DK169951B1 - Particulate immobilised lipase preparation, a process for producing it, and the use thereof - Google Patents

Description

DK 169951 B1

The invention relates to a particulate immobilized microbial lipase preparation, a process for its preparation, and its use.

Extensive research has been carried out to provide immobilized lipase preparations, given the growing utility of immobilized lipases for transesterification. Some of the first industrially useful immobilized lipase preparations for transesterification are described in U.S. Patent No. 4,275,081, column 7, lines 28-35, which show that the immobilized lipase preparations are prepared by acetone precipitation of an aqueous lipase solution on Ceiite® diatomaceous earth.

However, the half-life of these immobilized lipase preparations is relatively small. In addition, the achievable specific I in the passport is relatively small. In addition, there is a dust problem during storage and filling in the columns and solvents are needed using these known immobilized lipase preparations.

15 The research work has thus been more or less focused on the immobilization of lipase on carriers with hydrophobic binding positions. Reference can be made to Biotechnology and Bioengineering, Vol. XXIV, pp. 1007-1013 (1982), which states that it is generally believed that a carrier with hydrophobic binding positions was more or less a necessary condition in the preparation of high half-life immobilized lipase preparations. and high activity for industrial use. Reference can also be made to Danish Patent Specification No. 152763, which describes an immobilized lipase composition with a carrier consisting of a particulate macroporous weak anion exchanger of resin, e.g. a resin belonging to the Duolite® series. Carrier resins of this kind have hydrophobic bonding positions and the compositions exhibit a high half-life and are suitable for industrial transesterification.

Even these anion exchange resins have disadvantages. First, they are very expensive and this greatly affects the price of the immobilized lipase preparations. Secondly, when used in organic media, it has been found that extractable constituents from the anion exchange resins are transferred to the organic medium, and although the amount of the extractable constituents is small, this often represents a serious disadvantage, especially if it is sought to the end product of the enzymatic process DK 169951 B1 2 must be for human consumption. It is possible to wash the resins with organic solvents before immobilization, but this is an expensive step.

Thus, there is a need for an inexpensive, immobilized lipase preparation with a high half-life and the possibility of obtaining high specific activity, in connection with which the possibility of transferring extractable ingredients from the carrier to an organic phase can be excluded.

It has now been found in accordance with the invention that the stated purpose can be fulfilled if - against the prejudice created in connection with the previously stated carrier of diatomaceous earth - a carrier selected from a carefully defined class is used of inorganic carriers.

The particulate immobilized lipase composition according to the invention, which is of the kind set forth in the preamble of claim 1, is characterized by the characterizing part of claim 1.

In an article in Advances in Colloid and Interface Science, 25 (1986) 235-15248, "Macroporous Silica in Chromatography and Immobilization of Biopolymers1 shows that the maximum capacity for enzyme immobilization exists in silica materials, Surprisingly, it has been found that the maximum capacity for lipase immobilization occurs with silica materials 20 or silicates with pore sizes between 10 and 45 times the size of the lipase molecules. The invention is directed solely to particulate immobilized lipases and lipases are unique enzymes in the sense that enzymatic activity acts on an intermediate surface between two phases. which means that the immobilization of the lipases is a very sensitive problem, which greatly limits the utility of known immobilization techniques in the field comprising lipase immobilization, cf. J. Lavayre et al., Preparation and Properties of Immobilized Lipases, Biotechnology and Bioengineering, Vol. XXIV, * pages 1007-1013 (1982) John Wiley & Sons.

The term "macroporous" means that the diameter of the pores is at least 250 Å. The pore diameter is measured by the B.ET. method.

The carrier material used for the immobilized lipase according to the invention consists of at least 65% by weight of silica and / or silicates, preferably at least 90% by weight of silica and / or silicates. Further, "silica or silicates" in this specification of claims is understood to mean a genuine silica or genuine silicates, i.e., silicas or silicates, not of derivatives.

The diameter of the lipase molecules can be measured by X-ray diffraction analysis and other methods, as set forth in "Biochemistry" by Albert L. Lehninger, 1970, Worth Publishers Inc., pages 142-143.

The diameter of the lipase molecule is usually about 50 Å. Thus, the 10 particulate silica gels described in Grace Information Sheet SG BC1 E / June 1978 (from Grace, Grace Plaza, 1114 Avenue of the Americas, New York, NY 10036-7794) are suitable for the purpose of the invention because they are all has a pore diameter over 500 Å. Although it appears from the information sheet that the silica gels can be used to immobilize cells and enzymes, there is no indication at all in the information sheet that the silica gels can be used to immobilize lipases, and lipases are exceptional enzymes that, in comparison with other enzymes, exhibits outstanding properties in terms of immobilisation, cf. the explanation given above.

