JP2000166549A - Heat-stable enzyme and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a heat-stable enzyme capable of improving the heat-stability of lipoxidase for conducting its stable reaction at high temperatures, to provide an enzyme production method for producing a stable enzyme powder hardly reduced in enzyme activity by heat even in heat pulverization. SOLUTION: This heat-stable enzyme is obtained by adding iron salt and calcium salt to a lipoxidase with a metal content of <=0.5 wt.%, wherein a lipoxidase subjected to demineralization treatment is pref. used. The usages of the iron salt and calcium salt are 0.01 to 50 wt.% and 0.01 to 70 wt.% based on the enzyme, respectively. The other objective method for producing an enzyme power is conducted by drying a lipoxidase solution containing an iron salt and calcium salt by, pref., a spray drying method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the thermostability of lipolytic enzymes. More specifically, the present invention relates to a method for producing a stable enzyme while stabilizing the heat of the enzyme by adding a metal salt to the deionized enzyme solution.

[0002]

2. Description of the Related Art Enzymes can be catalyzed under mild conditions and have high substrate specificity compared with chemical reactions. Therefore, enzymes have recently been used in the fields of food industry, chemical industry, pharmaceutical industry, etc. The use range of is expanding. In general, enzymes are unstable, and particularly the stability of the enzyme in a solution is low. Therefore, as a method for concentrating and powdering the enzyme, a freeze-drying method, a vacuum-drying method, etc., to which heat is hardly applied, have been mainly used. However, these drying methods have problems in using them in the industrial field due to enormous capital investment for mass production and high running costs such as utility. In addition, among enzymes, when many fats and oils-related enzymes exceed about 35 ° C. in an aqueous solution, thermal denaturation starts to occur, and it becomes difficult to industrially produce enzymes with stable and high yield. Therefore, it is necessary to treat the enzyme solution in a low temperature range, and the concentration / pulverization method is limited to the above method. Therefore, in order to improve the thermal stability of the enzyme in an aqueous solution, attempts have been made to add a stabilizer or the like. For example, in an enzyme solution, proteins such as albumin and casein, amino acids such as sodium glutamate, reducing agents such as mercaptoethanol and cysteine, polyols such as glycerol, sucrose and sorbitol, and water-soluble polymer substances such as dextran are subjected to enzyme stabilization. Methods for adding as an agent have been generally studied. JP-A-6
284886 discloses adding a magnesium ion and a calcium ion to a lipoprotein lipase solution as a method for stabilizing an enzyme in a solution. It has been reported to add at least a stoichiometrically equivalent amount of divalent cation to the amount of lipase. However, in these methods, the thermal stability of the lipolytic enzyme in an aqueous solution is limited to about 37 ° C. to 80 ° C., and application to enzyme synthesis using a substrate having a high melting point has been difficult. In addition, the method of powdering an enzyme by spray drying, which is a typical drying method, has a drawback that the enzyme activity is reduced by spray drying because the enzyme is dried in a hot air stream after heat concentration.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a lipid-related enzyme capable of improving the thermal stability of an enzyme and performing a reaction at a high temperature.
An object of the present invention is to provide a production method capable of stably producing an enzyme powder without causing heat-induced deactivation.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, after deionizing a lipolytic enzyme solution, an iron salt and a calcium salt are added. The inventors have found that the thermal stability of the enzyme is dramatically improved, and completed the present invention. That is, the present invention relates to a thermostable enzyme comprising adding an iron salt and a calcium salt to a lipolytic enzyme having a metal content of 0.5% by weight or less. The lipolytic enzyme is preferably subjected to a deionization treatment. The iron salt is preferably 0.01 to 50% by weight based on the weight of the enzyme, and the calcium salt is preferably 0.01 to 70% by weight based on the weight of the enzyme. The present invention also relates to a method for producing an enzyme by spray-drying a lipolytic enzyme solution containing an iron salt and a calcium salt to obtain an enzyme powder.

[0005]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. The present invention provides a method for producing an enzyme powder in which enzyme deactivation by heat is unlikely to occur even in a method for heat stabilization of an enzyme by adding an iron salt and a calcium salt to a deionized lipolytic enzyme solution and powdering by a heat drying method. This is a method for producing a lipolytic enzyme that can produce

Although the lipolytic enzyme used in the present invention is not particularly limited, commercially available enzyme preparations, microbial culture solutions,
A plant extract, an animal cell extract, an animal cell extract, and the like can be used, and further, a culture solution, a concentrate of the extract, and the like can be used.

