DE2819384B2 - Microbiologically produced lipase and the process for making it - Google Patents

Description

The invention relates to a microbiologically produced lipase and a process for its production.

The invention relates to a microbiologically produced lipase with the following properties:

1) an optimal pH for the activity of about 9 ± 0.5;

2) an optimal temperature for activity of from about 40 to about 48 ° C;

3) a lipase activity which is activated or potentiated by bile salts;

4) a cholesterol esterase activity and

5) a molecular weight of about 30 χ 10 ⁴ to about 4OxIO ⁴ .

The lipase of the invention has properties that have not yet been described in the literature.

Processes for the production of lipase using microorganisms, in particular bacteria, have long been known in such processes are for example microorganisms of the species Pseudomonas, Chromobactcrium, Achromobacterium, Staphylococcus, Brevibacterium and Corynebacterium are used.

Studies have already been carried out to develop a lipase with an optimal pH value for the activity in the alkaline range / ti, which should be activated by bile salts. In these earlier investigations, the strain Alcaligenes Sp ₁ PL-266 Meito Strain FERM-P No. 3187 (deposited at the Fermentation Research Institute, Agency of Industrial Science *: Technology, Japan) was discovered. It is a microorganism of the species Alcaligenes, who has been isolated from the earth. This strain has the ability to produce a lipase with an optimal pH for activity in the alkaline range, which can be activated by bile salts. This procedure and the

ίο microbiological properties as well as the enzymatic properties of the lipase produced in this way are in of JP-OS 21387/77.

In further investigations, different strains of the same species have now been found in the earth

rs around Tokyo, Japan, was isolated found that these microorganisms have the ability to produce a lipase having an optimal Has pH for activity in the alkaline range and which can be activated by bile salts. at however, the new lipase is a different type of lipase which is more suitable than the above-mentioned known lipase. Also the separation of those formed by this new tribe Lipase succeeded. This new type of lipase has in addition to the lipase activity, a cholesterol esterase activity, the lipase described above does not own. Because of this activity, it is also useful as a diagnostic tool for various purposes, for example for determining the cholesterol level, suitable

The amount of lipase produced by the newly found microorganism is, for example, like this as high as about 2000 units / ml of filtrate of the culture broth. This amount is considerably larger than that Amount of lipase produced by the microorganisms according to

r> of the above patent application is generated. This is namely only about 320 units / ml of filtrate the culture broth.

The invention therefore provides a lipase with an optimal pH in the alkaline range

4i) Disposed The new lipase will when given the Humans or animals, for example, is administered orally as a digestive aid, through the bile in the Gut activated (alkaline intestinal juice). Another object of the invention is a

4T method for producing the above-mentioned lipase, which is characterized in that a microorganism with properties that make its identification as a strain Alcaligenes sp. PL-679 Meito allow, in einvm nutrient culture medium, which a

■ "> <) a carbon source, a nitrogen source, and minerals contains, at a pH of about 5.5 to about 11 and cultivated aerobically at a temperature of about 5 to about 40 ° C, and that the microbiologically produced Lipase is obtained from the resulting culture broth.

Yi The strain used to produce the lipase according to the invention was named Alcaligenes sp. PL-679 called Meito. It has been deposited with the German Collection of Microorganisms under the depository number DSM 1239, and he

bo is referred to herein as Alcaligenes sp. DSM 1239 designated. In addition, he became involved in Fermentation Research Institute, Agency of Industrial Science & Technology, Japan, under the accession number FERM-P No. 3783 and at the American Type Culture Collection,

b ^r > USA, on deposit under ATCC 31371.

The microbiological properties of the .strain Alcaligenes sp. DSM 1239 are as follows:

Λ. Morphological properties

Rods, 0.5 to 0.7x1.0 to 4.0 μτη, which are individually and Occasionally occur in chains of 2 to 4 cells on the nutrient agar medium or in the nutrient broth. ^

Non-pleomorphic.

They are movable by means of 1 to 6 hostages lashed around the mouth.

No spore formation.

Grief negative. Abundant growth and red colons formed on crystal violet agar.

Not acid-proof

B. Cultivation Properties

Food agar colonies: circular, rough, fringed, translucent, yellowish brown, brown in the middle; no formation of a diffusible pigment.

Food agar slant culture: spread or prickly, translucent, resinous, orange to yellowish brown.

Nutrient broth: lush growth, yellowish brown: i, cloudy with Membranes and presence of sediment.

Gelatin experiment: filiform, no liquefaction, abundant growth, yellowish brown on medium surface.

Litmus milk: alkaline. No peptonization.

Potato culture: lush growth, orange, turning light brown.

C. Physiological properties

Formation of nitrites from nitrates.

No formation of gas in nitrate broth under anaerobic conditions.

Methyl red test negative.

