The invention relates to an improved method for immobilizing and drying proteins, particularly enzymes, in particular lipases.
EP382767 describes a method for immobilizing lipases. In this case, an aqueous solution of a given lipase is mixed by rotation with a resin (for example Lewatit®) at fixed pH at room temperature. The resin with immobilized lipase was then collected by filtration, followed by washing with water and drying under reduced pressure.
The immobilization of enzymes is generally accompanied by loss of enzyme as a result of non-binding on the support or by desorption (“bleeding”) of enzyme already bound. Furthermore, the immobilized enzyme frequently experiences a loss of enzyme activity during the method steps of immobilization, which leads to significant yield losses and associated increased costs, especially on an industrial scale.
The object of the invention, therefore, was to find a method which allows an efficient immobilization of the protein on the support to be achieved, which signifies that as much as possible of the entire amount of enzyme present on the support is permanently bound and remains and, in the case of an enzymatically active protein, as far as possible the same high enzymatic activity is obtained after immobilization as before immobilization.
A method for immobilizing proteins on a support has been found, wherein the protein is incubated with the support in an aqueous phase in a discontinuous contact vacuum mixer dryer and the immobilized protein is then immediately dried, optionally following an optional washing step, in the same contact vacuum mixer dryer.
It has now been found that the method defined at the outset leads to particularly advantageous results since the overall steps of the immobilization, such as efficient incubation of protein with support, optionally washing the immobilized protein and drying the immobilized protein, are carried out in a single apparatus to give a stable product which can be readily stored and transported. This method is particularly well suited to an industrial scale, if immobilized protein is to be produced on a scale of hundreds of kilograms up to a tonne scale.
Using the method according to the invention, numerous proteins may be immobilized. It is particularly suitable for immobilizing enzymes such as oxidoreductases, hydrolases, isomerases and transferases.
It is particularly well suited for immobilizing hydrolases, in particular lipases.
Within the lipases, those lipases from Candida antarctica can be particularly effectively immobilized, particularly Candida antarctica lipase B (CALB) or such enzymes which are structurally derived therefrom. Such lipases structurally derived from CALB are lipases having at least one, preferably two or more amino acid alterations such as insertions, deletions or substitutions, compared to the CALB polypeptide sequence.
Examples of such lipases structurally derived from CALB are described in WO 2009/080676 (“CALB muteins”), wherein the disclosure content with respect to the CALB muteins in WO2009/080676 is explicitly incorporated by reference.
The enzymes to be immobilized may be isolated from the original organism by known methods or also may be produced by recombinant DNA techniques in suitable host organisms such as Bacillus, E.coli, Pichia, Chrysosporium, Aspergillus and Saccharomyces.
Suitable supports are various organic or inorganic materials such as silica gel, activated carbon or polymeric supports. Suitable polymeric supports are macroporous crosslinked polymers having a particle size of 100 to 1000 ∥m and an average pore radius of 10-20 nm. Particularly suitable are macroporous crosslinked acrylate polymers such as poly(meth)acrylates crosslinked with divinylbenzene, which may comprise, for example, acrylic acid, acrylic ester, methacrylic acid and methacrylic ester. Such polymers are sold, for example, by Lanxess under the name Lewatit® VP OC 1600 or by DOW under the name Amberlite® XAD-7.
Suitable discontinuos contact vacuum mixer dryers are known to those skilled in the art from the literature (e.g. Friedrich Kneule, “Das Trocknen” [Drying], Sauerländer A G, Aarau, 1975, ISBN 3-7941-0429-3). Particularly advantageous is the use of a vacuum tumble dryer or double-cone dryer. Well suited are tumble dryers having an internal volume of greater than 10 liters, preferably greater than 100 liters, preferably greater than one cubic meter.
The protein to be immobilized is incubated with the support in an aqueous phase adjusted to a particular pH, generally by buffer.
The pH depends on the nature of the enzyme to be immobilized, mainly on the isoelectric point of the enzyme. A pH range of 3 up to 11, particularly from 4 to 8, has proven to be advantageous.
A pH range of 4.8-5.2 is recommended for CALB lipases.
Suitable buffers for the aqueous phase are, for example, phosphate and acetate buffer.
The incubation is carried out generally at a temperature from 0° C. to 40° C., in particular from 4° C. to 30° C. If the enzyme to be immobilized is particularly tolerant to temperature, even temperatures above 50° C. may be suitable.
After the incubation, the enzyme-containing starting solution is separated from immobilized enzyme. This is most simply carried out by filtration through a sieve plate which is installed in the tumble dryer. The immobilized enzyme can subsequently be purified, if desired, in a washing step. Such washing steps are also carried out by mixing the immobilized enzyme with the washing solution, generally water, and subsequently separating from the immobilized enzyme via an integrated filtration unit.
The immobilized enzyme is subsequently dried also without further transferring into the discontinuous contact vacuum mixer dryer in which a reduced pressure is set in the dryer of less than 1013 mbar, preferably less than 100 mbar.
The jacket temperature is adjusted to less than 100° C., preferably less than 65° C., wherein it must be ensured that the product temperature does not exceed 50° C., preferably 40° C.
It is generally recommended not to set the temperature of the immobilized enzyme to be dried above 50° C. in order to avoid thermal inactivation of the enzyme. In cases of particularly temperature-insensitive enzymes, however, they can even be dried at above 50° C.
Under the conditions specified above, the drying time is typically from 10 to 30 hours, preferably from 15 to 20 hours.
The aim generally is to provide an immobilized product having a residual moisture content of less than 5%, preferably less than 2% and particularly preferably from 0.5 to 1.5% water.
If the product is excessively dry, this results in static charge which partially impedes the handling.
A product produced using the method according to the invention is easy to store at room temperature without notable losses of activity occurring. Furthermore, it is easy to handle, i.e. it can be readily filled and transferred.
The novel method is described in more detail in the example below.
Immobilization of CALB on Lewatit® on a 500 kg scale
The T0055 tumble dryer used for this method has the following technical data:
The following components are required for the experimental procedure:
Lewatit® VP OC 1600 moist (Lanxess) [544 kg]
According to protocol, a total of 544 kg of moist Lewatit should be filled. This 544 kg of moist Lewatit correspond to about 233 kg of dry Lewatit.
A specific activity of 460 TBU/mg of protein and a purity of 61% for the lipase is apparent.
The whole amount of 21 kg of enzyme is used for the immobilization.
Feeding of the Tumble Dryer with Lewatit® and First Washing Step
A theoretical absolute activity of 9 460 000 000 TBU was calculated at the start
An immobilization efficiency of 96.3% was achieved.
With the aid of the infrared balance, a triplicate measurement of the average dry content of the sample, taken from the filled Big Bag, was determined.
The measurements gave the following result (infrared balance, end point: 30 sec. scale stability, triplicate measurement: 5.3 g/9.3 g/4.6 g):
The average dry content of the filled product is 99.11%.
This corresponds in turn to a residual moisture content of 0.89%.
Number | Date | Country | Kind |
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13197469.3 | Dec 2013 | EP | regional |
Filing Document | Filing Date | Country | Kind |
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PCT/EP2014/076844 | 12/8/2014 | WO | 00 |