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

• the invention relates to a process for the preparation of water-insoluble enzyme preparations and enzyme preparations produced thereafter. It is known to immobilize enzymes by binding to organic or inorganic carriers, ie to render them water-insoluble, so that they are reusable and can be used in continuously operating processes.
• 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, causing the bond 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 is given organic functional groups (for example alkylamine) which contain organic sub punch a covalent bond.
• Aluminum oxide, nickel oxide, iron oxide, titanium oxide, zirconium oxide, hydroxylapatite, silicates and porous glass have been proposed as the material for inorganic carriers, the pore structure of which ensures the accessibility of the enzyme and the substrate to the inner surface, in addition to their other desirable properties, e.g. optimal pore distribution and surface size, however, there are very different information.
• the invention is therefore based on the object of improving the known processes for the preparation of water-insoluble enzyme preparations, in which an inorganic carrier having functional groups for covalent bonding is brought into contact with an enzyme solution, so that a maximally active preparation is obtained with minimal enzyme expenditure.
• This object is achieved according to the invention by selecting a carrier with such a most common pore diameter which, regardless of the amount of enzyme bound to it, results in a preparation with maximum activity, and by bringing the carrier selected in this way into contact with an enzyme solution which contains only enough enzyme that the specific activity of the preparation produced with it reaches or comes close to the specific activity of the enzyme in the free state.
• 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 covalent binding of the enzyme to the coupling member and thus to the carrier by known methods.
• Determination of the activity of the preparations thus obtained shows that 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 best influences its absolute value.
• the grain size of the carrier has therefore only of secondary importance for the teaching according to the invention and largely depends on the intended use, for example the viscosity of the substrate, the process control and the like.
• 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 optimum carrier 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 0 C to 850 ° C, preferably 570 0 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 (eg amylases, glycosidases, proteases), oxidoreductases (glucose oxidase, catalase), Isomerases (glucose isomerase), transferases (dextran sucrase).
• hydrolases eg amylases, glycosidases, proteases
• oxidoreductases glucose oxidase, catalase
• Isomerases glucose isomerase
• transferases dextran sucrase
• the optimal preparation is obtained when the optimal carrier is brought into contact with a solution which is 25 to 75 mg , preferably 50 mg, contains amyloglucosidase per gram of carrier.
• the optimal preparation is obtained if the optimal carrier is brought into contact with a solution containing 20-50 mg, preferably 25 mg, of glucose isomerase per Grams of carrier contains.
• the carrier 1 For the preparation of the carrier 1 a precipitated from sodium silicate solution with sulfuric acid was dried Si0 2 gel having a Na 2 0 content of 0.3 wt .-% at 180 0 C in water vapor saturated air for three hours. 1 kg of this material was annealed for six hours at 730 ° C. in an air stream of 2 l / min, which had a relative moisture content of 80%. After this treatment, the SiO 2 had a 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.
• Si0 2 gel having a Na 2 0 content of 0.3 wt .-% as in Example 1 dried. 1 kg of this material was annealed for 6 hours at 680 ° C in an air flow of 2 1 / min, which had a relative moisture content of 80%. After this treatment, the SiO 2 has a mode pore diameter of 340 ⁇ had. The carrier 2 was separated into fractions by sieving. The further preparation was carried out with the fraction 0.25 to 0.5 mm.
• 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 CN analysis of the finished sample 2.2 showed a protein content of 17.4 mg / g.
• Si0 2 gel having a Na 2 0 content of 0.3 wt .-% as in Example 1 dried. 1 kg of this material was annealed for six hours at 640 ° C in an air flow of 2 l / min, which had a relative moisture content of 60%. After this treatment, the Si0 2 had a common pore diameter of 180 ⁇ . The carrier 3 was separated into fractions by sieving. The further preparation was carried out with the fraction 0.25 to 0.50 mm.
• 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.
• a further 10 g of the silanized carrier 3 were suspended in 20 ml of a solution of 0.5 g of 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 3.2 showed a protein content of 12.7 mg / g.
• the fraction 0.25-0.5 mm was sieved from the carrier 2 (most common pore diameter 340 ⁇ ) 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 C1 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 2 0 5 in vacuo. 10 g of this carrier were suspended in 20 ml solution of 1 g amyloglucosidase (Merck 1330) in 0.05 m phosphate buffer (pH 7) and prepared as described in Example 1.
• the finished sample 4.1 had a protein content of 29.8 mg / g after the C-N analysis.
• 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 5 10 g of the carrier mentioned in Example 4 (most common pore diameter 340 ⁇ , 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 after the C-N analysis.
• 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 was used according to the dinitrosalicylic acid method (see Rick, W., Stegbauer, HP in: Bergmeyer, HU "Meth. D. Enzymatic Analysis", Verlag Chemie 1970 S. 848 ff).
• One activity unit (U) corresponds to the amount of enzyme that releases 1 ⁇ equivalent reducing groups (calculated as glucose) per minute under incubation conditions.
• 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 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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• 1977
  • 1977-06-10 DE DE2726188A patent/DE2726188C2/de not_active Expired
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