HK1085747B - Methods for refining concentrated enzyme solutions - Google Patents

Description

Method for refining concentrated enzyme solution

The present invention relates to a process for purifying concentrated industrial enzyme solutions, the products obtained by this process, and compositions based on such solutions, more particularly detergents and cleaning compositions.

Enzymes currently produced, particularly for those industrial applications, are mostly produced by microbial fermentation followed by purification from the medium used. Concentrated enzyme solutions obtained via usually several sequential process steps are often also referred to as "liquid enzymes". Liquid enzymes can be considered as a purified starting material which is either used in liquid form or is sometimes converted to dry form for use in the appropriate application.

Important industrial applications for enzymes, in particular in liquid form, are detergents and cleaning compositions, which are increasingly marketed in liquid or gel form. Other applications include, for example, the cosmetic field, where enzymes are used as activators, as they are used in detergents and cleaning compositions, textile and food manufacture and processing, where the main raw materials are converted into the end product by using enzymes.

Purification or enrichment methods to obtain concentrated enzyme solutions are described in detail in the prior art. Important aims in this connection are the removal of biomass, i.e. components of the host organism, more particularly high molecular weight components, the removal of low molecular weight foreign bodies and impurities, more particularly mediator components and metabolites, and the removal of other proteins, more particularly enzymes. At the same time, it is intended to obtain a target product in large quantities, of high purity and with a high active content. On the other hand, the culture supernatant usually contains factors, usually peptides or proteins, the recognition of which has not been usually reported yet, but which provide stabilization of the target enzyme. Thus, it is of particular advantage to obtain an enzyme solution that is not completely pure, i.e. that contains a certain percentage of such stabilizing factors.

Techniques based on filtration, sedimentation or precipitation are techniques commonly used for purification.

For example, processes have been developed for removing biomass, usually by continuous application, and are now recognized in the art. Such methods include, for example, separation, microfiltration and ultrafiltration. Only then in the context of the present invention may actually be mentioned enzyme concentrates.

For example, international patent application WO01/37628a2 describes a process for recovering a biotechnologically produced useful substance from a culture and/or enzyme solution, said process comprising separating water-insoluble solids from an aqueous solution containing the useful substance, followed by filtration of the resulting solution, and concentration of the solution containing the useful substance by ultrafiltration. The known method is characterized in that the removed solids are subjected to a washing step using the filtrate from the concentration step as washing liquid.

However, there has been no solution to the problem that the enzyme concentration removed from the biomass contains, in particular, the following impurities:

1. precipitation of solids, more particularly irreversibly denatured proteins comprising the target protein,

2. colored, mostly brown compounds which are formed during the pre-fermentation sterilization of the media components, more particularly of the nitrogen source (Maillard compounds), and

3. factors that increase the stability of the target protein,

conventional refining processes are not sufficiently suitable for removing denatured proteins and colored compounds from enzyme concentrates separated from biomass or removing most of the stabilizing factors together with denatured proteins and colored compounds. In either case, the result is an unacceptable product quality: either the enzyme concentrate is dark in color, streaky and/or contains suspended matter up to including precipitated matter, or the color is light to form a clear solution, but the enzyme stability is not satisfactory and is generally improved only to some extent by the addition of (expensive) stabilizing compounds. These disadvantages affect above all the products containing the concentrate in question.

For example, liquid detergents and cleaning compositions should contain a high proportion of active, i.e.stable, enzymes throughout the storage period. However, the water content and stability are inversely proportional to each other. At the same time, the composition in question should have a color and transparent appearance that is appealing to the consumer.

Methods for decolorizing concentrated enzyme solutions are described in the prior art. They involve precipitation procedures, for example using organic solvents or polymers, but more particularly salting out the target protein with sodium sulfate (described in H.Ruttloff (1994): "Industrial rielle Enzyme", Behr's Verlag, Hamburg, Chapter 6.3.3.6, pp 376-379). For example, US5405767 discloses certain compounds which are said to be added to a protein solution for precipitation to obtain a favorable precipitate. The protein was precipitated and the foreign material remained in the supernatant. However, part of the protein is irreversibly denatured, which overall impairs stability by precipitation and resuspension, the reason being of considerable importance for the removal of the stabilizing compound. Values of about 50% are mentioned as yields, as obtained in the table cited on page 378 of the textbook.

Another embodiment is to purify the Enzyme by adsorption, for example, using an ion exchange resin (H.Ruttloff (1994):, "Industrial Enzyme", Behr's Verlag, Hamburg, Chapters 6.3.3.7and 6.3.3.8, pages 379 to 396). In this method, the target protein is bound to the chromatography material and then eluted with additional media. However, the yield obtained is often poor due to deterioration and fold effects. Thus, yields of up to 60% are mentioned for different chromatography in the page table 378 (except for affinity chromatography). Specific chromatography materials, more particularly affinity chromatography materials, are generally more effective, but are sensitive and expensive to manufacture. Thus, affinity chromatography materials are mainly used in the analytical and medical fields, but are almost never used for the production of enzymes on an industrial scale.

For example, patent application WO89/05863A1 describes the recovery of extracellular enzymes from the fermentation broth of a Bacillus strain. This document describes an experiment for removing cell wall polymers by ion exchange chromatography, more particularly in the preparation of amylases. The solution containing the enzyme and the polymer is first applied to the column, then washed with buffer and subsequently eluted with the elution medium. In other words, the alpha-amylase to be purified is initially bound to the chromatography material in the same way as the foreign material, and is then only washed downwards as the ionic strength increases.

The opposite approach, i.e. selectively removing impurities from the liquid solution via the carrier, has so far only been selected for food quality raw materials. Thus, US5972121 discloses the removal of dyes from sugar liquors via weakly acidic or weakly basic adsorption chromatography. In particular, the decolorization of sugars by means of successive different ion exchange chromatography steps is described in the guide "Manual of ionexchange resins and synthetic adhesives, Vol.II ", Mitsubishi Kasei Corp. (Tokyo, Japan), 2nd printing, 1.5.1993, pages 93 to 100. The relative method essentially comprises several purification steps each with extreme selectivity, in which specific removable impurities remain on the respective mass.

