WO1997023604A1

WO1997023604A1 - Method for harvesting a protein

Info

Publication number: WO1997023604A1
Application number: PCT/US1996/020035
Authority: WO
Inventors: Christophe Andre
Original assignee: Genencor International, Inc.
Priority date: 1995-12-22
Filing date: 1996-12-20
Publication date: 1997-07-03
Also published as: AU1422697A; BE1009844A3

Abstract

The invention concerns a method for harvesting a microbial protein using a nonionic surfactant and an alcohol. More particularly, the invention relates to a method for harvesting a microbial protein from the culture of a microorganism which produces this protein, this method comprising the following steps: a) the aqueous fraction and the soluble impurities are eliminated from the culture at the end of the fermentation, the solid fraction is kept; b) a nonionic surfactant and an alcohol are added to the solid fraction, resulting in a suspension; and c) the suspension is subjected to a treatment allowing the elimination of the residual impurities and the microorganisms, the result being a composition containing the purified and concentrated microbial protein, the nonionic surfactant and the alcohol.

Description

METHOD FOR HARVESTING A PROTEIN

The invention relates to a method for harvesting a microbial protein from the culture of a microorganism which produces this protein.

The invention also concerns a composition containing the microbial protein obtained by the harvesting method.

Industrially, the microbial proteins are produced by culturing a microorganism in a fermenter containing an adequate liquid nutrient medium. The culture obtained at the end of the fermentation contains a certain quantity of the desired protein, but also other proteins, as well as the cellular biomass, various impurities and products of the degradation of the nutrient medium. Once the fermentation is finished, it is thus necessary to separate these different products to recover the desired protein. In addition, the protein is treated so as to obtain a commercial preparation which meets the desired criteria of purity and stability.

These treatments are known operations such as centrifugation, filtration, evaporation, precipitation, drying. These treatments are adapted as a function of the characteristics of the desired protein.

In this context, various harvesting methods have already been proposed. Thus, a method for harvesting an enzyme from the culture of a microorganism is described in U.S. Patent No. 4,673,647. The precipitation agent is added to the culture at the end of the fermentation. A polyol is added to the precipitate formed which contains the enzyme. The result is a solution containing the polyol and the enzyme. A method for harvesting a lipase produced by a strain of Fusarium oxysporum is also described in European Patent Application No. 0,130,064. The pH of the culture, which is obtained at the end of the fermentation, is adjusted to 8.0. Then the culture is flocculated by the addition of calcium chloride, flocculation agents and Triton X-100 (0.5% weight/volume). The flocculated culture is centrifuged. Then the centrifugation pellet is resuspended, the pH of this suspension is adjusted to 4.5. A precipitate forms. After a repeat centrifugation, Triton X-100 is added to the centrifugation supernatant. The preparation is again centrifuged. The centrifugation pellet is resuspended, and then diafiltered. The suspension obtained is then ultrafiltered and concentrated. The filtrate obtained is again filtered, and then precipitated with acetone. This method is relatively complicated. The very numerous steps inevitably result in product loss, and thus a low yield, but also in alterations of the lipase, such as alterations of the structure of the protein and of the properties of the lipase. The final product obtained has a low and deteriorated enzymatic activity and it is, in addition, unstable.

Similarly, another method for harvesting an enzyme from the culture of the microorganism which produces this enzyme is described in European Patent

Application No. 0,574,050. To the culture, at the end of the fermentation, at least two nonionic surfactants are added, such as Triton X-114, a flocculation agent and a salt, such as chloride, magnesium, calcium or sodium sulfate or phosphate. Three phases are obtained: an aqueous phase, a detergent phase containing the enzyme and a solid phase containing the biomass. To separate these phases, it is necessary to be in possession of a special separation apparatus capable of separating three phases, such as a decanter equipped with two liquid outlets and a solid outlet. In addition, the detergent phase containing the enzyme and the surfactants is particularly viscous because it contains a large quantity of surfactants (more than 50 g/L of surfactants are in fact used), and it is therefore difficult to treat. This method is necessarily followed by other separation steps to obtain an industrially acceptable enzymatic composition. Moreover, this method presents the drawback of generating large volumes of wastes, containing nonnegligible quantities of useful substance such as, notably, the surfactants and organic solvents. Consequently, this method does not allow the production of an enzyme- rich composition in an economical and ecological manner.

