SU1518373A1 - Method of producing immobilized tripsine - Google Patents

Abstract

This invention relates to biotechnology, in particular to the preparation of immobilized trypsin. The purpose of the invention is to simplify the process and increase the stability of the immobilized trypsin. The method consists in treating ferromagnetite with a pore diameter of 80 ... 100A with J-aminopropyltriethoxysilane until the concentration of primary amino groups reaches 140 ... 200 micron / mol per 1 / g of dry carrier, modifying the resulting derivative with glutaric aldehyde, taken in 25 ... 30 - fold excess in relation to the concentration of the primary amino groups of the carrier, binding of trypsin to the carrier. The method allows to simplify the process of obtaining immobilized trypsin, as well as to increase its stability.

Description

one

(21) 430876A / 31-13

(22) 01.10,87

(46) 10/30/89. Bksh. I- 40

(71) Vilnius State University V. Kupsukas

(72) V.G. Bendikene, A.A., Raeiūnas and B.A. Juodka

(53) 577.15 (088.8)

(56) Introduction to the applied encyclopes, diu / Ed. I.V. Berezina

and K. Martinek, M., Moscow State University, 1982, p. 112

E. van Leemputten and M, Horisbe rger. Immobilization of enzymes on magnette particles. - Biotechnol, Bioeng. Vol. XYI, p. 385-396, 1974.

(54) METHOD FOR OBTAINING IMMOBILIZED TRIPSINE

(57) The invention relates to biotechnology, namely to the preparation of immobilized trypsin. The purpose of the invention is to simplify the process and increase the stability of the immobilized trypsin. The method consists in treating phromomagnetite with a pore diameter of 80-100 A-aminopropyltriethoxysilane to achieve a concentration of primary amino groups of I40-200 µmol per 1 g of dry carrier, modifying the resulting derivative with glutaric aldehyde, taken in 25-30-fold excess relative to the concentration of the primary amino groups of the carrier, the binding of trypsin to the carrier. The method allows to simplify the process of obtaining immobilized trypsin, as well as increase its stability.

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The invention relates to biotechnology, in particular, to methods for producing immobilized proteins, and may find application in the chemical, pharmaceutical, food industry and medicine in dp carrying out enzymatic reactions.

The purpose of the invention is to simplify the process and increase the stability of the immobilized trypsin.

The method consists in treating ferromagnetite with a pore diameter of 80-100 X aminopropyltriethoxysilane (-APTES) to achieve a primary amino group concentration of 140-200 µmol per 1 g of dry carrier, modification of the resulting derivative with glutaric

aldehyde, taken in a 25-30 fold excess relative to the concentration of the primary amino groups of the carrier, binding of the trypsia and removal of the unbound protein, which has been washed out. The use of these methods allows immobilizing trypsin on a carrier with ferromagnetite properties and obtaining a very stable preparation that retains unchanged activity for 24 months and 50% of activity after 36 months.

The proposed method is easier known, since the stage of activation of the carrier is reduced by one stage and the use of toxic substances is excluded in the activation process.

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Example 1. Production of ferromagnetite. Ferromagnetite particles of colloidal sizes are obtained by precipitating aqueous solutions of a mixture of salts of bivalent and trivalent iron selected from the groups of sulfates and chlorides, solutions of alkali by known methods,

28 g of ferrous sulphate (FeS04, 0) are dissolved in 750 ml of water and 55.5 g of TCI ferric chloride () are dissolved in 750 ml of water. The resulting solutions are mixed at room temperature and the mixture is introduced into 50 ml (an excess of 27% from an equivalent amount of ammonia solution) of a 25% ammonia solution. The resulting mixture is stirred and settled for 1.5 hours, the liquid above the precipitate poured in an amount of 1.15 liters, which constitutes 70% of the mother liquor, is quickly washed with two portions of water in 500 ml each of 300 ml of acetone, portions in 150 ml of a mixture of acetone and i lvol (4: 1, 3: 2, 2: 3, i: 4) and twice toluolo in 150 ml and quickly used for further experiments. Ferromagnetite pore diameter 80-100 A.

Silanization of ferromagnetite. The resulting magnetite is dried (holding it with a permanent magnet) with filter paper, weighed and transferred into a round-bottom three-neck flask, equipped with a mechanical stirrer :), thermometer and cooler. The y-ALTES solution in toluene (ratio of magnetite magnetite ,} -LATES and toluene 1: 1: 10) and boil at the boiling point of the solvent for 10-25 hours (intermittently). After the mixture is cooled, the magnetite is held with a permanent magnet, the solution is drained, the precipitate is washed with toluene ( 100 ml 5-6 times ), then thoroughly with ethyl alcohol (100 m by 3-5 times) and dried at room temperature in a Petri dish,

In silanized magnetite samples (depending on the prolongation of the reaction time with y-APTES), determine the concentration of primary amino groups in 1 g of dry magnetite, measure UV

absorption of ethanol solution of salicylic aldehyde before reaction with samples and after it. Then calculate the concentration of free primary Amis

5 0 5 Q

c 0 5 Q

five

foot groups in µmol on I g of dry magnetite. ,

If the silanization reaction continues up to 30 hours, the number of primary amino groups on the surface of the carrier does not increase. Therefore, prolonging the reaction in excess of 25 hours is impractical from an economic point of view, 15 depending on the processing time of the v-APTES con1; the concentration of amino groups varies from 70 to 200 µmol per 1 g of carrier.

