Phospholipase C enzyme

Abstract

A phospholipase C enzyme having the ability to hydrolyze a phospholipid in both acidic and around neutral ranges and in a citrate buffer solution, as well as having a degree of heat stability, and having a property not to hydrolyze phosphate esters not containing lipid moieties. The phospholipase C enzyme shows activity from acidic to neutral pH and does not substantially hydrolyze any phosphate esters except for phospholipids.

1 . A phospholipase C enzyme having activity at from acidic to neutral pH which has hydrolyzing activity for the substrate phosphatidylcholine, wherein said enzyme fulfills at least one of the following criteria (a) to (c):
 (a) the hydrolyzing activity of phosphatidic acid is 30% or less compared to the hydrolyzing activity of phosphatidholine;   (b) the hydrolyzing activity of glycerophosphorylcholine is 15% or less compared to the hydrolyzing activity of phosphatidylcholine;   (c) the hydrolyzing activity of p-nitrophenylphosphate is 10% or less compared to the hydrolyzing activity of phosphatidylcholine.   
     
     
         2 . The phospholipase C enzyme according to  claim 1 , which has a high hydrolyzing activity for any one of the substrates phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol compared to the hydrolyzing activity for phosphatidylinositol. 
     
     
         3 . The phospholipase C enzyme according to  claim 2 , wherein an optimum pH is within a range of from pH 3 to pH 6. 
     
     
         4 . The phospholipase C enzyme according to  claim 2 , wherein the activity at pH 7 is 20% or more when compared to the maximum hydrolyzing activity at any pH. 
     
     
         5 . The phospholipase C enzyme according to  claim 3 , wherein the activity of pH 7 is 20% or more compared to the maximum hydrolyzing activity of any pH. 
     
     
         6 . The phospholipase C enzyme according to  claim 1 , which is produced by a filamentous fungus. 
     
     
         7 . The phospholipase C enzyme according to  claim 6 , wherein the filamentous fungus is  Aspergillus.    
     
     
         8 . The phospholipase C enzyme according to  claim 7 , wherein the filamentous fungus is selected from the group consisting of  Aspergillus oryzae  and  Aspergillus tamarii.    
     
     
         9 . The phospholipase C enzyme according to  claim 8 , wherein the filamentous fungus is a strain selected from the group consisting of  Aspergillus oryzae  strain FERM ABP 10200, strain NBRC 4190 and  Aspergillus tamarii  strain IAM 13907. 
     
     
         10 . The phospholipase C enzyme according to  claim 6 , which has the following properties:
 (a) a molecular weight of approximately 87,000 determined by SDS-PAGE electrophoretic analysis;   (b) hydrolyzes phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylglycerol;   (c) hydrolyzes egg yolk lecithin within a range of from pH 3 to pH 9; and   (d) the hydrolyzing activity in (c) being within a range of from 0° C. to 890° C.   
     
     
         11 . A phospholipase C enzyme which is one of the following proteins (a) to (e):
 (a) a protein consisting of the amino acid sequence of SEQ ID NO: 5;   (b) a protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4;   (c) a protein consisting of the amino acid sequence of (a) or (b), having one or several amino acids deleted, replaced, or added, and characterized in having phospholipase C activity;   (d) a protein comprising the amino acid sequence of SEQ ID NO: 5; and   (e) a protein comprising the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4.   
     
     
         12 . A phospholipase C enzyme which has a high hydrolyzing activity for any one of the substrates phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol compared to the hydrolyzing activity of phosphatidylinositol and which is produced by filamentous fungi, having activity at from acidic to neutral pH and substantially not hydrolyzing any phosphate esters except for phospholipids, wherein a optimum pH is within a range of from pH 3 to pH 6 and wherein the activity at pH 7 is 20% or more when compared to the maximum hydrolyzing activity at any pH. 
     
     
         13 . The phospholipase C enzyme according to  claim 12 , wherein the filamentous fungus is selected from the group consisting of  Aspergillus oryzae  and  Aspergillus tamarii.    
     
     
         14 . The phospholipase C enzyme according to  claim 12 , wherein the filamentous fungus is a strain selected from the group consisting of  Aspergillus oryzae  strain FERM ABP 10200, strain NBRC 4190 and  Aspergillus tamarii  strain IAM 13907. 
     
     
         15 . The phospholipase C enzyme according to  claims 12 , which has the following properties:
 (a) a molecular weight of approximately 87,000 determined by SDS-PAGE electrophoretic analysis;   (b) hydrolyzes phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylglycerol;   (c) hydrolyzes egg yolk lecithin within a range of from pH 3 to pH 9; and   (d) the hydrolyzing activity in (c) being within a range of from 0° C. to 80° C.   
     
     
         16 . The phospholipase C enzyme according to  claim 13 , which has the following properties:
 (a) a molecular weight of approximately 87,000 determined by SDS-PAGE electrophoretic analysis;   (b) hydrolyzes phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylglycerol;   (c) hydrolyzes egg yolk lecithin within a range of from pH 3 to pH 9; and   (d) the hydrolyzing activity in (c) being within a range of from 0° C. to 80° C.   
     
     
         17 . A phospholipase C enzyme which is produced by  Aspergillus oryzae  strain FERN ABP 10200, strain NBRC 4190, or  Aspergillus tamarii  strain IAM 13907, and which has the following properties:
 (a) a molecular weight of approximately 87,000 determined by SDS-PAGE electrophoretic analysis;   (b) hydrolyzes phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylglycerol;   (c) hydrolyzes egg yolk lecithin within a range of from pH 3 to pH 9; and   (d) the hydrolyzing activity in (c) being in a range of from 0° C. to 80° C.   
     
     
         18 . An enzyme preparation for foods, which comprises the phospholipase C enzyme according to  claim 1 . 
     
     
         19 . A method for treating a food material, which comprises adding the phospholipase C enzyme according to  claim 1  to a food material and hydrolyzing lecithin contained in the food material.