Consequently, there is currently a need for a method for harvesting a microbial protein which is simple to apply, economical, rapid, and which allows the obtention of a high yield of protein. There is also a need for a method for harvesting a microbial protein which allows the obtention of this microbial protein without alteration of its structure and of its properties and with a high activity, notably, in the case of an enzyme, with a high enzymatic activity. The invention is intended to overcome these drawbacks of the known methods described above by supplying an improved method which permits the obtention, in an economical manner, from the culture of a microorganism, of a microbial protein without generating voluminous waste materials and without prohibitive losses of useful substance.

The first purpose of the present invention is to supply a method for harvesting a microbial protein from the culture of a microorganism which produces this protein, this method being simple to use and requiring no special separation apparatus. Another purpose of the present invention is to supply a method for harvesting a microbial protein which allows the obtention of the protein at a high yield.

Another purpose of the present invention is to supply a method for harvesting a microbial protein, which method is economical, requiring no complicated recycling and using an industrially and economically reasonable quantity of products.

A purpose of the present invention is to supply a method for harvesting a microbial protein which is at the same time a method for the concentration and the purification of the protein. In addition, a purpose of the invention is to supply a method for harvesting a microbial protein which allows the obtention of a practically pure microbial protein, that is, one containing practically no contaminating proteins.

A purpose of the present invention is to supply a method for harvesting a microbial protein allowing the direct obtention of a stabilized and concentrated protein composition.

Another important purpose of the present invention is to supply a method for harvesting a microbial protein which allows the direct obtention of the finished product, that is, a protein composition which can be sold as is without subsequent treatment. The purpose of the present invention is to prepare and to supply a protein composition obtained by the harvesting method described above.

For this purpose, the invention concerns a method for harvesting a microbial protein from the culture of a microorganism which produces this protein, characterized in that the method comprises: a) a first step in which at least a part of the aqueous fraction and a part of the soluble impurities contained by the culture is eliminated from the culture of the microorganism, and a suspension is obtained which contains the microorganisms and the protein, b) a second step in which at least one nonionic surfactant and an alcohol are added to the suspension obtained in the first step, and a suspension is obtained which contains the microorganisms, the protein, the nonionic surfactant and the alcohol, and c) a third step in which the microorganisms are eliminated from the suspension obtained in the second step, and a composition is obtained which contains the microbial protein, the nonionic surfactant and the alcohol.

Thus, the invention allows the obtention of the purified and concentrated microbial protein.

A microbial protein is defined as any protein produced by a microorganism. Generally, the microbial protein is a hydrophobic protein. Usually, the microbial protein is an enzyme. Preferably, the microbial protein is an enzyme selected from the hydrolases, transferases, oxidoreductases and isomerases. It is particularly preferred for the microbial protein to be a hydrolase.

It is very particularly preferred for the microbial protein to be an esterase, such as a lipase, phosphatase, cutinase, a lipoprotein lipase; a glycosidase, such as glucosidase, a dextranase, a cellulase, an amylase, a galactosidase, a pullulanase, a xylanase, [or] a peptidase, such as a protease. Excellent results were obtained with a lipase.

This definition includes the natural proteins and the modified proteins, such as the proteins whose nucleotide or amino acid sequence was modified by the techniques of genetic engineering or by the techniques of mutagenesis.

Generally, the microbial protein originates from a bacterium, a fungus or a virus. Usually, the microbial protein originates from a bacterium or a fungus.

