NO139353B - PROCEDURE FOR PREPARING LIPASE - Google Patents

Description

The present invention relates to a method for producing a new lipase whose enzyme activity is increased considerably in the presence of bile.

Different types of microbial lipases have been described so far. For example, lipases from Candida cylindracea and from Propionibacterium shermanii are described in "Agricultural and Biological Chemistry", vol. 30, no. 6, pages 576-584 (1966) and "Applied Microbiology", vol. 20, no. 1, pages 16-22 (1970), respectively. However, these lipases are known to be inactivated by bile.

From British Patent No. 1,084,431 is. the known lipases

from algal fungi of the genera Mucor and Rhizopus; they have an optimal pH value for enzyme activity at pH 3.5 bg pH 6-7 and, in contrast to those mentioned above, are activated by bile. The lipase produced by the present method only has an optimum pH value, namely pH 6.0, and is activated to a higher degree by bile than those known from British patent no. 1,084,431. Their enzyme activity

tet rises with the addition of diluted bile to a maximum of 123% of the original, while the enzyme activity of the lipase produced according to the invention rises to approx. 200% of the original activity when diluted bile is added.

A lipase is known from German patent no. 1,442,093

which is produced by growing Rhisopus delemar. The relationship of this lipase to bile is not known, but the enzyme activity inhibits

is remarkably enhanced by Fe++ and disappears completely or almost completely at concentrations of Fe<++> of 5.0 ppm. In contrast, the lipase produced by the present method is inhibited, when it has a specific activity of 3750 units/mg,

not at a concentration of 120 ppm Fe<++> (1 x> 10 3 mol) by

measurement at pH 5-8 at 4°C for 60 minutes.

From US patent no. 13,634,195, a lipase is known which is produced from algal fungi of the genus Åbsidia. A closer comparison with the lipase produced according to the invention is not possible since this US patent does not contain information about the properties of the isolated and purified* enzyme. The patent also does not contain information that the enzyme's activity should be enhanced by bile.

During research and evaluation of lipase-producing microorganisms, it has been found that microorganisms belonging to the genus Phycomyces produce a lipase, whose enzyme activity increases considerably in the presence of bile.

The main purpose of the present invention is to provide an industrially favorable method for the production of a new microbial lipase with an enzyme activity which is increased considerably in the presence of bile. This lipase can be used in a pharmaceutical preparation together with a bile powder which optionally also contains other digestive aids, and to provide types of fatty food, such as butter, with a taste that is improved by means of the lipase.

According to the present invention, there is thus provided a method for the production of lipase by growing a microorganism in a culture medium, after which the accumulated lipase is separated from the culture medium, and this method is characterized by using as microorganism a strain of the species Phycomyces nitens or Phycomyces blakesleeanus.

Typical examples of microorganisms that can be used

is Phycomyces nitens IFO-5'694, Phycomyces nitens IFO-569 5, .Phycomyces nitens NRRL-2444, Phycomyces nitens NRRL-2678, Phycomyces blakesleeanus IFO-5822, Phycomyces blakesleeanus IFO-5823, Phycomyces blakesleeanus IFO-5870, Phycomyces blakesleeanus IFO -5871. All these species are known. The species with IFO numbering are stored in the "Institute for Fermentation". Osaka, Japan, and the organisms with NRRL numbering are deposited at the "Northern Utilization Research and Development Division",

United States Department of Agriculture. . The cultivation of the microorganism can be carried out either on solid or liquid medium and it is recommended to use liquid medium for industrial purposes. When using a liquid medium, cultivation can take place under aerobic conditions with shaking or stirring.

The culture medium used includes assimilable carbon sources, digestible nitrogen sources, inorganic salts, growth promoting factors etc. :.

The carbon sources can be various known substances, e.g. glucose, sucrose, mannose, dextrin, cellulose, starch, soluble starch, glycerol, sorbitol, wheat bran, rice bran, molasses.

The nitrogen sources are also of a known type and can e.g.

be natural products or substances derived from these (e.g. peptone, meat extract, casein, corn soaking liquid, defatted soybean powder, soybean meal, soybean cake, dry yeast, yeast extract, cottonseed meal, gluten, casamino acid, ammonium salts of organic acids (e.g. .eg ammonium succinate, ammonium tartrate, ammonium acetate), inorganic nitrogen compounds (eg ammonium chloride, ammonium sulphate, ammonium phosphate, sodium nitrate).