It has been found that the inorganic powder used in the previously cited U.S. Patent No. 4,275,081 (column 12, line 48) cannot be used in the process of the invention (cf. the particle size limitation of claim 1) and that acetone precipitation with all the attendant drawbacks seems to be the only way to fix the lipase on the Celite® powder used in US Patent 4,275,081.

From the previously cited article in Biotechnology and Bioengineering, Vol. 25 XXIV, pages 1007-1013 (1982), it appears that lipases can be immobilized on a carrier of derivative Spherosil®, which is a hydrophobic porous support having a pore diameter of 1250-3000. Aa. However, Sphaerosil® is very expensive. One of the most important aspects of the particulate immobilized lipase according to the invention is the possibility of producing an inexpensive, particulate immobilized lipase preparation for which derivation of the carrier is unnecessary.

DK 169951 B1 4 European Patent No. 147,914 discloses an immobilized lipase preparation, c 1 which lipase is immobilized on a glass carrier having a particle size of 30-45 mesh and an average pore size of 400 Å. However, it is also apparent from European Patent No. 147,914 that it is necessary to use a * 5 organotitanate type coupling agent to prepare the immobilized lipase preparation.

In Applied Microbiol. Biotechnol. 28, pp. 527-30 discloses a lipase preparation immobilized on a glass carrier PG 700-80 having a particle size of 20-80 mesh and a pore diameter of 700 Å. However, page 528 shows that the water was removed completely and this makes the known immobilized lipase preparation 10 out of the scope of this invention because the immobilized lipase of the invention contains 1-20% water. This difference means that the performance of the known product is inferior to the performance of the immobilized lipase according to the invention.

In an article in Enzyme Microbial Technology, 1984, Vol. 6, October, 443-446, it appears that various grades of diatomaceous earth can be used as carriers for the immobilized lipase intended as an agent for enzymatic transesterification of fats. However, these carriers have pore sizes far greater than the pore sizes of the carrier material of the particulate, immobilized lipase composition of the invention. This means that the activity of each introduced LU is significantly lower than that of the invention due to the significantly less specific surface area of the known immobilized lipase preparation. Thus, in the known immobilized lipase composition, there is a multilayer of lipase on the support, whereas in the immobilized lipase composition according to the invention a monolayer of lipase is present on the support.

A preferred embodiment of the immobilized lipase composition 25 of the invention is characterized by the characterizing portion of claim 2. If the particle size is less than 200 s, the pressure loss in a column will tend to be too large, and if the particle size is above 800 s, the diffusion inhibition will tend to be too large. *

A preferred embodiment of the immobilized lipase preparation 30 according to the invention is characterized by the characterizing part of claim 3 DK 169951 B1. In this range for pore sizes, the expressed lipase activity, measured in BLU / g, is particularly high.

A preferred embodiment of the immobilized lipase composition according to the invention is characterized by the characterizing part of claim 45. In this way, the immobilized lipase can be used in columns operating at high temperature, thereby obtaining at least two advantages. First, it is possible to use the immobilized lipase, e.g. for transesterification, without a solvent, because of the relatively low viscosity of the reaction mixture, secondly, the reaction rate will be relatively high, and thirdly, the diffusion rate 10 of substrate and products within the pores will be greater.

A preferred embodiment of the immobilized lipase composition according to the invention is characterized by the characterizing part of claim 5. These lipases have been investigated and the results obtained at high temperature in a column have all been good.

The invention also encompasses a method for preparing a particulate, immobilized lipase composition according to the invention, which is characterized by the characterizing part of claim 6. It has been found that washing the particulate immobilized lipase between the separation thereof from the aqueous phase and the drying thereof is advantageous.

The amount of time sufficient to bind the desired amount of lipase to the carrier material varies from lipase to lipase and may range from a few minutes to a few days.

A preferred embodiment of the method according to the invention is characterized by the characterizing part of claim 7. If the charge 25 is below 10,000 LU / g of carrier material, the rate of liquid reaction flowing through the column tends to become too low and it is very difficult to obtain a charge above 500,000 LU / g of carrier material.

A preferred embodiment of the method according to the invention is characterized by the characterizing part of claim 8. Thereby, the 30 expressed lipase activity, measured in BLU / g immobilized lipase, is as large as possible. Reference can be made to Figure 1, which will be explained in more detail below.

DK 169951 B1 6

A preferred embodiment of the method according to the invention is characterized by the characterizing part of claim 9. This is the simplest and cheapest way to perform the separation.