Voges-Proskauer-Tebt negative.

No formation of indole.

No formation of hydrogen sulfide.

No hydrolysis of starch.

Citratef are used as the only carbon source (Koser's Agar and Christensen's Agar).

Nitrates and ammonium salts are used as the only nitrogen sources.

A diffusible pigment is placed on nutrient agar, Nutrient gelatin or potato dextrose agar not formed. A diffusible ■! pale brown pigment is formed on King-A-Agar and Czapek-Dox-Agai. A diffusible pale yellowish brown Pigment is formed on King B agar.

Γι

No urease formation,
Oxidase positive,
Catalase positive,

pH range for growth: growth between 5.5 and ₁ U O. No growth below 5.0 or above 12.0.

Temperature range for growth: growth between 5 and 40 ⁰ C. Optimal growth between 15 and 35 ° C.
Aerobic.

OF test: both open and closed Neither an acid nor a gas is generated.

D-fructose and glycerine become acid (latent), but no gas in the oxidation-fermentation medium formed by Hugh and Leifson in open tubes. Neither acid nor gas is made from L-arabinose, D-xylose, D-glucose, D-mannose, D-galactose, maltose, Sucrose, lactose, trehalose, O-sorbitol, D-mannitol, inositol or starch in open and closed Tubes generated.

- >> D. Other properties

Formation of lipase.
No formation of 3-ketolactose.
Weak formation of ammonia from arginine.
jo G + C content of DNA: 66.7 Mot%.

The taxonomic status of the strain used according to the invention with those described above microbiological properties was considered in terms of

si information in Bergey's Manual of Determinative Bacteriology, 8th edition (1974) examined. It was confirmed that the strain belongs to the species Alcaligenes. In Bergey's Manual does not describe any known microorganism of the species Alcaligenes which is different from the above has microbiological properties. Furthermore, the strain used according to the invention differs from the strain Alcaligenes sp. PL-266 Meito, disclosed in the above-referenced laid-open patent application is described.

Γ) In Table I are the main differences between the Strain Alcaligenes DSM 1239, which is suitable for the production of the lipase according to the invention, and the Strain PL-266 Meito compiled according to the above patent application.

Table i

properties

Strain PL-266 Meito strain DSM 1239

(; use according to the invention.)

Nutragar Colony

Nutrient agar slant

Nutrient broth

Gclatinc trial

I. Sackmus milk

Nitrate reduction

Wcfelwiisscrstorfbi Idung

OF-Tcst

whole, gray-white or gray-yellow gray-white or gray-yellow not cloudy, without lliiutchcn

crater-shaped, becomes stratiform

Peptonization

negative

positive (weak)

oxidizing

fringed, yellowish brown or brown
jrange or yellowish brown
cloudy, with cap

no liquefaction
no peptonization
positive
positive
non-oxidicrend and

I igenscliaften Strain I ¹ I -2Mi Meilo Strain DSM I2.W (errindungsgcniiiU used) / ucker assimilation L-arabinose positive- negative D-xylosc positive negative D-glucose positive negative D-mannose positive negative D-galactose positive negative Inositol positive negative Maltose positive negative Sucrose positive negative I artnse positive neeative Trehalose positive negative D-sorbitol positive negative Mannitol positive negative G + C content of DNA 74.2 mole percent 66.7 mol%

The microbiologically produced lipase with the properties (1) to (5), which has not yet been described in the literature, can be produced as follows:

To produce the lipase according to the invention, the microorganism Alcaligenes sp. DSM 1239 aerobically in a nutrient culture medium containing carbon and nitrogen sources and minerals, at temperatures of about 5 to about 40 ⁰ C, preferably from about IO to about 40 ⁰ C, and a pH of about 5.5 to about 11, preferably about 6 to about 10, cultured, and the microbiologically produced lipase is recovered from the resulting culture broth.

The carbon source, nitrogen source, and minerals can be any materials that commonly used for growing microorganisms.

Examples of suitable carbon sources are glucose, fructose, maltose, soluble starch, starch, Dextrin, molasses, glycerin, oils and fats, wheat bran, lactose and sucrose. Examples of suitable Nitrogen sources are organic or inorganic nitrogen-containing compounds, such as soybean powder, defatted soybean powder, cottonseed residues, corza residues, peptones, meat extract, Yeast extract, gluten, corn steep liquor, casamino acid, urea, ammonium salts and nitrate salts. Examples of suitable minerals are phosphate salts, Magnesium salts, iron salts, potassium salts, sodium salts and calcium salts. If desired, various other organic or inorganic substances necessary for the growth of microorganisms or for enzyme production are suitable, e.g. B. silicone oils, anti-foaming agents, vitamins and phosphorus-containing compounds, can be added.

