WO2008151822A1 - Minéraux argileux modifiés thermiquement utilisés comme matériaux de support d'enzymes - Google Patents

Minéraux argileux modifiés thermiquement utilisés comme matériaux de support d'enzymes Download PDF

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Publication number
WO2008151822A1
WO2008151822A1 PCT/EP2008/004781 EP2008004781W WO2008151822A1 WO 2008151822 A1 WO2008151822 A1 WO 2008151822A1 EP 2008004781 W EP2008004781 W EP 2008004781W WO 2008151822 A1 WO2008151822 A1 WO 2008151822A1
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Prior art keywords
enzyme
carrier
solid
enzyme complex
solid enzyme
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PCT/EP2008/004781
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German (de)
English (en)
Inventor
Ulrich Sohling
Kirstin Suck
Friedrich Ruf
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Süd-Chemie AG
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Publication of WO2008151822A1 publication Critical patent/WO2008151822A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier

Definitions

  • the invention relates to a solid enzyme complex, a process for its preparation and the use of the solid enzyme complex.
  • the enzymes are bound on a carrier.
  • the binding can be done in different ways. Physical binding can be achieved by adsorption of the enzyme on the surface of the support. Binding occurs via hydrophobic interactions or by ionic forces, with charged groups of the enzyme interacting with oppositely charged groups on the surface of the support.
  • the advantage of this method is its ease of execution and the relatively low influence on the activity of the enzyme.
  • the disadvantage is that the enzymes can be displaced relatively easily from the surface of the carrier again.
  • An irreversible binding of the enzyme can be achieved by forming a covalent bond between the enzyme and the carrier.
  • the activity of the enzyme is lowered, since the enzyme may for example be fixed on the surface, that the active center is no longer accessible.
  • the stability of the enzyme / carrier complex can be further increased by the enzymes are crosslinked by at least bifunctional molecules. This results in larger aggregates which have a lower solubility.
  • the control of immobilization is very difficult in this method.
  • a significant deactivation of the enzyme usually has to be accepted, since its conformation is greatly altered or the active center is no longer freely accessible.
  • the enzyme is entrapped in a spherical or tubular matrix.
  • the matrix must be permeable to the educts and products of the catalyzed reaction, but not to the enzymes.
  • natural polymers such as alginates, gelatin or agar, or even syn- thetician polymers, such as polyacrylamide or Polyvmylalkohol used.
  • the enzyme is protected from being inactivated by the solvent in reactions in organic media.
  • the matrix can act as a diffusion barrier for the educts and products of the enzyme-catalized reaction.
  • DE 2 154 672 describes a process for preparing a solid enzyme complex, wherein an enzyme in an aqueous buffer solution is contacted with a support m which has been previously modified with a coupling agent.
  • the carrier used is preferably Bentomt, which has been treated with cyanuric chloride as the coupling agent.
  • the immobilization of the enzyme is very rapid, ie within a few minutes, and is carried out at temperatures of less than 40 0 C, preferably at about 0 0 C, so that the thermal load of the enzyme remains low.
  • sepiolite having a particle size of ⁇ 2 mm was first converted into the sodium form by repeatedly slurrying the sepiolite in a sodium chloride solution.
  • the enzyme is reacted in a phosphate buffer (pH 7) at 4 ° C. with the sepiolite present in the sodium form and the solid is separated by centrifuging. The separated pellet is washed with buffer, sodium chloride being added in the second washing step to separate weakly bound enzyme from the clay. Subsequently, the enzyme activity is determined.
  • the enzymes are immobilized on clay minerals, in particular bentonite, which are present in an aqueous buffer solution in colloidal form.
  • clay minerals in particular bentonite
  • such a finely divided suspension is not suitable for industrial applications because the fine particles are difficult to separate from the reaction medium after the end of the reaction.
  • Lipase which consists essentially of a thermostable
  • Lipase consists of a microorganism from the group of
  • the carrier consists of at least 65% silica gel or silicates, wherein at least 90% of the particles have a particle size between 100 and 1000 microns and wherein at least 80% of the pores of the particles have a diameter which is 12 to 45 times the diameter of a Lipasemole - kuls corresponds, and wherein the water content of the particulate immobilized lipase is between 1 and 20%.
  • the particulate carrier is placed in an aqueous solution of the lipase. The loaded carrier particles are separated, optionally washed and dried to the specified water content.
  • the invention had the object of providing a stored enzyme available, which can be produced easily and inexpensively, can be used in industrial processes and has a high activity and a long half-life in the application.
  • a heat-treated clay mineral is used in the solid enzyme complex according to the invention.
  • the heat treatment ensures that the swelling ability of the clay is significantly reduced or completely suppressed.
  • the heat-treated clay mineral can readily be agglomerated to form larger stable particles. It is therefore possible to easily separate the solid enzyme complex after completion of the reaction, for example, by allowing filtration or settling of the solid enzyme complex and decanting the liquid phase.