2.3 g of silanized magnetite with a concentration of primary amino groups of 70 µmol / g is washed with water, poured with a 25-fold excess (1.6 microns) (by note on the prayer of the primary lminogroups) of glutaraldehyde f 25-fold excess of glutaral apdehy- yes per mole NH2-rpyi n primary is chosen to avoid cross-linking -.) every day e) zoom by foot molecules: it teldehyde dials) ,; Then, 5.4 ml of water is added, stirred for i h at room temperature, washed with water by decantation, keeping it constant with Muri-shtom, then in two portions of 15-20 ml of 0, OL M Ca (CH tsOO) .j, K Wet modular airoma glutaral δdesm and m ai i nnH-marhGTH v irlbach: g b ml of trypsi-a solution (concentration of Zelk 5) in 0.002 M: a (di 3 -; o oV. Suspension is mixed with Hc i lel , 1st bath B for 2 hours, Magne;: ti.; Pmm1: 1 - initial initial 7PepII pi: TDOpO; 4 up to 1 npONii-ienyh vodschh -Jei-ka, measure emogs, with / VK-IHP rolls 280 them,

Both pimer 2, 2.3 g of succinic magnetite with primary amino groups co-centered with 140 µmol / g, washed with water, 4 ml of rjry-tallow aldehyde, 3 ml of water are poured, persisted for 1 h at room temperature, washed with water by decanting, holding the magnetite with constant Mgnitol, then in two portions of 15-20 nl each of 0.002 M Ca (CH3COO) g. To wet glutaraldehyde-modified magnetite, 6 ml of plant-tryfine rash (protein concentration 5 g / ml) in 0.002 M Ca (CH} COO) was added. The suspension is stirred in an ice bath for 2 h. Magnetites are washed with an initial buffer solution until protein is absent in industrial waters, measured at a wavelength of 280 nm.

51

PRI me R 3. 2.3 g of the staped-up magician) method (example 2) with a primary amino group concentration of 180 μmol / g, washed with water, 4.1 MJ1 glutaraldehyde was poured, 2.9 ml of water, mixed 7 The 1-hour is at room temperature, washed with water by decanting, holding the magnet with a permanent magnet, then in two portions of 15-20 ml of 0.002 M Ca (CH3COO) -g. To wet glutaraldehyde-modified magnetite, add 6 ml of trypsin solution (protein concentration 5 mg / ml) in 0.002 M Ca (CH-, COO) 2. The suspension is stirred in an ice bath for 2 hours. Magghetites are washed with an initial buffer solution until they are absent in the industrial waters of the protein measured at 280 wavelengths,

EXAMPLE 4, 2.3 SILANIZED; about magnetite (example 2) with a concentration of percyph amino groups of 200 μmol / g, they are pushed with water, 5.6 ml of glutaraldehyde are poured,), 4 ml of zod, mixed 1 at room temperature, washed with water by decantation, holding the magnetite with a permanent magnet, then in two portions of 15-20 ml of 0.002 M Ca (CH; | COO) 3. To a wet glutaraldehyde of modified magnetite, 5 ml of trypsin solution (5 mg / ml protein concentration) and 0.02 Ca (CH3COO) o are added, the suspension is stirred in an ice bath for 2 times. Magnetites are washed with an initial buffer solution until no. protein wash waters, measured at 280 nm.

Depending on the concentration of primary amino groups on the surface of the silanized magnetite (while maintaining identical immobilization conditions for all the samples), the amount of immobilized protein drastically changes. So, at a concentration of 70 µmol / g, only 0.45 mg / g is immobilized, and at a concentration of 140, 180 and 200 μmol / g - 1.8; 2.5 and 2.8 mg / g protein, respectively. Thus, the optimal amount of primary MH2 groups for binding the largest amount of protein is 140-200 µmol per 1 g of dry carrier. In addition, the upper limit of the concentration is just 200 µmol / g, since during continuation

The scientific research institute of silanization time is more than 25 hours; the amount of amino groups introduced does not change. The efficiency of immobilization with the addition of a 25-or 30-fold excess of glutaraldehyde is the same.

Esterase activity is determined with a specific substrate 4-nitro-Q anilide-N-benzoyl-DL-arginine (BApNA).

The specific activity of trypsin is determined by the number of moles of p-nitro5 aniline formed during the enzymatic hydrolysis of the substrate for 2 minutes at 37 ° C, at a pH of 8.2 per 1 mg of protein.

The resulting preparation of immobilized trypsin is stored for two years without loss of activity, and after three years its activity is reduced by 50%.

The thermostability of the obtained preparation is higher than at temperature for 1 h, the activity of preparations decreases by 50 and 70%, respectively.

Thus, the use of the proposed method significantly simplifies the process of obtaining the target product by eliminating one stage of carrier activation and carrying out the process of using toxic substances. In addition to this, the obtained preparation of immobilized trilles; ina has thermal stability and high storage stability.

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Claims (1)

Invention Formula The method of obtaining immobilized trypsin, including processing , (Ferromagnetite-aminopropyl-tristoxysilane, modification of the obtained derivative with chemical reagents, addition of trypsin and washing of unbound protein, which is used to simplify the process and increase the stability of the target product, use 80% ferromagnetite 100 A, treatment of ferromagnetite with aminopropyltristoxysilane is carried out until the concentration of primary amino groups reaches 140-200 µmol per I g of dry carrier, and the modification of the obtained derivative is carried out deeply. 715183738 container aldehyde, taken in a 25-concentration of primary amino groups and a 30-fold excess with respect to the quencher.