EP3083699A1

EP3083699A1 - Method for immobilizing and drying enzymes

Info

Publication number: EP3083699A1
Application number: EP14808645.7A
Authority: EP
Inventors: Steffen Maurer; Robert Bayer; Michael Budde; Michael Kerber; Farzad FARIVAR-MEMAR; Jürgen Däuwel; Thomas Berg; Sascha ROLLIE
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 2013-12-16
Filing date: 2014-12-08
Publication date: 2016-10-26
Also published as: WO2015091046A1; US20160312209A1; CN105849130A; CA2933661A1; JP2016540515A

Abstract

Disclosed is a method for immobilizing proteins on a carrier, characterized in that the protein is incubated with the carrier in an aqueous phase in a discontinuous contact vacuum mixer dryer and in that the immobilized protein is then immediately dried, if necessary following an optional washing step, in the same contact vacuum mixer dryer.

Description

Process for the immobilization and drying of enzymes

DESCRIPTION OF THE INVENTION The invention relates to an improved process for the immobilization and drying of proteins, in particular of enzymes, in particular of lipases.

Prior Art EP382767 describes a process for the immobilization of lipases. In this case, an aqueous solution of a given lipase is mixed by rotation with a resin (for example Lewatit®) at a fixed pH at room temperature. Then, the immobilized lipase resin was collected by filtration, followed by washing with water and drying in vacuo. The immobilization of enzymes is generally accompanied by loss of enzyme due to unbonding to the carrier or by desorption ("bleeding") of already bound enzyme Furthermore, the immobilized enzyme often undergoes a loss of enzyme activity due to the immobilization steps, which is precisely the case technical scale leads to significant yield losses and thus increased costs.

Description of the invention

The invention therefore an object of the invention to provide a method that allows to achieve efficient immobilization of the protein to the carrier, which means that as possible lent the entire amount of the enzyme present on the carrier is permanently bound and remains and that im In the case of an enzymatically active protein after immobilization, the same high enzymatic activity as possible is obtained as before immobilization.

A process has been found for the immobilization of proteins on a carrier, characterized in that the incubation of the protein with the carrier takes place in an aqueous phase in a discontinuous contact vacuum mixer dryer and immediately thereafter optionally after an optional washing step, the drying of the immobilized protein in It has now been found that the method defined at the outset leads to particularly advantageous results, since the entire steps of immobilization, such as efficient incubation of protein with carrier, optionally washing of the immobilized protein and drying of the immobilized protein to a stable , good storage and transportable product in a single apparatus are to be carried out. This method is particularly well suited on an industrial scale when immobilized protein is to be produced in the three-digit kilogram to the ton scale. With the method according to the invention a multiplicity of proteins can be immobilized. It is particularly suitable for the immobilization of enzymes, such as oxidoreductases, hydrolases, isomerases and transferases.

It is particularly suitable for the immobilization of hydrolases, in particular of lipases.

Within the lipases, it is particularly possible to immobilize those lipases from Candida antarctica, in particular the Candida antarctica lipase B (CALB) or those enzymes which are structurally derived therefrom. Such structurally CALB-derived lipases are lipases that have at least one, preferably two or more, amino acid changes, such as insertions, deletions or substitutions, to the CALB polypeptide sequence.

Examples of such lipases which are structurally derived from CALB are described in WO 2009/080676 ("CALB muteins"), reference being made expressly to WO2009 / 080676 with regard to the disclosure of the CALB muteins.

The enzymes to be immobilized can be isolated from the original organism by known methods or also prepared by recombinant DNA techniques in suitable host organisms such as Bacillus, E. coli, Pichia, Chrysosporium, Aspergillus, Saccharomyces.