  • the reactions can also be carried out in the flow, if the erfmdungsgeBOOKe solid enzyme complex is provided as a column or in a cartridge.
  • the carrier is obtained by heating the clay mineral used as starting material until the desired properties are achieved. On the heat-treated clay material can then be immobilized by conventional methods, the enzyme of interest.
  • a solid enzyme complex comprising at least one enzyme which is immobilized on a carrier which has been obtained by heating a clay mineral to a temperature and for a duration that the carrier has a swelling capacity in water of less than 15 ml / 2 g.
  • the carrier obtained by heating a clay mineral has a swelling capacity which substantially corresponds to the sediment volume. Even with prolonged standing in water or an aqueous reaction medium, the carrier does not swell and also shows no delamination, so that, for example, it does not cause an increase in the viscosity of the reaction medium.
  • the erfmdungsgeINEe solid enzyme complex can therefore be easily added in large quantities to a liquid, especially aqueous medium, without thereby, for example, the viscosity of the suspension increases dramatically.
  • the solid enzyme complex according to the invention can be separated again from the liquid medium without difficulty by conventional methods, such as filtration or centrifugation. The enzymes remain predominantly adsorbed on the carrier, so that the carrier can optionally be used in a further reaction or batch.
  • the properties of the carrier can be controlled by the temperature treatment.
  • High ion exchange capacity generally correlates with good Bmdekapazitat for enzymes.
  • the cation exchange capacity is preferably at least 5 meq / 100 g, preferably at least 10 meq / 100 g, more preferably at least 15 meq / 100g.
  • the carrier preferably has the highest possible ion exchange capacity.
  • the carrier has a cation exchange capacity of at least 40 meq / 100 g, more preferably at least 60 meq / 100 g. Most preferably, the cation exchange capacity is in the range of 65 to 130 meq / 100g.
  • the ion exchange capacity of the carrier prepared by a heat treatment from a clay mineral is an important criterion for distinguishing, for example, the carriers of clay materials contained in the solid enzyme complex of the present invention, such as those obtained by extracting with hot acid from natural clay minerals. The latter show little or no ability for ion exchange. High-digestion bleaching earths that no longer swell typically exhibit a cation exchange capacity in the range of less than 30 meq / 100 g. Due to the heat treatment, the inner surface of Ton ⁇ ii- nerals is significantly reduced.
  • the carrier has a specific surface area of less than 300 m 2 / g, particularly preferably less than 200 m 2 / g, more preferably less than 120 m 2 / g.
  • the specific surface of the carrier is at least 30 m 2 / g, particularly preferably at least 60 m z / g.
  • the carrier contained in the inventive solid enzyme complex has almost no swelling capacity in water more.
  • the swelling volume in this case corresponds to the sediment volume. Even with prolonged storage in water, no or almost no increase in sediment volume is observed.
  • the carrier After storage in water for three days, the carrier preferably has a sediment volume of less than 15 ml / 2 g, preferably less than 10 ml / 2 g, particularly preferably less than 8 ml / 2 g.
  • the inventive solid enzyme complex can be provided as a fine powder or else in the form of large-sized moldings. Since the carrier has no or only a very low swelling capacity in water, for example, granules of any size can be prepared, on which then the enzyme is immobilized. In order to achieve sufficient stability for large-sized molded articles, so that the solid enzyme complex does not decompose, for example, in an aqueous reaction medium, the thermal treatment is preferably carried out only after shaping.
  • the enzyme is preferably selected from the group which is formed from lipases, oxidoreductases, such as glucose oxidase or pyruvate dehydrogenase, transferases, such as hexokmase or glycogenophosphorylase, hydrolases, such as amylases, chymotrypsm, peptidases or esterases, isomerases, such as glucose isomerase, ligases such as carboxylases, lyases such as aldolase or catalase.
  • the enzyme is particularly preferably a lipase, in particular from the enzyme class of the hydrolases.
  • Lipases are enzymes that cleave lipids, such as triglycerides or diglycerides, to free fatty acids and glycine. Lipases (Tn acylglycerm hydrolases) catalyze the hydrolysis as well as the synthesis of esters of glycerm and long-chain fatty acids. Other lipase-catalyzed reactions include the transesterification of lipids and the esterification of primary, secondary and tertiary alcohols. The industrial use of these enzymes is very versatile. Addition to detergents is the largest field of application for lipases. Approximately 1000 tons of enzyme are added to detergents every year. In the cosmetics industry, lipases are used to make emulsifiers and flavorings.
  • lipases are used for the synthesis of glycerides and flavorings, as well as for the modification of fats.
  • a further large field of application for lipases is the production of consumer and feminine chemicals.
  • the use of the enzymes for stereoselective reactions is becoming increasingly important.