US5565348 relates to the recovery of certain alkaline bacitrases and comprises an example describing the purification of those enzymes by a purification process consisting of several steps. These include, in particular, the steps of precipitation of the protein by salting out, absorption of a further medium and dialysis before carrying out the ion exchange chromatography. This is followed by affinity chromatography, a chromatography step, in which the protease binds specifically to the column packing material. With respect to the ion exchange chromatography step, the literature in question indicates that the particular protease described does not bind to the particular chromatography material used and therefore penetrates. However, this does not appear to have a general applicable teaching of purifying the enzyme, since no specific column packing material is specified, no mention is made of the concentration of the enzyme, the actual removal of impurities in the preceding precipitation step, or the assurance by subsequent affinity chromatography. Thus, the purification of enzymes, more particularly proteases, via column packing materials which are not bound to themselves, has hitherto been regarded in the prior art as being essential, above all, as a precipitation step.

The problem underlying the present invention is therefore to liberate enzyme concentrates, more particularly irreversibly denatured proteins, from solids to decolorize the concentrates, so that selectively they remain extremely stable on storage.

This problem is solved by a process for refining a concentrated enzyme solution, said process comprising the steps of:

(a) preparing a concentrated enzyme solution,

(b) scavenging solid phase, more particularly foreign proteins and/or inactive enzymes, and

(c) strongly basic anion exchange chromatography.

No precipitation occurred. In contrast, the target enzyme protein remains in solution throughout the entire process, and a concentration range specified for this purpose is particularly advantageous for the yield to be obtained, as illustrated in the examples below.

Process step (a) is followed by processes known per se and described in the prior art for preparing an enzyme-rich aqueous solution which is substantially free of biological total. In general, this involves several constitutive steps such as cell disruption, granulation of cell debris, decantation and optionally further centrifugation steps. Isolation, microfiltration, ultrafiltration or sterile filtration (see below) and concentration, i.e.removal of the solvent, can be used to obtain the enzyme in the medium concentration range. For the other processes (see below), enzyme concentration values in the range of about half the optimum working concentration as described were considered to be optimal. Also advantageous is a target suspended particle or solids amount of less than 1 vol%, as demonstrated, for example, by centrifugation at 7,000G for 10 minutes on a bench top centrifuge. In addition, the particular solution should be adjusted to a pH tolerable for the enzyme at which the solution has a positive charge.

Step (a) referred to as "concentration" in FIG. 1 is likewise based on methods known per se to the person skilled in the art. For example, a rotary evaporator or a thin-layer evaporator can be used for the concentration. In a particularly advantageous embodiment, the pH is at a substantially constant pre-adjusted value, while the solids content of the enzyme concentrate is kept as low as possible (see below). In addition, the losses in the following step (b) undergo a considerable undesired increase.

Step (a) should be controlled in a known manner, in particular by stopping the concentration process at the correct time, in such a way that the enzyme concentrate obtained still shows just no (quantitative) protein precipitation. The optimum working range must be determined for each enzyme individually, not only with regard to temperature, pH and ionic strength, but in particular with regard to the optimum enzyme concentration range. Examples 1 and 2 of the present application the above-described conditions were tested for the alkaline protease from B.lentus and the alpha-amylase from Bacillus species A7-7(DSM 12368). Therefore, the optimum concentration range for alkaline proteases is defined as 700,000-800,000 HPU/g and 35,000-45,000 TAU/g for alpha-amylase. Beyond these values, the percentage of solids precipitated increases shortly, depending on the activity, over the normal ratio, which is accompanied by a significant increase in the loss of useful product. These effects on the enzymes mentioned are illustrated by way of example in FIG. 2. The dependence of the solids content on the concentration of the protein, in particular on the concentration of the enzyme activity, is to be expected for virtually all industrial enzymes. It must be determined experimentally in each individual case and process which is thus modified by concentration, optionally dilution methods known per se.

As also shown in the examples, this operating range is preferably maintained throughout the process, on the one hand in order to operate the process at high concentrations and thus with high efficiency, and on the other hand with as little enzyme as possible being lost due to spoilage and precipitation. In this way, yields of up to 95% are obtained overall for the process.

As discussed below, the optional step (a) is illustrated in fig. 1 followed by deodorization (a').

The removal of the precipitate (solid) formed in the concentration step in step (b) is particularly applied to the foreign protein and/or the inactive enzyme, particularly in the vicinity of the solubility product. This step is referred to as "separation" in the block flow diagram of fig. 1, and is likewise carried out in a known manner, for example via a separator (see below), also in the manner disclosed in the embodiments of the present patent application.

The supernatant containing the target enzyme should be essentially free (see below) of suspended particles, i.e.solids, which can be determined as described above by a bench top centrifuge. This is because the solid protein precipitate cannot be dissolved twice by dilution without considerable losses (see above) and without a substantial reduction in concentration. In addition, like other solids, they impair the following chromatography steps due to clogging of the column.

Step (c), i.e. the essentially solid supernatant-free decolorization of step (b) via strongly basic anion exchangers (adsorbents), represents the core of the present invention. In this step, the colored impurities, more particularly the Maillard compounds, are adsorbed on the resin, while the positively charged proteins do not bind to the resin due to the strong positive charge of the exchanger under correspondingly selected conditions, but give an eluate which is a substantially transparent solution. Thus, step (c) represents the selective separation of the dye from the concentrated enzyme solution.

The advantage of this process over the processes described in the prior art is that the useful substances in question, i.e.the enzyme proteins, remain in solution, i.e.without having to denature and recover them, whereby their three-dimensional structure is not modified. They therefore likewise remain in the phase to be further processed, are not discharged from the system, and therefore a high yield can be achieved as described above.

