Formation of Naringin-hydrolyzing Enzymes of Aspergillus niger (II)

Abstract

1. Selection of naringinase-secreting strains and naringinase formation. From fruits and vegetables were separated several strains of Aspergillus niger which secreted much naringinase and hesperidinase on the solid medium of rice bran and soybean cake. The addition of a small amount of naringin or rhamnose to the culture medium of Aspergillus niger promoted the formation of both enzymes. The effectiveness of naringin or rhamnose for naringinase formation was occasionally examined by the suspension of the washed cell of GrM-03,a strain of Aspergillus niger. Soybean cake showed a similar effect. The fraction containing saponin was separated from defatted soybean flour and was hydrolyzed with sulfuric acid. The sugar constituents of this fraction were detected by paper chromatography. These gave galactose, rhamnose, arabinose, and a small amount of glucose. The soybean saponin is effective, but a commercial saponin, not containing rhamose, is not effective. These facts are due apparently to the adaptive effect of rhamnose. 2. Crystallization of naringinase and hesperidinase and their enzymic properties. Naringin and hesperidin were hydrolyzed to aglycons and sugars by the water extract of the solid culture medium of Aspergillus niger GrM-03. The enzymes were purified from this culture extract by salting-out with ammonium sulfate, adsorption on acrinol, decoloring by Duolite A-2,paper electrophoresis, and precipitating with acetone. Naringinase and hesperidinase were precipitated by the addition of acetone to a concentration of 30-40% and 40-60%, respectively. Crystalline naringinase was most active at pH 4.5 and at 50° and stable at pH 5.5-8.0,and hesperidinase was most active at pH 3.5 and at 60° and stable at pH 3.0-8.5. From the actions of both enzymes on some flavonoids, disaccharicles, and derivatives of hesperetin, naringinase and hesperidinase were assumed to be a kind of rhamnosidase which was specific for β-1.4 and β-1,6 linkages in flavonoids, respectively. Naringin or hesperidin was hydrolyzed only 50 % by crystalline naringinase and hesperidinase. After splitting various flavonoids by both enzymes, the bond between glucose and aglycons in glucosides was attacked by flavonoid glucosidases. The flavonoid glucosidases were purified from the water extract of GrM-03 culture medium containing rutin. And some enzymic properties of these glucosidases were investigated.