Background of the invention In the recovery of soluble organic compounds from a fermentation broth, many recovery or purification techniques are known such as freeze concentration, organic freezing or lyophilisation, which are all based on cooling techniques.

Freeze concentration may be used to concentrate an organic compound, for example an enzyme solution can be concentrated by crystallizing ice in the solution by cooling below the freezing point. Ice can be removed to obtain concentrated enzyme solution.

Cryogenic freezing is used when a composition has to be frozen much more quickly than is possible in a conventional freezer. In general liquid carbon dioxide or liquid nitrogen is sprayed in a freezing chamber or directly onto the composition.

Lyophilisation, also referred to as freeze drying is a process of removing water from a composition by sublimation and desorption. In general the composition is brought in a frozen state and the product is dried using a vacuum system to reduce the pressure near the product. The temperature of the composition during lyophilisation is below the eutectic temperature. In a phase diagram, the eutectic temperature is the temperature at which at least two distinct solid phases are in equilibrium with a mother liquor.

However, freezing and freeze-drying processes may cause loss of functional properties and conformational changes of organic compounds such a proteins.

Care must be taken to avoid any damaging effect on proteins and other organic compounds in solution.

Other recovery or purification techniques are for example precipitation, crystallisation, membrane technology (such as ultra filtration) and chromatography.

Description of the invention The present invention relates to a process for the recovery and/or purification of at least one organic compound from a solution which solution comprises the at least one organic compound and a solvent, the process comprising the steps of (a) adjusting the temperature of the solution to a temperature at which both the solvent is at least partially in the solid phase and the at least one organic compound is at least partially in the solid phase ; and (b) recovering the solid phase of said at least one organic compound.

Adjusting the temperature is preferably done by lowering the temperature for example by cooling the solution. The solid phase of said at least one organic compound is preferably recovered by separation from the mother liquor.

The process of the present invention in a surprisingly advantageous combination of freeze concentration and a controlled selective crystallization of an organic compound. Hereafter this new process, is called Low Temperature Organics Crystallisation (LTOC).

According to the invention this technique can be used to obtain a pure organic compound and pure ice. Compared to cryogenic freezing in which the complete solution is solidified instantaneously by putting the sample for instance into liquid nitrogen, LTOC is operated in much milder conditions, whereby ice and the organic compound are crystallized relatively slow, and a concentrate remains which is in general not in the solid phase. This slow crystallisation rate will result in a high purification of the organic compound in question. Surprisingly we have found that organic compounds like enzymes did not loose activity in the LTOC process. In general organic compounds such as macromolecules having one or more, sometimes even multiple, chiral centers, cannot be processed without degradation at evaluated temperatures. Advantageously these compounds can be purified and/or recovered with the process of the invention without a substantial loss in activity.

It should be understood that, depending on the composition of the solution, there may be more than one eutectic point. For example, in case of a fermentation broth there might be an eutectic point for each organic compound present.

Therefore, by selecting the operational temperature during the LTOC process the crystallisation rate can influenced as well as which specific organic compounds will crystallize.

By organic compound is meant a carbon containing molecule having at least 2 carbon atoms and hydrogen atoms, whereby at least one hydrogen is attached to a carbon atom, and having a iv ! W (mofecufar weight) of more than 50 D, preferable a MW of more than 100 D and more preferably having a VIW of more than 500 D. In general the organic compound will be in the solid phase at 0°C.

The organic compound is preferably producible by or obtainable from a cell, more preferably producible by a microbial cell such as an eukaryotic or prokaryotic cell. Of course the organic compounds recovered and/or purified according to the process of the present invention can be produced otherwise, for example in a chemical or enzymatic production process. Organic compounds obtained from plant and animal sources can also be used in the present process. Preferred organic compounds are aminoacids, dipeptides, tripeptides, oligopeptides, polypeptides such as proteins (for example enzymes), protein hydrolysates, monoclonal antibodies, RNA, DNA, saccharides (mono, di, tri, oligo and poly saccharides, natural and artificial), secondary metabolites such as vitamins, carotenoids, antibiotics (for example natamycin) etc.

In general the purified organic compound will be formed in crystalline or amorphous form and will have a crystal density between 0.5 and 2.0 g/cm3, preferably the density is at least 0.7 g/cm3, more preferably at least 0.8 g/cm3. The density is preferably lower than 1.7 g/cm3 and more preferably lower than 1.5 g/cm3. The density is in general around 1.0 g/cm3 because most organic compounds produced by LTOC in water will contain a certain amount of water in their crystal matrix.