As is indicated in fig. 1 by the corresponding bold line arrows, the main color bound to the resin is subsequently washed out in the separation step, i.e. after discharge of the useful material phase (useful product) and optionally the heel. For example with a high ionic strength solution such as a concentrated NaCl solution. The anion exchanger material can be regenerated via the corresponding counter ions, for example NaOH. Other simple salts may be more appropriate depending on the chromatography material. The fact that the material is treated with such a compound, which is also inexpensive, results in a sterilization effect in addition to the cleaning effect. The system is therefore suitable for Cleaning In Place (CIP).

After process step (c), the liquid enzyme is substantially free from undesirable streaks, precipitates and coloration. It remains light, clear and bright even upon prolonged storage at different temperatures, while having a high level of stability. Examples of color values obtained according to the internationally accepted CIE color scale (defined in DIN5033-3 and DIN 6174) are described in the tests of the examples of the present application.

Process step (c) is optionally followed by step (d), described in detail below, in which the highly concentrated chromatography product is mixed with a solvent. This is also illustrated in fig. 1 ("mixing").

The purified concentrated enzyme solution obtained after step (c) or (d) is clearly depleted especially for colored impurities, but still contains essentially colorless impurities, which are not necessary nor desired to be removed from the concentrated enzyme solution due to their partial stabilizing effect, which is extremely favorable. In addition, intermediate steps can be carried out, which can be carried out beforehand, inserted, added or carried out together with the steps mentioned, depending on the separation problem. Examples of three such optional intermediate steps are described below.

Further possibilities are, for example, the selective removal of further impurities from the concentrated enzyme solution by one or more further chromatography steps, more particularly using other carriers which are sufficiently described in the prior art (see above). This can be carried out at any stage of the process, it appears to be appropriate in each individual case, and it is advantageous to carry out the chromatography step described in (c) immediately before or after it, optionally separated from one another by such intermediate steps as filtration or resolubilization.

The change of solvent according to the invention can be carried out at different stages of the process, preferably before or instead of step (d), as disclosed, for example, in German patent application DE19953870A 1. This application describes a process for producing an enzyme preparation containing an organic solvent which is substantially free of water, wherein the aqueous enzyme preparation is mixed with an organic solvent having a boiling point of more than 100 ℃ and the water is subsequently distilled off.

The liquid enzyme obtained by the process of the invention can be used or further processed in a known manner. Of particular importance are as starting materials for incorporation in detergents and cleaning compositions, more particularly in liquid form. The required balance between clear color and sufficiently satisfactory stability of such products according to the invention is illustrated in example 3, the results of which are also shown in fig. 3. It can be seen that the product purified in this way is only slightly less stable than the unpurified enzyme, but is substantially colorless, and on the other hand it still has a very good stability than the commercial product which has been conventionally decolorized, i.e.treated by precipitation.

Preferred embodiments of the present invention and other subject matter are described below.

As mentioned above, the aqueous enzyme concentrate enriched in biomass by the methods described in the prior art known per se is introduced into process step (a). Several building steps are usually required for this purpose. The preferred process according to the invention is characterized in that the ultrafiltration takes place as a previous step, immediately followed by process step (a), whereby the ultrafiltration concentrate according to the invention is introduced into step (a). One such method is described, for example, in WO01/37628A 2. In this way a relatively pure low-solids enzyme solution is obtained which has been concentrated to a moderate concentration value (cf. example 1).

As already mentioned, methods known per se are used for the concentration step (a), for example using a rotary evaporator or a thin-layer evaporator, preferably a thin-layer evaporator.

In a further preferred embodiment, step (a) is carried out via the particular parameters to be adjusted, more particularly the duration of the concentration process or optional dilution, in such a way that the enzyme concentrate obtained contains at most 4% to 20% by weight, preferably at most 4.5% to 15% by weight, more particularly at most 5% to 10% by weight of dry matter.

In contrast to the undesirable solid impurities described above, dry matter is the total content in the solid matter-concentrated enzyme solution, for example obtained by completely evaporating the solution. These values are determined by methods known per se, for example by drying aliquots or by absorption measurements and comparison with a calibration curve. The values mentioned prove to be particularly suitable values for further processing, since on the one hand the solution should be highly concentrated to avoid losses and on the other hand because excessively high viscosities lead to difficulties in constant delivery.

In a further preferred embodiment, the process according to the invention is characterized in that after step (a), the concentrated enzyme solution is deodorized in step (a'). Corresponding deodorization methods which can be integrated into a continuous process are known in the prior art. The microorganisms used for the preparation of the enzyme proteins are particularly preferred when they are microorganisms which form malodorous olfactory impurities or secreted proteins which very rapidly reduce other components.

Process step (b) -isolation of the solids-followed by (a) or by a deodorization step (a'), likewise based on methods known per se, for example filtration. However, mechanical separation methods preferably based on gravity or centrifugal separation are preferred.

Such processes are, above all, based on the use of separators, preferably continuous separators, which can be integrated into a continuous process. Separation processes involving periodic discharge deposition are particularly preferred. Such a method is also disclosed in the examples of the present application.

The aim of step (b) is to reduce the suspended matter or solids content of the enzyme concentrate to as low a value as possible. Preferred processes are characterized in that at most 1 vol%, preferably at most 0.7 vol%, more particularly at most 0.5 vol% of solids is obtained in the concentrated enzyme solution by step (b). The adjustment is made by adjusting the particular equipment used in a known manner. The separator mentioned above provides in particular a correspondingly advantageous solution.

The core of the process according to the invention is step (c), i.e.strongly basic anion exchange chromatography. The success of the process is therefore critically dependent on the type of chromatography material selected and the experimental procedure, e.g. sampling. As already mentioned, it is essential that the colored impurities should be adsorbed on the material, while the positively charged protein is not bound to the resin completely under the correspondingly selected conditions. The invention is therefore based on strongly basic anion exchangers. In view of the fact that most natural water-soluble, more particularly secreted proteins are more soluble in water at intermediate pH values, it is a particular advantage for the strongly basic anion exchanger of step (c) to have a maximum exchange capacity at pH values of 5-9, preferably 6-8.

