NL9300715A - Method for carrying out an enzymatic and/or catalytic reaction, respectively, to effect a separation of a mixture of optical isomers, membrane on and/or within which enzyme and/or catalyst is immobilized, and membrane reactor containing one or more of such membranes - Google Patents

Abstract

The invention relates to a method for carrying out an enzymatic and/or catalytic reaction, respectively, to effect a separation of a mixture of optical isomers, wherein a first liquid phase which contains starting material is brought into contact with a side A of a membrane, in or on which membrane enzyme and/or catalyst is immobilized, and at the same time a second liquid phase which may or may not contain starting material is brought into contact with the other side B of the membrane, said method being characterized in that a membrane having an anisotropic membrane structure is used, side A of the membrane containing pores and side B of the membrane either containing substantially smaller pores than those of side A or comprising a tight (impervious) homogeneous membrane surface. The invention also relates to a membrane and a membrane reactor.

Description

Process for carrying out an enzymatic and / or catalytic conversion or for separating a mixture of optical isomers. membrane on which and / or in which etc. and / or catalyst are immobilized, as well as membrane reactor. containing one or more such membranes.

The invention relates to a method for carrying out an enzymatic and / or catalytic conversion or for performing a separation of a mixture of optical isomers, wherein a first liquid phase, containing starting material, is brought into contact with a side A of a membrane, in or on which membrane enzyme and / or catalyst has been immobilized, while simultaneously contacting a second liquid phase, which may or may not contain starting material, with the other side B of the membrane.

Such a method for performing an enzymatic conversion is known from a publication by W. Pronk et al. In Biotechnology and Bioengineering, Vol. 32, 512-518 (1988). Candidavugosa lipase is immobilized on hollow fiber membranes, which are then used for the hydrolysis of fats. Symmetrical membranes of cellulose fibers (cut off = 5000) are used for this. Lipase is immobilized on the side of the membrane, which comes into contact with the hydrolysing grease (starting material). The membrane is impermeable to the fat phase. During the hydrolysis, water penetrates through the membrane into the enzyme layer, so that reaction water is supplied. Glycerol diffuses back through the membrane into the water phase, the fatty acids remain in the fat phase. The hollow fiber membrane systems used have a large surface area / volume ratio. Since the fat phase cannot penetrate Mem-Draan, the pressure caused by the circulation of the lipid phase is sufficient to maintain good phase separation so that no precise control of the pressure is necessary. A pressure of more than 1 bar is therefore not applied.

The use of so-called membrane reactors offers the possibility of enzymatic and / or catalytic conversions, respectively. optical isomer separations can be carried out in a relatively simple and safe manner. An additional advantage of membrane reactors is that the continuous and / or selective withdrawal of a product or the addition of reagents makes it possible to favorably influence the progressing processes. The apparent equilibrium can be shifted, making processes more specific and / or faster. However, it has been found that the productivity of the known methods for carrying out enzymatic and / or catalytic conversions by means of membrane reactors still leaves much to be desired.

This problem is solved according to the invention.

The invention relates to methods of the type mentioned in the preamble, characterized in that a membrane with an anisotropic membrane structure is used, in which side A of the membrane contains pores and side B of the membrane or substantially smaller pores than that of side A, or comprises a dense homogeneous membrane surface.

For the purposes of this disclosure, "a dense homogeneous membrane surface" is understood to mean a membrane surface containing very small pores, for example of the order of 1 A, the pore size generally being in the range of 0.1-5 A.

Incidentally, a general description of conventional membrane reactors and conventional conversions or separations (including the resolution of racemic mixtures of optically active compounds) using them may be taken from U.S. Patent 4,800,162, the contents of which are to be incorporated herein by reference. Inspected. All types of reactors and reactions resp. separations can be mentioned within the scope of the present invention.

If hollow fiber membranes are used in the process according to the invention, the pores in the fiber wall widen from the inside out (anisotropic) or from the outside in (inversely anisotropic). Standard practice of the invention is that the smallest pores are on the outside of the fiber. Of course, the first or second liquid phase can be allowed to flow on the outside or inside of the fibers as desired. Because the enzymes can adhere to the entire wall of the pores up to the outside of the fibers, they have a much larger effective membrane surface than with the symmetrical membranes. It has been found that this can increase productivity many times, even tens of times.