The inorganic salts can e.g. be different•fos-

fats, sulphates, hydrochlorides.

The growth factors can e.g. be vitamins, nucleic acids

and their related compounds.

Depending on the method and conditions during cultivation, the

increasing the accumulation of the relevant enzyme and adding a natural or synthetic antifoam agent such as soybean oil, silicon oil, etc., to the culture medium. 1

It is beneficial to make one pre-culture on a small scale and then inoculate the fermentation medium with this pre-culture.

The culture medium's temperature, cultivation time, pH, degree of aeration, stirring speed and other cultivation conditions will vary. share. depending on factors such as the microorganism used, the composition of the medium, etc., and everything else. need, to do is to choose and maintain such cultivation conditions that the production of the desired lipase is maximal..

E.g. the cultivation can take place at a temperature between 15 and 45°C, preferably 20-40°Ci. 10-240 hours, preferably 15-168. hours, at a pH value in the range 4-10, preferably - 5, 5-8 - ... and preferably 6-8, aeration 0.5-5 l/min./l medium, preferably 0.5 -3 l/min./l medium, stirring at 50-400 revolutions -per minute..

Cultivated in the above manner, the lipase-producing species will develop and accumulate a lipase in the culture medium. When using liquid medium, the lipase is mostly collected in the liquid phase of the culture medium. In this case, it is beneficial to remove the cells using; a filter press or centrifuge and work up the lipase from the resulting filtrate or clear liquid.

The lipase solution in the form of filtered culture or

-extract can be concentrated under reduced pressure.

For extraction of the lipase, the known separation and purification processes can be used and the lipase can be obtained in the desired purity. The relevant product can be isolated by subjecting the lipase-containing solution to fractional precipitation with an inorganic salt such as ammonium sulphate, sodium sulphate or sodium chloride or alternatively by adding a suitable hydrophilic organic solvent such as alcohols (eg methanol, ethanol or isopropanol) or acetone to the same solution. The droppings can be further cleaned in otherwise known manner. You can also use such cleaning methods as adsorption-desorption on calcium phosphate gel, aluminum oxide, bentonite, ion exchange resin, etc.; chromatography on diethylaminoethyl cellulose, carboxymethyl cellulose or other cellulose derivatives; molecular sieving (eg dextran such as "Sephadex" or "Biogel"), etc; precipitation with a protein precipitant such as a nucleic acid, tannin, phosphorus tungstate, etc.; isoelectric precipitation; dialysis; electrodialysis; electrophoresis; removal of contaminants with a heavy metal salt such as lead acetate, zinc acetate, barium acetate, calcium acetate, etc. In this way, lipase preparations can be obtained in the desired degree of purity as <solution, powder or crystals.

The produced lipase differs from other known lipases by the considerable increase in activity in the presence of bile, e.g. increases activity to not less than 150% of original activity in the presence of dog bile diluted to 1/200, measured under conditions at 37°C in 0.M phosphate buffer at pH 7.0 against olive oil (Japanese pharmaceutical grade, abbreviated J.P.) as substrate after 50 minutes of enzyme reaction.

The produced lipase is suitable, e.g. as an ingredient in digestives for humans or animals. The lipase is used for the treatment of e.g. dyspepsia, functional dyspepsia, acute post-operative dyspepsia and chronic pancreatitis. Preparations can contain a larger or smaller portion of lipase together with or without other digestive agents. Digestive agents can be given in the manner that is well known for the administration of known lipase-containing digestive agents. The usual daily dose of the lipase is in the range of 20-6000 units per adult. •- -'' r

For further illustration of the invention, the following examples are given, where "parts" are based on weight, unless otherwise stated, and the ratio between "parts" and "volume parts" corresponds to the ratio between grams and milliliters. Millimicron is abbreviated "my" and micron is abbreviated "y".