Alkaline proteins, such as in particular alkalophilic microorganisms, more in particular proteases, have an isoelectric point in the alkaline range and are therefore positively charged in this preferred pH range and therefore do not bind to particular materials. As already mentioned, the ideal pH of the process is, of course, experimentally determined for each protein and adjusted for said step (c). In examples 1 and 2, these values are approximately 7.5 and 7 for the selected alkaline protease and the selected alpha-amylase.

Strongly basic anion exchangers contain quaternary ammonium groups as functional groups, preferably those in which the groups are substituted by at least two alkyl groups, more preferably by at least two C₁Or C₂Those substituted by alkyl, optionally with C₁Or C₂Hydroxyalkyl-substituted ones, which have proven to be particularly suitable for step (C) in view of their chemical nature.

This requirement is met by using strongly basic anion exchangers containing the functional groups trimethylammonium or dimethylethanolamine. The latter are slightly more weakly basic than the former, so that the corresponding proteins can be optimized in particular by this change.

Accordingly, the corresponding chromatography materials characterize the preferred embodiments.

A further characteristic of chromatography materials is their exchange capacity, expressed as mol equivalents per unit volume. It indicates how densely the material is occupied by the functional group. Particularly suitable processes are characterized in that the strongly basic anion exchanger of step (c) has an exchange capacity of from 0.7 to 1.2meq/mL, preferably from 0.8 to 1.1meq/mL, preferably from 0.9 to 1.0 meq/mL.

An additional criterion that affects the separation efficiency of a chromatography column is the effective pore size. Measured in this way, the retention is sufficient that the substance, more particularly the protein, is not washed through, holding very firmly or even blocking the material. Two globular proteins which have been investigated in this example, Bacillus lentus alkaline protease and alpha-amylase from Bacillus species 7-7(DSM12368), which have molecular weights of about 27kD and about 58kD, respectively, have proven themselves as chromatography materials with an effective pore size of 0.45 mm. For significantly larger or smaller proteins, the chromatography material selected should have a correspondingly larger or smaller effective pore size.

A preferred process is therefore characterized in that the strongly basic anion exchanger has an effective pore size of from 0.2 to 0.7 mm, preferably from 0.3 to 0.6 mm, more preferably from 0.4 to 0.5 mm.

Suitable supports for the strongly basic anion exchangers to be used in accordance with the invention are, in principle, the materials described in the prior art for this purpose, including, for example, supports in gel form. In contrast, strongly basic anion exchangers based on porous polymers are preferred for step (c) because of their processing properties. Strongly basic anion exchangers based on styrene/divinylbenzene copolymers have proven particularly advantageous.

Chromatography materials having the properties just discussed are described in detail in the prior art. FromThose descriptions of series are, for example, in the guide "Manual of ion exchange resins and synthetic additives, Vol.1 ", Mitsubishi Kasei Corp (Tokyo, Japan), June 1995, pp.104 to 108 and in" Product line Brochure"1.6.2001, pp.4 to 6, available from the manufacturer or from Summit Chemicals Europe GmbH, Dusseldorf, Germany. Strongly basic anion exchangers described there comprise seriesSA，PA andHPA. A representative example isPA308L, successfully used in the examples of the present application.

Materials chemically similar for use in step (c) of the chromatography process may be produced by one of ordinary skill in the art through experimentation with the preferred properties described above, or may be obtained from other commercial manufacturers, as well as characterizing preferred embodiments. For example DOW MSA for Dow ChemicalsAnd Rohm&Of Haas900CL gave comparable results.

The chromatography step (c) is advantageously carried out under certain conditions. These include in particular a specific bed volume, which is the ratio of the volume of the applied substance to the column volume, a specific residence time, which is advantageously expressed by the enzyme solution.

It has proven to be particularly advantageous and characterizes correspondingly preferred embodiments to carry out step (c) at a bed volume of from 1 to 10, preferably from 1.5 to 7, more preferably from 2 to 4. These bed volumes represent the optimum conditions determined experimentally in the examples, in order to clean the filtrate while concentrating it as much as possible.

Suitable average residence time values in step (c) characterizing a correspondingly preferred embodiment are from 0.01 to 0.2g, preferably from 0.025 to 0.1g, more preferably from 0.04 to 0.06g, most preferably 0.05g per minute per g of support material.

Particularly suitable methods are characterized in that they are largely automated. A particularly convenient way of controlling the integration is based on determining the conductivity (quantifiable as μ S/cm) of the material being processed in the critical state and using the result to regulate the process. After the chromatography stage, it can be used, for example, to separate a useful material (useful product) containing fraction from other fractions. Thus, in a preferred embodiment, the process of the invention is characterized in step (c), more particularly in that the separation between the initial distillate and the useful product and/or the useful product and the tail fraction during distillation is controlled by the conductivity of the eluate.

In order to increase the yield, patent application WO01/37628A2 has proposed the use of the filtrate for an additional washing step. The process according to the invention is therefore likewise preferably characterized in that step (c) is carried out with recycling of the initial distillate and/or the tail fraction of at least a partial distillation of the ion exchange chromatography. In this way, the fraction in question is additionally enriched with enzyme molecules which, near the end of the peak, have not yet been washed out of the column. In principle this step is limited by possible impurities that may be washed out of the column. In each individual case, a balance is struck between the concentration obtainable and the quality, i.e. purity, of the product.

During the process described in this connection, the use of high enzyme concentrations is considered in efficiency terms. However, concentrated enzyme solutions do not generally require such high concentrations for their particular industrial applications. Accordingly, a correspondingly preferred process according to the invention is characterized in that after step (c) a relatively low concentration is set in step (d). Mixers known from the prior art, more particularly of the type which can be integrated into continuous systems, can be used for this purpose.

On the other hand, all liquid enzymes have a tendency to denature during storage, thus losing their activity. Especially proteases which hydrolyze other enzyme molecules. Accordingly, a preferred process according to the invention is characterized in that a stabilizer or stabilizer mixture is added after step (c). Such compounds are known per se from the prior art and include compounds which have a stabilizing effect against biophysical temperature changes, for example by adjusting the water activity, such as polyols, and compounds which reversibly deactivate proteases or provide protection against oxidation.