The solution when cooled to or under the eutectic point, will in general consist of ice crystals, crystals of at least one organic compound and a liquid concentrate of non-crystallized compounds including the solvent. The ice crystals can be separated rather easy due to the well-know low density of ice which will cause the ice to float. The ice crystals formed may become very large and spherical, having a size of between 100 and 5000 um (average diameter). The crystals of the organic compound in general have a smaller size, typically between 10 to 200 um average diameter, and depending on their density and concentration will be present as a suspension and/or precipitation.

The crystals of the organic compound can be separated from the concentrate or mother liquor using well-known techniques such as filtration or centrifugation. The ice crystals and/or the separated crystals of the organic compounds are advantageously washed to improve their purity and avoid loss of valuable

compounds.

Preferably the same solvent as present in the starting solution of the organic compound is used for washing. In the present process water is preferably used as solvent. Other solvents especially organic solvents, combinations of organic solvents, or a combination of one or more organic solvent and water can also be used in the process of the present invention. For example methanol, ethanol, aceton can be used as solvent.

The process of the present invention can also be used in combination with other precipitation techniques. For example, precipitation by salting out can be used. Salts which improve the precipitation or crystallisation of the organic compound are preferably used. Chaotropic salts that may be used in the present invention are for example salts of perchlorate (Cl04-), thiocyanate (SCN-), hydrogensulfate (HSO4-), dihydrogenphospate (H2PO4-), hydrogencarbonate (HC03), iodide (I-), chloride (CI-), nitrate (NO3-), guanidinium chloride, urea or trichloroacetate. Also kosmotropic salts or lyotropic salts can be used, an example is a sulphate.

Preferably, the chaotropic salt is a thiocyanate (SCN-) or perchlorate (CI04). Preferably, the thiocyanate salt is a sodium or a potassium salt.

These salts can be added before or during the cooling of the solution.

Preferably the salts are added before the starting solution is cooled. By using this salting out technique together with the process of the invention, in general less cooling is needed compared to a process without salting out. In the other hand, less salt is needed compared to salt-out techniques without cooling below the eutectic freezing point.

Eutectic freeze concentration is advantageous from economical perfective due to the relatively low energy consumption as well as the very pure end products such as the desired purified organic compound and the ice formed.

The purified organic compound obtained with the process of the invention preferably has a purity of at least 70%, more preferably at least 80%, even more preferably at least 90% and still more preferably at least 95% and most preferably at least 98% (based on wt% dry matter).

The ice obtained in this project will in general contain less than 10wt% of other compounds, preferably less than 5wt%, more preferably less than 2wt%, even more preferbly les thn 1wt%, still more preferably less than 0. 5wt% and still more preferably less than 0. 2wt% of the impurities, and most preferably less than 100 ppm impurities.

The process of the present invention can be done as a continuous, batch, fed batch or repeated fed batch process. In case of a batch process during the cooling of the starting solution, this starting solution can be concentrated by removal of ice in case the temperature is below the freezing point of the solvent, but still is above the eutectic point.

In all processes such a concentration step (freeze concentration) can be used as a pre-treatment step before the process of the present invention.

The cooling rate of the solution, the temperature during the process of the invention, the presence of other components in the solution, for example the impurities as well as salts added, the pH and addition of anti-solvents during the crystallisation of the organic compound will determine the crystallisation rate of the organic compound. In case of low crystallisation rates, in general crystals will be formed.

In case of high crystallisation rates in general the solid phase formed can be completely or partly amorphous.

Brief description of the LTOC technology The basic principle of LTOC is presented in figure 1. In order to explain the principle, a possible cooling trajectory is indicated with an arrow. When a solution of an organic compound with a mol fraction Xs (starting at T=T1) is cooled to T=T2, ice starts to crystallize. Decreasing the temperature even further to T=TE will result in the formation of larger quantities of ice. At T=TE (the so-called eutectic temperature), also the organic compound starts to crystallize. The corresponding composition of the liquid concentrate (mother liquor) is the eutectic composition XE. Further cooling to T=T3 would result in complete solidification of the mixture (i. e. a mixture of ice crystals and crystals of the organic compound). Note that in reality the starting solutions such as fermentation broths are multi-component systems and therefore, the phase-behaviour is much more complex compared to the pure two-component system shown here. However, the basic principle is the same.