  • Lipases are also used in the production of lubricants, hydraulic oils and biodiesel.
  • the enzyme can be bound to the carrier via a covalent bond or else via a non-covalent bond.
  • coupling agents are Molecules having at least two reactive groups, one group reacting with a hydroxy group provided on the surface of the support and the at least one further reactive group reacting with a corresponding group on the enzyme, such as a hydroxy group, an amino group or a thiol group, so that the enzyme is over the coupling agent molecule is fixed on the surface of the support by covalent bonds.
  • the enzyme is cross-linked by corresponding at least bifunctional molecules, so that the molecular weight of the enzyme is increased and thus its solubility in the reaction medium is deteriorated.
  • the enzyme is bound to the carrier via a non-covalent bond.
  • the carrier has a sufficiently high cation exchange capacity even after the thermal treatment, so that adsorption of the enzyme on the carrier can be carried out via ionic bonds.
  • an immobilization of the enzyme via hydrophobic interactions between enzyme and Trager take place. The noncovalent binding of the enzyme to the carrier lessens the structure of the enzyme so that the immobilization does not interfere excessively with the activity of the enzyme.
  • the amount of the enzyme immobilized on the carrier is preferably selected to be high enough to achieve a sufficiently high activity of the solid enzyme complex.
  • the proportion of the enzyme based on the weight of the solid enzyme complex, between 3 and 35 wt .-%, particularly preferably 5 to 20 wt .-%, particularly preferably 8 to 15 wt .-%.
  • the enzyme contained in the solid enzyme complex is, as already explained, not subject to any particular restrictions.
  • the enzyme has a molecular weight in the range of 10 to 200 kDa.
  • the erfmdungsgedorfe solid enzyme complex can be produced in a simple and cost-effective manner, so that larger amounts of erfmdungsgedorfen solid enzyme complex are accessible.
  • the invention therefore also relates to a process for the preparation of a solid enzyme complex as described above.
  • the erfmdungsgeloise method comprises the following steps:
  • the carrier is provided by subjecting a clay mineral to a thermal treatment so that it obtains a certain, very low swelling capacity. Since the clay mineral used as starting material is obtained from a natural source and therefore variations in the properties of the various Tonmmeralien may occur, it is preferred m the way that initially determined by screening the optimum temperature for the clay mineral, wherein the desired low swelling capacity of the carrier is set. Preference is given to ⁇ as low as possible selected temperature and optionally adjusted the duration of the temperature treatment. Are preferred for the heat treatment temperatures of at least 400 0 C, preferably at least 450 0 C, particularly preferably Kursm- least 500 0 C chosen. However, the clay mineral should also not experience too high a thermal load, otherwise the adsorption capacity will drop.
  • the temperatures are preferably less than 1000 0 C, preferably lower than 900 0 C, and particularly preferably chosen below 800 ° C.
  • the duration of treatment depends on the amount of clay mineral used and the type of furnace. Rotary kilns are preferably used for the heat treatment, since they allow uniform heating of the clay mineral.
  • the treatment time is preferably chosen as short as possible in order to avoid excessive thermal stress on the clay mineral. In an industrial implementation of the method treatment times of at least 10 minutes, preferably at least 20 minutes are selected. In order to avoid a thermal overload, the treatment time is preferably chosen shorter than 2 hours, particularly preferably shorter than 1 hour.
  • the appropriate duration of treatment can be easily determined by one skilled in the art by periodically taking samples during the thermal treatment of the clay mineral and examining their swelling capacity. Further parameters, such as the ion exchange capacity, the specific surface or the pore volume, can also be used to assist in the monitoring of the thermal treatment.
  • the carrier can be used in any clay minerals, which are accessible from natural sources. Preference is given to using clay minerals which have a high ion exchange capacity and a high swelling capacity.
  • Clay materials are preferably used which have an ion exchange capacity of at least 5 meq / 100 g, preferably at least 10 meq / 100 g, more preferably at least 20 meq / 100 g, especially preferably at least 40 meq / 100 g, and clay minerals are particularly preferably used.
  • their cation exchange capacity is in the range of 50 to 130 meq / 100 g, more preferably in the range of 70 to 120 meq / 100 g.
  • clay minerals which have a high swelling capacity are preferably used.
  • the clay minerals used as starting material have a swelling capacity in water after one hour of at least 5 ml / 2 g, preferably at least 8 ml / 2 g, particularly preferably at least 10 ml / 2 g.
  • the clay minerals used as administratmate ⁇ alien a swelling capacity in the range of 6 to 80 ml / 2 g, more preferably 9 to 40 ml / 2 g, particularly preferably 10 to 20 ml / 2 g.
  • sodium bentonites or calcium bentonites are suitable.
  • the clay minerals used for the preparation of the carrier preferably have neutral to weakly basic properties.
