DE2619571A1 - Acylase enzyme immobilisation - on lamellar aluminium hydrosilicates modified with amino-silanes - Google Patents

Abstract

In a new process for immobilizing acylases to silicaceous carrier masses by linking via cpds. contg. amino gps. as the silicaceous carrier mass there are used Al hydrosilicates having a lamellar lattic structure which are modified with silanes contg. amino gps. Immobilized N-acyl-L-amino acid amidohydrolases (amino-acylases) are useful for optical resolution procedures involving selective deacylation of the L-isomer of a racemic N-acyl-aminoacid. The immobilized acylases obtained by the new process retain practically constant activity after prolonged (>1000 hrs) use, whereas preparations obtained in exactly the same way but using porous glass instead of aluminium hydrosilicates are inactive or lose their activity after only brief use in enzymatic optical resolution.

Description

Method for fixing soluble acylases to inorganic

Support material The present invention relates to a method for fixing soluble acyases to inorganic, silicate 1 carrier material. The acylases are activated via compounds containing amino groups or their derivatives connected to the carrier material.

N-acyl-L-amino acid amidohydrolases, mostly abbreviated amino acylases or called acylases, are able to selectively deacylate N-acyl-L-amino acids. These Property of the acylases can be used to synthesize L-amino acids from Evin racemates of the corresponding N-acyl derivatives: After separating the two The L-form released by the asymmetric hydrolysis becomes the non-split N-acyl-D-form racemized and reapplied the cleavage process on this mixture. To this Way, the total amount of synthetically produced amino acid in the L configuration is derived from the naturally occurring amino acids Predominantly occurs.

The e acylases, which e.g. from animal organs, such as Pig kidneys or bovine pancreas, isolated or from microorganisms such as Aspergillus orycae, are prepared are water-soluble proteins. Your insolubilization under possible little loss of enzyme activity and stability is a prerequisite for a advantageous technical implementation of the above resolution: Lie insoluble Enzyme-active solids can be used as satellite phases, e.g. in reaction columns will. A laborious recovery process that is effective in very low concentrations Biocatalysts from mostly large substrate volumes is not required if they are used in -water-insoluble form. Contamination of the cleavage solutions with Pro4. one or their breakdown products is avoided.

To fix cleavage-active proteins on carrier material has already been suggested, modified polysaccharides or polymers on the carrier material To be used based on acrylamides.

However, these hydrophilic carriers have several disadvantages: Because of their irreversible swellability in the event of changes in the pH value and / or the ion concentration of the substrate solutions, they have poor dimensional stability. Farther the mechanical strength, especially when wet with water, is insufficient.

If, on the other hand, less hydrophilic materials are used as carrier substances such as porous glass according to DT-AS 1 949 418, then increases with increasing hydrophobic character of the carrier depends on the activity of the enzyme fixed on it. It is therefore possible to bind acylases to porous glass, where, if necessary, the adhesion is reinforced by organosilanes; the activity such preparations takes but already after brief contact with the neutral or alkaline pH values to be cleaved substrate solutions to such an extent that these materials are out of the question for a technical application.

Hydrophobic carrier materials also have the disadvantage that they are not sufficiently resistant to hydrolysis in aqueous, weakly alkaline solutions.

The task was therefore to produce carrier-bound acylases, the activity and, above all, durability that are sufficient for technical use the activity be 'continuous use among those to be observed in the resolution Have conditions, have good mechanical and hydrodynamic properties, are not very susceptible to attack by microbes and are present in a form that is optimal Reaction procedure permitted, and readily available starting materials for the carrier require.

In fulfilling this task, a method for fixing has now been made more enzyme-active Acylases on silicate carriers by linking these acylases via amino groups Found compounds with the carrier, which is characterized in that one aluminum hydroxylicates with layered lattice structures that are modified with amino group-containing silanes.