Suitable carriers are various organic or inorganic materials such as silica gel, activated carbon or polymer carrier. As the polymeric support, macroporous crosslinked polymers having a particle size of 100 to 1000 μm and an average pore radius of 10 to 20 nm are suitable. Particularly suitable are macroporous crosslinked acrylate polymers, such as, for example, poly (meth) acrylates which are crosslinked with divinylbenzene, which may comprise, for example, acrylic acid, acrylic acid esters, methacrylic acid and methacrylic acid esters. Such polymers are sold, for example, by the company Lanxess under the name Lewatit® VP OC 1600 or by the company DOW under the name Amberlite® XAD-7. Suitable discontinuous contact vacuum mixer dryers are known to the person skilled in the art from the literature (eg Friedrich Kneule, Das Drying ", Sauerland AG, Aarau, 1975, ISBN 3-7941 -0429-3.) Particularly advantageous is the use of a vacuum tumble or double cone dryer. Taumeltrockner are well suited with an inner volume of greater than 10 liters, preferably greater than 100 liters, preferably greater than one cubic meter.

The incubation of the protein to be immobilized with the carrier takes place in an aqueous phase, which is generally adjusted by buffer to a specific pH.

The pH value depends on the nature of the enzyme to be immobilized, mainly the isolelectic point of the enzyme. It has proven to be advantageous to have a pH range of from 3 to 1, in particular from 4 to 8.

For CALB lipases, a pH range of 4.8 - 5.2 is recommended.

Suitable buffers for the aqueous phase are, for example, phosphate and acetate buffers. The incubation is usually carried out at a temperature between 0 ° C and 40 ° C, in particular between 4 ° C and 30 ° C. If the enzyme to be immobilized is particularly temperature-tolerant, temperatures above 50 ° C. may also be considered.

After incubation, the enzyme-containing starting solution is separated from the immobilized enzyme. This is most easily done by filtration through a sieve plate mounted in the tumble dryer. If desired, the immobilized enzyme can then be further purified in a washing step. Such washing steps are also carried out by mixing the immobilized enzyme with the washing solution, usually water, and then separating the immobilized enzyme via an integrated filtration device.

The subsequent drying of the immobilized enzyme also takes place without further transfer in the discontinuous contact vacuum mixer dryer, wherein a negative pressure in the dryer of less than 1013 mbar, preferably less than 100 mbar is set. The jacket temperature is set to less than 100 ° C., preferably less than 65 ° C., care being taken that the product temperature does not exceed a value of 50 ° C., preferably a value of 40 ° C.

As a rule, it is not recommended 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, can be dried at over 50 ° C. The drying time under the conditions given above is usually between 10 and 30 hours, preferably between 15 and 20 hours. The aim is generally an immobilized product having a residual moisture content of less than 5%, preferably less than 2% and more preferably between 0.5 and 1.5% water.

If the product is dried too much, static charge may occur, which may make handling somewhat difficult.

A product prepared by the process according to the invention is readily storable at room temperature without significant loss of activity. Furthermore, it is easy to handle, i. it is easy to transfer and transfer. More specifically, the new method is described in the following example.

Example:

Immobilization of CALB on Lewatit® in 500 kg scale

The tumble dryer T0055 used for this process has the following technical data:

• Volume: 6.3 m ³

• Heating surface: 17 m ² 1. Raw materials used

The following components are required for the experiment:

• Lewatit® VP OC 1600 damp (Lanxess) [544 kg]

• Lipase ultrafiltrate CALB-2012-01 [21 kg protein]

• DI water (for 3 washes) [3 x 2000L]

· N ₂ - stripping gas [5-20 Nm ³ / h]

According to regulations, a total of 544 kg of wet Lewatit should be filled. These 544 kg of wet Lewatit correspond to about 233 kg of dry Lewatit. The lipase has a specific activity of 460 TBU / mg protein and a purity of 61%. For immobilization, the entire amount of 21 kg of enzyme is used. 2. Experimental procedure

Charging the tumble dryer with Lewatit ® and first wash

· At 8:15 am, the Lewatit barrels were weighed and then placed in the tumble dryer via a funnel

• In total, 544.1 kg of wet Lewatit VP OC 1600 were added to the dryer. A mixed sample of all barrels was put together