By incorporating a non-ionic surfactant into the nutrient culture medium according to According to the invention, the amount of microbiologically produced lipase can be increased significantly. the Yield of the components of the invention microbiologically The lipase produced can also be increased by adding salts which are capable of iron ions submit, such as B. iron chloride or iron sulfate, used together with sodium salts of organic acids such as Na'numacetat or sodium citrate.

The amount of the nonionic surfactant used is about 0.01 to about 2 jo% by weight, based on the weight of the nutrient culture medium. Examples of suitable non-ionic surfactants are

Sorbital alkyl ester, polyoxyethylene sorbital alkyl ester, ^J> polyoxyethylene alkyl ether,

Polyoxyethylene alkylaryl ether, Polyoxyethylene alkyl ester,

Polyoxyethylene / polyoxypropylene copolymers and Fatty acid monoglycerides.

The cultivation is carried out under aerobic conditions. It can be both a diving and a Surface cultivation technique can be applied. However, an immersion culture is usually suitable. For

In technical operations, it is advantageous to carry out immersion cultivation with aeration and stirring. A suitable approach is e.g. B. the implementation of a small-scale pre-cultivation and the inoculation of the resulting culture

5n broth in a culture medium for the main cultivation.

The cultivation conditions vary somewhat according to, for example, the strain and strain used the composition of the culture medium. It is recommended to select conditions that are suitable for the Production of the lipase as a final product on vorteilhaftesten are The cultivation temperature is generally about 5 to 40 ³ C, preferably about 10 to 40 ° C temperatures from 20 to 30 ⁰ C are particularly preferred. The cultivation time varies accordingly the conditions, but is usually about 1 to about 4 days. Cultivation can be finished at a time when the amount of accumulated lipase has reached a maximum The pH of the culture medium can change at the time of manufacture development of the culture medium from weakly acidic to Extend alkalinity. It is usually not necessary to adjust the pH separately. The pH of the Culture medium is generally about 5.5 to

about II. preferably about 6 to about 10. more preferably about 7 to about 10. The cultivation can, if necessary, with adjustment of the pH to the aforementioned areas are carried out.

The microbiologically produced ί lipase according to the invention can be obtained from the resulting culture broth by known procedures such as those used for the separation and purification of lipase. For example, in the case of surface culture, the culture broth is extracted with in water or another extraction agent to form an extract. In the case of immersion culture, the microbial cells or solids are separated from the culture broth by known filtration or centrifugation to obtain an extract or a filtrate. The extract or the filtrate can be used either as such or after concentration. By treating the extract, the filtrate or its concentrated product by known techniques such as e.g. B. precipitation, spray drying or lyophilization, the microbiologically produced lipase according to the invention can be obtained in the form of a solid product.

Suitable precipitation agents for the precipitation method are e.g. B. soluble salts such as sodium chloride, ammonium sulfate and sodium sulfate, and hydrophilic organic solvents such as ethanol, methanol and acetone. Suitable stabilizers to be used in spray drying are e.g. B. maltodextrin, lactose, carboxymethyl cellulose, polyethylene glycol , skimmed milk, casein, sorbitol and sodium chloride.

The lipase product according to the invention with the desired purity and lipase potency can by a or several known purification techniques can be obtained, for example by adsorption and EIu-J5

Table II

tion methods using ion exchange resins, calcium phosphate gel, aluminum oxide or bentonite. by molecular sieve methods with Sephadex or Biogel, by chromatographic methods with cellulose or Sephadex ion exchangers, by precipitation methods using a protein precipitation agent such as tannin and calcium salts, by an isoelectric precipitation method, by a dialysis method and an electrophoretic method. The lipase produced microbiologically according to the invention has the following properties:

(1) an optimal pH for activity of about 9 ± 0.5,

(2) an optimal temperature for the activity of about 40 to about 48 ° C,

(3) a lipase activity that is activated or potentiated by bile salts,

(4) a cholesterol esterase activity and

(5) a molecular weight of ^{about 30 × 10 4} to about 4O × 10 ⁴ .

This lipase has not yet been described in the literature. Lipases (fractions I and II), the in JP-OS 21 387/77 come closest to the lipase according to the invention. the microbiologically produced lipase according to the invention can be differentiated from lipases I and II primarily with regard to the properties (2), (4) and (5) described above can be distinguished. Furthermore, she also shows different inhibitory properties for the activity. Table II shows the properties of the microbiologically produced lipase according to the invention in comparison to the properties of lipases I and II compiled according to JP-OS 21 387/77.

properties

Lipase according to the invention Lipase according to JP-OS 21 387/77 Lipase I Lipase II

Lipase activity Cholesterol esterase activity Lipoprotein lipase activity Optimal pH for activity. Optimal temperature for activity Molecular weight