The aluminum hydrosilicates to be used as basic products according to the invention with layer lattice structures are widespread in nature and are used for various Purposes obtained by special processes. A characteristic Leristic mineral this group is the montmorillonite. Ideally, its formula is Al2 [(OH) 2SI4O10] + x H20. In this formula some of the aluminum can be replaced by magnesium and aluminum can be exchanged for silicon. When dried, corresponds to the montmorillonite of the formula (OH) 2 (Mg, Al) 2 [(Al, Si) 2O10]. like montmorillonite. and related minerals, have become an inner crystalline, reversible Cuelling enables: You take water and aqueous solutions between the silicate layer packages on, whereby the distance between the layer planes changes lawfully and reversibly. You can, depending on the requirements of the reaction in technical use, can be used in the form of powders, extrudates or, preferably, spheres.

The chemical modification of the aluminum hydrosilicates with layer lattice structures for the purpose of introducing reactive groups is carried out by treatment with organosilanes containing amino groups. These silanes correspond to the general formula In this formula, R stands for an alkyl radical with 1 to 4 carbon atoms, which can optionally be interrupted by an oxygen atom, R ″ for an alkyl radical with 1 to 8 carbon atoms or for the radical R denotes an alkylene radical with 1 to 4 carbon atoms or a phenyl radical, Z1 represents H or - (CH2) n NH2 (n = 2-4) and Z2 = H or Z ,. Examples of corresponding aminosilanes are γ-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane, ß-aminoethyl-trimethoxysilane, N-aminoethyl-γ-aminopropyltriethoxysilane or p-aminophenyltriethoxysilane. The alkoxy groups of the silanes react with hydroxyl groups of the layered lattice silicates to form —Me — O — Si bonds (Me = Al, Si). The content of amino groups in the carrier modified by means of organosilane can be varied within wide limits. The optimum degree of substitution of the silicate hydroxyl groups for a particular aoylase can be produced by appropriate selection of the ratio of the reactants.

For linking the carrier containing amino groups with layer lattice structures with reactive, enzymatically inactive groups of the acylases are all suitable Reactics known per se which lead to a firm, covalent bond. Examples Such reactions are e.g. formation of an acid amide bond or addition of nitriles or isocyanates, the corresponding reaction components being bound to the enzyme available.

Because acylases are very sensitive to chemical interference and theirs Activity in reactions with aggressive reagents, such as acid chlorides, is considerable impaired, if not entirely eliminated, the coupling with the carrier must under as possible gentle and mild conditions. One A particularly gentle method is the conversion of the acylases with a carrier material, which contains diazo groups. Therefore it is active in the production of the cleft Immobilisate according to the invention preferably used.

If the aluminum hydrosilicates aminated according to the invention with layer lattice structures contain aliphatic amines, those are necessary for the linkage with the acylase Diazonium salts by aminoarylation and diazotization in a manner known per se won. The aminoarylation 9sr γ-aminopropyl group can, for example by dissolving with p-nitrobenzoyl chloride and subsequent reduction of the nitro groups take place.

The generation of diazonium salts is very simple in the case of the p-aminophenyl groups modified carriers with layered lattice structures through direct conversion with nitrous Acid under the usual conditions for diazotization reactions.

The coupling of the acylatically acting proteins with the pretreated Carrier material takes place in aqueous, preferably buffered, solutions at pH values near neutral point and at temperatures generally between OOC and the thermal.

the stability limit of the enzyme. It can be both highly purified Acylases as well as technical acylasse concentrates, e.g. from Aspergillus orycae cultures, The reaction time depends on how much time for a complete Sales of the couplable groups is required. she can up can be extended to several days.

After the coupling reaction has ended, there are enzyme-active solids before, which apart from the chemically tightly bound acylase, broader protein and possibly Contain accompanying material. To remove this sticky cder only loosely tied Proteins and any enzyme-inactive accompanying substances that may be present become the solids Washed using buffer solutions or substrate solutions until they are all soluble Parts are completely removed; the buffer solutions used should in general have a pH between 6.0 and 8.0. Ice for their use will be the carrier-fixed Acylases are expediently stored at temperatures up to about 0C without drying.

