CS201786B1 - Process for preparing imobilized beta-galactosidase - Google Patents

Description

The invention relates to a method of immobilizing a yeast β-galactosidase comprising anchoring the entire producer cell by covalent bonding to a solid support.

The industrial application of bound β-galactosidase finds considerable application in the milk treatment process as well as in the degradation of lactose-containing waste products. In this context, the use of yeast β-galactosidase is more advantageous because the optimum pH of the enzyme is closer to pM of milk and that the activity of the yeast β-galactosidase is more stable in the milk environment.

In the development of methods for the preparation of bound enzymes, the immobilization of whole cells of their producers represents a new direction, which is currently being addressed primarily for considerable operational advantages.

The known methods for the preparation of bound microbial cells are generally based on methods of immobilizing pure enzymes and can be divided into the following categories:

a) binding the cell by physical entrapment in a gel, membrane or microcapsule;

b) binding the cell by adsorption to an ion exchanger or biospecific adsorbent of biological nature;

c) binding the cell by covalent binding to a solid support.

The advantages and disadvantages of these known methods are relatively few to evaluate the application of bound cells. It will be appreciated, however, that immobilizing whole cells by covalent binding may yield an anchored enzymatic system whose activity will not be adversely affected by the diffusion barrier or other negative effects that occur when methods of cell binding are performed by physical capture or adsorption on an ion exchange resin. For these reasons, cell immobilization by covalent binding to a solid support is very promising.

According to the invention, the method of immobilizing a yeast β-galactosidase consists in anchoring the permeabilized yeast cell to a hydroxyalkyl methacrylate gel via triethylenetetramine which has been activated by glutaraldehyde or carbodiimide. Synthesis (S-galactosidase) was induced in these cells prior to immobilization.

The effect of this method of imbolization of β-galactosidase is to eliminate isolation and purification of the enzyme and to maintain it in the intracellular environment of the cell, which contributes to the stability of its activity, guarantees optimal conditions for the enzymatic reaction itself and isolates the enzyme from negative effects.

The method according to the invention is explained in more detail below by means of several exemplary embodiments,

Example 1

The epichlorohydrin-treated hydroxyalkyl methacrylate gel (8-13 µm) was modified by triethylenetetramine binding according to the following procedure: 1 g of the gel was suspended in 10 ml of 1 M triethylenetetramine dihydrochloride, which was adjusted to pH 9.0 with 1 M NaOH. After stirring at room temperature for 24 hours, the suspension was transferred to a column and the gel was washed with water until the conductivity was equalized. Then, the gel was washed with ten times the volume of ethanol, butanol and ethanol again and dried. The triethylenetetramine content was calculated based on the Kjeldahl nitrogen determination of 256 pmol / g dry gel. The modified gel (1 g) was washed with multiple volumes of water after 3 hours of swelling in water. The aspirated gel was suspended in twice the volume of water and glutaraldehyde (25%) was added to the resulting suspension to a final concentration of 5%. After stirring for 1 hour, the gel was transferred to a column and washed with water until a negative reaction was observed to monitor the presence of glutaraldehyde in each wash. After washing free glutaraldehyde, the gel was washed with 300 ml of 0.05 M phosphate buffer (pH 7.0). The gel (0.5) thus prepared was added to a 5 ml cell suspension containing 10 ⁹ permeabilized cells of Kluyveromyces lactis strain in 1 ml of the same buffer. In view of the transition of (? -Galactosidase activity to the carrier and its stability), it was noted that immobilization of the cells by solid covalent bond was completed at about 20 ° C under stirring suspension conditions after about 100 hours.

The utilization of the intracellular enzymatic activity of immobilized cells is conditioned by their permeabilization, which in this case was carried out as follows: the cell suspension was applied in 0.1 ml volumes to the center of the membrane filter, each subsequent deposition being performed after complete media aspiration. ml volumes were transferred to a glass plate and the captured cells were exposed to a warm air flow at a constant temperature of 60 ° C for 70 sec. The permeabilized cells were washed off the surface of the filters with 0.05 M phosphate buffer (pH 7.0).

Example 2

Preparation of the embedded permeabilized cells was performed as described in Example 1 except that hexamethylenetetramine was used for binding instead of triethylenetetramine.

Example 3

Preparation of the anchored permeabilized cells was carried out as described in Examples 1 and 2, except that dimethylsulfoxide permeabilized cells were used. The cell suspension in 0.05 M phosphate buffer (pH 7.0) was exposed to dimethylsulfoxide (40%) at 20 ° C. After 30 min, the cells were washed, resuspended in said buffer and immobilized. Example 4

Non-permeabilized cells were anchored as described in Examples 1 and 2. The cells so anchored were additionally permeabilized as described in Example 1 except that the cell suspension was used.

Example 5

Preparation of the anchored cells was performed as described in Example 1 except that the modified gel was activated during the immobilization by the presence of 100 mg of N-ethyl N '- (3-dimethylaminopropyl) carbodiimide per 100 mg of carrier.

Claims (1)

SUBJECT OF THE INVENTION A process for the preparation of immobilized β-galactosidase by covalent binding of an intact yeast cell to a solid support, characterized in that the permeabilized or non-permeabilized yeast cell is anchored at 20 ° C with triethylenetetramine to the surface of the hydroxylalkyl methacrylate gel particles.