Activation inhibition by Hg ⁺⁺ , Fe ⁺⁺ , Zn ⁺⁺ , Sn ⁺⁺ , Cd ⁺⁺ or Cr ⁺⁺⁺ (concentration, 10 " ³ mol)

Activity inhibition by sodium ascorbate (concentration, 10 ~ ² mol)

Activity inhibition by monoiodoacetic acid (concentration, 10 " ³ mol)

9 ± 0.5

40 to 48X

34 X 10 ^ 37XlO ⁴

Inhibition

(more than 50%)

Inhibition (more than 50%)

Inhibition (more than 50%) 9 ± 0.5

70 to 75 ° C

18 ± 1X1O ⁴

no inhibition

no inhibition no inhibition

10 ± 0.5 70 to 8O = C 3.2X10 ⁴ -3.7X10 ⁴ Inhibition by Cd ⁺⁺ (10 to 20%), but not by the other metal ions

no inhibition no inhibition

The enzymological properties of the microbiologically produced lipase according to the invention are described in more detail below.

Preparation of the samples

The mycelia and other solids are separated from the culture broth by filtration. The filtrate is cooled to about 5 ° C and ammonium sulfate becomes

65 with stirring to the filtrate to saturation of 40% given. The mixture is allowed to stand at about 5 ° C for 24 hours. The precipitated enzyme is collected and dialyzed in running water at about 18 ° C for 2 days using a dialytic cellophane membrane. The dialysate is then dialyzed for 24 hours at about 5 ° C against a Tris buffer solution (concentration 0.01 mol; pH 9.0) dialyzed further. The dialyzed enzyme solution

Solution is applied at about 5 ^° C. to a column of DEAE cellulose ion exchanger which has been sufficiently equilibrated with a Tris buffer (concentration 0.01 mol; pH 9.0). The column is then washed with a Tris buffer (concentration 0.01 mol; pH 9.0) and eluted with a Tris buffer (concentration 0.01 mol; pH 9.0), the concentration of sodium chloride in the buffer is gradually increased to 0.4 mole. The activity of the effluent is measured and active fractions are collected. The active fractions are then placed in a cellophane dialytic membrane and concentrated using polyethylene glycol powder. The concentrate is dialyzed in running water at about 18 ° C. for 24 hours in the same manner as described above. It is then dialyzed further against a Tris buffer. The dialysate is re-chromatographed and the resulting active fractions are re-concentrated as described above. The concentrate is then centrifuged to remove the solids. The enzyme solution obtained is gel-filtered by passing it through a column of Sephadex G-200 using the same Tris buffer as above. The active fractions are collected. The resulting active fractions are concentrated and centrifuged as above. The resulting enzyme solution is gel-filtered in the same manner as above using a column of Sepharose 4B. In this way a purified sample is obtained.

Using the purified sample, the Properties (1) to (7) were measured. The determination is carried out according to the following methods.

(4) cholesterol esterase activity

The measurement of the cholesterol esterase activity is carried out according to the procedure described by Leschroue et al. in Z. Klin. Chem. Klim. Biochem., 12.403 (1974).

(5) molecular weight

The measurement and calculation of the molecular weight are carried out by the method described by P. Andrews in Biochem. J. 91, 222 (1964) described method. A column measuring 2.5 χ 100 cm is provided with packed with a gel from Sephadex G-200. Catalysis (molecular weight 240,000) and ferritin (molecular weight 540,000) are used. A 1/00 M Tris-HCl buffer (pH 8.7) is used as the elution buffer used.

(6) lipoprotein lipase activity

The lipoprotein lipase activity is determined according to the Method determined by Saiki et aL (Agr. BioL Chem. 32, 1459 [1968]). The human serum is fresh lyophilized human plasma (Miles Laboratories, Ina) Used Fatgen (product of Dainippon Pharmaceutical Co, Ltd.) is used as oil and fat

(7) Inhibition of the activity of sodium ascorbate and monoiodoacetic acid

An aqueous solution of the lipase sample (about 3 units / ml) is brought into contact with the individual inhibiting agents at 37 ° C. for 10 minutes. The lipase activity is measured. The concentration of sodium is 10 ~ ² moles of mono-iodoacetic acid and the iC.onzentration is I0 ~ ³ mol

Determination of the characteristic property

ten (I), (2) and (3) with regard to the enzymological properties of the lipase according to the invention are carried out in the manner described below.

(1) Mode of operation

The microbiologically produced lipase according to the invention is an enzyme that converts oils and fats to glycerol and Fatty acids hydrolyzed, taking large amounts of monoglyceridesii as intermediates during the Hydrolysis can be obtained.

(2) substrate specificity

The lipase activities of the microbiologically produced lipase according to the invention with respect to oils and Fats and various esters are given in Table III, expressed as a percentage of activity based on the Activity towards olive oil.

Table H!