• 2000 L of demineralised water (via water meter) were then fed via a hose into the dryer through the hopper at a flow rate of 80 - 90 l / min

-> The funnel was freed from Lewatitresten

• the filling process was finished at 8:55

• Looking into the dryer, one could see that the water had turned slightly milky and that some proteinaceous foam had formed on the surface

• now the sieve fitting (160 μηη) was placed on the manhole cover (9.20 o'clock)

• then the dryer was evacuated to 78.6 mbar for leak testing

• after that, the pressure was restored to normal pressure (1020 mbar) (9.30 am)

• then mixed for 2 hours at atmospheric pressure, room temperature and 0.33 rpm (for this, the drive motor on the tumble dryer was set to 5.5 Hz, which corresponds to 0.33 rpm

-> manually measured: 1 U / 2.43 min = 0.41 rpm)

-> the heating bath stood on full cooling

• at the end of the 2 hours (1 1 .26 o'clock) the T0055 was positioned with the lid downwards and the washing water was filtered through the built-in 160 μηη sieve in IBCTs -> it was filtered with an overpressure of 100-200 mbar

-> The overpressure was generated by the supply of nitrogen

• after 20 L, 1000 L and 1700 L, one sample each was taken

-> purely optically, the first sample showed a slight haze, but sat down on

Ground nothing

-> the second and the third sample were clear; against the light were small ones

Suspended particles recognizable

• a total of 1700 liters of water were drained (1 1:30 am - 12:25 pm)

• The IBCTs have now been rested for 2 hours

• then it was checked whether floating particles had settled on the ground; But this was not the case

• The IBCTs filled with wash water were weighed IBC 1: 1003 kg net

IBC 2: 717 kg net

then the water was drained into the bbA Second wash

• the manhole was positioned up again

• Subsequently, 2000 L of water (2000 kg), which were already in two 1000 L IBCTs, were packed with 800-700 mbar abs. sucked in (12:40 - 13:22 clock)

• The filling process took about 20 minutes per container

Thereafter, the tumbling dryer was mixed for 2 hours at 0.33 rpm (5.5 Hz), room temperature and normal pressure

• at the end of the 2 hours, the manhole was positioned downwards and the wash water was filtered through the built-in 160 μηη sieve in IBCTs

-> This was filtered again with an overpressure of 100-200 mbar

After 50 L, 1000 L and 2000 L one sample each was taken

• a total of about 2050 liters of water were drained

Filtration took about 30 minutes per container (i.e., per 1000 L)

• The IBC ^'s filled with wash water were then weighed

IBC 1: 1002 kg net

IBC 2: 1043 kg net

• then the wash water was drained into the bbA

• then a sample of the washed Lewatit was pulled

-> it was noticeable that the filter cake was slightly inclined to the inner wall after stopping the rotation of the dryer

The tumble dryer was rotated once every hour once an hour to avoid the formation of chunks

Lipase addition and immobilization

• The cooled lipase casks were weighed once more

-> 1749.6 kg lipase net

• then a 2 L sample was drawn from each barrel (9.30 am - 9.50 am)

• The 9 barrels (200 L each) with Lipase CALB-2012-01 were filled into the tumble dryer

• The contents of 3 drums each were sucked into the dryer

· For this purpose, a negative pressure of 300 mbar was applied

• first the containers were filled in 7,8,9 (10.19 o'clock - 10.32 o'clock) • In the barrel, only a very small amount of enzyme solution remained

-> After adding the contents of 3 barrels each, the dryer was rotated for 10 minutes at 0.33 rpm

• Then the contents of the containers 3, 2, 1 (1 1:06 am - 1 1 .23 o'clock) were sucked in and the 10-minute rotation at 0.33 rpm

• last the contents of the containers 6,5,4 were sucked in (1 1 .52 o'clock - 12.07 o'clock)