Preferably, the protein originates from a bacterium selected from Bacillus, Pseudomonas, Alcaligenes, Streptococcus and Streptomyces, or from a fungus selected from Aspergillus, Geotrichum, Humicola, Trichoderma, Penicillium, Rhizopus and Fusarium. It is particularly preferred for the protein to originate from a bacterium selected from Bacillus, Pseudomonas and Alcaligenes. Good results were obtained when the microbial protein originates from a Pseudomonas sp. Excellent results were obtained when the microbial protein originates from a strain of Pseudomonas wisconsinensis, notably from the Pseudomonas wisconsinensis strain T 92.677/1 (LMG P- 15151).

The definition includes the microbial proteins originating from microorganisms which are derivatives or mutants. A derivative of a microorganism is any naturally modified microorganism, that is, one modified by a natural selection. The mutant of a microorganism is any artificially modified microorganism. The mutants can be obtained by known modification techniques, such as ultraviolet radiation, X-rays, mutagenic agents or genetic engineering. These techniques are known to a person skilled in the art and described notably in SAMBROOK et al. , 1989, Chapter 15. Examples of mutagenic agents are notably described by R. Scriban, Biotechnologie [Biotechnology], (Technique et Documentation Lavoisier), 1982, pp. 365-368.

In general, the microbial protein is extracellular, intracellular or bound to the cell membrane. Usually, the microbial protein is bound to the cell membrane. According to the invention, the first step comprises the elimination of at least a part of the aqueous fraction and a part of the soluble impurities contained in the culture of the microorganism.

This first step allows the elimination of a large part of the aqueous fraction, and preferably a majority of the aqueous fraction. This first step allows the elimination of a large part of the soluble impurities, and preferably of a majority of the soluble impurities.

The suspension obtained in the first step is in the form of a pellet or a cake, depending on the technique used.

This first step can be carried out by techniques known to a person skilled in the art such as centrifugation, filtration, precipitation, drying or a combination of these techniques. Examples of such techniques are described notably by R. Scriban, Biotechnologie, (Technique et Documentation Lavoisier), 1982, pp. 267- 276.

Generally, the first step is carried out using a technique selected from centrifugation, filtration, precipitation, or a combination of these techniques. Usually, the first step is carried out using a technique selected from centrifugation, filtration which includes ultrafiltration, microfiltration and/or diafiltration, or a combination of these techniques. Preferably, the first step is carried out by a centrifugation or filtration.

Advantageously, the first step comprises the addition of a flocculation agent to the culture. Generally, the flocculation agent is selected from polymers of acrylamide, copolymers of acrylamide and acrylate, quaternary polyamines and polysaccharides. Usually, the flocculation agent is selected from the quaternary polyamines. Preferably, the flocculation agent is selected from the quaternary polyamines sold under the trade name OPTIFLOC. Good results were obtained with the flocculation agent sold under the trade name OPTIFLOC FC205 (SOLVAY).

According to the invention, the second step consists of the addition of at least one nonionic surfactant and an alcohol to the suspension obtained in the first step.

A nonionic surfactant is an amphiphilic compound which does not dissociate into ions in an aqueous solution. The nonionic surfactant is selected from ethoxylates, esters of fatty acids and polyhydroxylated compounds, and amine oxides. Generally, the nonionic surfactant is selected from ethoxylates.

Ethoxylates are generally known under the name of poly ethoxylated aliphatic alcohols (Ullman's Encyclopedia of Industrial Chemistry, 1994, 5th edition, Vol. A25, pages 785-787) or ethoxylated fatty alcohols.