Substrate

relative activity

olive oil 100 Methyl n-butyrate 1 Methyl-n-ceproate 3 Methyl n-caprylate 6th Methyl n-caprate 6th Methyl laurate 6th Methyl myristate 7th Methyl palmitate 34 Methyl stearate 33 Methyl oleate 18th Tributyrin 60 Tricaproin 52 Tricaprylin 123 Tricaprine 100 Triplet 141 Monolein 86 Monolaurin 40 Dilaurine 75 Trilaurin 128 Chip 85 29 Tween 85 20th castor oil 85 Buffered 53

From Table III it can be seen that the microbiologically produced lipase according to the invention not only on , ο Glycerol esters of higher fatty acids, such as natural oils and fats, but also acts on water-soluble ones Esters, such as Tween 85.

The microbiologically produced lipase according to the invention also acts on cholesterol esters of fatty acids and converts them into fatty acids and cholesterol.

(3) Optimal pH range for stability [Determination of property (I)]

The lipase according to the invention is dissolved at a concentration of 2.5 to 5 U / ml and the optimum pH value is determined. The results obtained are shown in FIG. 1, which shows that the maximum activity is obtained at a pH of about 9 ± 0.5

H-. The lipase according to the invention is kept in a concentration of 50 to 100 U / ml in buffers of different pH values at 5 ° C. for 24 hours. Then the pH is adjusted to 8.6 with a glyein buffer

set. The remaining Lipa ^ e activity is measured. The values are shown in FIG. Thus, the lipase is shown to be essentially stable at a pH in the range from about 6.5 to about 10.5 ^; st. A Mcllvaine buffer (VioM citric acid. V) M-Na ₂ HPO.!) Is used for the pH range from 2.5 to 8.0, a '/ iM trismaleate buffer for the pH range from 5.5 to 8.5, a '/ 10M-Na ₂ CO ₃ -H ₃ BO ₄ -KCI buffer for the pH range from 8.0 to 10.5 and a Vi-Na ₂ HPO ₄ -NaOH buffer for the pH -Range from 11 to 12 used.

(4) Appropriate activity temperature [determination of property (2)]

The suitable activity temperature range of the lipase according to the invention is shown in FIG. Of the optimal temperature range is about 40 to about 4S ° C. The determination is carried out using the following method:

f \ L. ' Λ Al * '' · "*» · 1 * »U An tint ·

Uäl LfCl U (UI nMITIiai3Ullll.l3U <.tlUII5JIIIVtlluuv »*.,

The turned reaction substrate is preheated to various temperatures for 10 minutes. 1 ml one Enzyme solution (concentration 0.01 mg / ml) is added and it is allowed to stand for 10 minutes at the temperature of the Preheating implemented. The activities at the different temperatures are expressed as a percentage Activities expressed in terms of the activity of the lipase at 45 ° C.

(5) Inactivation by pH and temperature conditions

The lipase is dissolved in water at a concentration of 4 U / ml and the solution is kept at a temperature of 70 to 8O ⁰ C. Changes in the activity of the lipase are periodically determined by the analytical method described below. The results obtained are shown in Fi g. 4 shown. It can be seen that the lipase is almost completely inactivated by heat treatment at 80 ° C. for 30 minutes.

The lipase is dissolved in water at a concentration of 25 to 50 U / ml and mixed with an equal amount of a Mcllvaine buffer (pH 3.0) or a '/ 4-Na ₂ HPO ₄ -NaOH buffer (pH 12, 0) mixed. The resulting mixtures are kept at 37 ° C. and then samples are taken at predetermined times. The sample mixtures are each diluted 5-fold with a 1 M glycine buffer (pH 8.7) and the activity of the lipase is measured. At a pH of 3.0, more than 95% of the lipase activity is lost in 10 minutes. At a pH of 12.0, more than 95% of the lipase activity is lost in 120 minutes.

(6) inhibition

The lipase is contacted at a concentration of about 3 U / ml for 10 minutes with various inhibiting reagents at 37 ° C. The lipase activity is then measured. When the concentration of Inhibierungsreagenses ΙΟ- is ³ mol, then, that the lipase activity + + + is inhibited more than 50% by Hg ⁺ +, Fe + + +, Zn ++, Sn ++, Cd ++ and Cr is detected.

The inhibition is more than 50% with 10- ² mole sodium ascorbate, about 100% at 10 ~ ³ MoI mono-iodoacetic acid, about 10 to about 20% with MoI IQ ² potassium ferricyanide, pyrophosphate salts, sodium azide and disodium ethylenediaminetetraacetate, about 95% with ΙΟ- ² moles iodine and about 70% to 10 ^-2 mol Äthoxyameisensäureanhydrid.

(7) Activation f determination method of property (3)]

Use the determination method described under (8) below for lipase activity, various bile salts are dissolved in distilled water and the Lipase activity is measured as in TabeK'e IV is shown, the lipase of the invention is apparently activated by bile salts.