The process according to the invention may, for example, concern the hydrolysis of fats or the formation of esters.

The dimensions of the pores in the membranes used according to the invention naturally depend on the conversions to be carried out. Generally, side A will have macropores, generally with a diameter of 1 nm - 50 µm, preferably 10 nm-100 µm, and silk B will have micropores, mainly with a diameter of 0.1 nm - 10 µm, preferably 1 nm - 1 µm. Although these areas appear to overlap, it is noted that in the process of the invention it is a condition that the pores on side B of the membrane are generally smaller than those on side A of the membrane.

In particular, in the membranes used in the method according to the invention, the membrane on side A comprises an underlayer with a continuous network of pores of substantially a diameter of 10 nm - 1000 µm and a porosity of 1 - 90%, and that the membrane side B comprises a top layer with a contiguous network of pores substantially in diameter of 0.1 nm-1 µm and a porosity of 0.1-80%.

In general, membranes thinner than membranes used in the prior art can be used in the process according to the invention. For example, the total thickness may be in the range of 0.1-2000 µm, for example, in the range of 1-60 µm. In the latter case, it is preferred that the thickness of the layer of the membrane on side A, where the large pores are located, be 1-50 µm and the thickness of the layer on side B, where the small pores are located or that have a dense membrane surface contains 0.1-10 ym.

The two liquid phases used in the process according to the invention may, depending on the conversion or separation, be an organic phase and an aqueous phase. It is preferred that the membrane contains the micropores on the water phase side and the macro pores on the organic phase side. Incidentally, it is also quite possible to use systems without an organic phase, i.e. systems with two water phases, in which different polymers are dissolved in the two separate water phases, for example polyethylene glycols (PEG) and dextrans. For example, mixtures of optical isomers can be separated using such aqueous systems.

It has been found that by applying pressure the reaction rate or. can control the separation speed in the method according to the invention. In the method according to the invention a positive pressure difference of the first liquid phase with respect to the second liquid phase can therefore be applied across the membrane, preferably a pressure difference of 1 - 100 kPa.

In the process of the invention, the enzymes and catalysts used generally have a much longer service life than in conversions in membrane reactors of the prior art. However, if the enzymes or catalysts gradually become "depleted", by controlling the pressure the total activity resp. keep the reaction rate constant. Accordingly, in a preferred embodiment of the process of the invention, the productivity of the conversion is controlled by adjusting a differential pressure of the first liquid phase relative to the second liquid phase across the membrane.

The membrane to be used in the process according to the invention can be provided in advance or in situ with the enzyme or catalyst to be used with the desired conversion required enzyme. The enzyme or the catalyst is applied by methods conventional in the art. If the first resp. second liquid phase an organic reaction. water phase is used, the enzyme resp. the catalyst for immobilization in the membrane fed from the large pore side to the membrane.

The invention therefore also relates to a membrane, as defined above, on which and / or in which enzyme and / or catalyst has been immobilized.

The invention further relates to a membrane reactor containing one or more such membranes. Such reactors can not only be designed as devices with very many parallel hollow fibers, but they can also be flat membranes, optionally designed as so-called "spiral wound" reactors and "plate-end-frame" reactors.

The invention is further illustrated in the examples below. Use is made of the terms "lumen silk" and "shell silk". These expressions relate to the inside, respectively. outer side of hollow fiber membranes.

Example 1

Asymmetrical anisotropy hydrophilic membranes, produced by X-Flow BV, total wall thickness 0.5 mm, outer diameter fiber 2 mm, cut-off 80000 Dalton, skin on the lumen side, were used as a reactor for the hydrolysis of rapeseed oil using the enzyme Candidarugosa. High productivities, expressed in molar free fatty acid per m2 membrane per hour, were found for this reactor (see table 1) .These productivities are significantly higher than those of systems described in the literature, performed with a comparable enzyme, but with symmetrical porous membranes (Cuprophane).

