Methods for the isolation of proteinase inhibitor from potato tubers

Abstract

A rapid and simple method of isolating heat stable proteinase inhibitor proteins from plant tissues such as potato tubers is disclosed. The method comprises three steps. Proteins from potato tubers are extracted in an aqueous/alcohol extraction medium to form an alcohol extract. The alcohol extract is heated to a first temperature then cooled to a second temperature to form an insoluble precipitate phase containing debris and a soluble phase that contains the heat stable proteinase inhibitor proteins. The heat stable proteinase inhibitor proteins are precipitated from the soluble phase by dialysis against a suitable dialysis medium. The precipitated proteins may include a single inhibitor protein, or a mixture thereof.

We claim:  
     
         1 . A method for obtaining a heat stable proteinase inhibitor protein from plant tissues containing the same, comprising the steps of: 
 extracting the proteinase inhibitor protein from the plant tissues in an aqueous/alcohol solvent to form an alcohol extract containing a solubilized proteinase inhibitor protein;    heating the alcohol extract to a first temperature followed by cooling to a second temperature to form an insoluble precipitate phase and a soluble phase; and    precipitating the proteinase inhibitor protein from the soluble phase by dialysis against an acidic dialysis solution.    
     
     
         2 . The method of  claim 1  wherein the plant tissues are potato tubers.  
     
     
         3 . The method of  claim 1  wherein the proteinase inhibitor protein is potato proteinase inhibitor II.  
     
     
         4 . The method of  claim 1  wherein the proteinase inhibitor protein is a mixture of potato proteinase inhibitor II, a Kunitz-family chymotrypsin/trypsin inhibitor having a dominant chymotrypsin inhibitory activity, and a Kunitz-family trypsin/chymotrypsin inhibitor having a dominant trypsin inhibitory activity.  
     
     
         5 . The method of  claim 1  wherein the aqueous/alcohol solvent is comprised of a final concentration of about 0.2% formic acid, about 0.3 M NaCl and about 20% ethanol; the first temperature is about 70° C. or above; the second temperature is about 50° C. or below; and the aqueous dialysis solution contains about 0.22% formic acid.  
     
     
         6 . The method of  claim 1  wherein the aqueous/alcohol solvent comprises of about 0.2% formic acid, about 0.3 M NaCl and about 20% ethanol; the first temperature is about 50° C.; the second temperature is about 27° C. or below; and the aqueous dialysis solution is initially comprised of water; and wherein after a period of dialysis against water, at least one of the soluble phase or the aqueous dialysis solution is adjusted to comprise about 0.88% formic acid.  
     
     
         7 . A method for obtaining heat stable proteinase inhibitor protein from potato tubers containing the same, comprising the steps of: 
 extracting the potato tubers by homogenizing the potato tubers in a medium comprising about 0.2% formic acid and approximately 0.3 M NaCl to form crude extract;    adding about one volume of ethanol to four volumes of crude extract to form an alcohol extract;    filtering the alcohol extract to form a filtered extract;    heating the filtered extract to a first temperature of 50° C. or higher followed by cooling to a second temperature of 50° C. or lower, to form a heat-treated extract containing an insoluble precipitate phase and a soluble phase;    separating the insoluble precipitate phase from the soluble phase by at least one of, filtering the heat-treated extract through a porous filter, or centrifuging the heat-treated extract at about 4000× g for about 5 minutes, to form a clarified soluble phase;    removing impurities from the clarified soluble phase by dialysis using a dialysis membrane having a molecular weight cutoff of approximately 12,000 daltons, against a dialysis medium comprising either tap water, or about 0.2% formic acid, to form a dialyzed extract;    precipitating the proteinase inhibitor proteins from the dialyzed extract by at least one of; including about 0.22% formic acid in the dialysis medium, or adding formic acid to about 0.88%, to form a precipitation mixture;    recovering the precipitated proteinase inhibitor proteins by at lease one of, filtering the precipitation mixture through a porous filter, or centrifuging the precipitation mixture at about 4000× g for about 5 minutes, to obtain a mixture of proteinase inhibitor protein.    
     
     
         8 . A heat stable proteinase inhibitor protein obtained according to the method of any one of claims  1 - 7 .