Definition of lipase unit: It should be noted that the strength of lipase in the examples is determined according to the following procedure: A substrate is prepared by mixing 2% polyvinyl alcohol and olive oil (J.P.). in the ratio 3:1 (v/v) and homogenizing the mixture in a homogenizer at 4°C for 10 minutes. An L-tube is then filled with 5 ml of this substrate and 4 ml of 0.1 M phosphate buffer (pH 7.0), followed by the addition of 1 ml of the lipase solution to be examined. The reaction is allowed to take place in a Monod shaker at 37°C for 50 minutes. After this time, the reaction is terminated by adding 20 ml of a mixture of acetone and ethanol (1:1 v/v) and the reaction system is immediately titrated with a 0.05 N aqueous solution of NaOH against 1% phenolphthalein as indicator. A lipase unit (1 U) is defined as the amount of enzyme that produces an amount of free fatty acids equivalent to one micromole of oleic acid per minute, and the lipase's specific activity is expressed as units per mg lipase (U/mg).

Example

A fermentation tank containing 200 parts by volume is filled with 40 parts by volume of liquid medium (pH 6.0) containing 2.0% wheat bran, 0.5% rice bran, 8.0% corn soaking water, 0.5% KH2P04# 0.2% CaCl2, 2H20 and 0.1% "Actcol" (polymer of glycerol and propylene oxide). After sterilization, the tank is inoculated with one of the species listed below and incubated at 24°C with stirring at 240 revolutions per minute. minute and ventilation in a quantity of 1.0 parts of air per minute per 1.0 part medium. The supernatant of the culture medium is measured for lipase activity. The results are listed below. It is found that all the aforementioned microorganisms form and accumulate lipase which showed high activity, at approx. pH 7.

Each of the species indicated below is cultivated for 72 hours in a similar manner as described above and ammonium sulphate is added to the above liquid to a concentration of 50%. The precipitate is desalted by dialysis against distilled water at 4°C and the dialysis solution is tested for lipase activity with regard to optimal pH and temperature and, with regard to an increase in activity when dog bile is added. The results appear below. One can see that the enzymes formed and collected from the organisms are all identical.

A medium (pH .6, 0, 20,000 parts by volume) containing 2% dextrin, 2% corn steeping water, 0.5% KH2PO4, 0.05% MgSO4-7H2O

and 0.1% "Actcol", is inoculated with 500 parts by volume medium of the same

composition in which Phycomyces nitens had been suspended.

NRRL 2444.

In a container with a volume of 50,000 parts, the inoculated medium is incubated at 28°C for 24 hours. 6,000 parts by volume of the above grafting should be added to 100,000 parts by volume of medium

(pH 6.0) containing 1% wheat bran, 1% rice bran, 5% po 1 y pep ton>;;.'

0.5% KH2P04, 0.2% CaCl2-2H20 and 0.1% "Actcol" in a tank with

volume content 200,000 parts by volume. The medium is incubated during the following

conditions: Incubation temperature: 24°C, ventilation 2 l/minute/,

medium, stirring at 200 revolutions per minute and 66 hours.

The pH values of the medium and the production of lipase measured with . ■ . certain intervals during cultivation appear below.

After 66 hours of cultivation according to the above method, the culture medium is cooled to approx. 5°C and the cells are removed through a filter press with added diatomaceous earth ("Hyflo Super-cel"), and you get approx. 80,000 parts by volume filtrate. To the filtrate is added '. ammonium sulphate to a concentration of 50% and the precipitate centrifuged at 9000 revolutions per minute for 10 minutes.-.The sediment is desalted by dialysis against cold water, using a fish skin membrane, and the dialysate is freeze-dried to 178 parts of crude enzyme as a grey-white powder. The lipase activity for this sample is 55 U/mg.