Table 1,

Productivity of membrane reactors: Candida ruaosa membrane, etc. Productivity (mol m-2 “*) with degree of hydrolysis type 0?! 20% 40 *

Cuprophane 0.04 L 0.029 0.017

Exp. 1 0.21 0.092 0.086 (0.86 hours)

Exp. 2 0.12 0.053 0.053 (0.215 hours)

The experiments listed in Table 1 exp. 1 or exp. 2 are designed according to the invention with the oil on the skin side and identical enzyme loading. That is, immobilization was done once, before the first reaction (exp. 1). Starting from a solution of Ccmdida rugosa, 0.5g in 100 ml of water, recirculated for 15 hours, then 100 ml of rapeseed oil passed through. After 40 hours of hydrolysis, 500 ml of oil is rinsed, then exp. 1 started. The first reaction was carried out for 86 hours, then 1000ml of new oil single pass was passed and then experiment 2 was started. In total, therefore, the same enzyme was worked for 301 hours. 1 and 2, inactivation may occur, perhaps due to air-enzyme contact during the switchover from experiment 1 to experiment 2.

Other experiments with the bottom side oil yielded products comparable to the values given in Table 1 (pre-exp. 1 and exp. 2).

It can be seen from Table 1 that the productivity of the membrane reactors mentioned herein using the asymmetric anisotropic membranes mentioned are 10 to 2 times more effective than the reactors described in the literature. If activities are compared at the same degree of hydrolysis (Table 1), the ratio (reactor productivity according to the invention / productivity literature) is approximately 5 ~ 2.

With a progressive degree of hydrolysis, the productivity of the reactor according to the invention stabilizes, while that of the Cuprophane reactor decreases further. The difference between the two reactor types becomes larger with increasing hydrolysis.

Better results are obtained with membranes with skin on the outside, cut off at least 80,000 daltons, large pores on the lumen side (pore radius in the order of microns), total wall thickness approx. 100 μm, external fiber diameter 0.5 mm. The surface of these fibers is higher, while diffusion distances are smaller. Productivity increases.

Example 2

Various membranes are used in this example, which are explained below:

Table 2

Characteristics of membrane modules used module surface pore fiber wall thickness (m2) size1 diameter (mm) (nm) (mm) cuprophane 1.0 5-10 0.25 0.009 invention 0.11 10-50 type 1 invention 0.156 10-50 2 0.5 type 2 invention 0.066 400 type 3 1 indicated is the smallest pore diameter in the membrane, in case of anisotropic membranes (invention) the pores are in deskin.

Type 1 and type 2 differ only in hydrophilicity. That is to say the fiber is manufactured through the same process, but the post-treatment which induces the hydrophilic character is different. As a result, type 2 is more hydrophilic than type 1. The structure of the fibers is such that the pores on the bore side (lumen, inside of the fiber) are smaller than those on the outside of the fiber. The cutoff of the membranes is 80 kDa (> size of inner pores), outer pores are approximately 0.4 micron. These membrane types are available from X-Flow.

Type 3 has an 'inverted' structure compared to types 1 and 2. The pores on the outside of the membrane are smaller than those on the lumen side. This smallest pore (on the outside) has a diameter of 0.4 micron, on the lumen side the diameter is significantly larger (a few microns).

The hydrolysis of rapeseed oil with Candida rugosa has been used as a model system. The enzyme was immobilized by ultrafiltration of an enzyme solution and the system was rinsed with oil. The experiment was then started. The Cuprophane reactor was based on a 2 g / 100 ml enzyme. A standard solution of 1 g / 100 ml was used for the reactors according to the invention (other solutions 0.5 and 2 g / 100 ml were also tested). With a few exceptions, the hydrolysis batch experiment was performed. All hydrolysis experiments were performed at 30 ° C.

No significant differences in reactor activities were observed for the different concentrations of enzyme solution. This indicates that the membrane surfaces are saturated with (physically adsorbed) enzyme.

Oil-fatty acid samples were checked for enzyme activity (e.g. tributerine essay) to ensure 'emulsion free operation'. After it was determined that "emulsion activity" could not be measured, an experiment was started. The values given in Table 2 refer to the activity of enzyme immobilized on the membrane. Table 2 summarizes the productivities for a degree of hydrolysis of 50% of the different reactors.