It is most surprising that the invention is based on of the hydrosilicates modified by aminosilanes with layer lattice structures Acylase Immobiilsate in continuous use for over 1000 hours practically constant activity show that in exactly the same way from porous glasses with primary amino groups Acylase preparations obtained show no activity or after a short period of use become completely inactive for the resolution.

Example 1 250 g of montmorillonite balls (catalyst "KA" from the company SÜDCHEMIE, Munich), which after chemical modification by means of -Äiiinopropyltriäthoxisilan 1.4% nitrogen were obtained by reaction with a solution of 25 g of p-nitrobenzoyl chloride in chloroform with the addition of 12.5 g of triethylamine at reflux temperature for one hour acylated. The spheres were washed with chloroform at 40 ° C. and dried the reduction of the nitro groups by heating the balls in 2.5 1 one percent for one hour Sodium dithionite solution on the reflux condenser. Then was with ice cooling means Sodium nitrite / 2N hydrochloric acid diazotized in the usual way. After washing with 332.5 g of moist, diazotized spheres were present in ice water.

The pretreated montmorillonite balls were in a solution of 50 g of soluble acylase from Aspergillus orycae (specific activity 10 U / mg) in 300 ml Phosphate buffer stored at pH 7 for four days at 40C. Then the soluble Parts are washed out thoroughly with phosphate buffer solution (pH 7). The washed out The resolution of N-acetyl-D, L-phenylalanine yielded product in 10 pointers aqueous solution at pH 7 and 370C in a cleavage column within 48 hours 25 g L-phenylalanine / 1 1 substrate solution. This value was after 1000 hours of use of the spheres with fixed acylase still unchanged. During this time the Substrate solution is conveyed through the column at 300 ml / hour. After reaching the specified Split state either fresh cleavage solution was used or that after separation of the L-phenylalanine by adding the equivalent amount of the acetyl compound back to the initial concentration supplemented solution returned to the cleavage. Example 2: (Comparative example) In Exactly the same way as described in Example 1, porous glass, which contained reactive amino groups by reaction with aminosilane (CORN; NG Code No. GAO-7920; Commercial product of PIERCE CHEMICAL CO., Rockford, III .; Product no.

24108) acylated with p-nitrobenzochloride, then with sodium dithionite reduced, diazotized and with the same soluble acylase from Aspergillus orycae (specific activity 10 U / mg) coupled. In N-acetyl-D, L-phenylalanine solution at pH Incubated 7 and 370C, the preparation showed no cleavage activity.

Example 3: 10 g chemically functionalized by p-aminophenyltrimethoxysilane Montmorillonite balls with a nitrogen content of 0.43% were made using sodium nitrite / 2 n Hydrochloric acid-completely diazotized in the usual way. After thorough washing with ice water, the diazotized spheres were 72 hours in a solution of 1 g soluble acylase from Aspergillus orycae (spec.

Activity 10 Uímg) in 10 ml phosphate buffer solution at pH 7 and 40C kept. After that, all soluble parts were -by To wash removed with phosphate buffer solution (pH 7). The weight of the moisture attached freed but not completely dried acylase immobilizate spheres was 15 g. They showed in the resolution of N-acetyl-D, L-phenyleanine in 10% aqueous Solution at pH 7 and 370C both in continuous, stirred etching with 24 hours Change of substrate than the same permanent activity when used in a split column like the spheres with fixed acylase described in Example 1.

Claims (4)

P a t e n t a n s p r u 'c h e 1. Method for fixing enzyme-active Acylases to Silicatisone Tragernlasserl by linking via amino groups Compounds that are not identified as siliceous Carrier mass aluminum hydrosilicates with layered lattice structures used with amino group-containing silanes are modified. 2. The method according to claim 1, characterized in that the aluminum hydrosilicate is a montmorillonite. 3. The method according to claim 1 or 2, characterized in that the amino groups are diazotized and the resulting diazonium compounds to the Arvlasen binds. 4. The method according to any one of claims 1 to 3, characterized in that that if aliphatic amino groups are present, these are known per se Way connects with an aromatic diazotizable amine and then the Linking with the acylase via the corresponding diazonium compound carries out.