Separation and properties of alpha and beta-galactosidase and other glycosidases from jack bean meal

Abstract

a and  b-Galactosidases were extracted from jack bean meal; they were partially purified by precipitation with ammonium sulfate at 60 % saturation and fractiona­ tion with Bio-Gel-P-200 or Sephadex-G- 200; the purification factor was about 40. Other fractionations with CM-, DEAE- and TEAE-cellulose, calcium phospate gel and ethanol were also investigated; the last procedure gave the best possibility of separation between a-galactosidase (which is found in the 0-30 % fraction) and ^-ga­ lactosidase (which remains in the super­ natant of the 70 % fraction).

Using p-nitrophenyl-a-D-galactopyranoside as substrate, the optimal pH for a-ga­lactosidase, in citrate buffer, was 6.2 at 25°; for b-Galactosidase, in the same con­ditions, it was 4.6 using the /3-nitrophenyI derivative. a-Galactosidase split natural substrates such as melibiose and raffinose; b-galactosidase hydrolyses lactose.

a-D-Fucosidase and b-Fucosidase activi­ ties were detected in enzymic preparations, parallel with a and b-D-galactosidase, using p-nitrophenyl-a and b-fucoside as substrates. We could not separate either fucosidases from the respective galactosi­ dase activities.

With preincubation at 62° for 15 min, a-D-galactosidase is completely destroyed, but b-D-gaIactosidase retains 80 % of its original activity. Both galactosidases were completely inhibited by D-galactose and y-galactono-Iactone, and b-D-galactosidase was inhibited by D-xylose as well.

b-D-Galactosidase and b-D-N-acetylglucosaminidase activities were also detected in calf serum and in mouse fibroblasts.