Table λ

Membrane reactor productivities «nonjbraan type P (molm" 2h-1) comments cuprophane 0.017 literature *) (conventional type) 0.018 exp. Of applicant invention type 1 0.06 0.2-0.3 bar invention type 2 0.11 0, 08 and 0.4 bar invention type 3 0.07 0.07 har 0.4 0.4 bar *) W. Pronk et al., Biotechnol. Bioeng., Vol. 32, pp. 512-518 (1988)

The productivity of the cuprophane reactor found corresponds to the literature value. Furthermore, the experiments of Example 1 are confirmed, i.e. these modules are more effective than the Cuprophane modules.

Differences in activity between type 1 and type 2 membranes are thought to be caused by differences in hydrophilicity, although the physicochemical consequences of hydrophilicity are difficult to imagine. Surface roughness may also play an important role in this; The after-treatment of the fibers mainly influences the structure and composition of the fiber surface.

The high productivity of the type 3 fibers at 0.4 bar oil pressure is striking. All the more so because the productivity at low pressure is roughly equal to the type 1 and type 2 membranes. In fact, the pressure dependence of membrane activity confirms the idea that the oil-water interface can be displaced in the membrane, with a suitable pore structure. In contrast to the case where denser membranes are used, the enzyme which is present in the internal pore structure of the membrane is immobilized to be utilized. Provided that a suitable pore size gradient is present in the membrane structure, the productivity of the module is in fact controllable by the applied pressure.

There are two main aspects that determine the productivity and stability of a reactor equipped with type 3 membranes. The activity is determined by the amount of enzyme immobilized for the oil. This amount is determined by the size of both the internal surface and the size of the pores. The size of the surface determines the amount immobilized enzyme, the pore size determines whether the oil can penetrate the pore to where the enzyme is located at moderate pressures up to 50 kPain.

All examples below relate to the method according to the invention.

Example λ

Phenoxyacetic acid methyl ester (FAME) was synthesized by refluxing phenoxyacetic acid in methanol with sulfuric acid as the catalyst. An enzyme solution was prepared by dissolving 1 gram of Ccmdida vugosa lipase in 200 ml of water. This solution was filtered through a 0.1 m2 membrane module from the lumen side of the membrane to the shell side for 30 minutes, the permeate was recycled. The shell side filtrate was then collected. The water on the lumen side was replaced by the water-immiscible organic phase in which FAME + octanol is present. Then the shell side of the membrane was rinsed with 1 liter of water for 15 minutes.

A solution of 66.6 g of FAME in 200 ml of 1-octanol was hydrolyzed by circulating this organic phase on the lumen side of the membrane at 25 ° C. On the shell side, a 500 ml 0.1 molar phosphate buffer, pH 7., was flown at a flow rate of 50 ml / min. During the experiment, the organic phase was kept at an overpressure of 10 kPa. After 7 hours, a significant amount of FAME was no longer present, demonstrating the hydrolysis activity of the enzyme.

Example 4

Racemic 2-octane acetate was synthesized by reacting 2-octanol metacetic acid at 25 ° C. Undiluted racemic 2-octane propionate was used for the enzymatic reactions. Candida rugosa lipase was immobilized on a membrane as described in Example 3

After loading the membrane with enzyme, 200 ml of racemic 2-octane propionate was circulated. The circulation rate was 40 ml per minute. The gauge pressure was set at 4 kPa and did not change significantly during the experiment. The temperature was 30 ° C. The shell side was rinsed recirculatingly with a phosphate buffer solution with pH = 7.4. After 40 hours the experiment was stopped, the conversion based on the racemic ester phase was 50%. The optical rotation of the substrate of the ester phase was + 0.8%. This means that enrichment of the chiral component has occurred in the organic mixture.

Example 5

A racemic 2-chloropropanoic acid 1-octyl ester mixture was synthesized by heating an equimolar mixture of 2-chloropropanoic acid and 1-octanol with a catalytic amount of sulfuric acid to 250 ° C for 15 minutes.

450 g of Ester were then hydrolysed using an immobilized membrane reactor system, as described in Example 3. The organic phase was circulated on the lumen side for 400 hours, on the shell side a phosphate buffer phase was circulated (0.25 molar ). The pressure difference between organic phase and buffer was set at 4 kPA, the temperature was 30 ° C. After 24 hours, the rotation of the organic phase was -0.14 °. This demonstrates the chiral resolution of the membrane reactor system. After 4000 minutes, 50% of the organic phase was converted.