Usually the nonionic surfactant is selected from the polyethoxylated aliphatic alcohols containing approximately 10-20 carbon atoms and having an ethoxylation degree of approximately 2-40. Preferably, the nonionic surfactant is selected from the polyethoxylated aliphatic alcohols containing approximately 10- 18 carbon atoms and having an ethoxylation degree of approximately 2-20. It is particularly preferred to select the nonionic surfactant from the polyethoxylated aliphatic alcohols containing approximately 12-14 carbon atoms and having an ethoxylation degree of approximately 3-11. It is particularly preferred to select the nonionic surfactant from the polyethoxylated aliphatic alcohols containing approximately 12-14 carbon atoms and having an ethoxylation degree of approximately 3-9. Good results were obtained with a nonionic surfactant selected from the polyethoxylated aliphatic alcohols containing approximately 12-14 carbon atoms and having an ethoxylation degree of 3, such as the product sold under the trade name MARLIPAL 24/30 by Huls AG; the polyethoxylated aliphatic alcohols containing approximately 12-14 carbon atoms and having an ethoxylation degree of 6, such as the product sold under the trade name MARLIPAL 24/60 by Huls AG; the polyethoxylated aliphatic alcohols containing approximately 12-14 carbon atoms and having an ethoxylation degree of 7, such as the product sold under the trade name MARLIPAL 24/70 by Hiils AG, and the polyethoxylated aliphatic alcohols containing approximately 12-14 carbon atoms and having an ethoxylation degree of 9, such as the product sold under the trade name MARLIPAL 24/90 by

Huls AG.

Excellent results were obtained with the polyethoxylated aliphatic alcohols containing approximately 12-14 carbon atoms and having an ethoxylation degree of 9, such as the product sold under the trade name MARLIPAL 24/90 by Huls AG. Generally, less than 10 g of nonionic surfactant are used per liter of culture. Usually, less than 5 g of nonionic surfactant are used per liter of culture.

Preferably, less than

1 g of nonionic surfactant is used per liter of the culture.

Generally, more than 0.01 g of nonionic surfactant is used per liter of culture. Usually, more than 0.05 g of nonionic surfactant is used per liter of culture. Preferably, more than 0.1 g of nonionic surfactant is used per liter of culture.

It is particularly preferred to use 0.1-1 g of a nonionic surfactant per liter of culture. An alcohol is a compound containing at least one functional -OH group.

Generally, an aliphatic alcohol is used.

Usually, a polyol is used. Preferably, a diol or a triol is used. It is particularly preferred to use 1 ,2-ethanediol, 1 ,2-propanediol, 1 ,3-butanediol or glycerol.

Excellent results have been obtained with 1,2-propanediol (propylene glycol). Generally, less than 75% (volume) of alcohol is used per volume of culture. Usually, less than 60% (volume) of alcohol is used per volume of culture. Preferably, less than 50% (volume) of alcohol is used per volume of culture.

In general, more than 5% (volume) of alcohol is used per volume of culture. Usually, more than 10% (volume) of alcohol is used per volume of culture. Preferably, more than 15% (volume) of alcohol is used per volume of culture.

It is particularly preferred to use 10-50% (volume) of alcohol per volume of culture.

In a preferred variant of the invention, at least one salt is added during the second step of the method.

A salt is a compound consisting of an anion and a cation.

Generally, the salt is a mineral salt.

Usually, the salt is selected from the group of compounds in which the cations are sodium, potassium, magnesium and ammonium. Preferably, the salt is selected from the group of compounds in which the cations are sodium and magnesium.

Usually, the salt is selected from the group of compounds in which the anions are sulfates, chlorides, phosphates, carbonates, citrates and a mixture thereof. Preferably, the salt is selected from the group of compounds in which the anions are sulfates, chlorides, phosphates and mixture thereof.

It is particularly preferred for the salt to be a calcium chloride, a sodium chloride or a mixture thereof. Excellent results were obtained with a mixture of calcium chloride and sodium chloride.

Generally, less than 100 mM of salt per volume of culture are used. Usually, less than 80 mM of salt per volume of culture are used. Preferably, less than 70 mM of salt per volume of culture are used.

Generally, more than 5 mM of salt per volume of culture are used. Usually, more than 10 mM of salt are used per volume of culture. Preferably, more than 20 mM of salt per volume of culture are used.

It is particularly preferred to use 20-70 mM of salt per volume of culture. Good results were obtained using 20-40 mM of sodium chloride and 10-20 mM of calcium chloride per volume of culture.