The stabilizer or stabilizer mixture can optionally be added simultaneously, before or after the diluent (d). In a particularly advantageous embodiment, step (d) is used to add a stabilizer solution and a diluted solution, or a solution which exerts both effects.

The added stabilizer is preferably selected from liquid compounds containing hydroxyl groups, for example polyols such as glycerol, in a particularly preferred embodiment propane-1, 2-diol. The liquid compound in question can likewise be a mixture of water and/or other stabilizing compounds.

In order to produce a stabilizing effect, it has proven particularly advantageous to add from 40 to 70 vol.%, preferably from 45 to 65 vol.%, more preferably from 50 to 60 vol.%, based on the final volume, of a polyol stabilizer mixture.

If dilution at this point results in a very weak concentrated solution, a concentration step may optionally be inserted between steps (c) and (d) before the solution passes through the mixer. In principle, any method known in the art, preferably the method described above, can be used for this purpose.

Due to the strong influence of dilution, the use of highly concentrated enzyme solutions is also additionally supported in the processes known to date in order to obtain enzymes in highly concentrated liquid form which are still sufficient for the intended industrial application.

The dilution step is also used to adjust the final product of the process to a dry matter content of 2 to 15 wt%, preferably 5 to 13 wt%, more preferably 8 to 12 wt%. It may also be useful to adjust the viscosity number at 25 ℃ of the end product of the process to a value of 1 to 20mPas, preferably 1 to 15mPas, more preferably 1 to 10mPas, and/or to a silt content of less than 1 vol%, preferably less than 0.75 vol%, more preferably less than 0.5 vol%. These values are usually linked to one another and have proven to be particularly suitable for further storage and/or handling of the enzyme in liquid form. As mentioned above, adjustments have to be taken into account after the chromatography stage or before the addition of the stabilizer. Therefore, the method of the present invention satisfying these requirements is preferable.

The present specification refers to an enzyme, said concentrated enzyme solution being purified by the process of the present invention. Enzymes represent a preferred embodiment, since on the one hand they are of particular industrial importance and on the other hand can be detected by their specific activity, which can be used in particular for determining the optimum working range of examples 1 and 2 and FIG. 2. Nevertheless, the method can be applied to any water-soluble protein that provides a suitable solvent system, and their chromatography material determined, to form a suitable detection reaction. This applies, for example, to peptides, such as peptide hormones, pharmacologically significant oligopeptides, and antibodies. Antibodies are also suitable, for example, for detection. All such proteins are intended to be understood as enzymes in the context of the present invention.

However, industrially useful enzymes in the conventional sense are of central consideration, preferably hydrolases or oxidoreductases, more preferably proteases, amylases, cellulases, hemicellulases, lipases, cutinases or peroxidases. The design of the process, in particular for the treatment of such enzyme solutions, by determining and establishing appropriate process conditions (see above) represents a correspondingly preferred embodiment of the present invention.

Proteases are of major interest, more particularly for the production of detergents and cleaning compositions, alkaline proteases being preferred because of their particular activity and inclusion in alkaline formulations. Accordingly, correspondingly preferred processes are those which are characterized by the use of suitable proteases.

Among such processes, those which are characterized in that step (c) is carried out at a pH of 5 to 9, preferably 6 to 8.5, more preferably 7 to 8, are preferred due to the biochemical properties of the alkaline proteases mentioned.

One such protease was also investigated in examples 1 and 3, where it was shown that it is necessary to maintain a critical activity value in order to carry out the process of the invention in order to minimize the solids content present. Accordingly, a correspondingly preferred process for refining the concentrated protease solution mentioned is characterized in that the product of step (a) is adjusted to an activity value of 600,000 to 900,000, preferably 650,000 to 850,000, more preferably 700,00 to 800,000 HPU/g. As is known in the art, the parameters of the above-mentioned constitution steps are adapted to the adjustment, i.e.concentration or optionally dilution.

According to the above experiments, the end product designed for subsequent use advantageously has a relatively low activity value, which can be obtained in particular by dilution with a stabilizing solution. The method of adjusting the final product to an activity value of 150,000 to 500,000, preferably 175,000 to 300,000, more preferably 200,000 to 260,000HPU/g, based on the protease, represents a preferred embodiment.

Another industrially important enzyme type is the alpha-amylase. For example, they are used in the food industry for the production of confectionery or, owing to their starch-hydrolyzing activity, are added to detergents and cleaning compositions. Alkaline alpha-amylases are particularly preferred, as are proteases. Accordingly, correspondingly preferred methods are those designed for the treatment of alpha-amylases, characterized in that they are preferably used for or by treatment under alkaline pH-optimum conditions.

As described in examples 2 and 3, the preferred process of the invention for treating alpha-amylase is characterized in that the product of step (a) is adjusted to an activity value of 30,000 to 50,000TAU/g, preferably 35,000 to 45,000 TAU/g.

Since alpha-amylases are generally likewise used in correspondingly low concentrations and, in addition, should likewise be stabilized, a further preferred embodiment of the process according to the invention is characterized in that the end product is adjusted to an activity value of from 4,000 to 14,000, preferably from 6,000 to 12,000, more preferably from 8,000 to 10,000 TAU/g.

Further particularly important enzyme types are cellulases, which are used, for example, in the detergent industry and in textile production for the surface treatment of textiles. Thus, the method characterized by a cellulase, preferably with an alkaline pH optimum, represents a preferred embodiment corresponding to the present invention.

All process parameters mentioned so far are reflected in the specific process product, for example the type of enzyme, the purity level, the nature of the isolated compound, the activity or the stability value obtained. Therefore, the products obtained by these processes of the invention are likewise preferred. Thus, in addition to this method, the present invention also relates to the concentrated enzyme solution obtained by the above-described method.

The invention likewise relates to a composition comprising the enzyme as an intermediate product in the form of a concentrated enzyme solution obtained according to the invention. For example, the concentrated enzyme solution obtained according to the present invention need not be used in liquid form, but may instead be converted into a dry, highly pure form. This can be achieved, for example, by freeze-drying or by incorporation in solid particles. The corresponding method is described in detail in the prior art. In this form, they can be stored for long periods of time or included in other solid compositions, for example in solid detergents and cleaning compositions.