Example 6

In a membrane module, a 1.3 specific lipase was immobilized by passing an enzyme solution of 2 g Rhizopus javanicus lipase in 100 ml water along the lumen side of the membrane (0.1 m2) for 20 minutes without permeation of the solution through the membrane occurred.

This membrane reactor was used to produce dimorpholic acid from Dimorphoteca oil. To this end, the enzyme-containing solution on the lumen side was replaced by Dimorphoteca oil. 150 ml of crude methhexane extraction, unrefined undiluted dimorfoteca oil, was circulated on the lumen side of the membrane. The oil phase pressure was kept between 30 and kO kPa. A phosphate buffer pH 7 was applied to the shell side. After 100 hours, about 50% of the dimorphecolic acid was released.

Example 7

A membrane module as described in Example 6 was used for the specific hydrolysis of Crambe oil. Crambe oil contains approximately 60% erucic acid, which is mainly in the α-position (i.e. 1.3 position) of the triglyceride. 100 ml of Crambe oil was hydrolyzed in 10 hours, after which a conversion of 50% of the possible fatty acids was reached.

Example 8

A membrane module with a surface of 1 m2 was provided with Candida rugosa lipase. The enzyme was immobilized by filtering a solution of 10 g of Candida rugosa in 500 ml of water from lumen to shell side for 60 minutes. The permeate was then recycled. The filtrate was then removed and collected on the shell side. The membrane was then rinsed (not circulating) with 5 liters of water for 15 minutes.

The substrate, sunflower oil, was continuously hydrolyzed by flowing 0.1ml per minute along the lumen side of the membrane. The pressure on the lumen side was set at 30 kPa, the temperature in the reactor was 30 ° C. 15 liters of water were recycled on the shell side. Degree of dehydrolysis of the substrate at the outlet of the reactor, based on the fatty acids, was 70%. The reactor was in continuous operation for 105 days. The enzyme half-life based on this data is 160 days.

Claims (10)

1. Process for carrying out an enzymatic and / or catalytic conversion or for separating a mixture of optical isomers, whereby a first liquid phase containing starting material is brought into contact with a side A of a membrane , in or on which membrane enzyme and / or catalyst has been immobilized, while at the same time contacting a second liquid phase, which may or may not contain starting material, with the other side B of the membrane, characterized in that a membrane with a anisotropic membrane structure, in which side A of the membrane contains pores and side B of the membrane either contains substantially smaller pores than that of side A or comprises a dense homogeneous membrane surface. Method according to claim 1, characterized in that silk contains amacropores, mainly with a diameter of 1 ni - 50 µm and silk B contains micropores, mainly with a diameter of 0.1 nm - 10 µm. Method according to claim 2, characterized in that silk contains Amacropores, mainly with a diameter of 10 nm - 100 nm and Silk B contains micropores, mainly with a diameter of 1 nm - 1 µm. 4. A method according to any one or more of the preceding claims, characterized in that the membrane on side A comprises a bottom layer with an interconnected network of pores substantially having a diameter of 10 nm - 1000 µm and a porosity of 1-90%. Method according to one or more of the preceding claims, characterized in that the membrane on side B comprises a top layer with a contiguous network of pores substantially in diameter of 0.1 nm-1 µm and a porosity of 0.1-80% . Method according to one or more of the preceding claims, characterized in that the thickness of the layer of the membrane on side A, where the large pores are located, is 1-50 µm and the thickness of the layer on side B, where the small pores or containing a dense membrane surface is 0.1-10 µm. Method according to one or more of the preceding claims, characterized in that a positive pressure difference of the first liquid phase is applied over the membrane relative to the second liquid phase, preferably a pressure difference of 1-100 kPa. Method according to one or more of the preceding claims, characterized in that the productivity of the conversion or the speed of the separation is controlled by adjusting a pressure difference of the first liquid phase with respect to the second liquid phase over the membrane. Membrane as defined in any of the preceding claims, on which and / or wherein enzyme and / or catalyst is immobilized. Membrane reactor containing one or more membranes as defined in claim 9.