CS238544B1 - Immobilized amyloglucosidase and method for its preparation - Google Patents

Abstract

Immobilized amyloglucosidase on a carrier based on pearl cellulose and a method for its preparation. The essence is immobilized amyloglucosidase bound in an amount corresponding to an activity of up to 1,300 units/g on a carrier based on regenerated pearl cellulose containing diethylamine groups in a concentration of 0.1 to 0.2 mmol/g of cellulose. The method for preparing immobilized aminoglucosidase according to the invention is carried out with a carrier based on regenerated pearl cellulose containing diethylamine groups in the 0H" form suspended in an enzyme solution in water in the presence of 0.5 to 2% by weight of maltose or starch with a pH of 4.0 to 4.7 at a temperature of 0 to 50 °C or with a carrier placed in a column with the flow of the enzyme solution under the same conditions. The higher effect compared to the current technical state lies primarily in the orderly higher activity of the immobilized amyloglucosidase, saving of the native enzyme during immobilization, longer life of the immobilized enzyme and easy regenerability of the carrier.

Description

The invention relates to an immobilized amyloglucosidase on a carrier based on pearl cellulose and to a process for its preparation.

The current status of amyloglucosidase immobilization is characterized by the following review. In the vast majority of works, immobilization is carried out by chemical bonding or polymerization. Amyloglucosidase was immobilized by sorption on supports based on synthetic ion exchangers, cellulose derivatives and dextran gels (M. Moulond, J. Boundrant, J. C. Cheftel: Fr. Demande 2,372.653 (1978)). Amberlite IRA. 93 », which is a macroporous styrene-divinylbenzene copolymer with diethylaminomethyl groups, was used to immobilize under static conditions by suspending the ion exchanger in an enzyme solution at pH 5.2 and 4 ° C for 5 hours to obtain an immobilized enzyme activity of 60.4 µl. G.

The hydrolysis of a 25% solution of maltodextrans (DE 4-5) in a flow reactor with a residence time of 50 minutes resulted in a 70 $ conversion at pH 4 and 40 ° C (DE stands for glucose equivalent expressed as percentage of glucose in starch, oligosaccharide) or their hydrolysis products).

On a multifunctional weakly basic anion exchange resin (Amberlit IR 45), it absorbed amyloglucosidase YK Park (j. Ferment. Technol. 52,140 (1974)) with an activity of 1000 j / g for 4% starch in 0.1 mol / l acetate buffer pH 4 at temperature 60 ° C. 12.6 mg amyloglucosidase / g copolymers were immobilized on 2,2-dimethylaminoethyl methacrylate copolymers (v. S. Nithianandam, M. Santappaí Biotechnol. Bioeng 23,2273 (ΐ9θΐ))

Better results were obtained by sorption of amyloglucosidase on cellulose derivatives. For example, Brouillard (US Pat. 4,029,546 (1977)) regenerated cellulose into large cuckle particles into which he introduced 2-diethylamionoethyl groups at a concentration of 0.97 mmol / g.

238 544

On this material, the enzyme absorbed an activity of 24.3 IGU / g. The DE product 60 was obtained in a column at a flow rate of 30% DE 35 maltodextrin of 4 ml / min at 58.9 ° C and pH 4.2.

Smiley (Biotechnol. Bioeng 13,399 (197l)) adsorbed amyloglucosidase on fibrillar cellulose with 2-diethylaminoethyl groups at 4.3 J / g. 10 g of immobilized enzyme hydrolyzed a 30% liquefied starch solution in a 4 L continuous stirred tank reactor at 55 ° C and a flow rate of 36-70 ml / hour. He had problems with the mechanical stability of the carrier, the conversion to glucose was 84 92%.

An object of the present invention is an immobilized amyloglucosidase characterized in that it is bound in an amount corresponding to an activity of up to 1300 units / g on a regenerated bead cellulose carrier containing diethylamine groups at a concentration of 0.1 c, 2 mmol / g t

cellulose.

Diethylamino groups are introduced into pearled regenerated cellulose by a suspension process by reaction of (3-chloro-2-hydroxypropyl) -diethylamine hydrochloride, respectively. (2-chloroethyl) -diethylamine chloride, respectively. of a mixture of diethylamine with 1-chloro-2,3-epoxypropane in 1-10 mol / l NaOH, at a temperature of 0 ° C to 70 ° C for 0.5 to 48 hours according to cf. 199 420 (Hradil, Musil, Mlejnek).