In a preferred variant of the invention, at least one buffer is added during the second step of the method. Generally, the buffer is a buffer having a pH of approximately 7-12. Usually, the buffer is a buffer having a pH of approximately 7.5-11. Preferably, the buffer is a buffer having a pH of approximately 8-10.5. It is particularly preferred for the buffer to be a buffer having a pH of approximately

8.5-10. Good results have been obtained with a buffer having a pH of approximately 9.5. Excellent results were obtained with 2-amino-2-hydroxymethyl- 1,3-propanediol (tris at pH 9.5).

In the particularly preferred variant of the invention, a nonionic surfactant, an alcohol, a salt and a buffer are added during the second step of the method.

Preferably, the nonionic surfactant is a polyethoxylated aliphatic alcohol containing approximately 12-14 carbon atoms and having an ethoxylation degree of 9. Preferably, the alcohol is 1,2-propanediol. Preferably, the salt is calcium chloride and sodium chloride. Preferably, the buffer is a buffer at pH 9.5, such as 2-amino-2-hydroxymethyl- 1 ,3-propanediol .

During the second step, the suspension can be stirred or not. If the suspension is stirred, the stirring is preferably axial stirring. Usually, the stirring is at approximately 5-200 rpm. Preferably, the stirring is at approximately 10-100 φm. The second step is carried out at a temperature which does not alter the microbial protein. Generally, the second step is carried out at a temperature of approximately 2-40°C. Usually, the second step is carried out at a temperature of approximately 5-30°C. Preferably, the second step is carried out at a temperature of approximately 10-20°C. According to the invention, the third step comprises the elimination of the microorganisms, from the suspension obtained in the second step, resulting in a composition containing the microbial protein, the nonionic surfactant and the alcohol.

During this third step, the suspension obtained is subjected in the second step to a treatment allowing the elimination of the microorganisms. Such a solid- liquid separation treatment is known to a person skilled in the art. Examples of treatment are notably centrifugation, filtration, or a combination of these techniques. Examples of such techniques are described by R. Scriban, Biotechnologie, (Technique et Documentation Lavoisier), 1982, pp. 267-276.

This treatment can be carried out by a technique selected from centrifugation, filtration which includes ultrafiltration, filtration through a filter press, microfiltration and/or diafiltration, or a combination of these techniques. Preferably, this treatment is carried out by centrifugation or filtration.

The invention also concerns a composition containing the microbial protein obtained by the harvesting method. The composition according to the invention also contains additives.

The additives, included in the composition according to the invention, are known to a person skilled in the art and they are selected depending on the use for which the composition is considered. They must be compatible with the microbial protein and not affect its activity. In the case of an enzyme, they must not affect the enzymatic activity of this enzyme. Usually, these additives are stabilizers, preservation agents and formulation agents.

However, in most cases it is no longer necessary to add stabilizers. In fact, one of the advantages of the invention resides in the obtention of an already stabilized composition, since it contains a nonionic surfactant, an alcohol and salts, the latter being known to be compounds which stabilize the proteins.

Examples of additives, added to detergent compositions, are described in particular in United Kingdom Patent Application No. 1,372,034, and European Patent Application Nos. 0,218,272, 0,430,315, Nos. 0,341,999, International Patent Application WO 94/03578, WO 94/25556 and WO 91/00910. The composition according to the invention has multiple outlets in various industries, such as, for example, the food, pharmaceutical and chemical industries. The composition according to the invention containing the lipase can notably be used in detergency. The present invention also concerns the use of the composition containing the lipase, as defined above, in detergency. In this context, it is a part of detergency compositions.

The present invention thus also concerns the detergency compositions containing the lipase. The components of the detergency compositions are known to a person skilled in the art and they are adapted depending on the use for which the composition is considered. Such components are notably enzymes, such as, for example, proteases, amylases and/or cellulases, fillers, such as sodium tripolyphosphate, bleaching agents, such as perborate; formulation additives, surfactants. The detergency compositions of the invention can be used, depending on their formulation, as a washing powder, granules or liquid to wash clothes; as a spot-removing product to remove spots or degrease the objects or to remove spots from linen before cleaning; and as a powder, granules or liquid for dishwashing. The detergent compositions according to the invention can also contain other substances selected as a function of the special field of application of the detergent composition. They include optical brighteners, tarnishing inhibitors, antiredeposition of soiling agents, disinfectants, corrosion inhibitors, perfumes, dyes, pH regulators.