  • a 2% by weight suspension of the clay mineral in distilled water preferably has a pH in the range of 6 to 11.
  • the carrier smectitic sheet silicates are preferably used. These include, for example, charged clay minerals of the 2: 1 layer type with a negative charge of 0.2 to 0.6 per formula unit.
  • the cation exchange capacity of the clay minerals used as starting materials for the preparation of the carrier can be determined by exchange with ammonium chloride and subsequent nitrogen determination and is preferably in the above-described range of 10 to 130 meq / 100 g.
  • suitable smectite clay minerals are bentonite, montmorillonite, beidellite, nontronite, hectorite and saponite.
  • Another suitable smectite clay mineral is stevensite. Its structure is derived from talc, with the positions of the magnesium ions being replaced by vacancies.
  • the proportion of charge interactions and hydrophobic interactions, with The selected enzyme is fixed on the surface of the carrier.
  • supports made from montmorillonite-containing clay minerals with high cation-exchange capacities of preferably more than 65 meq / 100 g, preferably more than 85 meq / 100 g, by thermal treatment are more suitable for adding the enzymes to be immobilized via charge interactions whereas carriers made, for example, from stevensites, kerolites or saponites are particularly suitable for the immobilization of enzymes which are bound to the carrier via hydrophobic interactions.
  • Suitable saponites have, for example, a cation exchange capacity of preferably 5 to 60 meq / 100 g, and preferably a specific surface area of 100 to 170
  • clay minerals for the thermal treatment which have a cation exchange capacity of less than 80 meq / 100 g, preferably less than 75 meq / 100 g, particularly preferably in a range of 40 to 70 meq / 100 g.
  • these clay minerals have a high specific surface area.
  • the clay minerals preferably have a specific surface area of more than 100 m 2 / g, preferably more than 120 m 2 / g, particularly preferably more than 150 m 2 / g.
  • the clay minerals used for the preparation of the carrier may optionally be dried before the heat treatment, preferably to a moisture content of less than 20 wt .-%, in particular to a moisture content in the range of 5-15 wt .-%. Further, the raw clay material may be ground prior to the heat treatment, preferably to a mean grain size D 50 of less than 3 mm, more preferably less than 1 mm, most preferably less than 0.2 mm.
  • the clay minerals, in particular smectitic clay minerals, used as starting material for the preparation of the carrier may be in both the sodium form and in the calcium or magnesium form.
  • monovalent alkali metal ions and divalent alkaline earth metal ions are present as intermediate layer ions in the clay minerals.
  • the proportion of monovalent or divalent cations can be adjusted before the heat treatment by reacting the clay mineral with a corresponding alkali metal salt, for example sodium carbonate or sodium bicarbonate.
  • a corresponding alkali metal salt for example sodium carbonate or sodium bicarbonate.
  • a naturally moist calcium bentonite can be sprayed with a soda solution or kneaded with solid sodium carbonate.
  • the clay mineral with a suitable Erdalkaliverbmdung such as calcium chloride, can be implemented.
  • the heat-treated Tonmmeral used in erfmdungsgespecializeden method is prepared from a raw clay material having at least a proportion of divalent alkaline earth metal ions, preferably calcium ions on pet harshplatzen.
  • the proportion of divalent cations in the ion exchange capacity is preferably at least 15%, particularly preferably at least 30%, particularly preferably at least 70%.
  • the proportion of divalent cations, in particular calcium and magnesium, in the cation exchange capacity is preferably less than 95%, in particular less than 85%. It has been observed that a high proportion of exchangeable divalent cations can enhance the tendency of enzymes to bind to the heat-treated clay material. Without wishing to be bound by this theory, the inventors assume that the alkaline earth metals arranged in intermediate layers exert a support function during the thermal treatment of the clay mineral so that the layers of the clay mineral do not sink together as much as with pure natural bentonites.
  • the proportion of divalent or monovalent cations can be adjusted accordingly before the heat treatment of the clay mineral.
  • this can be reacted with a suitable sodium salt, such as sodium carbonate, by spraying the clay mineral, for example with a soda solution, or by kneading it with solid sodium carbonate.
  • the exchanger according to the thermal treatment preferably has a specific surface area of less than 300 m 2 / g, more preferably less than 200 m 2 / g, more preferably less than 120 rn 2 / g.
  • the surface of the carrier preferably amounts to at least 30 m 2 / g, more preferably at least 35 m 2 / g, particularly preferably at least 60 m 2 / g.
  • the spe- In addition to the swelling capacity already described, the cif- ic surface can therefore be used to monitor the thermal treatment of the clay mineral.
  • the carrier has almost no swelling capacity in water after the thermal treatment.
  • the swelling volume in this case corresponds to the sediment volume. Even with prolonged storage in water, no or almost no increase in sediment volume is observed.
  • the carrier has a swelling volume of less than 15 ml / 2 g, preferably less than 10 ml / 2 g, more preferably less than 8 ml / 2 g.