Table IV concentration 0.4 of bile salt 0.8 Bile salts 0.2 229 0.6 226 221 221 224 224 Taurocholate 218 221 232 229 Glycocholate 197 300 211 303 Cholate 289 305 Deoxycholate

The activities shown in the table above are - '"> percentage activities related to lipase activity in the absence of a bile salt.

(8) Determination of the activity (a) Composition of the reaction mixture

Enzyme solution 1.0 ml

Glycine buffer (1 mole, pH 8.7) 2.0 ml

Olive oil emulsion 2.5 ml

distilled water 0.5 ml

i'i The olive oil emulsion is prepared in such a way that 20 g of olive oil are added to 180 ml of a 10% aqueous solution of gum arabic, that the mixture is homogenized at 11,000 rpm for 15 minutes while it is on 5 bi. ^c 10 ° C is cooled, and that the pH of the homogenized mixture is adjusted to 8.7.

(b) Procedure

The reaction mixture is poured into a 25 ml vjlasstop-

4) inserted and held for exactly 10 minutes 37 ° C implemented. The reaction is then terminated by adding 1 ml of 2N sulfuric acid and 15 ml of a mixture of n-heptane and isopropanol in the ratio of 11: 4 are added. The mixture

it is shaken vigorously for 30 seconds and more than 30 minutes ditched. 5 ml of the upper layer are collected and poured under nitrogen gas with a 0.01 N ethanol Potassium hydroxide solution titrated using thymol blue as an indicator. A 2N sulfuric acid solution is added to a reaction mixture having the same composition as above and the enzyme solution is then added. The mixture is added in the same way as described above, treated and the resulting product is titrated with a potassium hydroxide solution. The measured value obtained is subtracted from the reading obtained above. The amount of fatty acid released is determined by the The resulting titration value is calculated using a calibration line, which is determined using a known Amount of palmitic acid has been established

A Upase unit is defined as the amount of enzyme that 1 μΜοΙ fatty acid in 1 min in the case of the above described experimental conditions releases

(9) Cleaning method

The lipase according to the invention can be prepared by salting out by cellulose ion exchange chromatography and gel filtration, suitable combinations of these measures also being used can be. An embodiment is shown in example 5

As already stated, the properties of the Lipase according to the invention is very similar to that of a pancreatic lipase of an animal with regard to the optimal pH (in alkalinity) for activity

Table V

10

and activation with bile salts.

The lipase according to the invention also has the ability Break down cholesterol (i.e., a cholesterol esterase activity). It is not yet known been that a lipase having such property is produced by a microorganism of the Alcaligenes species will.

Table V shows the properties of the lipase according to the invention in comparison with the properties of known microbiologically produced lipases that can be activated with bile salts

Micro Optimal Optimal Activation Choleste- Molecular Lipopro Inhibition literature t organism PH value Tempe rungsfakior rin-este- weight tein- by credentials 56 rature by rush Lipase Reagents I. Bile salts f (Q

Chromobacterium viscosum var. paralipolytica

7.0

1.5

Pseudomonas 9 to 9.5 -

trim (ATCC

19154)

Phycomyces sp. 6 to 7 40

6 to 10 2

Mucor javanicus

7.0

2 to 5

Alcaligenes sp. PL-266 Meito

Alcaligenes

sp. PL-679

Meito

(inventive

according to

used)

18.5-9.5 70-75 2 II 9.5-10.5 70-80 2

8.5-9.5 40-48 2

120000 no _ Agr. Biol. 30000 Chem. 37, 999 (1973); JA-PS 29787/71 and J. Biochem. 81, 1639 (1977) JP-PS 10754/69 _ _ JP-PS 17 559/75; Together no 180000 yes version of the 26500 yes ± 10000 Writing down ± 500 no 32 000- of the annual 37,000 Meeting of Agricultural yes J (K) 000 yes Chemical So 400,000 ciety of Japan in 1974; Together versions of the Writing down of the meeting of Western Divi sions of the Agricultural Chemical So ciety of Japan in 1977 Yakuzaigaku (»Pharma ceutical Science «) 27, 314 (1967); Eisoi Kagaku (»Hygienic Chemistry «), 13, 257 (1967) no JP-OS 21 387/77 Inhibition by ascorbine acid and mono- iodoacetic acid Inhibition by ascorbine acid and Monoiodine vinegar

acid

As can be seen from Table V, lipase produced by Chromobacterium viscosum, Phycomyces sp, and Mucor javanieus is that of the present invention Similar to lipase in terms of the activation factor by bile salts, but the optimal ones are pH values for these lipases are in the neutral range and thus differ from the corresponding ones Values for the microbiologically produced lipase according to the invention. The pruning by Pseudomonas The lipase produced differs from the lipase according to the invention with regard to the activation factor by bile salts and its optimal pH curve differs considerably from that of the lipase according to the invention (cf. FIG. 1).