The detergent compositions of the invention can be used, depending on their formulation, as a washing powder, granules or liquid to wash clothes; as a spot-removing product to remove spots or degrease objects or to remove spots from linen before washing; as a powder, granule or liquid for dishwashing. The present invention is illustrated by the following examples.

Example 1 : Production of the lipase by the Pseudomonas wisconsinensis strain T

92.677/1

The Pseudomonas wisconsinensis strain T 92.677/1 is cultured in a Petri dish containing the agar medium A at 30°C for 24 h.

The Pseudomonas wisconsinensis strain T 677/1 was deposited at the collection called BELGIAN COORDINATED COLLECTIONS OF

MICROORGANISMS (LMG culture collection) under No. LMG P-15151 on October 12, 1994. The agar medium A contains 10 g/L of tryptone (DIFCO), 5 g/L of yeast extract, 5 g/L of NaCl, and 20 g/L of agar, 2.5 g/L of NaHCO₃, 7.5 g/L of Na₂CO₃. The tryptone, yeast extract, NaCl, and agar which comprise the medium A are mixed with 900 mL of distilled water and then sterilized for 30 min at 121 °C. The pH is adjusted at 9.5 by the addition of 100 mL of carbonate buffer which has first been sterilized (containing 25 g/L of NaHCO₃ and 75 g/L of Na₂CO₃).

Then, from this culture, a culture is prepared in 25 mL of a liquid medium B. The medium B contains 10 g/L 7.0 of tryptone (DIFCO), 5 g/L of yeast extract, 10 g/L of NaCl. The medium is sterilized for 30 min at 121 °C, the pH of the medium is adjusted at 9.4 by adding 10% (vol/vol) of a carbonated buffer which contains 53 g/L of Na₂CO₃ and 42 g/L of NaHCO₃. The culturing is carried out in 250-mL flasks, at 30°C with orbital stirring at 200 rpm with an amplitude of approximately 2.54 cm.

After 16 h of incubation, 1 mL of this culture is introduced into a 2000-mL flask containing 200 mL of sterilized liquid medium B. The culturing is carried out at 30 °C with orbital stirring at 200 rpm with an amplitude of approximately 2.54 cm.

After 16 h of incubation, this culture is introduced into 20-L fermenter containing 13 L of sterilized liquid medium C.

Medium C contains K₂HPO₄ 2.5 g/L, KH₂PO₄ 2.5 g/L, MgSO₄ ^■ 7H₂O 1 g/L, (NH₄)₂SO₄ 2 g/L, (NH₂)₂CO 2 g/L, CaCl₂ 1 g/L, soybean meal 20 g/L, yeast extract 2 g/L, glucose 10 g/L, antifoaming agent Mazuόl (MAZES

CHEMICALS) 1 g/L. The pH is adjusted at 8.5 with 5N sodium hydroxide after sterilization in the fermenter (30 min at 121 °C). The medium is sterilized in the fermenter for 30 min at 121 °C. The glucose is sterilized, separately, at a pH of 4.0 (pH adjusted with normal phosphoric acid) for 30 min at 121 °C. The pH of the medium is obtained at 8.5 with 5N sodium hydroxide.

Culturing in the fermenter is carried out at a temperature of 30 °C and at a pressure of 0.15 x 10⁵ Pa (Pa = Pascal) (0.15 bar) with an aeration of 0.3 VVM (volume of air per volume of culture medium per minute) and with an actual stirring of 200 rpm, the regulation of dissolved oxygen being fixed at 10% (vol/vol) of saturation by regulation of the stirring speed.

