DD150628A1 - PROCESS FOR THE REACTIVATION OF ADSORPTIVED POLLUTED IMMOBILIZED ENZYMES - Google Patents

Abstract

The invention relates to a process for the reactivation of adsorptively contaminated immobilized enzymes. The aim of the invention is to restore the activity of immobilized enzymes after they have suffered a loss of activity due to prolonged use. The immobilized enzymes are used as heterogeneous biocatalysts in the production or conversion of organochemical compounds, for example in enzymatic starch degradation. For the reactivation of immobilized enzymes, the contaminated enzyme-carrier complex is treated with the addition of suitable solvents with ultrasound. Due to the cavitative effect of the ultrasound, a reactivation of the enzyme-carrier complex occurs.

Description

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the invention

Process for reactivating adsorptively contaminated immobilized enzymes

b) Field of application of the invention

The invention relates to a process for the reactivation of immobilized enzymes which are used as heterogeneous biocatalysts in the food and beverage industry, for the production of pharmaceuticals and fine chemicals, in analytics and in medicine.

c) Char akteristik the known technical solutions

It is well known that the use of immobilized enzymes as heterogeneous biocatalysts over time results in significant decreases in activity caused by the adsorption of suspended matter, lipid components and other impurities of the substrate solution on the support matrix. To eliminate these impurities

13.MAI1B80 * So9313

It is necessary to subject the enzyme carrier particles to extensive cleaning operations. On the one hand there is a risk of damage to the enzyme structures, on the other hand, both the impurities and the enzyme can be washed out. However, both the pore structure of the carriers and the sensitivity of the functional protein structure have, in most cases, caused the enzyme carrier particles to be discarded after a single use. It is known that the binding ability of organic carrier materials for enzymes can be positively influenced by treatment with ultrasound. Using the example of polystyrene carriers, ultrasound treatment has shown that the binding capacity for glucoamylase is increased by 50 % ( ¹ JTP JtH SS *).

d) Object of the invention

The aim of the invention is to substantially or completely regenerate the original activity of enzyme-carrier complexes, which have lost their catalytic activity as a result of prolonged practical use by adsorbed impurities, in order to increase the efficiency of enzyme reactors.

e) loan of the essence of the invention

The object of the invention is to achieve a substantial reactivation by physical treatment of an adsorptively damaged enzyme-carrier complex, without the enzyme

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to destroy or wash out of the carrier.

It has been found that ultrasonication of the contaminated enzyme-carrier complexes leads to the desired result. The carrier material used was, for example, a polystyrene-based weakly basic anion exchanger prepared for enzyme binding (Wofatit Y 58, grain diameter

0.5 - 0.7 mm, inner surface 40 m, mean pore radius 700 &), to which glucoamylase from Asp. Niger (ITovo)! Was covalently bound. This glucoamylase-polystyrene complex allows the enzymatic hydrolysis of starch solutions of technical purity. After the original activity of the loaded carrier material (240 U / g carrier) has been reduced to approximately 30 % of the initial activity, the enzyme-carrier complex is subjected to an ultrasound treatment. The sound is applied at frequencies between 10 and 800 kHz, and a sound exposure up to one hour. The action of the ultrasound causes the separation of the predominantly adsorptively bound impurities without appreciably damaging the bound enzyme. In the present Pail, the impurities blocking the matrix consisted predominantly of protein particles, lipids, and other, difficult-to-filter, suspended solids. To dissolve the lipids, it proved to be advantageous to add chloroform in appropriate concentrations to the sonication medium. On other loaded substrates on Glasbzw. Silicate base, similar results are achieved

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f) Example

The invention will be explained in more detail by the embodiment set forth below:

1. Preparation of the enzyme-carrier complex

The anionic-containing polystyrene carrier Wofatit Y 58 used was activated by addition of excess 1,4-benzoquinone and, after thorough washing, loaded with glucoamylase (Asp. Niger, Novo).

2. Activity measurement

For activity measurement, 50 mg of enzyme resin dissolved by stirring with 20 ml of 1% starch solution in 0.025 M acetate buffer, pH 5.5, incubated at 37 ⁰ C for 20 minutes. The increase in reducing groups was determined with 3,5-dinitrosalicylic acid. (P. Bernfeld, Methods of Enzymology 2 »149-158, 1955).

3. Regeneration of the contaminated enzyme carrier particles by means of ultrasound

The activity of a glucoamylase carrier complex of the starting activity of 240 U / g had fallen to about 75 U / g after 6 v / öchigem operation by adsorbed impurities. For regeneration, 400 ml of carrier volume were suspended in 1.5 1 v / ass. With the addition of 50 ml of chloroform, the sonication was carried out with constant stirring at 20 ^° C. and 46 kHz over a period of 45 minutes. The sound intensity used was at

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\ va 5-10 W / cm. After sonication, the support material was again tested for enzymatic activity.

The activity of the regenerated carrier was 135 U / g. Compared to the contaminated carrier, this means an increase in activity to 180 %.

Further results of embodiments are included in Table 1:

Sonication time carrier activity

without chloroform with chloroform

10 min. '20 min. 45 min. 60 min.

86 U / g 113 U / g 86 120 98 135 102

Claims (3)

2 103 Inventory of application 1. A process for the reactivation of adsorptively contaminated immobilized enzymes ge tnzeichne t by the fact that the contaminated enzyme-carrier complex · is treated with ultrasound. 2. The method according to item 1 is characterized in that the sonication of the contaminated enzyme-carrier complex takes place at 10 to 800 kHz for 1 to 60 minutes. 3. The method according to the items 1 and 2 geken nzeichnet characterized in that the sonication of the contaminated enzyme-carrier complex with the addition of a solvent or, the impurities binding substance takes place.