  • the swelling volume may also take on lower values, for example values of less than 5 ml / 2 g
  • the carrier is preferably no longer subjected to further activation. It can still be adjusted by physical process steps, such as grinding and sieving, a desired grain size. Preferably, the carrier is adjusted to a mean grain size D 50 in the range of 10-200 ⁇ m. Granulation can also produce larger particles. The larger particles can be stabilized in their shape by a, possibly renewed, thermal treatment.
  • the particulate carrier in particular the granules, is heated to a temperature in the range from 400 to 750 ° C. for 2 minutes to 5 hours. Preferably, however, proceeding in such a way that the clay mineral is first granulated or molded and only then the thermal treatment is carried out, at the same time a stabilization of the granules or Formkorpers is achieved.
  • the granulation of the clay mineral which is preferably carried out before the thermal treatment, takes place in such a way that the clay mineral is introduced into a high-speed mixer and that an aqueous binder, such as For example, water or water glass is added in a short period of time in its entire amount.
  • the granulation can be carried out both in a batch process and in a continuous process.
  • a so-called egg mixer and for continuous granulation for example, a continuously operating ploughshare mixer, as offered for example by the company Lodige, or a ring-layer mixer, such as a Lodige CB mixer, can be used.
  • the dry powdered thermally treated clay mineral can be pressed under high pressure into molded articles which, if appropriate, can also be comminuted again to the desired size after shaping.
  • a highly compacting shaping can be carried out, for example, in a tableting press, a disk press or a briquetting press.
  • This plastic mass can then be extruded, for example, into a strand which is comminuted to a granulate of the desired size.
  • an enzyme is provided in erfmdungsge speciallyen process, which is to be immobilized on the carrier.
  • the enzyme is preferably provided in the form of an aqueous solution.
  • the enzyme is preferably in a buffered solution provided.
  • the pH of the buffer is selected depending on the enzyme to be immobilized and is preferably in the range of 3 to 9. The ideal range for the pH depends on the specific enzyme.
  • the pH of the buffer is preferably selected in the range of 3 to 5.
  • the buffer is suitably selected in such a way that its buffer effect is as large as possible at a pH which is approximately one unit below the isoelectric point of the enzyme in question.
  • the enzyme then has a positive total charge and can be fixed by an ionic bond on the carrier.
  • the concentration of the buffer is suitably adjusted in the range of 10 to 300 mmol / l, preferably 50 to 200 mmol / l.
  • the concentration of the enzyme in the solution is preferably selected in the range of 1 to 10 mg / ml.
  • the immobilization is preferably carried out at a temperature in the range of 0 to 37 ° C, particularly preferably 4 to 10 0 C.
  • the enzyme and the carrier can be brought into contact in any desired manner.
  • the carrier can be suspended in a solution of the enzyme. But it is also possible to spray the solution of the enzyme on the carrier while it is being moved, for example.
  • the time required for the immobilization of the enzyme depends on the carrier used and the enzyme used. Preferably, the reaction time is chosen in the range of 10 minutes to 5 hours.
  • reaction medium can still be separated from the solid enzyme complex. This can be done by conventional methods, for example by filtration or Zent ⁇ fug Schl.
  • the solid enzyme complex can then be washed to remove unbound enzyme.
  • the same buffer can be used for washing as has been used in the reaction of enzyme and carrier. Is relatively little solvent, especially water in the solid enzyme complex
  • the solvent can also be evaporated.
  • the solvent can be distilled off under reduced pressure. Again, the temperature is chosen as low as possible in order to avoid premature deactivation of the enzyme.
  • the carrier is preferably before the task of the enzyme to a pH of preferably 3.0 to 7.0 equilibrium.
  • the carrier can be slurried, for example, in a suitable buffer medium.
  • the enzyme is immobilized by non-covalent bonds on the surface of the carrier.
  • the carrier and the enzyme are reacted with a coupling agent which has at least two reactive groups, so that one of the groups with, for example, hydroxyl groups on the surface of the carrier and the other group with a suitable group can react on the enzyme, such as a hydroxyl, a mino- or a thiol group.
  • a suitable coupling agent is for example Cyanurchlo ⁇ d.
  • Particularly suitable as coupling agents are functionalized silanes, as are commonly used for the fixation of enzymes on to ⁇ organic Tragern. This binds first functionalized alkoxy or chlorosilanes on the carrier. As alkoxy groups, for example, methoxy or ethoxy groups are used. The attachment of the functionalized silane takes place for example via the formation of Si-O-Si bonds with elimination of alcohol or HCl. If one uses functionalized silanes, which carry functional groups, which can react directly with groups on the enzyme, so in a second - -
  • Step the enzyme to be bound directly to the carrier A typical example of this are epoxysilane compounds, such as epoxyalkyltrialkoxysilanes.
