phase distribution system with cationic surfactant present
Utilizing the aqueous two-phase partitioning system , the enzyme is preferentially distributed to the phase to which the water-soluble polymer is distributed, and the enzyme is separated together with the water-soluble polymer and dried. A method for producing an enzyme-containing composition. 2. The method for producing an enzyme-containing composition according to claim 1, wherein the cationic surfactant is a quaternary amine salt.