Classifications

• • 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

• Organic materials e.g. cellulose, nylon, polyacrylamide
• cellulose, nylon, polyacrylamide have considerable disadvantages as carriers because they do not have sufficient mechanical stability, can be attacked by the solvent, are sensitive to changing pH values and ionic strengths, and in some cases tend to attack microbes, which leads to binding to Enzyme can be dissolved.
• inorganic substances have been proposed as carriers on which enzymes are bound by adsorption or covalently.
• the preferred type of binding depends on the type and conditions of use of the enzyme and the nature of the substrate. If, for example, the substrate is in a high salt concentration, the adsorption method cannot be used because desorption of the adsorbed enzyme molecules is possible. Therefore, the covalent binding of the enzyme to the carrier is preferred.
• the carrier surface must then contain specific functional groups that ensure binding of the enzyme. Since the carrier does not have these functional groups in most cases, the surface must be pretreated. For example, the coating of inorganic material with silanes is known, as a result of which the surface receives organically functional groups (e.g. alkylamine) which form a covalent bond with organic substance.
• organically functional groups e.g. alkylamine
• Aluminum oxide, nickel oxide, iron oxide, titanium oxide, zirconium oxide, hydroxylapatite, silicates and porous glass have been proposed as materials for inorganic carriers, the pore structure of which ensures the accessibility of the enzyme and the substrate to the inner surface, but their other desirable properties such as optimal pore distribution and surface size, however, vary widely Information is available.
• the invention is therefore based on the object of the known processes for the preparation of a water-insoluble enzyme preparation, in which an inorganic carrier which has functional groups for the covalent binding of an enzyme is brought into contact with a solution of an enzyme by means of processes known per se binds to the carrier and isolates the enzyme preparations obtained, to improve them so that a maximally active preparation is obtained with minimal enzyme expenditure.
• This object is achieved according to the invention in that firstly carriers which differ in their most common pore diameter are brought into contact with enzyme solutions which differ in their enzyme concentration, the enzyme is bound to the carriers, the enzyme preparations are isolated and their activity is isolated determined and selected from the different carriers used the carrier with the most common pore diameter which, regardless of the amount of enzyme bound to it, gave the preparation with the highest activity, and then brings the selected carrier into contact with enzyme solutions which differ in their enzyme content, the enzyme binds to the carrier, isolates the enzyme preparations, their activity and specific activity is determined and, from the different enzyme solutions used, selects the one which gives the preparation with the highest activity and a specific activity close to or equal to the specific activity of the enzyme in the free state n has.
• carriers with different common pore diameters are first provided with coupling agents according to known methods, which both adhere sufficiently firmly to the carrier, in particular form a covalent bond to the carrier, and are capable of covalent binding with the enzyme.
• coupling agents according to known methods, which both adhere sufficiently firmly to the carrier, in particular form a covalent bond to the carrier, and are capable of covalent binding with the enzyme.
• silanization has become the most common method for inorganic carriers, but, as already mentioned, other coupling agents can also be used.
• the number of coupling links on the carrier must be sufficiently large and largely depends on the surface of the carrier.
• the carriers pretreated in this way are then offered different amounts of enzyme by bringing them into contact with enzyme solutions of different concentrations and by known methods covalently binding the enzyme to the coupling member and thus to the carrier.
• the activity of the preparations depends on the most common pore diameter regardless of the amount of enzyme bound to them and runs through a maximum.
• the grain size of the carrier is irrelevant to the position of the maximum and at most influences its absolute value. The grain size of the Carrier therefore has only a subordinate meaning for the teaching according to the invention and largely depends on the intended use, for example the viscosity of the substrate and the procedure.
• the optimal carrier determined in this way with regard to the most common pore diameter is then again offered different amounts of enzyme. It is shown that preparations are obtained at certain enzyme concentrations, the specific activity of which approaches or reaches the specific activity of the enzyme in the free state, i.e. the relative activity of the preparation reaches a value of 100%.