Thus, embodiments of the present invention also encompass all the different possible types of cleaning compositions-concentrates and compositions that can be used without dilution-for use in industrial scale washing equipment or for use in hand washing or cleaning. These include, for example, detergents for textiles, carpets or natural fibers, for which the term "detergent" is used in the description of the invention; dishwashing detergents for dishwashing equipment or manual dishwashing detergents or cleaning agents for hard surfaces such as metal, glass, porcelain, ceramic, tile, stone, painted surfaces, plastics, wood or leather, for which the present description uses the term "cleaning composition". Any type of detergent or cleaning composition represents an embodiment of the present invention, provided that it is enriched with enzymes that have been purified by the process of the present invention and further processed according to the specific aspects of the present invention described.

Embodiments of the present invention include all known and/or suitable supplies of formed detergent or cleaning compositions according to the present invention. These include, in particular, solid compositions in powder form, optionally with several phases, concentrated or not; an extrudate; particles; tablets or sachets, either in bulk containers or in portions. Embodiments in liquid, paste or gel form are likewise included, provided that the enzyme treated according to the invention is introduced in further processed form for such embodiments.

In a preferred embodiment, the detergent or cleaning compositions according to the invention comprise active enzyme in an amount of from 2. mu.g to 20mg, preferably from 5. mu.g to 17.5mg, more preferably from 20. mu.g to 15mg, most preferably from 50. mu.g to 10mg, per gram of composition.

In addition to the enzymes prepared according to the invention, further enzymes, detergents or cleaning compositions which are possible according to the invention optionally comprise further ingredients, such as enzyme stabilizers; surfactants such as nonionic, anionic and/or amphoteric surfactants; a bleaching agent; a bleach activator; a bleach catalyst; a synergist; a solvent; thickeners and-optionally as further typical ingredients-sequestering agents, ionogens, optical brighteners, redeposition inhibitors, dye transfer inhibitors, foam inhibitors, dyes and/or perfumes, bactericides and/or uv absorbers, to mention only the most important classes of ingredients. The corresponding formulations are described in detail in the prior art.

In contrast, compositions comprising suitably concentrated enzyme solutions are preferred due to the advantageous properties of the liquid enzymes obtained according to the invention, in particular due to their transparent appearance and their use without further manipulation. Compositions in the form of liquids, pastes or gels in general are of particular importance. They are easy to dose, contain the enzyme in the desired activity, and are aesthetically pleasing, at least as long as the enzyme component is of interest. Emphasis is placed on the beginning as desired.

This applies in particular to detergents and cleaning compositions intended for the end user. Thus, in a particularly preferred form, the composition of this embodiment is a detergent or cleaning composition. These fall under the above definition and may contain the substances mentioned therein. In addition, in this embodiment, the composition is in general of liquid, gel-like or paste-like consistency, wherein the refined product according to the invention can easily be included by methods known per se.

Examples

Example 1

Refining concentrated protease solution

Isolating the biomass

After production of the useful material protease by a fermentation process as described in 91/02792a1, the biomass can be almost completely isolated by known separation, microfiltration and sterile filtration methods. It is subsequently concentrated, as described in WO01/37628A2, by removing the solvent by ultrafiltration until the protease concentrate has an activity of 300,000 to 400,000HPU/g, as described in van Raay, Saran and Verbeek, entitled "Zur Bestimung derproteolischenin Enzymkonzentraten und enzymhaltigenWasch-，Spül-und Reinigungsmitteln[Determining Proteolytic Activity inEnzyme Concentrates and Enzyme-containing Laundry Detergents，Dishwashing Detergents and Cleaners]"in Tenside (1970), Vol.7, pp.125-132. In addition, the pH was adjusted to 7.5 with 30% calcium chloride solution. The solution solids content was less than 1 vol%, as determined by centrifugation using a tabletop or laboratory centrifuge measuring 10 minutes at 7,000G. In addition, the protease obtained showed a positive charge and an ionic strength of 20 mS/cm.

Determining an optimal working range

Samples of the solution obtained after separation of the biomass were taken during the ultrafiltration step (values up to 400,000HPU) or further concentrated by a separator as described below and the amount of solids was determined as a function of the particular activity as described above. The activity measurements were carried out at a pH of 7.5, at a temperature of 20 ℃ and an ionic strength of 10 mS/cm. The dependence of the concentration of active protease on the solids content is illustrated in Table 1 and FIG. 2.

TABLE 1

Dependence of solid content on active protease concentration

Activity [1000HPU/g]	Solids content of the solution [ vol% ]]
		0	0.2
200	0.5
		400	1.2
600	1.5
		800	3.0
1000	7.5

As can be seen from Table 1, the solids content of the concentrated protease solution increased beyond the normal ratio, over about 900,000HPU/g, so that a range of about 700,000 to 800,000HPU/g was considered the optimum working range, with maximum activity and minimum solids content.

Step (a): concentration of the enzyme solution to working range

Based on this result, the enzyme solution obtained at the end of the preceding step by ultrafiltration was concentrated in a thin layer evaporator to a value of 800,000HPU/g under the following conditions: the product temperature was greater than 35 ℃ and the vacuum was about 20 mbar, the substantially constant pH was 7.5, and the solids content of the enzyme concentrate remained less than 3 vol%, so losses in the following steps were still minimal.

Step (b): separating the (solid) precipitate formed

The separation of the solids formed by concentration (precipitate) is carried out by mechanical separation based on the principle of gravity or centrifugal separation. The solid was separated using a separator with periodic discharge of the precipitate (ALFA LAVAL BTPX 205, sigma 11,700m2, operating at a G value of 12,800 and a throughput of 200 l/h). Thus an enzyme concentrate is obtained which is largely free of solids (solids content less than 0.2 vol%, determined as described above). The activity was still approximately 800,000 HPU/g.