  • the epoxy group of the silane after application to the support can react directly with free NHo groups of the enzyme.
  • the functionalized silane is first reacted after application on the support with an activating agent.
  • groups are introduced into the silane bound on the surface of the support which can react with a group on the enzyme, for example an amino group.
  • the enzyme be attached in a further reaction step to a group of the activating agent.
  • the above-described carrier obtained by thermal treatment of a clay mineral can be reacted with an aminoalkoxysilane.
  • the ammoalkoxysilane reacts with a hydroxy group on the surface of the carrier.
  • amino groups can then be reacted with a bifunctional activating agent, for example a dialdehyde or a diisocyanate.
  • a bifunctional activating agent for example a dialdehyde or a diisocyanate.
  • One functional group reacts with the amino group provided on the surface of the support and the other functional group is available for reaction with a group on the enzyme.
  • a suitable bifunctional aldehyde is, for example, glutaraldehyde.
  • the invention also relates to the use of the above-described solid enzyme complex for enzyme-catalyzed reactions.
  • the reactions can be carried out per se under known conditions and in known devices.
  • the reactions can be carried out in suspension, including the solid enzyme complex in a, preferably buffered, solution the substrate is slabbed, to which other conventional components such as coenzymes, ATP, etc. may be added.
  • Fig. 1 an adsorption isotherm recorded for a lipase at pH 4 in a 50 mM acetate buffer of Bentomt I 500 ;
  • FIG. 2 an adsorption isotherm recorded on Bentomt 3 500 for a lipase at pH 4 in a 50 mM acetate buffer
  • FIG. 3 shows an adsorption isotherm, which was recorded for a lipase at pH 4 in a 50 mM acetate buffer at saponite l 6 oo.
  • the surface area and the pore volume were determined using a fully automatic nitrogen porosimeter from the company Mikromeritics, type ASAP 2010.
  • the sample is cooled in a high vacuum to the temperature of liquid nitrogen. Subsequently, nitrogen is continuously metered into the sample chamber. By detecting the adsorbed amount of gas as a function of pressure becomes more constant - -
  • the pore volume is also determined from the measurement data using the BJH method (EP Barret, LGJoiner, PP Haienda, J. Am. Chem Soc 73 (1951, 373).) This procedure also takes into account effects of capillary condensation Pore volumes of certain pore sizes are determined by summation of incremental pore volumes obtained from the BJH adsorption isotherm analysis.
  • the total pore volume according to the BJH method refers to pores with a diameter of 1.7 to 300 nm.
  • the measurements are carried out with a device "Mastersizer” from Malvern Instruments Ltd., UK, according to the manufacturer's instructions.
  • the measurements are carried out with the intended sample chamber ("dry powder feeder") in air and the values related to the sample volume are determined.
  • the water content of the products at 105 0 C is determined using the method DIN / ISO-787/2.
  • This analysis is based on the total resolution of the clay materials or the corresponding product. After releasing the festival Substances, the individual components are analyzed and quantified using conventional specific analysis methods, such as ICP.
  • the clay material to be tested over a period of 2 hours at 105 0 C is dried. Thereafter, the dried clay material is reacted with an excess of aqueous 2N NH 4 Cl solution for 1 hour under reflux. After a service life of 16 hours at room temperature, it is filtered, whereupon the filter cake is washed, dried and ground and the NH 4 content in the clay material is determined by nitrogen determination (CHN analyzer "Vario EL III" from Elementar in Hanau) according to the manufacturer's instructions. The proportion and type of exchanged metal ions is determined in the filtrate by ICP spectroscopy.
  • a graduated 100 ml measuring cylinder is filled with 100 ml of distilled water. 2 g of the substance to be measured are added slowly and in portions, each about 0.1 to 0.2 g, with a spatula on the surface of the water. After a drop of added portion, the next portion is added. After the 2 g of substance have been added and dropped to the bottom of the measuring cylinder, the cylinder is allowed to stand for one hour at room temperature. Then the height of the sediment volume in ml / 2 g is read off the graduation of the measuring cylinder. For the determination of the sediment volume after 3 tagiger storage in water of the sample preparation is sealed with Parafilm ® and vibration-free for 3 days at room temperature, allowed to stand. Then the sediment volume is read off the graduation of the measuring cylinder. Determination of the montmonellite content via methylene blue adsorption
  • the methylene blue value is a measure of the inner surface of the
  • the test of the clay wxrd was carried out in the same way as for the test bentonite. From the used amount of Methylenblaulosung can calculate the inner surface of the clay material.
  • 381 mg of methylene blue / g of clay correspond to a content of 100% montmorillonite according to this method.