• the method according to the invention is therefore a double selection process, i.e. a process by means of which one can select from a number of carriers which are suitable with regard to their pore diameter and from a plurality of enzyme solutions which are possible with regard to their enzyme concentration, the carrier and the enzyme concentration which give a preparation with the highest possible activity with the least possible use of enzyme.
• the optimal carrier determined is reacted with differently concentrated enzyme solutions, the absolute activity and the specific activity, i.e., of the enzyme preparations obtained.
• the activity per milligram of enzyme is determined, and the values obtained are compared. It is initially confirmed that, with increasing concentration of the enzyme solution, the absolute activity of the preparations obtained also initially increases, but then remains the same from a certain concentration. Any increase in concentration beyond the concentration determined in this way is therefore a waste of enzyme. This is in contradiction to the known experiences, which act according to the motto "A lot helps a lot".
• the bound enzyme is just as active as the enzyme in the free state, i.e. the specific activity of both the bound and free enzyme are the same or the relative activity of the enzyme preparation is 100%.
• the specific activity rapidly decreases from a certain concentration and the relative activity becomes less than 100%, i.e. an increasing proportion of the bound enzyme is in inactive form.
• the invention provides the teaching set out in the claims for the production of a water-insoluble enzyme preparation, which is both the optimal carrier with regard to maximum activity and the optimum concentration of the enzyme solution on the one hand and in the interest of maximum space-time yield on the one hand and avoiding enzyme waste on the other can be determined.
• the optimum support can be obtained according to a further development of the invention if the gel, after setting an alkali content, calculated as Na 2 0 and based on dry substance, from 0.1 to 0, 5 wt .-%, and drying, 5 to 10 hours in a steam-containing air stream at 400 ° C to 850 ° C, preferably 570 ° C to 750 ° C, glows.
• the drying is expediently carried out in water vapor-saturated air at 180 ° C. to 200 ° C.
• a water vapor-containing air stream with a relative humidity of 40 to 80% has proven to be advantageous for annealing.
• the carrier thus produced has a most common pore diameter of 175 to 3,000 ⁇ , preferably 250 to 600 ⁇ , optimally about 340 ⁇ .
• the immobilization method according to the invention can be used for all technically and analytically important enzymes, for example for hydrolases (e.g. amylases, glycosidases, proteases), oxidoreductases (glucose oxidase, catalase), isomerases (glucose isomerase), transferases (dextran sucrase).
• hydrolases e.g. amylases, glycosidases, proteases
• oxidoreductases oxidoreductases
• oxidoreductases oxidoreductases
• isomerases glucose isomerase
• transferases dextran sucrase
• the optimal preparation is obtained when the optimal carrier is brought into contact with a solution containing 25 to 75 mg , preferably 50 mg, contains amyloglucosidase per gram of carrier.
• the optimal preparation is obtained if the optimal carrier with a Solution is brought into contact containing 20-50 mg, preferably 25 mg, of glucose isomerase per gram of carrier.
• a SiO 2 gel precipitated from sodium silicate solution with sulfuric acid and having a Na 2 O content of 0.3% by weight was dried at 180 ° C. in water-saturated air for three hours. 1 kg of this material was annealed for 6 hours at 730 ° C in an air flow of 2 l / min, which had a relative moisture content of 80%. After this treatment, the Si0 2 had a most common pore diameter of 1400 ⁇ . Carrier 1 was separated into fractions by sieving. The further preparation was carried out with the fraction 0.25 to 0.5 mm.
• sample 1.2 10 g of silanized carrier 1 were suspended in 20 ml solution of 0.5 g amyloglucosidase (Merck 1330) in 0.05 m phosphate buffer (pH 7). The further procedure corresponded to the preparation of sample 1.1. The C-N analysis of the finished sample 1.2 showed a protein content of 9.0 mg / g.
• Carrier 2 calculated on the basis of the mean value of the C and N determination, contained 0.19 m eq of silane / g.
• silanized carrier 2 Another 10 g of the silanized carrier 2 were suspended in 20 ml solution of 0.5 g amyloglucosidase (Merck 1330) in 0.05 m phosphate buffer (pH 7) and treated as described in Example 1 (sample 1.2).
• the C-N analysis of the finished sample 2.2 showed a protein content of 17.4 mg / g.