The lipolytic enzymes include lipases, phospholipases, esterases and the like. The lipases include lipoprotein lipase, monoacylglycerolipase, diacylglycerolipase, triacylglycerolipase, galactolipase and the like.
Phospholipases include lysophospholipase, phospholipases A1, A2, B, C, D and the like. Esterases include cholinesterase, cholesterol esterase, pectin esterase, tropine esterase, acetylcholinesterase, acetylesterase, carboxyesterase, allylesterase and the like.

The microorganism used in the present invention is not particularly limited, such as bacteria, yeasts, filamentous fungi, actinomycetes, etc.
Pseudomonas sp., Alcaligenes sp., Asrobacter sp.
bacter sp.), Staphylococcus
s sp.), Torulopsis sp., Escherichia sp., Mycotorula sp., Propionibacterium sp.
pionibacterum sp.), Chromobacterium (Chromo
bacterum sp.), Xanthomonas s
p.), Clostridium sp., Candida sp., Geotricum sp.
richum sp.), Saccharomycos sp.
psis sp.), Nocardia sp., Fusarium sp., Aspergillus (Asper)
gillus sp.), Penicillium sp.,
Genus Mucor sp., Rhizopus s
p), Phycomyces sp., Puccinia sp., Bacillus s
p.) and Streptomyces sp.

The medium for producing lipase is not particularly limited, but is preferably soybean powder, peptone,
Corn stap liquor, K ₂ HPO ₄ , (NH ₄ ) ₂ S
O ₄ and MgSO ₄ .7H ₂ O can be used. As for the addition amount, soybean flour is 0.1 to 20% by weight, preferably 1.
0 to 10.0% by weight. Peptone is 0.1 to 30% by weight, preferably 0.5 to 10% by weight. corn·
The stap liquor is 0.1 to 30% by weight, preferably 0.5 to 10.0% by weight. K ₂ HPO ₄ is 0.01
-20% by weight, preferably 0.1-5% by weight.
(NH _{4) 2} SO ₄ is 0.01 to 20% by weight, preferably 0.05 to 5 wt%. MgSO ₄ · 7H ₂ O is 0.
It is from 0.01 to 20% by weight, preferably from 0.05 to 5% by weight. Regarding the culture conditions, the culture temperature is 10 to 40C, preferably 20 to 35C. Ventilation rate is 0.1 to 2.0
VVM, preferably 0.1 to 1.0 VVM. The rotational speed of the stirring is 100 to 800 rpm, preferably 200 to 400 rpm. The pH is between 3.0 and 10.0, preferably between 4.0 and 10.0.
9.5.

The method for extracting the enzyme is not particularly limited.
However, in the case of exocrine enzymes, centrifuge the cells,
It is preferable to remove by filtration or the like. Centrifuge 200
20,000 xg, membrane filtration is MF membrane, filter
Pressure of 3.0kg / m ^TwoControl below
Is preferred. In the case of intracellular enzymes, a homogenizer,
Waring blender, ultrasonic crushing, French press,
Crush cells with a ball mill, etc.
It is preferable to remove cell residue. Homogenizer is 5
00 to 30,000 rpm, preferably 1,000 to 1
5,000 rpm. Waring blender is 500
5,000 rpm, 0.5-10 minutes, preferably 100
10,000 rpm, 1-5 minutes. Ultrasonic crushing is 1
５０50 KHz, preferably 10-20 KHz. Ball ball
Use small glass balls with a diameter of about 0.1 to 0.5 mm
Is preferred.