  • a 5% suspension is prepared by stirring an appropriate amount of the clay material to be examined at about 930 rpm for about 5 minutes in water. The suspension is stirred for a further 15 minutes at about 1865 rpm and the suspension is then poured through a sieve of the desired mesh size. The residue is washed with tap water until the wash water runs clear. The screen with the residue is then placed in an ultrasonic bath for 5 minutes to remove residual females. The remaining residue is briefly washed with tap water and the sonication is repeated, if necessary, until during the sonication - -
  • the protein quantification according to the modified Lowry protocol is based on the reaction of proteins with copper sulfate and copper tartrate in alkaline solution, which leads to the formation of a four-toothed copper-protein complex.
  • 100 ⁇ l of the protease / enzyme solution are pipetted into a cavity of a 96-well plate.
  • 100 ⁇ l Lowry reagent is pipetted in and the mixture is homogenized.
  • 50 ⁇ l of Folm-Ciocalteu reagent are added and the mixture is rehomogenized.
  • 30 minutes after addition of Folm-Ciocalteu reagent the absorbance is measured against a blank value at 750 nm in the plate photometer.
  • Bicinchoninic acid (BCA) is used as the detection system in the BCA method. Initially, complex formation of protein with Cu 2+ ions in alkaline solution occurs. The Cu 2+ ions of the complex are reduced to Cu + ions, which can be detected by complex formation with BCA by absorption measurement at 562 nm.
  • 25 ⁇ l of the enzyme / proteme solution are pipetted into a cavity of a 96-well plate.
  • 200 ⁇ l of working reagent are pipetted in and the mixture is homogenized. After incubation for 30 minutes at 37 ° C., the absorbance at 550 nm is measured in the plate photometer.
  • Bentomt 1 is a natural calcium / Natn-umben- tonit.
  • Bentonite 2 was prepared by blending Bentomt 1 with 4.3% by weight soda, then kneading, drying and milling.
  • the bentonites had a dry sieve pressure of ⁇ 15 wt .-% on a sieve of mesh size 45 microns and a residue of ⁇ 7 wt .-% on a sieve of mesh size 75 microns.
  • Table 1 The properties of the bentonites 1 and 2 used as starting materials are shown in Table 1.
  • Table 3a Physical properties of the thermally treated and untreated bentonite 1
  • Table 3b Screen pressure levels of the thermally untreated and treated bentonite 1
  • Bentonite 4 is obtained from bentonite 3 by a stochiomet ⁇ sche activation with soda. Bentonite 3 is a natural Ca / Na bentonite.
  • Table 5 The properties of Bentomte 3 and 4 used as starting material are summarized in Table 5.
  • Table 6 The swelling volumes for the thermally untreated and the treated at different temperatures Bentomte 3 and 4 are summarized in Table 6.
  • the saponite characterized in Table 9 was subjected to a temperature treatment at 500 ° C. (saponite I 500 ) and at 600 ° C. (saponite I 600 ) analogously to Examples 1 and 2.
  • the physical properties of the thermally untreated and thermally treated saponite are shown in Table 10.
  • Table 10 Physical properties of the thermally treated and untreated saponite 1
  • a lipase from Candida rugosa (Sigma-Aldrich GmbH, Taufkirchen, DE) was used.
  • the lipase from the organism Candida rugosa a yeast fungus, has a size of about 60-65 kDa and an isoelectric point of 5.2.
  • Lipases from the organism Candida rugosa belong to the nonspecific lipases, which have no regiospecific properties and thus hydrolyze all ester bonds.
  • a stock solution of the lipase is made at a concentration of 2 mg / ml in the appropriate buffer.
  • the amount of enzyme adsorbed on the carrier was calculated from the difference between the amount of enzyme used and the sum of the amount of enzyme measured in the supernatant or in the wash water. All experiments are carried out in triplicate. The loadings determined for bentonite 5 oo are given in Table 13.
  • Table 13 pH dependence of the adsorption of lipase (Candida rugosa) on thermally treated bentonite I 500
  • the carrier loaded with the enzyme is taken up in 1 ml of distilled water and the suspension used for the activity tests.
  • Activity determination of the stored lipase (Candida rugosa)
  • the activity of the enzyme is determined by the NaOH consumption. Unit-Definition
  • One unit hydrolyzes 1.0 ⁇ M fatty acid (from triglycerides) for one hour at pH 7.7 and 37 ° C.
  • the lipase activities determined are given in Table 14 in comparison to the unlabelled lipase.
  • adsorption isotherms for the adsorption of the lipase to various supports of thermally treated clay minerals were determined.
  • 25 mg of the carrier equilibrated with 50 mM Na acetate buffer at a pH of 4 in 10 ml of enzyme solution were added.