• from 12:20 to 13:00 was with 200 mbar abs. and rotated 0.33 rpm

• at 1 pm, the tumble dryer was lowered to 900 mbar abs. degassed

• then the mixture was further mixed until 1.15 pm at 100 - 200 mbar vacuum

-> the internal temperature was 20 ° C; for this reason, the cooling was done on

24 ° C was set, turned off

• Then a slight overpressure of 100 - 200 mbar was applied in the dryer

• Under these conditions, the dryer was run at 0.33 rpm for 15 hours

• every hour pressure and temperature were noted

· At the end of the first 3 hours (15.15), the first sample (250 ml) was taken from the supernatant of the immobilizate and stored in a refrigerator at 8-14 ° C

• total samples were taken after 3, 5, 7, 9, 12 and 15 hours of immobilization

• at the end of the 15 hours the dryer was switched off and only turned once every hour (1 turn) (until next day, 7:00 am)

Filtration of the enzyme-Lewatit solution after immobilization

• At 7:00 am the tumble dryer was turned again and positioned with the manhole cover down

• now an overpressure of about 100 mbar was applied in the dryer

· Then the mother liquor was drained through the 160 μηη sieve in IBCTs (7.10 o'clock - 8.20 o'clock)

• a first sample was drawn after about 300 L in a glass bottle

Hereby it was noticeable that the sample was cloudier and not quite as dark green as the day before and also foamed

-> The sample was so visually lighter and cloudier than the previous day, also it smelled different now (after butyric acid)

• The second sample was also removed after about 900 L into a glass bottle

• Since the mother liquor foamed so much when it was filled into the IBC, the first IBC could only be filled with 900 L mother liquor

· After the second IBC was also filled with about 700 L of mother liquor, nitrogen was blown out for the first time • For this reason, the taps were closed and waited 10 minutes to allow the mother liquor to settle down

• then the remainder of the mother liquor was added to the IBC

• a third sample was drawn into a glass bottle

· Overall, the second IBC was filled with 750 L of mother liquor at the end

• Finally, the two ^IBC's were weighed

-> IBC 1 + 2 = 1683 kg net

Washing the immobilized lipase

· After draining the mother liquor, the tumble dryer was first turned and positioned with the slider upwards

• A sample of the damp, unwashed property was then removed via the slide

-> The Immobilisat had settled again on the side of the dryer

-> the Immobilisat had a slight green cast

• then the dryer was turned again so that the manhole cover was up

• In the dryer, a vacuum of about 300 mbar was applied

• then 2000 L of demineralised water (filled in 2 IBCs) were sucked into the tumble dryer (8.55 am - 9.30 am)

Followed by 2 hours mixing at 0.33 rpm, room temperature and normal pressure

• after the 2 hours, the manhole cover was positioned downwards

• In the dryer, an overpressure of 400-580 mbar was applied and the washing water in IBC ^' s drained (1 1.45 pm - 1.15 pm)

· After 50 l the first, after 1000 l the second and after 1825 l the third sample was taken

-> all samples were slightly greenish cloudy; but no sediment settled down -> but you could not see a big difference between the samples

• From about 1750 L drained wash water N ₂ was first blown with

· From about 1850 L of drained washing water, a third IBC was put outside and the line was put outside, as very large amounts of nitrogen were blown out with it

• now an overpressure of 900 mbar was placed on the dryer to really filter out all the water

· Under this pressure was blown dry for 15 minutes (towards the end came only nitrogen from the line and a few drops of washing water) • then the IBCTs were weighed

-> IBC 1 + 2 = 1921 kg net

• then a 1 L sample of the solid was pulled over the manhole

-> also this sample was very slightly greenish (but not as strong as the previously drawn solid sample)

vacuum drying

• The tumble dryer was first evacuated while rotating at 0.33 rpm

• then the rotation was gradually increased to 2.4 rpm (1/1, 5 / 2.0 / 2.4 rpm)

-> viewed from the outside, the dryer ran very quietly, which in turn indicates a steady slipping (slipping) of the immobilizate from the dryer wall (at that time, therefore, no larger chunks have formed)