• Carrier 3 calculated on the basis of the mean value of the C and N determination, contained 0.51 m eq of silane / g.
• the finished sample 3.1 had a protein content of 26.2 mg / g after the C-N analysis.
• silanized carrier 3 Another 10 g of the silanized carrier 3 were in 20 ml solution of 0.5 g of amyloglucosidase (Merck 1330) suspended in 0.05 m phosphate buffer (pH 7) and treated as described in Example 1 (sample 1.2).
• the C-N analysis of the finished sample 3.2 showed a protein content of 12.7 mg / g.
• the fraction 0.25-0.5 mm was sieved out of the carrier 2 (most common pore diameter 340 A) and 50 g thereof in 500 ml 12.5 % aqueous glutardialdehyde solution stirred for 5 minutes at room temperature. 500 ml of saturated NH 4 CI solution were then added. After four hours of stirring at room temperature, the sample was washed with water until free of chloride and dried over P Z 0 5 in vacuo.
• the finished sample 4.1 had a protein content of 29.8 mg / g after the C-N analysis.
• the C-N analysis of the finished sample 4.2 showed a protein content of 17.9 mg.
• the reaction time was 30 minutes at room temperature. After every 10 minutes, the reaction vessel was evacuated and, after the reaction had ended, the residual solution was suctioned off. Then 3 washes with water and 0.05 m phosphate buffer (pH 7).
• the finished sample 5.1 had a protein content of 4.8 mg / g after the C-N analysis.
• the C-N analysis of the finished sample 5.2 showed a protein content of 2.0 mg / g.
• the further treatment corresponded to Example 5.
• the finished sample 6.1 had a protein content of 22.0 mg / g after the C-N analysis.
• sample 6.2 a further 10 g of carrier 2 were suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7) which contained 0.25 g of glucose isomerase.
• the further treatment corresponded to example 5.
• the C-N analysis of the finished sample 6.2 showed a protein content of 10.2 mg / g.
• the further treatment corresponded to Example 5.
• the finished sample 7.1 had a protein content of 11.2 mg / g after the C-N analysis.
• the C-N analysis of the finished sample 7.2 showed a protein content of 5.1 mg / g.
• Example 4 10 g of the carrier mentioned in Example 4 (most common pore diameter 340 A, treated with aqueous glutardialdehyde solution) were suspended in 40 ml of a 0.05 M phosphate buffer solution (pH 7) which contained 0.5 g of glucose isomerase.
• the further treatment corresponded to example 5.
• the finished sample 8.1 had a protein content of 21.3 mg / g.
• sample 8.2 a further 10 g of the same carrier were suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7) which contained 0.25 g of glucose isomerase. The further procedure corresponded to preparation 5.1. The C-N analysis of the finished sample 8.2 showed a protein content of 9.8 mg / g.
• the activity of the preparations 1.1, 1.2 described in Examples 1 to 4; 2.1, 2.2; 3.1, 3.2; 4.1, 4.2 and that of the enzyme used for fixation (amyloglucosidase, Merck 1330) was used according to the dinitrosalicylic acid method (cf. Rick, W., Stegbauer, HP in: Bergmeyer, HU "Meth. D. Enzymatic Analysis", Verlag Chemie 1970 S. 848 ff).
• One activity unit (U) corresponds to that Amount of enzyme that releases 1 ⁇ equivalent of reducing groups (calculated as glucose) per minute under incubation conditions.
• Carrier-fixed preparations were suspended under the above conditions in a 40 ml reactor at a stirring speed of 600 min -1 (product formation rate regardless of stirring speed).
• the protein content of the preparations was determined on the basis of the mean values of the C-N determination.
• the most common pore diameter of the carriers was determined from the pore distribution (measured with a high pressure porosimeter).
• the carrier-fixed preparations were suspended as described in Example 9 under standard conditions in the stirred reactor.
• the protein content of the preparations was determined on the basis of the mean values of the C-N determination.
• the method according to the invention has for the first time made it possible to use expensive enzymes technically, since the method allows the use of carrier according to the invention to optimize the amount of enzyme required for maximum activity.

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