The invention also encompasses an use of the immobilized lipase composition according to the invention, and this use is characterized by the characterizing part of claim 10. In connection with this application, it is not necessary to use any solvent, but a solvent may be used if desired. The application can be carried out continuously, e.g. in columns, or discontinuously.

The immobilized lipase of the invention can also be used to hydrolyze fats by contacting an emulsion of triglycerides and water with the immobilized lipase composition of the invention. The immobilized lipase composition of the invention can also be used for the synthesis of triglycerides or other fatty acid esters by contacting a mixture of glycerol or substituted glycerol or 15 other types of alcohols and free fatty acids with the immobilized lipase composition of the invention. For these applications, it is not necessary to use any solvent, but a solvent may be used if desired. The applications can be carried out continuously, e.g. in columns, or discontinuously.

The lipase activity unit (LU) is determined as described in publication 20 AF 95.1 / 2-GB of 83-01 -03, which can be obtained from Novo Nordisk A / S, Novo Allé, 2880 Bagswaard, Denmark.

The lipase activity expressed in BIU (Batch Interesterification Units) is determined as described in the publication AF 206-2, which can be obtained from Novo Nordisk A / S,

Novo Allé, 2880 Bagswaard, Denmark.

FIG. Figure 1 illustrates the dependence relationship between expressed lipase activity (BLU / g) and pH during loading of a lipase from Humicola lanuginosa (see below). carrier materials with different pore sizes.

FIG. Figure 2 shows the flow rate versus time logarithm of a Humicola lanuginosa lipase (see below) on carrier materials of various 30 pore sizes.

DK 169951 B1 7

The figures of FIG. 1 showed the following. The silica support, which is a Grace carrier product described in Biocatalyst Supports SG BC 1E / June 1987, is washed with a buffer at the pH to be used during the following lipase adsorption step, i.e. at pH 4, 4.5, 5, 6 and 7, cf. FIG. 1, for half an hour, then filter. The desired amount of lipase, which is the lipase activity sufficient to produce a charge of 186,000 LU / g, is dissolved in 5 ml of deionized water and added to 1 g of carrier. The lipase is prepared as set forth in Example 1 of DK Patent Application No. 4117/86, i.e. using Humicola lanuginosa. The pH is adjusted and the carrier and lipase solution are slowly stirred by rotation 10 for 2 hours, followed by vacuum filtration. The filtrate is analyzed for hydrolytic activity (LU / ml) to determine the amount of adsorbed (charged) lipase. The immobilized lipase is air dried, the moisture content is adjusted to 10% by weight and the sample is analyzed for discontinuous transesterification activity (BIU / g). It is apparent from the figure that the silica carrier having pore size 1500 A (i.e. 25 times the diameter of the lipase molecule, i.e. within the range of pore sizes specified in the main claim) exhibits an excellent expressed lipase activity at the optimum pH.

In FIG. 2, the three of the four immobilized lipase preparations of FIG. 1, which is within the scope of the invention, examined in a column as follows.

A system consisting of a column containing 20 ion exchange resins saturated with water and an enzyme column (containing 4.5 g of immobilized lipase preparation) was mounted in series. The function of the column was saturation of the substrate with water. An oil mixture consisting of 28.6% (w / w) lauric acid and 71.4% (w / w) soybean oil was pumped through the columns. The temperature in the columns was maintained at 60 ° C. The flow rate was set to maintain a constant conversion of 14% of lauric acid incorporated into the soybean oil. Samples were taken 3-5 times per week and analyzed by removing the free fatty acids and partial glycerides by column chromatography and by methylating the triglycerides, followed by capillary gas chromatography of the methyl esters. The flow rate proportional to the activity was then plotted against time when the conversion was 14% ± 1% incorporated lauric acid.

DK 169951 B1 8 Based on the figure, the following half-lives and initial activities can be estimated:%

Type of carrier Initial flow rate halving (activity) (g TG * / g lipase time, 5 preparation) hours GRACE 4 3.6 700 GRACE 5 8.0 750 GRACE 6 12.0 850 10 _ * TG is an abbreviation for triglyceride.

In this case, too, the sieve carrier having a pore size 1500 Å functions well with respect to the initial flow rate.

To demonstrate the importance of varying water content in the immobilized lipase preparation, the following experiments were performed. Immobilized lipase preparations were prepared with Grace 6 as the carrier. The lipase was the Humicola lanuginosa lipase. With the exception of the drying, the immobilized preparations were prepared as shown in connection with FIG. 1, at pH 4.5.