• at 2.20 pm the heating was set to 50 ° C

· At 5.30 pm it was closed again and the stripping nitrogen was switched on instead

Regulated 25 m ³ / h

• at 2:15 am, the cooling was stopped to pull the first sample

• After a cooling time of 2 h, the first sample of the immobilizate was drawn at 4.15 o'clock -> the internal temperature had dropped from 39.3 ° C to 23 ° C

· The sample was given to the in-house laboratory to determine residual moisture

-> Residual moisture determination at 105 ° C / 60 minutes: 35.76%

• At 8:25 pm, the dryer was positioned with the slider down and allowed to stand for 5 minutes for temperature measurement

-> The internal temperature has a temperature difference of 5 ° C, depending on whether the tumble dryer is rotating or if the sensor is submerged in the solid

• At 1:45 pm, the cooling was stopped in order to be able to draw a second sample

• after a cooling time of 30 minutes, the sample was drawn

-> it was noticeable that there were also smaller, darker (brown) particles between the white spheres of the immobilizate

· In the company's own laboratory, the residual moisture was determined

-> Residual moisture determination at 105 ° C / 60 minutes: 9.72%

• Store the sample in the refrigerator

• at 17:28, the internal temperature rose to 46 ° C-> bath taken to 50 ° C

• at 17.47 o'clock the indoor temperature had fallen back to 45. 5 ° C-> bath to 53 ° C

• From 18.00 clock was started to cool • From 7:00 pm to 7:20 pm, the sample was taken and taken to the in-house laboratory for residual moisture determination

-> Residual moisture determination at 105 ° C / 60 minutes: 1, 4%

• since the residual moisture was below the setpoint of 3%, drying was stopped

· At 20.30 the vacuum was released

-> while the temperature rose from 25 ° C to 31, 7 ° C.

• At 20.50 the drive was switched off at an internal temperature of 25.8 ° C

• From this point on, the dryer was turned once every 2 hours

The dryer was positioned with the slider down

then the sieve insert was removed again

-> slight product residue on the sieve

Manhole cover left for filling a piece open

· Nibbler / fitting piece mounted

then inertized with about 2.4 m ³ / h N ₂

Big Bag No. 67291 120 hung on the filling device and inflated with N ₂ and grounded exhaust air at filling device half open

use a manual tap to let the product run gently; later turned to half position

-> The Immobilisat is easily drizzled in the suspended Big-Bag, without hanging on the film or in the dryer itself

-> The Big-Bag had inflated only once briefly, when for a short moment a little more product came

· Then the remains, which were still in the tumble dryer, knocked off -> all in all no large residues remained in the tumble dryer (only 3 about DIN A3 large places, where a perhaps 2mm thick layer had formed, otherwise the

Dryer inner wall only covered by a very fine layer of dust)

· From the big-bag, another 1 L sample was drawn in a plastic bottle

• Last, the Big-Bag was weighed and then stored dry

-> Tare: 25 kg

-> gross: 271 kg

• The filling itself took only about 5 minutes (together with the preparation it took about 20 minutes) 3. Analysis results

A theoretical absolute activity of 9,460,000,000 TBU at startup was calculated

Measured values of activity / total protein in the course of immobilization:

Total volatilestrogenicity [TBU] US [%] [g / L] protein [TBU / m abs. Calc. from CooPlus [%] L] Start / BGG CooPlu worth s

/ BGG

Mixed sample Concentrate 5390 9,270,800,000 8.63

from barrels CALB-2012- 01

mixed accordingly

jew. masses

(kg net) of the barrels

Supernatant after 3h Immo2215 4,474.3 million 48

balancing, native

Supernatant after 3 hours Immo2225 4,494,500,000 48 3,47 40,16 bilation, filtered (PVDF

0,2μιη)

Supernatant after 5h Immo1730 2,975,600,000 32

balancing, native

Supernatant after 5 hours Immo1760 3.027.200.000 33 3.13 36.23 stabilization, filtered (PVDF

0,2μιη)