The metal of the present invention refers to a metal element in the periodic table of elements, and is a group I except for hydrogen, a group II except for hydrogen, a group III except for boron, a group IV except for carbon and silicon, a group VIII and V, VI, VII It is an element belonging to each subgroup a of the group.
Other examples include antimony, bismuth, and polonium. The deionization treatment specifically removes or reduces metal ions from the enzyme solution, and as a result, the metal content in the lipolytic enzyme is reduced. The type of metal to be removed may be any type, and the reduction ratio is a problem. That is, the metal remaining in the enzyme after the deionization treatment is 0.5% by weight or less, preferably 0.25% by weight.
Or less, and particularly preferably 0.05% by weight or less. If the metal is present in an amount exceeding 0.5% by weight, a thermostable enzyme cannot be obtained even when iron salts and calcium salts are added. The method of deionizing the enzyme is not particularly limited, but a method by molecular weight fractionation using a membrane such as a semipermeable membrane and an anisotropic membrane, a method of depositing a metal salt by EDTA, a method of molecular sieve chromatography And electrodialysis. Examples of the material of the semipermeable membrane include a cellophane membrane, a collodion membrane, and a cellulose acetate membrane. Also,
Examples of the material of the anisotropic film include a polymer electrolyte, polysaccharide, cellulose triacetate, cellulose acetate, cellulose nitrate, polyacrylonitrile,
Examples thereof include polyvinylidene fluoride polyamide and polyvinylidene fluoride. Examples of the membrane include a flat membrane and a hollow fiber membrane. The molecular weight cut off of the membrane is 3,000-100,
000, preferably 6,000 to 50,000.
In the case of concentration deionization using a membrane, the enzyme solution is buffered, distilled water, ion-exchanged water, tap water, etc., to a volume of 1 to 1,
Dilute 000 times, preferably 2 to 50 times. The diluted enzyme solution is concentrated by a membrane, and concentrated to 2 to 1,000 times the weight of the diluted enzyme solution. The membrane module inlet pressure during concentration is preferably 0.5 to 2.0 atm, and the outlet pressure is preferably 0.1 to 1.5 atm. Molecular sieve chromatography was performed on Sephadex G-25 as a gel,
Gel particle size medium or coarse is preferred. Further, it is preferable to adjust the amount of enzyme protein or the amount of gel so as to be about 30% by weight or less of the bed volume of the column. The ionic strength of the enzyme solution is preferably set to 0.02 or more so that the protein is not adsorbed to a dissociating group such as a carboxy group of a gel such as Sephadex. In the electrodialysis method, a method in which a cation exchange membrane and an anion exchange membrane are alternately stacked as a diaphragm is preferable. ED
The concentration of TA is 0.01 to 100 mM, preferably 0.1 to 100 mM.
1 to 50 mM.

The iron salts used for stabilizing enzymes include ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, iron nitrate, iron phosphate, iron oxalate, iron lactate, and fumaric acid. It is preferable to use one or more of iron acid and iron citrate. Calcium salts include calcium chloride, calcium sulfate, calcium nitrate, calcium acetate, calcium oxalate, calcium citrate, calcium ascorbate, calcium benzoate, calcium propionate,
It is preferable to use one or more of calcium salicylate, calcium stearate, calcium tartrate, calcium thiocyanate, calcium hydrogen phosphate, and calcium silicate.

As a method for adding an iron salt and a calcium salt to an enzyme, the enzyme is dissolved in water to form an enzyme solution, and the enzyme to be treated is first treated at 0 to 25 ° C., preferably 0 to 5 ° C. Is to add 0.01% iron salt to the weight of the enzyme.
To 50% by weight, preferably 0.05 to 30% by weight, and in the case of a calcium salt, the calcium salt is added to the enzyme at 0.01 to 70% by weight, preferably 0.05 to 70% by weight.
50% by weight is added, and the mixture is stirred with a stirrer for 1 minute to 48 hours, preferably 30 minutes to 10 hours. Further, iron salts and calcium salts can be added directly to the deionized enzyme solution.

The method of pulverizing the enzyme is not particularly limited, but usually, the enzyme solution is concentrated and then dried to obtain an enzyme powder. As the concentration method, all methods such as evaporator, flash evaporator, UF membrane concentration, MF membrane concentration, salting out with inorganic salts, precipitation method with solvent, adsorption method with ion exchange cellulose, etc., and water absorption method with water absorbing gel are used. It is possible. Preferably, UF membrane concentration and evaporator are good. As the UF membrane concentration module, the molecular weight cut-off is 3,000 to 100,000, preferably 6,000.
It is preferable to use a polyacrylonitrile-based or polysulfone-based flat membrane or hollow fiber membrane of 00 to 50,000. The evaporator has a heating temperature of 90 ° C. or less and a reduced pressure of 40 cmHg or less, preferably a heating temperature of 80 ° C. or less and a reduced pressure of 60 cmHg or less.

Examples of the drying method include vacuum drying, freeze drying, spray drying and the like, and spray drying is preferred. Examples of the spray dryer include a nozzle countercurrent type, a disk countercurrent type, a nozzle cocurrent type, and a disk cocurrent type. A disk parallel flow type is preferable, and the atomizer rotation speed is 4,000.
0-20,000 rpm, heating at inlet temperature 100-200
And the outlet temperature is controlled at 60-100 ° C.