In 2,000 parts by volume of a 10% aqueous solution of ammonium sulphate, 178 parts of crude enzyme prepared above are dissolved and the solution is filled onto a column containing "Asmit 173 NP"

(styrene resin). The enzymes retained in the column are eluted with 6,000 parts by volume of 10% aqueous ammonium sulphate solution and the flow through and the eluate are combined. Ammonium sulphate is added to the total solution to a concentration of 50% and the resulting precipitate is dialysed against 0.02 M acetate buffers (pH 5.0). The dialysis residue is then applied to a column of diethylaminoethyl cellulose ("Serva Entwicklungslabor") in equilibrium with 0.02 M acetate buffer, pH 5.0, and ammonium sulfate is added

to the flow-through liquid to a concentration of 60%. The enzyme fraction is obtained in this way as a precipitate and the precipitate is dialysed

■ -3

against 0.02 M acetate buffer containing 2 x 10 M CaCl2 (pH 5.0). The dialysis residue is adsorbed on a column filled with dextran (carboxymethyl cellulose, "Sephadex C-50") set in equilibrium with the same buffer. The column is then eluted with 0.15 M NaCl solution in the same buffer and 600 parts by volume are obtained with 0.15 M NaCl solution in the same buffer and 600 parts by volume of lipase-rich fraction are obtained. The eluate is desalted by dialysis with distilled water through a cellophane membrane at 4°C and the dialysate is freeze-dried to 0.7 parts of purified enzyme. This product's specific activity is 2400 U/mg. The general characteristics of this product are listed below:

A) Color and form: white powder

B) Typical elementary analysis:

C, 46.12% ±0.26 H, 6.51% ± 0.07 N, 13.80% ± 0.20 C) Typical molecular weight (Andrew's method, Biochem. J. 91, 222 (1964): 25 000 ± 1000.

D) UV absorption spectrum: As can be seen from figure 1, max

1%

absorption at 275 to 280 my, E_0l_ . = 7.7.

J p' 280my, 1 cm

E) IR spectrum: As can be seen from Figure 2 (KBr disc), there are clear IR absorption peaks, in microns;

3.0 (strong), 3.38 (medium), 6.05 (broad,.strong),

6.55 (broad, strong), 6.88 (weak), 7.15 (broad, medium), 8.13 (medium) and 9.3 (broad, weak). r?;-/ F) Isoelectric point (electrofocusing method): pH 6.3 to 7.0. G) Solubility: Soluble in water and aqueous salt solutions with an ionic strength of 0.01 to 0.1: insoluble in alcohols, acetone and ether.

H) Effect of pH on the activity:

Optimal pH lies within 5 to 8, measured at 37°C i

.50 minutes against olive oil (J.P.) as substrate. The effect of pH on the activity is measured in the same way as indicated earlier under "definition of lipase unit" and can be seen from Figure 3.

I) Effect of temperature on the activity:

Optimal temperature is; from 35 to 41°C, which is measured in 0.1 M phosphate buffer at pH 7.0 for 50 minutes against olive oil (J.P.) as substrate. The effect of temperature on stability is measured in the same way as previously stated in "definition of

lipase unit" and appears in figure 4.

J) Effect of pH on stability.

Residual lipase activity (%) after standing for 2 hours at 23°C at different pH can be seen in figure 5. The enzyme is

stable over the range pH 4 to pH 10.

K) Thermostability:

Residual lipase activity (%) after 10 minutes of heating in the presence of 0.1 M phosphate buffer at pH 7 and different temperatures is shown in Figure 6. The enzyme is stable at temperatures up to 40°C, but is inactivated at 30% at 45°C

and is almost completely inactivated at 50°C.

L) Effect of bile:

The lipase activity measured against olive oil (J.P.) increases up to no less than 150% compared to the intrinsic activity when you add dog bile diluted in a ratio of 1/200.

Typical experiments are listed below.

The bile is collected directly from the gallbladder of a crossbred dog (male dog) with a syringe and diluted to .1/50, 1/100 and 1/200, after which these solutions are added to

sample lots of the lipase-substrate system for. to compare the changes in enzyme activity.

As can be seen from the table below, the activity of the present lipase is clearly increased by the addition of bile. Conversely, the activity of a comparative lipase extracted from Candida cylindracea (Agr. Biol. Chem. 30, No. 11, 1097

(1956) inhibited by bile.

H') Effect of pH on the activity: Optimal pH lies within

5 to 8, measured at 37°C for 50 minutes against olive oil (J.P.)

as substrate. The effect of pH on the activity is measured as described in "Definition of lipase unit" and can be seen in Figure 9.

