Plant-derived alkaline alpha-galactosidase

Abstract

There are provided novel plant-derived enzymes for hydrolysis of sugars and particularly an alkaline alpha-galactosidase which hydrolyzes a broad spectrum of galactosyl-saccharides such as melibiose, raffinose and stachyose and guar gum, at neutral to alkaline pH conditions.

1 . An isolated, plant derived enzyme comprising, 
 (a) an alpha-galactosidase (E.C. 3.2.1.22, alpha-D-galactoside galactohydrolase) activity;    (b) a Vmax/Km ratio for the substrate raffinose at least 0.1 that of the Vmax/Km ratio for the substrate stachyose; and    (c) optimal activity in the range of pH 7.0 to pH 8.0.    
   
   
       2 . The isolated plant-derived enzyme of  claim 1 , wherein said Vmax/Km ratio for the substrate raffinose is about 0.5 the Vmax/Km for the substrate stachyose.  
   
   
       3 . The isolated plant-derived enzyme of  claim 1 , wherein said Vmax/Km ratio for the substrate raffinose is substantially equal to the Vmax/Km for the substrate stachyose.  
   
   
       4 . The isolated plant-derived enzyme of  claim 1 , wherein said Vmax/Km ratio for the substrate raffinose is greater than the Vmax/Km for the substrate stachyose.  
   
   
       5 . The isolated plant-derived enzyme of  claim 1  wherein said K m  for the substrate raffinose is less than about 10 mM.  
   
   
       6 . The isolated plant-derived enzyme of  claim 1  wherein said K m  for the substrate raffinose is less than about 5 mM.  
   
   
       7 . The isolated plant-derived enzyme of  claim 1  wherein said K m  for the substrate raffinose is less than or equal to about 1.5 mM.  
   
   
       8 . The isolated plant-derived enzyme of  claim 1  isolated from an alpha-galactosyl saccharide metabolizing plant.  
   
   
       9 . The isolated plant-derived enzyme of  claim 8 , wherein said alpha-galactosyl saccharide metabolizing plant is a member of a plant family selected from the group consisting of Cucurbitaceae, Lamiaceae, Piperaceae, Solanaceae, Leguminosae, Cruciferae and Gramineae family.  
   
   
       10 . The isolated plant-derived enzyme of  claim 9 , wherein said member of the Cucurbitaceae family is a melon plant.  
   
   
       11 . The isolated plant-derived enzyme of  claim 8 , wherein said alpha-galactosyl saccharide metabolizing plant is a member of the Lamiaceae family.  
   
   
       12 . The isolated plant-derived enzyme of  claim 1 , wherein the enzyme is isolated from a plant tissue selected from the group consisting of a fruit, a leaf, a seed, a stalk, a root, a tuber, a flower and a runner of the plant.  
   
   
       13 . The isolated plant-derived enzyme of  claim 12  wherein the enzyme is isolated from a germinating seed.  
   
   
       14 . The isolated plant-derived enzyme of  claim 1 , wherein the enzyme is a protein monomer.  
   
   
       15 . The isolated plant-derived enzyme of  claim 1 , wherein the enzyme is an exo-alpha-galactosidase.  
   
   
       16 . The isolated plant-derived enzyme of  claim 1 , wherein the enzyme is a non-glycosylated plant-derived enzyme.  
   
   
       17 . The isolated plant-derived enzyme of  claim 1 , wherein the enzyme is further characterized having a molecular mass of about 70-100 kDa, as determined by gel electrophoresis.  
   
   
       18 . The isolated plant-derived enzyme of  claim 17 , wherein the enzyme is further characterized having a molecular mass of about 80-90 kDa, as determined by gel electrophoresis.  
   
   
       19 . The isolated plant-derived enzyme of  claim 18 , wherein the enzyme is further characterized having a molecular mass of about 84 kDa, as determined by gel electrophoresis.  
   
   
       20 . The isolated plant-derived enzyme of  claim 1 , wherein the enzyme is further characterized having a pI of 4.0-6.0, as determined by isoelectric focusing on high-speed gel electrophoresis.  
   
   
       21 . The isolated plant-derived enzyme of  claim 20 , wherein the enzyme is further characterized having a pI of 4.8-5.5, as determined by isoelectric focusing on high-speed gel electrophoresis.  
   
   
       22 . The isolated plant-derived enzyme of  claim 21 , wherein the enzyme is further characterized having a pI of 5.0, as determined by isoelectric focusing on high-speed gel electrophoresis.  
   
   
       23 . The isolated plant-derived enzyme of  claim 1 , wherein the enzyme is not inhibited by galactose, characterized by a Ki (galactose) of at least 13 mM.  
   
   
       24 . A method for removing alpha-galactose from a galactosyl-saccharide containing material comprising: 
 (a) providing a plant-derived alkaline alpha galactosidase of  claim 1;  and,    (b) contacting the galactosyl-saccharide containing material with said plant-derived alkaline alpha galactosidase of  claim 1  at a pH greater than 7.0 so as to remove alpha-galactose from said galactosyl-saccharide containing material.    
   
   
       25 . A method for seroconversion of blood group B erythrocytes to blood group O erythrocytes comprising: 
 (a) providing a plant-derived alkaline alpha galactosidase of  claim 1;  and,    (b) contacting the blood group B erythrocytes with said plant-derived alkaline alpha galactosidase of  claim 1  at a pH greater than 7.0, so as to remove the terminal a-linked galactose residue from the group B surface antigen;    thereby seroconverting the blood group B erythrocytes to blood group O erythrocytes.    
   
   
       26 . A method for facilitating the crystallization of sugar beet sucrose from sugar beet molasses comprising: 
 (a) providing a plant-derived alkaline alpha galactosidase of  claim 1;  and,    (b) contacting the sugar beet molasses with said the plant-derived alkaline alpha galactosidase of  claim 1  at a pH greater than 7.0, so as to hydrolyze the raffinose in the sugar beet molasses to galactose and sucrose;    thereby facilitating the crystallization of sugar beet sucrose from said sugar beet molasses.    
   
   
       27 . A method for reducing the capability of a foodstuff to cause flatulence during digestion of said foodstuff comprising: 
 (a) providing a plant-derived alkaline alpha galactosidase of  claim 1;  and,    (b) contacting the foodstuff prior to ingestion with said plant-derived alkaline alpha galactosidase of  claim 1  at a pH greater than 7.0, so as to hydrolyze an alpha-galactosyl saccharide contained in the foodstuff;    thereby reducing the capability of said foodstuff to cause flatulence during digestion.    
   
   
       28 . A method of modifying the Theological properties of alpha-galactosyl saccharide containing plant gums comprising: 
 (a) providing a plant-derived alkaline alpha galactosidase of  claim 1;  and,    (b) contacting the plant gum with the plant-derived alkaline alpha galactosidase of  claim 1  at a pH greater than 7.0, so as to hydrolyze an alpha-galactosyl saccharide contained in the plant gum;    thereby modifying the rheological properties of said alpha-galactosyl saccharide containing plant gum.