The lipase of the present invention also differs from the lipase produced by Alcaligenes sp. PL-266 Meito is generated in terms of the optimum temperature for the activity and that it has a cholesterol esterase activity. As shown in Table II, there are also differences, for example, in molecular weights and inhibition by metal ions.

The microbiologically produced according to the invention Lipase can be used for a wide variety of uses, such as pharmaceuticals and diagnostics. You can continue to Production of fatty acids, glycerine and monoglycerides, to enhance the milk aroma, to improve the quality of bread and biscuits and the like, for use in wastewater treatment and also leather tanning agents, hair rinsing agents, Bath preparations, face washing preparations and food can be used.

The microbiologically produced lipase according to the invention has an optimal pH value in the alkaline Area and because it is activated by bile salts, it has the advantage that when it is used orally, for example Digestive aid, which activates bile salts in the intestinal juice, and a shows increased activity. The microbiologically produced lipase according to the invention decomposes lipoprotein into Food as it has lipoprolein lipase activity. If the lipase according to the invention as Digestive aid is used, it can be taken orally at a dose of, for example, 1000 to 8000 U / day administered. When used as a diagnostic agent, the cholesterol spicgel of the blood can be measured using 0.1 to 2.0 U of cholesterol esterase in one To be determined individually. Furthermore, by using 100 to 1000 U of lipoprotein lipase in The triglyccride level of the blood can be determined on an individual basis.

As the lipase according to the invention, rats at a dose of 2.5 and 5.0 g / kg for 1 month and one Dose of 2.5 g / kg administered for 3 months by natural ingestion was to reduce subacute toxicity to determine there were no changes in general behavior, faeces, urine, the Blood composition, determined by autopsy findings, and weight of organs. Even with the no significant changes were found on histological examination.

The invention is illustrated in the examples.

R c i s ρ i c I I

Shell flasks with 500 ml were each filled with 50 ml of a liquid culture medium containing 3% soybean powder. 1% defatted soybean pellet, 1% corn liqueur water. Contained 0.5% dipotassium phosphate and 0.23% sodium nitrate. It was adjusted to a pH of 8.0 and the culture medium was sterilized with steam at 120 ° C. for 15 min. Alcaligenes sp, DSM 1239 was added to the media

emokkuliert was cultured 40 h with shaking at 30 ⁰ C. The culture broth was centrifuged. The lipase activity of the supernatant was measured and found to be 50 U / ml. Ammonium sulfate became one Saturation of 50% to 100 ml of the culture broth added. The resulting precipitate was collected and dried to give 1.5 g of crude Enzyme powders with a lipase activity of 2560 U / g were obtained.

Example 2

The procedure was as in Example 1, with the exception that 0.5% sorbitol oleyl ester was added to the culture medium described in Example 1. The lipase activity of the supernatant liquid of the culture broth was 351 U / ml.

An enzyme powder in an amount of 1.4 g was collected from the resulting culture broth as in Example. The lipase activity of the powder was r. 16,000 U / g.

Example 3

A liquid culture medium containing 3% soybean powder, 0.5% defatted soybean powder, 0.5%

jn corn steep water, 0.1% wheat starch, 0.2% dipotassium phosphate, 0.5% polyethylene stearyl ether, 1.0% sodium nitrate and 0.075% silicone, was adjusted to a pH of 9.5 set. 50 ml of the culture medium was placed in a 500 ml shake flask and held for 15 minutes

r> steam sterilized at 120 ° C. Alcaligenes sp. DSM 1239 was inoculated. Hs was ^{cultivated at 30 °} C. for 40 hours. The culture broth was filtered. The filtrate had a lipase activity of 500 U / ml, a cholesterol esterase activity of 3.7 U / ml and a lipopro-

4 <protein lipase activity of 155 U / ml. For 500 ml of filtrate cold acetone was added to a concentration of 80%. The precipitate was collected, washed with cold acetone and dried in vacuo at room temperature, yielding 124 g of a

4Ί raw enzyme powder were obtained. The powder had a lipase activity of 13,200 U / g, a cholestcrin esterase activity of 99 U / g and a lipoprotein lipase degree of 4100 U / g.

. B e i s ρ i e I 4

To '51 of the culture medium described in Example 3, 0.4% sodium citrate and 0.028% iron (II) sulfate were added and the mixture was poured into a 30 l cup of Fermentalor given. As an anti-foam agent

v, 5 g of silicone oil was added and the mixture was steam sterilized.

