CS261324B1 - A method of separating amylolytic enzymes - Google Patents

Abstract

The enzyme solution is sorbed batchwise or in a column onto cyclodextrins bound to an insoluble carrier and, after washing, the sorbed amylolytic enzymes are eluted with a cyclodextrin solution with a concentration of 5 to 25 mM. The advantage of the above solution is the high purity of the enzyme obtained.

Description

The invention relates to a process for separating amylolytic enzymes from a culture liquid after cultivation of fungi or bacteria, from an extract of plant or animal materials or from other solutions containing amylolytic enzymes.

A method is used to separate amylolytic enzymes in which the enzyme solution is precipitated with ammonium sulfate, tannin, ethanol, acetone, or mixtures thereof, and then the precipitate is dried.

Another possibility is to concentrate the enzyme solution by evaporation at reduced temperature or by ultrafiltration. The dry powdered enzyme may also be prepared from its solution by spray drying.

The disadvantages of these processes are that large amounts of heavily polluted waste water are generated during precipitation or that the precipitant must be regenerated by evaporation or distillation, which is energy intensive. Concentration of the enzyme by ultrafiltration is limited by the capacity of the device, which is generally low, and the investment cost of the device. All these methods, separation then give only technically enzyme concentrate, the quality of which is strongly dependent on the type of raw material used and on by-products which are not removed by any of the mentioned methods.

These disadvantages are overcome by the method according to the invention, characterized in that the enzyme is sorbed, batchwise or continuously, to the insoluble cyclodextrin derivative prepared by cross-linking the cyclodextrin or by binding them to an insoluble carrier, preferably pearl cejujlos.

After washing the sorbent with the enzyme with water, the enzyme is then eluted from the sorbent with a solution of cyclodextrin at a concentration of 5 to 25 millimoles per liter. This double affinity separation yields a high purity enzyme with an almost quantitative yield, the quality of which is not affected by the quality of the raw material used.

Example 1

The 1x12 cm column is packed with acylodextrin covalently bound to bead cellulose and washed with 0.1 M acetate buffer pH 5.5. Then the column is passed through the rye extract containing rye alpha and beta amylases. After passing 30 ml of extract containing 40 mg of protein, the column is washed with 0.1 M acetate pH 5.5 and then the amylases are eluted with 10 mM beta cyclodextrin solution in the same buffer. Concentrated amylolytic enzymes are obtained with a yield for alpha-amylase of 92.6%, for beta-amylase of 79.2%.

Example 2

The culture liquid after the cultivation of the fungus Aspergillus oryzae containing the fungal alpha-amylase is passed through the column as in Example 1 under the same conditions. In the same manner as in Example 1, a fungal alpha-amylase concentrate is obtained in a yield of 94.8%.

Example 3

The commercial technical preparation of bacterial alpha-amylase is diluted to 95 EU / ml and processed according to the procedure of Example 1 except that a buffer of pH 6.0 is used for all operations. A pure bacterial alpha-amylase preparation is obtained with a yield of 93.0%.

Example 4

Rye extract as in Example 1 is passed through a 1x12 cm column packed with beta-cyclodextrin covalently bound to Ultrogel 34 commercial agarose gel. All other conditions are identical to Example 1. An amylolytic enzyme concentrate is obtained with an alpha-amylase yield of 93.8% and for beta-amylase 77.6%.

Example 5

The bacterial amylase solution as in Example 3 is passed through a 1x12 cm column containing beta-cyclodextrin covalently bound to Spheron 1000 hydroxyalkyl methacrylate gel. All other conditions are the same as in Example 3. A pure bacterial alpha-amylase concentrate was obtained with a yield of 91.8%.

Claims (1)

SUBJECT OF THE INVENTION Method for separating amylolytic enzymes from a solution obtained by cultivating fungi or bacteria, or from a solution obtained from the extraction of plant or animal tissues or other solutions containing these enzymes, characterized in that the enzyme solution is sorbed, in batch or column, to cyclodextrins bound to insoluble carriers such as pearl or fibrous cellulose, polysaccharides, porous glass, ceramics, synthetic polymers, etc., and after washing, the sorbed amylolytic enzymes are eluted with a 5-25 mM cyclodextrin solution.