The abbreviation % (vol/vol) represents a percentage expressed in volume per volume. The abbreviation % (vol/wt) represents a percentage expressed in volume per weight. The abbreviation % (wt/vol) represents a percentage expressed in weight per volume. The abbreviation % (w/w) represents a percentage expressed in weight per weight.

After 50 h of fermentation, the enzymatic activity of the culture so obtained is measured by the technique described below:

The hydrolysis of the triolein is quantified by the neutralization of fatty acids released under the action of the lipase. This measurement is carried out using an automatic titration apparatus, which apparatus maintains the pH constant at the operational value by the addition of 0.01N NaOH. One lipase unit (LU) is defined as the quantity of enzyme which catalyzes the release of a micromole of fatty acid per minute under the standard conditions of the test described below.

10 g of Triolein (ROTH 5423.1) and 10 g of gum arabic (FLUKA 51200) are mixed in 100 mL of distilled water. This mixture is emulsified using an ULTRA-TURRAX mixer at 13,500 rpm (axial stirring) 3 times for 5 min, while maintaining the mixture under nitrogen and in an ice bath.

A dilution buffer is prepared which contains 2.34 g/L of NaCl, 2.94 g/L of CaCl₂ - 2H₂O and 0.61 g/L of tris(2-amino-2 -hydroxymethyl- 1,3-propanediol).

An automatic titration apparatus, equipped with a burette containing 0.01N NaOH, a temperature probe, a pH probe, and a thermostat-regulated reactor, is used.

In the reactor which is thermostat-regulated at 30°C, a magnetic stirring bar, 10 mL of emulsion of triolein and 20 mL of dilution buffer are introduced. The pH of the solution so obtained is adjusted to 9.5 with 0.1N NaOH. Then, 0.5 mL of the sample to be tested containing the lipase is added, the sample being optionally diluted so that it contains at most 5 LU. The pH is controlled during the first two minutes with 0.01N NaOH. Then, the consumption of NaOH is recorded between 2 and 4 min, while maintaining the pH constant (volume of NaOH consumed between 2 and 4 min = VI in mL).

Then, the same test is carried out replacing the sample containing the lipase with 0.5 mL of dilution buffer (volume of NaOH consumed between 2 and 4 min = V2 in mL).

A lipase unit (LU) is determined as follows:

1 LU/mL = (VI -V2) x optional dilution of the sample x 10

Using this method, lipase activity is detected in the culture.

Example 2: Harvesting of the lipase of the Pseudomonas wisconsinensis T

92.677/1 strain

To the culture, as obtained at the fermentation of Example 1 , 1 % (wt/wt) of flocculation agent OPTIFLOC FC205 (SOLVAY) is added in the form of a 10% solution (wt/wt). The mixture is gently stirred (axial stirring at 50 φm) for 1 h at l5°C.

The mixture is centrifuged for 10 min at 8500 φm (BECKMAN J21 , rotor JA10) at a temperature of 4°C. The centrifugation supernatant is eliminated. The centrifugation pellet is dissolved with an elution buffer, 1 mL of culture being dissolved in 1 mL of elution buffer. The elution buffer comprises 3mM tris, 24 mM NaCl, 12 mM CaCl₂, polyethoxylated aliphatic alcohol having

12-14 carbon atoms and an ethoxylation degree of 9 (ethoxylate) (EO (ethylene oxide) group number equal to 9) sold under the trade name MARLIPAL 24/90 (Company Huls AG) 0.3 g/L and propylene glycol 40% (vol/vol).

The mixture is gently stirred (axial stirring at 50 φm) for 15 min at 15 °C, to resuspend the centrifugation pellet.

Then, the suspension so obtained is centrifuged for 10 min at 8500 φm at a temperature of 4°C. The centrifugation pellet is eliminated. The enzymatic activity of the centrifugation supernatant is measured by the technique described in Example 1. The enzymatic activity of the centrifugation supernatant is equal to 116% of the enzymatic activity of the culture as obtained at the end of the fermentation of Example 1. 15

Example 3:

Harvesting of the lipase of the Pseudomonas wisconsinensis strain T 92.677/1

The method described in Example 2 is reproduced exactly, with the exception of the elution buffer.