A method for preparing immobilized amyloglucosidase is characterized in that the immobilization is carried out with a carrier based on a regenerated pearl cell of loza containing diethylamine groups in OH form suspended in an enzyme solution in water in the presence of 0.5 to 2% by weight. maltose or starch at a pH of 4.0 to 4.7 at a temperature of 0 to 50 ° C or a support placed in the column by flowing the enzyme solution under the same conditions.

Prior to immobilization, it is convenient to introduce the carrier groups by washing with an alkali metal hydroxide solution to form the free base.

The stability of the immobilized enzyme of the invention can be enhanced by crosslinking the glutaraldehyde reaction at 25 ° C for 2 hours at pH 6.8 after the enzyme has been adsorbed onto a regenerated bead cellulose-containing carrier containing diethylamine.

This carrier absorbs up to 407 J / g (the unit of activity represents

- 3,238,544 enzyme activity which releases from 1% starch solution in 1 minute at pH 4.7 at 30 ° C 1 / µmol glucose (0.18 mg based on 1 g dry carrier) and 370 mg protein /G.

Hydrolysis of starch or oligosaccharide solution (13% by weight)

DE 12-35) can be carried out by the above-described mobilized enzyme at a temperature of 40 (60 60 ° C at pH 3 already 7) in both a stirred batch reactor and a continuous stirred or column reactor. 30% starch solution) decreased only in the first 200 hours (from 1240 to 500 J / g), but stabilized at 300 J / g after 400 hours.

Advantageously, the immobilization can be carried out in a flow-through dynamic arrangement by a support placed in a column through which the enzyme solution is pumped, optionally repeatedly. In contrast to sorption under static conditions with a sorbent mixed in an enzyme solution, the above-described method provides, under comparable conditions, a mobilized enzyme with a higher specific activity of 29%.

A further advantage of the invention is that the carrier is fully regenerable with 0.1 to 1 mol / l sodium chloride solution and / or 0.1 to 0.5 mol / l hydrochloric acid solution and / or 0.1 to 0.5 mol / l solution sodium hydroxide solution. Pearl cellulose with diethylamine groups also has an affinity for impurities contained in oligosaccharide solutions. However, the sorption of these impurities does not reduce the enzymatic activity of the mobilized enzyme and does not prevent regeneration in the sense of the above.

The basic prerequisite for this universal use of the mobilized enzyme is sufficient strength and incompressibility of the carrier.

The carrier grains are sufficiently resistant to abrasion, less than 7% of the particles disintegrate when working in a stirred reactor for 473 hours.

The higher effect of the invention compared to the present state of the art consists mainly in the order of magnitude higher activity of the mobilized amyloglucosidase, the saving of the native enzyme during immobilization, the longer life of the mobilized enzyme and the easy regeneration of the carrier.

The present invention is characterized by the following non-limiting examples.

EXAMPLE 1 238 544 parts of wet aspirated cellulose regenerated in suspension form into pearls were washed with a solution of 5) 3 parts of sodium hydroxide (NaOH) in 5.3 parts of deionized water and introduced into the reactor in portions and 12.2 parts of 1 -chloro-2,3-epoxypropyl and 9.7 parts of diethylamine were stirred at 63-80 ° C for 3 hours. Then washed with ethanol (50 parts) and deionized water (250 parts). 30 parts of pearl cellulose with 3-diethylamino-2-hydroxypropyl (DEAHP) groups were obtained at a concentration of 1.2 mmol / g and a water retention of 5.12 g HgO / g (according to US-A-199 420). 100 parts aspirated pearl cellulose with 3-diethylamino-2-hydroxypropyl (DEAHP) groups converted 150 parts 0.5 mol / l sodium hydroxide (NaOH) to OH form, washed with 1000 parts dest. The water contained in the column was added dropwise with 2000 parts of the enzyme solution prepared by suspending 60 g of stabilized amyloglucosidase in 100 parts of distilled water, after filtering off and adding 6.8 parts of 0.01 mol / l maltose in pH 4.2 acetate buffer over 4 hours.