20 The following activities were found.

Activity% water j ————— _ BLU / g_% of maximum 0 15 29 _1 ^ 05__24__47_ 1 35 69 * 2 48 94

According to the invention, the invention is 10 51 100 12 50 98 15 49 96 __20 46 90 DK 169951 B1 9

The following examples further illustrate the immobilized lipase composition of the invention. Examples 1, 2 and 7 are production examples; Examples 3,4,5 and 6 are application examples and Example 8 is a comparative example.

EXAMPLE 1 2 g of carrier (Grace, pore diameter 700 Å, particle size 0.5-1 mm) is washed in 0.2M acetate buffer at pH 4.5 for half an hour and vacuum filtered.

400,000 LU (0.17 g of Rhizomucor miehei lipase prepared as disclosed in European Patent Specification No. 238,023 and having an activity of 2.4 x 10 6 LU / g) are dissolved in 8 g of deionized water and added to the carrier. The pH is adjusted to 4.5 and the slurry of carrier and enzyme solution is slowly stirred by rotating for two hours at room temperature, followed by vacuum filtration.

The filtrate contains 2.7 x 10 5 LU, corresponding to an adsorption of 32%. The immobilized lipase preparation is dried in a vacuum oven at 25 ° C overnight and the moisture content is adjusted to 10%. The sample was analyzed at 26 BlU / g.

Example 2 1 g of carrier (Grace 4, pore diameter 500 Å, particle size 0.5-1 mm) is washed in 0.2 M acetate buffer with pH 4.5 for half an hour and vacuum filtered. 50,000 LU (0.35 g lipase with an activity of 1.4 x 10 5 LU / g) of Candida antarctica lipase prepared as disclosed in European Patent Application No. 0287634 were dissolved in 5 ml of deionized water and added to the carrier. The pH was adjusted to 4.5 and the carrier and lipase slurry was slowly stirred by rotation for 12 hours at room temperature, followed by vacuum filtration. The filtrate contains 19,000 LU, corresponding to an adsorption of 62%. The immobilized lipase was air dried and the moisture content adjusted to 10%. The sample was analyzed for 24 BlU / g (60 ° C analysis without solvent).

Example 169951 B1 10 EXAMPLE 3 a

A

Hydrocarbons with Immobilized Lipase 2.5 g of Immobilized Lipase prepared as set forth in Example 1 of Danish Patent Application No. 4117/86 (i.e., using Humicola lanuginosa) 5 were added to a flask containing soybean oil and water at pH 4.5 and shaken. at 50 ° C. Samples were taken and the content of free fatty acids (degree of hydrolysis) was measured by titration with KOH. The experiments were performed at 25%, 50% and 75% soybean oil content. The total amount of oil and water was 50 g at all times.

The results are shown in FIG. 3. It is seen that the highest degree of hydrolysis was achieved with the highest water content.

EXAMPLE 4

Ester synthesis with immobilized lipase 0.05 mole (10 g) of lauric acid and 0.05 mole of alcohol was mixed with 5% (w / w) immobilized lipase prepared as described in Example 3. The reaction was carried out at 60 ° C at to stir the flask containing the substrate and enzyme. Four different alcohols were used: lauryl alcohol, pentanol, isopropanol and propanol. The results are shown in FIG. 4. The test with lauryl alcohol was carried out under reduced pressure (about 50 mbar). The figure shows that the largest conversion is achieved with lauryl alcohol and that the 20 enzyme cannot esterify secondary alcohols.

EXAMPLE 5

Continuous fixed-bed operation with immobilized lipase

Two columns, each containing 60 g of immobilized lipase, are prepared as described in Example 3. The columns were arranged in series and the temperature in the columns was 60 ° C. A similar column system was set up in parallel, containing 2 times 60 g of Lipozyme® (a commercially available DK 169951 B1 11 immobilized lipase from Novo Nordisk A / S) instead. Pure soybean oil was pumped through the systems and recycled. The height of the bearing in the two systems was measured at time zero. A fixed flow rate was used for 3 days, after which the pressure drop across the columns the bearing height of the columns was measured.

5 Then a new flow rate was used and the new values were measured after 3 days, etc. The results are shown in fig. 5. The results show that the lipase on a silica carrier is a better enzyme preparation than the commercially available Lipozyme® since the pressure drop is lower at the same flow rate. It was also observed that the bed containing the silica support was not compressed even after more than 300 hours of operation, whereas the columns containing Lipozyme® were compressed to a total of 13 mm.

Example 6 5 g of carrier (Grace 6, pore diameter 1500 Å, particle size 0.5-1 15 mm) was mixed with a solution containing 250,000 LU of the B component of Candida antarctica, as described in International Patent Application No. 88/02775, page 9. The pH was adjusted to 4.5 and the carrier and lipase slurry was slowly stirred by rotation for 4 hours at room temperature followed by drying under vacuum.