Legend to the Figure Figure 1: Melting point diagram for an ideal binary mixture of enzyme and water showing eutectic behaviour. Note that T1 is the melting temperature of pure ice and Tp is the melting temperature of pure protein.

Example 1 This example shows that the LTOC technology can be applied to recover relatively pure +-glucanase at a high yield (more than 80%) in a single step from Fromase 0, an aspartic protease used as rennet in cheese production.

First step : preparation of solutions for buffer : 2. 1g of citric acid (C6H807. 1 H2O) was dissolved in distilled water in a gauged flask, and further filled to 100ml. A 0. 1 fiv citric acid solution is obtained.

3.6g of disodium phosphate (Na2HP04. 2H2O) was dissolved in distilled water in a gauged flask, and further filled to 100ml.

Second step: preparation of salt solution: 0.16g of anhydrous sodium thiocyanate (NaSCN), 20ml of citric acid solution (0. 1M) and 5ml of disodium phosphate solution (0.2M), was added.

Third step: The salt solution prepared in the second step was added to 25g of Fromase0 (commercial available, DSM. N. V. , The Netherlands) with an aspartic protease activity of around 44001MCU/g. On lab scale the LTOC technology was implemented in a stirred vessel equipped with a cryostat. Separation of the ice crystals (which can become very large spherical crystals, size 100-5000pm) was rather easy due to the well-known low density of ice, which causes the ice to float. The enzyme crystals (small size, typically 10-50pm) were found to be suspended in the concentrate. The enzyme crystals were separated from the concentrate by centrifugation.

The LTOC procedure that was applied here is as follows : 1. A starting solution was brought to a temperature at 10°C.

2. The solution was cooled to-0. 5°C and maintained at-0. 5°C for 24 hours and ice was formed which was removed.

3. The solution was further cooled to-0. 7°C and maintained at-0. 7°C for 24 hours and ice was removed.

4. The solution was further cooled to-1. 2°C and maintained at-1. 2°C for 24 hours and ice was removed.

We have found that overall no detectable Fromase activity (protease and-glucanase) was lost during the LTOC procedure. The Fromase concentration was increased by a factor of almost 2. 5 times during the LTOC process.

These results show the possibilities of the LTOC technology for the recovery, purification and concentration of organic compounds such as enzymes. This experiment shows that no activity was lost during operation of the LTOC process during several days. The purity of the glucanase crystals obtained (around 93%) is rather high for a single crystallization step.

Example 2 A glass container containing 1 liter of a filtrated fermentation broth comprising 48 mg/l of nisine was stirred at 100 rpm. An amount of 33grams of ammoniumsulphate was added to the solution at 0 °C. The solution was continuously stirred and gradually cooled down to-2, 7°C in a period of 1 hour. An amount of 18 grams of ice flakes (diameter between 1 and 20 mm) was added to serve as seed crystals. The solution was then further cooled to-4, 0 °C in a period of 4 hours. Then the solution was put through a sieve of 750 um in order to separate the ice from the nisine solution. An amount of 469 grams of ice was retained on the filter, whereas 408 grams of filtrate was obtained. After centrifugation of the filtrate at 4000 rpm a pellet of 60 mg was obtained. Nisine contents of these fractions were as follows : Nisine Filtrated fermentation broth 27.3 mg/I Ice 10, 4 mgll Filtrate 20, 8 mgll Pellet 3, 6% This experiments show that nisine can be effectively purified from a solution containing a manifold of other proteins such as a fermentation broth using LTOC.

Example 3 A glass container containing 1 liter of a filtrated fermentation broth comprising 48 mg/l of nisine was stirred at 100 rpm. The solution was continuously stirred and gradually cooled down to-1. 4°C in a period of 1 hour. An amount of 18 grams of ice flakes (diameter between 1 and 20 mm) was added to serve as seed crystals. The solution was then further cooled to-3.6 °C in a period of 4 hours. Then the solution was put through a pressure filter at 0.5 bar in order to separate the ice from the nisine solution. The ice was washed twice with 20 ml ice water. An amount of 333 grams of ice was retained on the filter, whereas 436 grams of filtrate and 104 grams of wash fluid was obtained. Nisine contents of these fractions were as follows : Nisine Filtrated fermentation broth 27.3 mg/l Ice 10, 0 mg/l Wash fluid 19, 2 mg/l Filtrate 57, 9 mg/i