[0016]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto. The methods for evaluating the thermal stability and residual activity of the enzymes obtained in Examples and Comparative Examples are shown below. [Thermal stability] In the case of the enzyme powder, a 10% by weight solution is used.
Sampling was performed at the lapse of 20 minutes and 30 minutes, and the residual activity of each enzyme solution was measured, and compared with a sample that had not been heat-treated. [Residual activity] In the case of lipase, the residual activity
5 mL of 2% polyvinyl alcohol (225 mL) was placed in a container of a homogenizer (manufactured by Nippon Seiki), and 4 mL of phosphate buffer pH 7.0 was added to 5 mL of an olive oil emulsion emulsified at 15,000 rpm for 10 minutes while cooling at 10 ° C. with ice. After placing in a test tube, 1 mL of the enzyme solution was added, and the reaction was carried out at 37 ° C. for 10 minutes, the reaction was stopped with 2N HCl, and the hydrolyzed free fatty acid was converted to 2N with phenolphthalein as a color developing solution.
Titrate with NaOH. The residual activities were calculated by comparing the titration amounts. For phospholipase, 1 mL of lecithin emulsion, 0.05 mL of 0.1MCaCL2.2
H2O solution, 0.1 mL citrate buffer (pH 5.
5), 0.15 mL of 7.5% Triton X-100
0.1 mL of the enzyme solution was added to the solution, and the reaction was carried out at 37 ° C. for 10 minutes, and the reaction was stopped by adding the solution to boiling water.
mL of 0.1 M Tris-HCl buffer (pH 8.0), 4 U
Choline oxidase of 4U Perox
tdase, 2mg 4-aminoantipyri
ne, 1 mg phenol, 20 mg Triton
X-100 was added, the reaction was further performed at 37 ° C. for 20 minutes, and the absorbance at 500 nm of the reaction solution was measured and calculated. 1 U of the enzyme was defined as an amount of the enzyme that produces 1 μmol of choline per minute.

Example 1 Alkaligenes species IFO14130 (Alcale
igenes sp.) 25 ° C in 50L fermenter (30L charge)
Culture was performed for 36 hours in 0, 5 VVM and Medium 1 to obtain a 26 L culture solution. The culture was centrifuged (4000 × g,
The resulting supernatant was diluted to 260 L with tap water, and the diluted solution was subjected to an ultrafiltration membrane (molecular weight cutoff: 3,000, material:
2.8 at the entrance using polyacrylonitrile)
m, and the outlet was adjusted to 1.0 atm, and deionized until the metal became 0.05% by weight with respect to the enzyme. further,
20% by weight of calcium lactate and 10% by weight of ferrous sulfate heptahydrate were added per enzyme weight, and the mixture was mixed by stirring with a stirrer for 30 minutes while cooling at 5 ° C. with ice. This mixture was spray-dried (inlet temperature: 190 ° C., outlet temperature: 95 ° C.) to obtain 432 g of lipase powder. The thermal stability was measured for the mixture and the powder. Table 1 shows the results. In addition,
326 g (control 1) of a lipase powder obtained by spray-drying a deionized enzyme solution to which calcium lactate and ferrous sulfate heptahydrate were not added, and a diluent before ultrafiltration (a metal containing 2. The same amount of calcium lactate and ferrous sulfate heptahydrate as above was added to the mixture, and 318 g of a lipase powder obtained by spray-drying the mixed solution (Control 2)
I got

Example 2 Ferrous chloride was added to 24 L of an aqueous enzyme solution cultured and deionized in the same manner as in Example 1 and 20% by weight of the enzyme weight was added thereto. It was spray-dried to obtain 410 g of lipase powder. The thermal stability was measured for the mixture and the powder. Table 1 shows the results.

Comparative Example 1 Copper chloride was added to 25 L of a deionized aqueous solution of an enzyme which had been cultured and deionized in the same manner as in Example 1, and 30% by weight of the enzyme and 20% by weight of magnesium sulfate were added. The mixed liquid mixture and the lipase powder 41 obtained by spray drying the mixed liquid.
5 g were obtained. The thermal stability was measured for the mixture and the powder. Table 1 shows the results.

Comparative Example 2 Zinc chloride was added to 26 L of the culture supernatant cultivated in the same manner as in Example 1 by weight of 10% by weight of enzyme and manganese chloride by 3%.
0% by weight was added and mixed in the same manner as in Example 1 to obtain 421 g of a lipase powder obtained by spray-drying the mixture. The thermal stability was measured for the mixture and the powder. Table 1 shows the results.