100 parts of the mobilized enzyme were placed in a column with a jacket tempered to 55 ° C. A liquefied starch solution (dry matter 29) 7% by weight was pumped through the column. %, DE 20.2) at a rate of 0.45 g / min. The glucose content of the effluent was 26.66 g / 100 g corresponding to a conversion of 88.7 $ ·

Example 2 parts of wet pearl cellulose were suspended in a solution of 5.3 parts of NaOH in 5.3 parts of deionized water and then 22 parts of (3-chloro-2-hydroxypropyl) diethylamine hydrochloride were added. The temperature gradually increased from 10 ° C to 80 ° C, to which it was maintained under stirring for 3 hours. After washing with ethyl alcohol (50 parts) and deionized water (100 parts), the prepared bead cellulose containing 3-diethylamino-2-hydroxypropyl (DEAHP) contained 1.07 mmol diethylamine groups / g. 10 parts of 3-diethylamino-2-hydroxypropyl (DEAHP) bead cellulose washed with 50 parts of 0.5 mol / L sodium hydroxide solution (NaOH) and 100 parts of distilled water were poured over 2200 parts of 2.2 parts of stabilized amyloglucosidase in acetate pu238 544

- 5 fru 0.01 mol / l and pH 4.2. The activity of the immobilized enzyme under standard conditions was 30 J / g.

This amount of mobilized enzyme was suspended in a reactor tempered to 55 ° C in 1240 parts of a liquefied starch solution (29.7 $ dry matter, DE 20.2). After 34.4 hours of heating and stirring, 81.5% conversion to glucose was achieved, the glucose content in the solution being 24.45 g / 100 g of solution. The apparent activity of the mobilized amyloglucosidase was 470j / g under these conditions.

Example 3 parts of the immobilized amyloglucosidase according to example 1 with a particle size of 0.3 mm were suspended in a flow stirred reactor tempered to 55 ° C in 1240 parts of liquefied starch (29.74% dry matter, DE 20.2). The product was withdrawn from the reactor at a variable rate of 0.3 αί. 3.2 dbl / minute, but mostly 1 part / minute, with the same amount of fresh substrate being added automatically. In this arrangement, the reactor was immobilized enzyme for a period of time

473.5 hours. The final apparent activity of the immobilized enzyme under these conditions was 310 j / g. At this level, activity decreased from a baseline of 1240 j / g.

The immobilized enzyme darkened after this use, but retained a regular spherical shape (7% of the particles were halved). Washing with 50 parts of 0.5 mol / l hydrochloric acid (HCl) 100 parts of distilled water, 50 parts of 0.5 mol / l sodium hydroxide (NaOH) and 100 parts of distilled water was prepared for repeated immobilization of the enzyme which under the conditions of experiment 1 was at a rate of 316 j / g.

Example 4 dzl of pearl cellulose with 3-methylamino-2-hydroxypropyl (DEAHP) groups in Example 1 converted to the OH form was suspended in a solution of 0.1 part amyloglucosidase in 100 parts 0.02 mol / l acetate buffer pH 4.2. Enzyme sorbed with occasional stirring for 47 hours at 4 ° C. The activity of the thus immobilized enzyme after washing with 200 parts of distilled water was 1448 j / g.

Claims (3)

SUBJECT OF THE INVENTION Immobilized amyloglucosidase, characterized in that it is bound in an amount corresponding to an activity of up to 1300 units / g on a regenerated bead cellulose carrier containing diethyl amine / new groups at a concentration of 0.142 2.0 mmol / g cellulose. 2. A process for the preparation of immobilized amylogylcosidase according to claim 1, wherein the immobilization is carried out with a carrier based on regenerated pearl cellulose containing diethylamine groups in OH form suspended in an enzyme solution in water in the presence of 0.5 to 2% by weight. maltose or starch at a pH of 4.0 to 4.7 at 0 to 50 ° C or a column supported in the column by flowing the solution under the same conditions. 3. A process for the preparation of immobilized amyjoglucosidase according to claim 2, characterized in that after the enzyme has been adsorbed on a carrier based on regenerated bead cellulose containing diethylamine groups, crosslinking is carried out by reaction with glutaraldehyde at 25 DEG C. for 2 hours and at pH 6.8.