Alcaligenes sp. DSM 1239 was inoculated into the culture medium produced in this way. It was 38 h cultured with aeration and stirring at 24 ° C. the

The culture broth was filtered, whereby 12.01 of a filtrate having a lipase activity of 800 U / ml was obtained. The absorption of this filtrate at 280 nm was 100. The filtrate had a cholesterol esterase activity of 6 U / ml and a lipoprotein lipase activity. vity of 250 U ml.

Ammonium sulfate was added to 2.4 l of this filirate to / u a saturation of 40% and the mixture was allowed to stand at 5 ° C for 24 hours. The over-

The liquid was decanted off and the precipitate was collected by centrifugal separation About 73% of the lipase activity was in the Recovered precipitate formed by the addition of ammonium sulfate,

Example 5

The precipitate obtained in Example 4 by the addition of ammonium sulfate was placed in a cellophane tube and in running water for 2 days and then for a further 24 hours at 5 "C against an O.OIM-Tris buffer with a pH of 9, 0 dialyzed The ritration of the dialysate yielded 1.31 of an enzyme solution with a lipase activity of 1020 U / ml and an absorption at 280 nm of 28.5.

The enzyme solution was applied to a column of a DEAE cellulose ion exchanger which was sufficiently mixed with an O.OIM-Tris buffer with a pH 9.0 had been equilibrated. The adsorbed enzyme solution was with the The same pnffir was washed and eluted with a Tris buffer (0.01 mol, pH 9.0), gradually the Concentration of sodium chloride in the buffer was increased to 0.4 mol. Fractions with lipase activity were eluted as the concentration of NaCl increased ranged from 0.05 to 0.2 moles. Your activities were measured and the active fractions were collected. The collected fractions were concentrated and gel filtered through Sephadex G-200 and Sepharose4B in that order. The active ones Fractions were collected

The collected active fractions were dialyzed again and rechromatographed on a Sau'ν of DEAE cellulose. The active fractions were eluted by the concentration gradient method using sodium chloride The fractions with a lipase activity were concentrated and lyophilized, whereby 293 g of a purified lipase Powder were obtained. A 0.1% aqueous solution of the purified enzyme powder had an absorption at 280 nm of 0.43. The enzyme powder had a lipase activity of 890 U / mg, a cholesterol ester

rase activity of 6.6 U / mg and a lipoprotein lipase activity of 275 U / mg, the lipase had an optimal pH range of about 9 ± 0.5 and an optimal temperature of about 45 ° C, you could be potentiated with 0.2% sodium taurocholate to twice their original borrowed activity. The molecular weight was about 35x10 *. The activity inhibition of the lipase by sodium ascorbate was about 52% and by monoiodoacetic acid about 98%.

Example 6

50 ml of a liquid culture medium containing 3% soybean powder, 1% fructose, 0.2% potassium phosphate, 1% sodium nitrate 0.6% sodium citrate 0.03% FeSO ₊ · 7H20.05% polyethylene glycol stearyl ether and 0.2% of an antifoam was poured into a 500 ml rocking flask and kept for 15 minutes 120 ° C steam sterilized.

The strain Alcaligenes sp. DSM 1239 was inoculated into the sterilized culture medium and it became

The cultivated for 2Ί42 h with shaking at 25 ° C Culture broth was centrifuged and the enzyme activity of the supernatant was measured. It the lipase activity was found to be 2000 U / ml, the cholesterol esterase activity 15 U / ml and the Lipoprotein lipase activity was 625 U / ml. To 100 ml of the resulting supernatant liquid 400 ml of cold acetone were added. The resulting precipitate was collected and washed with acetone dehydrated and vacuum-dried at 25 ° C 1.8 g of an enzyme powder with a lipase activity of 77,000 U / g, a cholesterol esterase activity of 585 U / g and a lipoprotein lipase activity of 24,000 U / g were obtained.

For this purpose 2 sheets of drawings

Claims (5)

Claims; 1. Microbiologically produced lipase with the following properties: 1) an optimal pH for activity of about 9 ± 0.5; 2) an optimal temperature for activity of from about 40 to about 48 "C; 3) a lipase activity which is activated or potentially activated by bile salts; 4) a cholesterol esterase activity and 5) a molecular weight of about 3Ox 10 ⁴ to about 40xl0 ⁴ . 2. Process for the preparation of the microbiologically produced lipase according to claim 1, characterized in that Alcaligenes sp. DSM 1239 in a nutrient culture medium which contains a carbon source, a nitrogen source and minerals, hsr a pH of about 53 to about 11 and at a temperature of about 5 to about 40 ⁰ C aerobically, and that the microbiologically produced lipase wins from the resulting culture broth 3. The method according to claim 2, characterized in that a culture medium is used which further contains a non-ionic surfactant 4. The method according to claim 3, characterized in that the amount of non-ionogenic riaß Surface-active agent about 0.01 to about 2 wt .-%,. Based on the C ^ weight of the nutrient culture medium, is.