.1'.);, Effect of temperature on the activity: Optimum temperature éf from 35 to 41°C, measured in 0.1 M phosphate buffer at pH 7.0 for 50 minutes against olive oil (J.P.) as substrate. Effect of temperature on activity is measured in the same way as described above under "Definition of lipase unit"

and appears in Figure 10.

J<1>) Effect of pH on stability: Residual lipase activity (%)

after standing for 2 hours at 23°C at different pH can be seen in Figure 11. The enzyme is stable in the range pH 4 to pH 10.

K') Temperature stability: Residual lipase activity after 10 minutes of treatment of the enzyme solution at pH 7 and different temperatures is shown in figure 12. The enzyme is stable at temperatures up to 40°C, but is inactivated by 30% at 40°C and

almost completely at 50°C.

L<1>) Effect of inhibitor: The lipase activity in solution containing 0.05% (w/v) lipase and 5 x 10 mol diisopropylfluorophosphate (DFP), methylenediaminetetraacetic acid (EDTA) or p-chloromercuribenzoate (PCMB) is measured by stand at pH 5 to 8 and 4°C for 1 hour. The results are as follows:

As stated above, the lipase is stable to these reagents. Therefore, the lipase is not a so-called metal enzyme, SH enzyme or serine enzyme.

M<1>) Results of the amino acid analysis:

(i) has no cysteine residue.

(ii) has two S-S bonds.

(iii) has two tryptophan residues.

In 24 parts by volume of distilled water, dissolve 2.4 parts of enzyme prepared as above. The solution is run through, a column filled with cellulose powder, 200 to 300 mesh, the cellulose is equilibrated with 0.02 M acetate buffer (pH 5.0) as in--3

holds 2 x 10 . M CaCl~. The column is then washed with 0.02 M acetate buffer (pH 5.0) containing 2 x 10 M CaCl,,, and 200 parts by volume of eluates are obtained. Ammonium sulphate up to 50% (w/v) is added to the eluate and a precipitate is formed. The precipitate is centrifuged (9000 revolutions per minute, 10 minutes) and dialyzed against the acetate buffer for 72 hours. 67 volume parts of the dialysis residue are passed through a column filled with dextran (carboxymethyl-"Sephadex C-50", in equilibrium with said acetate buffer) for adsorption of the enzyme.

The column is eluted with a solution of 0.1 M sodium chloride and 23 parts by volume of lipase fraction are obtained. The fraction is allowed to stand at 4°C overnight and 0.185 parts of crystals of the enzyme can be collected. The product's specific activity is 37 50 U/mg.

The general characteristics of this product are:

A'.) Color: white

B<1>) Typical elementary analysis:

C, 47.05% 0.05

H, 6.48% ± 0.02

N, 13.99% ±0.01

C) Typical molecular weight (Andrew's method, Biochem. J., 91, 222 (1964) and S.D.S. polyacrylamide gel electrophoresis, J. Biol. Chem., 244, 5074 (1969)

26,000 ± .1000

D1) UV absorption spectrum: As can be seen from figure 7, maximum

absorption at 275 to 280 my, ^280 my lem = 10.1.

E<1>) IR spectrum (as can be seen from Figure 8 (KBr disc):

absorption peaks in microns as follows:

3.0 (strong), 3.38 (medium), 6.05 (broad, strong),

6.55 (broad, strong), 6.88 (weak), 7.15 (broad, medium), 8.12 (medium) and 9.3 (broad, weak).

F') isoelectric point (electrofocusing method):

pH 5.8 to 6.7.

G') Solubility: Soluble in water and aqueous salt solutions with an ionic strength of 0.01 to 0.1, insoluble in alcohols,

acetone and ether..

(iv) has no saccharide moiety..

(v) has no lipid residue.

N<1>) Effect of bile: The lipase effect measured against olive oil (J.P.) is increased up to 150% compared to the activity -

without addition, by adding dog bile diluted in a ratio of 1/200.

The typical experiments are carried out in the same way as

previously described under "(L) action of bile".

The results appear below:

Note: Olive oil (J.P.) is used as substrate.

Claims (1)

1. Process for the production of lipase by cultivating a microorganism in a culture medium, after which the accumulated lipase is separated from the culture medium, characterized by using a strain of the species Phycomyces nitens or Phycomyces blakesleeanus as the microorganism.