[0021]

[Table 1]

Example 3 Penicillium cyclopium strain ATCC-34613 (Penicillium cychlopium) was cultured in a 30 L fermenter in culture medium 2.
And cultivation was performed at 250 rpm for 48 hours at an aeration rate of 1 VVM. 18 L of the supernatant obtained by removing bacteria from the culture by squeezing filtration was diluted to 200 L with ion-exchanged water. The diluted solution was deionized with an ultrafiltration membrane (fraction molecular weight: 10,000, material: cellulose acetate) until the metal was 0.1% by weight with respect to the enzyme. Further, 50% by weight of calcium sulfate per enzyme weight and 3% of ferrous chloride
0% by weight was added and mixed in the same manner as in Example 1 to obtain a mixed solution. The thermal stability of the mixture was measured.

Example 4 A culture supernatant cultured in the same manner as in Example 3 was
Using adex G-25 gel particle size medium, the metal was added to the enzyme by molecular sieve chromatography at 0.
Deionization was performed to 25% by weight. Further, 20% by weight of calcium phosphate and 10% by weight of iron citrate were added per enzyme weight, and a mixed solution was obtained in the same manner as in Example 3. The thermal stability of the mixture was measured. Table 2 shows the results.

Example 5 A culture supernatant cultured in the same manner as in Example 3 was prepared using a semipermeable membrane (molecular weight cut off: 10,000, cellulose acetate membrane) at an inlet of 1.5 atm and an outlet of 1.0 atm. Deionized until the metal was 0.3% by weight of the enzyme. Further, 50% by weight of calcium acetate and 20% by weight of iron nitrate were added per enzyme weight, and a mixed solution was obtained in the same manner as in Example 3. The thermal stability of the mixture was measured. Table 2 shows the results.

[0025]

[Table 2]

Example 6 Aspergillus niger strain NRRL-337 (Asperg
illus niger) in a 500 L fermenter (300 L charge)
At 25 ° C., 1 VVM, stirring speed 300 rpm, medium 2
For 48 hours. This culture solution was filtered by squeezing filtration, and 251 L of the supernatant obtained after removing the bacteria was recovered. The supernatant was diluted to 1,000 L with distilled water. The diluted solution was concentrated to 30 L by a UF membrane module (fraction molecular weight: 3,000, material: polyacrylonitrile, hollow fiber membrane). After dilution with 230 L of tap water, the metal was further deionized to 0.4% by weight of the enzyme in the same manner, and concentrated to 35 L. Furthermore, calcium citrate is added at 40% by weight per enzyme weight,
10% by weight of iron oxalate was mixed in the same manner as in the example. The mixed concentrated solution is spray-dried (entrance temperature: 195
° C, outlet temperature: 95 ° C, evaporation amount: 10 L / hr)
4.8 kg of lipase powder was obtained. The activity recovery of this powder was 76.9%. Table 3 shows the results of the thermal stability test of this lipase powder.

Comparative Example 3 A mixed concentrate was obtained in the same manner as in Example 6. An equal amount (1: 1) of acetone at −20 ° C. was added to the mixed concentrated solution, and the mixture was stirred for 2 hours with a stirrer. The precipitate was suction-filtered through a filter paper (type A), and the precipitate was collected. Vacuum drying (70cmHg
Above) to obtain 2.0 kg of lipase powder. The activity recovery was 30.6%. Table 3 shows the results of the thermal stability test of this lipase powder.

[0028]

[Table 3]

Example 7 Candida Cylindrasse NRRL Y-146
Nine strains (Canida sylindracea) were grown in a 50 L fermenter (30 L charged) at 25 ° C., 1 VVM, medium 3 and stirring speed 35.
The culture was performed at 0 rpm for 24 hours. The culture was centrifuged (3,000 × g, 15 minutes) to obtain 26 L of a supernatant. The supernatant was diluted with a 200 L phosphate buffer (pH 7.0) using an ultrafiltration membrane, and then the metal was added to the enzyme at 0.0%.
The solution was deionized to 5% by weight, and concentrated by an evaporator (heating temperature: 80 ° C., reduced pressure: 60 cmHg) to obtain a 3.2 L concentrated solution. Further, 1% by weight of calcium benzoate and 2% by weight of iron lactate were added to the concentrated solution per enzyme weight, and mixed in the same manner as in Example 1. This mixed concentrated solution is spray-dried (inlet temperature: 195 ° C., outlet temperature: 9
(0 ° C., evaporation amount: 9 L / hr) to obtain 430 g of lipase powder. Table 4 shows the results of the thermal stability test of the mixed concentrate and the powder.