Step (c): strongly basic anion exchange chromatography.

Using strongly basic anion exchangersThe decolorization by chromatography of the Pa308L type is carried out in a fixed bed using anion exchangers available from Mitsubishi, Tokyo, Japan, or from Mitsubishi Chemical Europe GmbH, Dusseldorf, Germany. The quality of decolorization and thus the stability of the enzyme is controlled by the bed volume ratio (BV-volume ratio of enzyme concentrate to resin) and the residence time. Adjusting the ratio of 2-5 BV and the dosage of 0.05 kg enzyme per minute per kg resin. The principle is based on the exclusion of the enzyme by the support material and entrainment into the liquid stream, while the dye is bound to the immobilization support. To increase the yield, part of the tail was also passed through the column. The compounds adsorbed on the column, more particularly the dyes, are then washed out by flushing with NaCl and NaOH solutions, in this way regenerating the fixed bed.

Step (d): mixing, stabilizing and adjusting the activity with solvents

The filtrate, having an activity value of approximately 700,000HPU/g, is mixed directly in-line in a static mixer with propane-1, 2-diol which simultaneously has a stabilizing effect. About 55 vol% of the solvent was withdrawn.

The liquid enzyme purified by these four steps has the following properties (activity is determined as defined above, colour is determined using the internationally adopted CIE colour scale defined in DIN5033-3 and DIN 6174):

activity: 260,000HPU/g

Color: l value > 96

B value < 14

Viscosity: < 10mPas

pH value: about 7

The resulting liquid was essentially transparent and did not show any precipitation immediately after the process. Additional stability is described in example 3.

Example 2

Refining concentrated amylase solution

An amylase solution purified according to the invention was prepared as in example 1, with the following differences. A fermenter batch containing the alpha-amylase described as useful material in application WO02/01036A2 was used. Since alpha-amylase has a different isoelectric point than protease, the pH is adjusted to 6.25 prior to step (a), and the pH is maintained at 6-6.5 throughout the process to keep the amylase positively charged.

Determination of Activity

The amylolytic activity, expressed as TAU, was determined using a modified p-nitrophenyl maltoheptaside (maltoheptaside), the terminal glucose unit of which is capped by a benzylidene-dichloro group; the p-nitrophenyl maltoheptaside is cleaved by amylase enzymes into free p-nitrophenyl oligosaccharides, which are subsequently converted to glucose and p-nitrophenol by the action of a coenzyme glucoamylase and an alpha-glucosidase. The amount of released p-nitrophenol is thus proportional to the amylase activity. The measurement can be made, for example, with Abbott Quick-Test chambers (manufacturer: Abbott, Abbottpark, Illinois, USA). The increase in absorbance (405 nm) of the test mixture was measured photometrically at 37 ℃ over 3 minutes relative to the blank value. Calibration is carried out on the basis of known activity standard enzymes (e.g.of Genencor, Palo Alto, Calif., USA)2900, Activity 2,900 TAU/g). Evaluation was performed by plotting the difference in absorption DE (405 nm)/min versus the standard enzyme concentration.

Determining an optimal working range

Samples of different concentrations were taken during the ultrafiltration step, as in example 1, and subsequently separated, also determining the solids amount according to the particular activity. The measurement was carried out at a pH of 6.25, at a temperature of 20 ℃ and an ionic strength of 10 mS/cm. The results are shown in table 2 and fig. 2.

TABLE 2

Dependence of solid content on concentration of active alpha-amylase

Activity [100TAU/g]	Solids content of the solution [ vol% ]]
		0	0.5
100	1.0
		200	1.5
300	2.5
		400	3.2
500	5.0

As can be seen from table 2, the amylase and protease are comparable in the dependence of the solids content on the active enzyme concentration (see above); this is shown in FIG. 2 as 100 TAU/g. Thus, the solids content of the concentrated amylase solution increased beyond the normal ratio, over about 50,000HPU/g, and thus a range of about 35,000 to 45,000HPU/g was considered the optimum working range, with maximum activity and minimum solids content. Therefore, the working range of the present invention should be lower than the range.

Thus, an activity value of 35,000 to 45,000TAU/g was adjusted by step (a) as in example 1, and a further step was carried out as in example 1, i.e.using the same anion exchange chromatography material. In step (d), the concentration value was adjusted analogously to 9,000TAU/g by mixing with propane-1, 2-diol.

The liquid enzyme purified by these steps has the following characteristics:

activity: 9,000TAU/g

pH: about 6.25

The resulting liquid was essentially transparent and did not show any precipitation immediately after the process as did the protease of example 1.

Example 3

Storage stability of proteases in liquid detergent matrices

To determine the storage stability of the protease purified according to the invention, the following three samples were prepared compared with the unpurified enzyme and the commercial product in the same liquid detergent matrix: (1.) following ultrafiltration in example 1 and prior to step (a), unrefined protease was present, (2.) fully refined commercial product available from Novozymes, Bagsvaerd, Denmark16.0LEX and (3.) the protease purified according to example 1. All three samples were placed in 55 vol% propane-1, 2 at 260,000HPU/g activity-a glycol in aqueous solution, in an amount of 0.4 vol% contained in a liquid detergent base of typical composition.

The protease samples introduced into the matrix had L values on the CIE color scale of (1.)78, (2.)99 and (3.) 97. In other words, the protease purified according to the invention is almost as transparent as the fully refined product. Samples were taken periodically over a 12 week period and residual activity was determined as described above. The values listed in table 3 were obtained and are shown as a graph in fig. 3.

TABLE 3

Storage stability of protease in liquid detergent matrix (expressed as% residual Activity of HPU)

It can be seen that the protease purified according to the invention is more stable than the fully refined product, despite the virtually identical color number, and that the protease purified according to the invention loses only a little activity in the detergent matrix compared with the dark unpurified enzyme.