  • the enzyme solutions were prepared by dilution of the enzyme stock solution (Na acetate 50 mM, pH 4) with Na acetate buffer (50 mM, pH 4). The following concentrations were provided: 0.25 mg / ml 0.5 mg / ml 1.0 mg / ml 2.0 mg / ml 3.0 mg / ml 4.0 mg / ml
  • Tables 13 to 16 show the results for the adsorption and activity of the supported enzyme lipase ⁇ Candida rugosa). Surprisingly, it has been found that the enzyme can be adsorbed with very good binding capacities of up to 1.1 mg of protein per mg of carrier. Usually, protein binding capacities or enzyme binding capacities of commercially available carrier materials or chromatography materials are in the range from 0.1 mg to 0.3 mg per milligram of carrier. In addition, it has been shown that the enzyme activity of the supported enzymes is still high, ie only slightly reduced with respect to activity in solution or to solution activity. This is especially true for bentonite I500, bentonite as well as bentonite 3soo and bentonite 3 6 oo the case.

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Abstract

L'invention concerne un complexe enzymatique solide comprenant au moins un enzyme immobilisé sur un support obtenu par chauffage d'un minéral argileux à une température et pendant une durée suffisantes pour que le support présente une capacité de gonflement inférieure à 15 ml/2 g. L'invention concerne en outre un procédé de préparation de ce complexe enzymatique solide, ainsi que son utilisation dans des réactions catalysées par des enzymes.
PCT/EP2008/004781 2007-06-13 2008-06-13 Minéraux argileux modifiés thermiquement utilisés comme matériaux de support d'enzymes WO2008151822A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3953292A (en) * 1974-02-01 1976-04-27 Engelhard Minerals & Chemicals Corporation Enzymes bound to heat-activated attapulgite clay
WO2006097326A2 (fr) * 2005-03-18 2006-09-21 Süd-Chemie AG Procédé pour séparer des biomolécules de milieux liquides
WO2007073893A1 (fr) * 2005-12-16 2007-07-05 Süd-Chemie AG Procede de separation de proteines a partir de milieux liquides
WO2007134816A2 (fr) * 2006-05-20 2007-11-29 Süd-Chemie AG Procédé de séparation de protéines de milieux liquides par utilisation de matériaux argileux modifiés thermiquement

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Publication number Priority date Publication date Assignee Title
FR2112634A5 (fr) 1970-11-03 1972-06-23 Elf
DK638688D0 (da) 1988-11-16 1988-11-16 Novo Industri As Partikelformet immobiliseret lipase-praeparat, fremgangsmaade til fremstilling deraf og anvendelse deraf
DK589389D0 (da) 1989-11-23 1989-11-23 Novo Nordisk As Immobiliseret enzympraeparat
EP0600128A1 (fr) * 1992-11-30 1994-06-08 En-Tech Research Institute Inc. Agent pour l'immobilization des déchets industriels
US6180378B1 (en) 1999-01-29 2001-01-30 The United States Of America As Represented By The Secretary Of Agriculture Immobilization of bioactive protein in phyllosilicates
WO2002018564A1 (fr) * 2000-08-29 2002-03-07 University Of Virginia Patent Foundation Supports de silicate de potassium derivatises

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3953292A (en) * 1974-02-01 1976-04-27 Engelhard Minerals & Chemicals Corporation Enzymes bound to heat-activated attapulgite clay
WO2006097326A2 (fr) * 2005-03-18 2006-09-21 Süd-Chemie AG Procédé pour séparer des biomolécules de milieux liquides
WO2007073893A1 (fr) * 2005-12-16 2007-07-05 Süd-Chemie AG Procede de separation de proteines a partir de milieux liquides
WO2007134816A2 (fr) * 2006-05-20 2007-11-29 Süd-Chemie AG Procédé de séparation de protéines de milieux liquides par utilisation de matériaux argileux modifiés thermiquement

Non-Patent Citations (4)

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Title
CAO L: "Carrier-immobilized enzymes: principles, applications and design", 2005, WILEY-VCH VERLAG GMBH & CO. KGAA, WEINHEIM, GERMANY, ISBN: 3-527-31232-3, XP002495090 *
KAMORI MASANOBU ET AL: "Immobilization of lipase on a new inorganic ceramics support, toyonite, and the reactivity and enantioselectivity of the immobilized lipase", JOURNAL OF MOLECULAR CATALYSIS B ENZYMATIC, vol. 9, no. 4-6, 21 April 2000 (2000-04-21), pages 269 - 274, XP002495088, ISSN: 1381-1177 *
SECUNDO ET AL: "Adsorption and activities of lipases on synthetic beidellite clays with variable composition", MICROPOROUS AND MESOPOROUS MATERIALS, vol. 109, no. 1-3, 21 December 2007 (2007-12-21), pages 350 - 361, XP022399287, ISSN: 1387-1811 *
TISCHER W ET AL: "Immobilized enzymes: crystals or carriers?", TRENDS IN BIOTECHNOLOGY, vol. 17, no. 8, 1 August 1999 (1999-08-01), pages 326 - 335, XP004172537, ISSN: 0167-7799 *

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