The charge of the particles was 28,100 LU / g dry carrier material. 125 mg (on a dry basis) of the immobilized lipase containing ca. 2% water was used to esterify 40 mmol of propanol and myristic acid at 60 ° C. Within 20 minutes, 29.2% ester was formed.

EXAMPLE 7 1 g of the Manville R-648 silicate support (with mean pore diameter 1400 Å) was washed in 0.2M acetate buffer at pH 4.5 for half an hour and vacuum filtered. 186,000 LU (0.06 g of lipase with an activity of 3.1 x 10 6 LU / g) of lipase, prepared as set forth in Example 1 of Danish Patent Application no.

4117/86, viz. using Humicola lanuginosa, was dissolved in 5 ml of deionized water and added to the carrier. The pH was adjusted to 4.5 and the slurry of carrier and lipase solution was stirred slowly under rotation for two hours followed by vacuum filtration. The filtrate contained 34,000 LU, corresponding to an adsorption of 82%. The immobilized lipase was air dried and the moisture content adjusted to 10%. The sample was analyzed for 14 BIUs.

EXAMPLE 8 10 This example is a comparative example as the pore diameter of the carrier is less than 5 times the diameter of the lipase molecules.

1 g of carrier material Amicon Matrex ™ silica Si Chromatography medium (pore diameter 100 Å, particle size 190-300 μηη) was washed in 0.2M acetate buffer with pH 4.5 for half an hour and then vacuum filtered.

15 186,000 LU (0.06 g lipase with an activity of 3.1 x 10 6 LU / g) lipase

(prepared as set forth in Example 1 of Danish Patent Application No. 4117/86) was dissolved in 5 ml of deionized water and added to the carrier. The pH was adjusted to 4.5 and the carrier and lipase slurry was stirred slowly by rotation for 2 hours followed by vacuum filtration. The filtrate contained 50 LU

20 corresponding to an adsorption of 100%. The immobilized lipase was air dried and the moisture content adjusted to 10%. The sample was analyzed for 6 BIUs. The low BlU value shows that the immobilized lipase is of poor quality.

*

Claims (10)

A particulate immobilized microbial lipase preparation immobilized on a macroporous support material consisting of at least 65% silica or silicates, characterized in that over 90% of the particles have a particle size 5 of between 100 and 1000 µπ the particles exhibit a diameter of between 10 and 45 times the diameter of the lipase molecules, and that the water content of the particulate immobilized lipase is between 1 and 20%, preferably between 2 and 20%, especially between 5 and 20%. Particulate immobilized lipase composition according to claim 1, 10, characterized in that over 90% of the particles have a size of between 200 and 800 µm, preferably between 200 and 400 µm. The particulate immobilized lipase composition according to claim 1 or 2, characterized in that over 80% of the pores in the particles exhibit a diameter of between 12 and 40 times the diameter of the lipase molecules. A particulate immobilized lipase composition according to claims 1 to 3, characterized in that the lipase is a thermostable lipase. The particulate immobilized lipase preparation according to claims 1-4, characterized in that the lipase is prepared by culturing a microorganism containing a gene encoding and expressing a lipase derived from a strain of a Humicola species, Candida antarctica or Rhizomucor miehei . DK 169951 B1 Process for the preparation of a particulate immobilized v lipase preparation according to claims 1 to 5, characterized in that an aqueous solution of a microbial lipase is contacted with a particulate macroporous support material consisting of at least 65% silica or silicates in which more than 90 %. . 5 of the particles have a size of between 100 and 1000 µπ \ and where more than 80% of the pores in the particles exhibit a diameter of between 10 and 45 times the diameter of the lipase molecules, for a time sufficient to bind the desired amount of lipase to the carrier material, after which the thus formed particulate immobilized lipase preparation is separated from the aqueous phase and the separated immobilized lipase preparation is dried to a water content of between 1 to 20%, preferably between 2 and 20%, especially between 5 and 20%. Process according to claim 6, characterized in that the ratio of the amount of the aqueous solution of the microbial lipase to the weight of carrier material corresponds to 10,000 - 500,000 LU / g of carrier material (dry weight). Process according to claim 6, characterized in that the pH during the contact between carrier material and aqueous solution does not deviate more than 1 pH unit from the optimum charge pH of the lipase in question in terms of expressed lipase activity. Process according to claims 6 - 8, characterized in that the separation is carried out by simple filtration. Process for the transesterification of fats, characterized in that liquid fats or fat mixtures, including free fatty acids or fatty acid esters, are contacted with the immobilized lipase preparation ifølge 25 according to claims 1 - 5.