Description of the drawings:

FIG. 1 shows a schematic view of a

Block flow diagram for purifying concentrated enzyme solutions according to the present invention

The following steps show that:

(a) concentrating the enzyme solution until the working range, and draining the supernatant solution;

(a' optionally deodorizing;

(b) separating the (solid) precipitate formed;

(c) strongly basic anion exchange chromatography, where the compound is adsorbed on a column, more particularly a dye, washed out by washing with a suitable medium in a separation step, after which the column is regenerated; and

(d) stabilization and adjustment of the activity by addition of solvents.

FIG. 2

Dependence of the solids content on the concentration of active enzyme as determined in examples 1 and 2

The solids content, expressed as vol%, was determined by measuring with a laboratory centrifuge at 7,000G for 10 minutes. Protease activity was expressed as 1,000HPU/g and alpha-amylase activity was expressed as 100 TAU/g. Emphasizing the range of operation considered optimal for the purposes of the present invention.

FIG. 3

Storage stability of protease in liquid detergent as determined in example 3

Determining:

1. unrefined proteases;

2、16.0LEX (products of Novozymes) and

3. the protease purified according to example 1.

Claims (40)

1. A method for refining a concentrated enzyme solution, characterized by the steps of:

(a) providing a concentrated enzyme solution comprising insoluble solids,

(b) separating the insoluble solids to produce a solids-free supernatant solution, and

(c) contacting the supernatant with a strongly basic anion exchange material in a bed volume of 1-10,

wherein the enzyme is retained in solution to produce an eluate comprising a protein solution containing colourless impurities which have a stabilising effect on the protein solution.

2. The process as claimed in claim 1, characterized in that the ultrafiltration concentrate is introduced in step (a).

3. A process as claimed in claim 1 or 2, characterized in that step (a) is carried out using a thin-layer evaporator.

4. The process according to claim 1 or 2, characterized in that in step (a) an enzyme concentrate is prepared containing at most 4 to 20 wt.% dry matter.

5. The process as claimed in claim 1 or 2, characterized in that after step (a), the concentrated enzyme solution is deodorized in step (a').

6. The process according to claim 1 or 2, characterized in that step (b) is carried out by mechanical separation methods or centrifugation.

7. The process of claim 6, characterized in that step (b) is carried out in a separator.

8. The process according to claim 1 or 2, characterized in that up to 1 vol% of solids is obtained in the concentrated enzyme solution by step (b).

9. The process as claimed in claim 1 or 2, characterized in that the strongly basic anion exchanger used in step (c) exhibits a maximum exchange capacity at a pH of from 5 to 9.

10. The process as claimed in claim 1 or 2, characterized in that the strongly basic anion exchanger used in step (c) contains quaternary ammonium groups as functional groups.

11. The process according to claim 10, characterized in that the strongly basic anion exchanger used in step (c) contains trimethyl-ammonium or dimethylethanol-ammonium groups as functional groups.

12. The process as claimed in claim 1 or 2, characterized in that the strongly basic anion exchanger of step (c) has an exchange capacity of 0.7 to 1.2 meq/mL.

13. The process as claimed in claim 1 or 2, characterized in that the strongly basic anion exchanger has an effective pore size of 0.2 to 0.7 mm.

14. The process as claimed in claim 1 or 2, characterized in that the strongly basic anion exchanger used in step (c) is based on a porous polymer.

15. The process according to claim 1 or 2, characterized in that step (c) is carried out at a bed volume of 1.5 to 7.

16. The process according to claim 1 or 2, characterized in that the average residence time in step (c) is 0.01 to 0.2g enzyme per g support material per minute.

17. A method according to claim 1 or 2, characterized in that step (c) is controlled by the conductivity of the eluate.

18. Process according to claim 1 or 2, characterized in that the initial distillate and/or the tail fraction in the at least partial ion exchange chromatography distillation is recycled in step (c).

19. A method as claimed in claim 1 or 2, characterized in that after step (c) a relatively low concentration value is formed by dilution in step (d).

20. Process according to claim 1 or 2, characterized in that after step (c) optionally a stabilizer is added during, before or after the dilution according to claim 19.

21. The process according to claim 20, characterized in that the stabilizer is a polyol.

22. The process according to claim 21, wherein the stabilizer is added in an amount of 40 to 70 vol.%, based on the final volume.

23. A process according to claim 19, characterized in that the dilution step is used to adjust the final product of the process to a dry matter content of 2-15 wt%.

24. The process according to claim 19, characterized in that the final product of the process is adjusted to a viscosity of 1 to 20mPas at 25 ℃.

25. The process according to claim 19, characterized in that the end product of the process is adjusted to a sediment content of less than 1 vol%.

26. The process as claimed in claim 1 or 2, characterized in that the enzyme is an industrially usable enzyme.

27. The method of claim 26, wherein the enzyme is a hydrolase or an oxidoreductase.

28. The process of claim 27 wherein the enzyme is a protease, an amylase, a cellulase, a hemicellulase, a lipase, a cutinase or a peroxidase.

29. The process as claimed in claim 26, characterized in that the enzyme is a protease.

30. The process as claimed in claim 29, characterized in that, in particular, in step (c), the reaction is carried out at a pH of from 5 to 9.

31. The process as claimed in claim 29 or 30, characterized in that the product of step (a) is adjusted to an activity of 600,000 to 900,000 HPU/g.

32. The process according to claim 22, characterized in that the final product is adjusted to an activity of 150,000 to 500,000 HPU/g.

33. The method of claim 26, characterized in that the enzyme is an α -amylase.

34. The process as claimed in claim 26 or 33, characterized in that the product of step (a) is adjusted to an activity of 30,000 to 50,000 TAU/g.

35. The process as claimed in claim 33, characterized in that the final product is adjusted to an activity of 4,000 to 14,000 TAU/g.

36. The method of claim 26, characterized in that the enzyme is a cellulase.

37. A concentrated enzyme solution obtained by the method of any one of claims 1 to 36.

38. A composition comprising an enzyme obtained as an intermediate product in the form of a concentrated enzyme solution according to claim 37.

39. A composition comprising the enzyme solution of claim 37 in bulk liquid, paste or gel form.

40. A composition according to claim 39, characterized in that it is a detergent or cleaning composition.