Supervision after 7h Immo1220 2,098,400,000 23

balancing, native

Supernatant after 7h immobilization, filtered (PVDF 1090 1 .874.800.000 20 1, 79 20.68 0.2μιη)

Supernatant after 9h Immo820 1 .410.400.000 15

balancing, native

Supernatant after 9 h Immo744 1 .279.680.000 14 1, 45 16.88 precipitation, filtered (PVDF 0,2μπΊ)

Supernatant after 12h Im502 863.440.000 9.3

mobilization, native

Supernatant after 12 h Im460 791 .200,000 8.5 0.85 9.88 mobilization, filtered

(PVDF 0,2μιτι)

Supernatant after 15h Im443 761 .960.000 8.2

mobilization, native

Supernatant after 15 h Im425 731,000,000 7,9 0,92 10,69 mobilization, filtered

(PVDF 0,2μιτι)

Supernatant after the end 204 41 1 .070.000 4.3

Immobilization, native

Supernatant after end 174 351 .480,000 3,7 0,43 4,97 Immobilization, filtered

(PVDF 0,2μιτι)

Wash water 3 after Im- 20.4

mob., sample 1 natively

Washing water 3 after Im 17,16 .06

mob., sample 1 filtered

(PVDF 0,2μιτι)

Wash water 3 after Im- 35.8

mob., sample 2 natively

Wash water 3 after Im 19.5 0.06

mob., sample 2 filtered

(PVDF 0,2μιτι)

Wash water 3 after Im- 28.0

mob., sample 3 natively

Wash water 3 after Im- 33.4 0.07

mob., sample 3 filtered

(PVDF 0.2μm)

An immobilization efficiency of 96.3% was achieved. Residual moisture determination in the course of vacuum drying 0 53.1 - 74.2

12 35.76

18 9.72

22 1, 40

Residual moisture of the bottled product

The infra-red balance was used in a triple measurement to determine the mean dry content of the sample taken from the filled big-bag. The measurements gave the following result (infrared balance, end point: 30 sec. Balance standstill, triple measurement: 5.3 g / 9.3 g / 4.6 g):

The mean dry content of the bottled product is 99.1 1%. This in turn corresponds to a residual moisture content of 0.89%.

Claims

claims

1 . A process for the immobilization of proteins on a carrier, characterized in that the incubation of the protein with the carrier takes place in an aqueous phase in a discontinuous contact vacuum mixer dryer and immediately thereafter, optionally after an optional washing step, the drying of the immobilized protein in the same Contact vacuum mixer dryer is performed.

2. The method according to claim 1, characterized in that a tumble dryer with an internal volume of more than 100 L is used as a contact vacuum mixer dryer.

3. The method according to claim 1, characterized in that an enzyme is used as the protein.

4. The method according to claim 1, characterized in that a lipase is used as the enzyme.

5. The method according to claim 4, characterized in that is used as lipase Candida antarctica lipase B (CALB) or a structurally derived lipase thereof.

6. The method according to claim 1, characterized in that a material having a hydrophobic surface is used as the carrier.

7. The method according to claim 1, characterized in that a crosslinked poly (meth) acrylate-containing resin is used as the polymeric support.

8. The method according to claim 7, characterized in that is used as a polymeric support crosslinked with divinylbenzene, macroporous poly (meth) acrylate resin in spherical bead form whose particles at least 60%, preferably at least 80% of the mass, a size of 50 μηη have up to 2000 μηη.

9. The method according to claim 1, characterized in that the incubation time of the protein with the carrier is 2-30 hours.

10. The method according to claim 1, characterized in that the incubation of the protein with the carrier is carried out at a temperature of 0 to 40 ° C.

1 1. A method according to claim 1, characterized in that the drying temperature of the immobilized enzyme is 30-60 ° C.

12. The method according to claim 1, characterized in that the drying is carried out under reduced pressure.

13. The method according to claim 12, characterized in that the drying is carried out in a range of 5 to 800 mbar.