Example 8 A 50 L fermenter (30 L charge) was prepared by using Streptomyces sp.
At 25 ° C., 1 VVM, 300 rpm, medium 4 for 48 hours. The culture is centrifuged (6,000 × g, 1
0 min) to obtain 26 L of a supernatant. The supernatant is
Dilute with 300 L phosphate buffer (pH 7.0) and use ultrafiltration membrane (molecular weight cutoff: 3,000, material: polyacrylonitrile) until the metal becomes 0.02% by weight with respect to the enzyme. After deionization, concentration was performed with an evaporator (heating temperature: 80 ° C., degree of reduced pressure: 60 cmHg),
A 3.2 L concentrate was obtained. Further, 0.1% by weight of calcium ascorbate per enzyme weight, 0.1%
Weight percent ferrous chloride was added and mixed in the same manner as in Example 1. This mixed concentrate is spray-dried (entrance temperature: 195
° C, outlet temperature: 95 ° C, evaporation amount: 10 L / hr)
10 g of phospholipase powder were obtained. Table 4 shows the results of the thermal stability of the mixed concentrate and the powder.

Example 9 Xanthomonas campestris NRRL-B145
9 strains (Xanthomonas campestris) in a 20 L fermenter
The cells were cultured at 0 ° C., 250 rpm, aeration rate of 1 VVM, and medium 1 for 48 hours. The culture is centrifuged (6,000 ×
g, 15 minutes), the supernatant was added to a 200 L Tris buffer (p
H8.0). The diluted solution was deionized with an ultrafiltration membrane (fraction molecular weight: 10,000, material: cellulose nitrate) until the metal became 0.01% by weight with respect to the enzyme. Further, 0.05% by weight of calcium sulfate and 0.05% by weight of iron nitrate per enzyme weight were added to the deionized solution while cooling with ice at 4 ° C.
The mixture was stirred with a stirrer for 24 hours while being cooled with ice to ℃, and mixed. This mixture is spray-dried (entrance temperature: 191
° C, outlet temperature: 95 ° C, water evaporation 5 L / hr) to obtain 850 g of esterase powdered enzyme. Table 4 shows the results of the thermal stability test of the mixed concentrate and the powder.

[0032]

[Table 4]

Example 10 Commercially available lipase lipase OF (manufactured by Meito Sangyo), paratase (manufactured by Novo Nordesk), lipase D (manufactured by Amano Pharmaceutical)
Was dissolved in tap water at 10% by weight, and 5 L of each enzyme solution was diluted with 100 L phosphate buffer (pH 7.5). This diluted solution is passed through an ultrafiltration membrane (fraction molecular weight;
Deionization was carried out using 10,000 (material: cellulose acetate) until the metal became 0.01% by weight with respect to the enzyme. To this deionized enzyme solution, 30% by weight of calcium nitrate and 10% by weight of ferrous chloride were mixed with a stirrer for 24 hours while maintaining the temperature at 4 ° C. A heat stability test was performed on each mixture using a deionized enzyme solution without addition as a control. Table 5 shows the results.

[0034]

[Table 5]

Medium 1

Medium 2

Medium 3

Medium 4

[0039]

According to the present invention, the thermostability of the enzyme is improved by adding an iron salt and a calcium salt to the deionized lipolytic enzyme solution, and sufficient enzyme activity is obtained at a high temperature. Is obtained and stable, so that enzyme synthesis using a substrate having a high melting point becomes possible. Furthermore, in the production of lipolytic enzymes, the method of concentration and pulverization through heating can be used, and an efficient method of producing an enzyme powder is possible, which is extremely useful.

Claims (5)

[Claims] 1. A thermostable enzyme comprising an iron salt and a calcium salt added to a lipolytic enzyme having a metal content of 0.5% by weight or less. 2. The thermostable enzyme according to claim 1, wherein the lipolytic enzyme is deionized. 3. The method according to claim 1, wherein the iron salt is present in an amount of from 0.01 to 0.01 wt.
The thermostable enzyme according to claim 1, wherein 50% by weight and calcium salt are 0.01 to 70% by weight. 4. The method for producing an enzyme according to claim 1, wherein an enzyme solution containing an iron salt and a calcium salt is dried to obtain an enzyme powder. 5. The method for producing an enzyme according to claim 4, wherein the drying is a spray drying method.