In this example, the elution buffer used comprises 4mM tris, 32 mM NaCl, 16 mM CaCl₂, MARLIPAL 24/90 0.4 g/L and propylene glycol 20% (vol/vol).

The enzymatic activity of the centrifugation supernatant is equal to 98% of the enzymatic activity of the culture as obtained at the end of the fermentation in Example 1.

Example 4R (comparison): Harvesting of lipase of the Pseudomonas wisconsinensis strain T 92.677/1

The method described in Example 2 is reproduced exactly, except for the elution buffer.

In this example, the elution buffer used comprises 3 mM tris, 24 mM NaCl, and 12 mM CaCl₂. It contains no propylene glycol.

The enzymatic activity of the centrifugation supernatant is equal to 14% of the enzymatic activity of the culture as obtained at the end of the fermentation in Example 1.

Claims

WHAT IS CLAIMED IS:

1. Method for harvesting a microbial protein from the culture of a microorganism which produces this protein, characterized in that this method comprises: a) a first step in which at least a part of the aqueous fraction and a part of the soluble impurities contained by the culture is eliminated from the culture of the microorganism, and a suspension is obtained which contains the microorganisms and the protein, b) a second step in which at least one nonionic surfactant and an alcohol are added to the suspension obtained in the first step, and a suspension is obtained which contains the microorganisms, the protein, the nonionic surfactant and the alcohol, and c) a third step in which the microorganisms are eliminated from the suspension obtained in the second step, and a composition is obtained which contains the microbial protein, the nonionic surfactant and the alcohol.

2. Method according to Claim 1, characterized in that the microbial protein is a hydrophobic protein.

3. Method according to Claim 1 , characterized in that the microbial protein is an enzyme.

4. Method according to Claim 1, characterized in that the microbial protein is a lipase.

5. Method according to Claim 1 , characterized in that the microbial protein originates from a bacterium or a fungus.

6. Method according to Claim 5, characterized in that the microbial protein originates from a Pseudomonas sp.

7. Method according to Claim 6, characterized in that the microbial protein originates from a strain of Pseudomonas wisconsinensis.

8. Method according to Claim 1, characterized in that the nonionic surfactant is selected from the ethoxylates, the fatty acid esters and polyhydroxy lated compounds, and amine oxides.

9. Method according to Claim 8, characterized in that the nonionic surfactant is selected from the polyethoxylated aliphatic alcohols containing approximately 10-20 carbon atoms and having an ethoxylation degree of approximately 2-40.

10. Method according to Claim 9, characterized in that the nonionic surfactant is selected from the polyethoxylated aliphatic alcohols containing approximately 12-14 carbon atoms and having an ethoxylation degree of approximately 3-9.

11. Method according to Claim 1, characterized in that less than 10 g of nonionic surfactant are used per liter of culture.

12. Method according to Claim 1, characterized in that more than 0.01 g of nonionic surfactant are used per liter of culture.

13. Method according to Claim 1, characterized in that 0.1-1 g of nonionic surfactant is used per liter of culture.

14. Method according to Claim 1, characterized in that the alcohol used is an aliphatic alcohol.

15. Method according to Claim 1 , characterized in that the alcohol used is a polyol.

16. Method according to Claim 1, characterized in that the alcohol used is a 1,2-propanediol.

17. Method according to Claim 1, characterized in that less than 75% (volume) of alcohol is used per volume of culture.

18. Method according to Claim 1 , characterized in that more than 5 % (volume) of alcohol is used per volume of culture.

19. Method according to Claim 1, characterized in that 10-50% (volume) of alcohol is used per volume of culture.

20. Method according to Claim 1, characterized in that at least one salt is added during the second step.

21. Method according to Claim 1 , characterized in that at least one buffer is added during the second step.