The method of obtaining inhibitor bromeline


(57) Abstract:

Usage: the invention relates to biotechnology and concerns a method for obtaining inhibitor bromeline. The essence of the method lies in the fact that proliferating the tops of the fruit of the pineapple is extracted with water in the ratio of raw materials - solvent 1:(2-4) and then hold clearing sepharose with immobilized cysteine proteinase type in acetate buffer pH of 4.4 to 4.6, - laundering of the sorbent was carried out in 0.1 M phosphate buffer pH of 7.6 to 7.8, containing 0.5 m sodium chloride, and the target product elute in 0.01 N sodium hydroxide solution containing 0.1 m sodium chloride. The technical result is to simplify the method.

The invention relates to biotechnology, namely to methods for enzyme inhibitors, in particular inhibitors of cysteine proteases from plant material inhibitor bromeline.

Inhibitors of cysteine proteins of plants find wide application in medicine and scientific research. These inhibitors found in the sources, both animal and vegetable origin. They were isolated from the epidermis, internal organs, from the blood plasma of animals and humans. Inhibitors found in the tubers of cards is hibitor bromeline (cysteine proteinase pineapple juice from the stems of pineapple), which consists in grinding the stems of pineapple, wringing from them juice with subsequent two-stage precipitation with acetone. Then the acetone powder of the stem of the pineapple is subjected to gel chromatography on Sephadex G-75 and ion-exchange chromatography on DEAE Sephadex at pH of 7.55 [1]

The known method is complex, involving two-stage precipitation with acetone and subsequent clearance of the drug inhibitor in two stages. In addition, the use of acetone increases the risk of production, especially in hot climates.

In connection with the above, the technical result will be to simplify the method.

The proposed method for inhibitor bromeline is the following. As a source of raw materials use waste from industrial processing of pineapples (proliferous the tops of the fruit of the pineapple), extraction of proteins is carried out by extraction with water. When this ratio of the raw material and the extractant is 1:(2-4) g/ml of Raw materials with the extractant is placed in a homogenizer, crushed and then extracted for one hour at room temperature, stirring with a mechanical stirrer. The obtained mass press, centrifuged and the clarified protein solution,, balanced 0.1 M acetate buffer, pH 4,60. Nonspecific adsorbed proteins remove 0.1 m phosphate buffer, pH of 7.6 to 7.8, containing 0.5 M NaCl. The evolution of the associated column of inhibitor implement 0.01 n NaOH solution, containing 0.1 M NaCl, pH of 11.5 to 12.0. It should be noted the importance of compliance with all these conditions allocation of the inhibitor. Numerous attempts to allocate inhibitor under conditions different from those described above did not lead to the obtaining of the inhibitor. Thus, the binding of the inhibitor bromeline carried out exclusively at pH of 4.4 to 4.6, and the elution of the inhibitor from the medium can be performed only in a narrow range of high alkaline pH of 11.5 to 12.0.

P R I m e R 1. To 250 g of crushed mass of proliferating tops pineapple add 500 ml of water and conducting further ground in a homogenizer. The extraction continued with stirring with a mechanical stirrer for one hour at room temperature with the addition of 500 ml of water. The resulting mass is squeezed through several layers of gauze, center at 3000 xg for one hour. Get 920 ml of a solution containing 1500 mg of protein with specific activity 0,0086 I. IU/mg. Per unit of inhibitory activity (I. units) made this quantity of inhibitor that th and applied on the column with 50 ml sepharose with immobilized bromelain, balanced 0.1 M acetate buffer, pH 4,60. Nonspecific adsorbed proteins remove 0.1 M phosphate buffer, pH of 7.60, containing 0.5 M NaCl. The evolution of the bound inhibitor with immobilized bromeline exercise 0.01 n NaOH solution, containing 0.1 M NaCl. The output of the inhibitor reaches 94% increase in inhibitory activity compared to the original 389 times. Molecular weight inhibitor of about 6 kDa purified inhibitor effect on papain, fitsin, bromelain, trypsin.

P R I m m e R 2. The method is carried out according to example 1, but the weight ratio of raw material:solvent is 1:4, and the sorbent using immobilized on sepharose 6V papain. As a result, the output of the inhibitor is 90% increase in inhibitory activity compared to the original 388 times.

Summary information about the efficiency of the method is reflected in the table.

The METHOD of OBTAINING INHIBITOR BROMELINE by extraction of the crushed waste from industrial processing of pineapples with subsequent chromatographic isolation of target product, wherein the proliferating use the tops of the fruit of the pineapple, the extraction was carried out with water at a mass ratio of raw material: solvent 1:2-4, and highlighted steinbaugh type in acetate buffer pH 4,4-4,6, laundering sorbent is carried out in 0.1 M phosphate buffer pH of 7.6 to 7.8, containing 0.5 M sodium chloride, and the elution of the target product of 0.01 N. the solution of sodium hydroxide containing 0.1 M sodium chloride at a pH of 11.5 to 12.0.


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