Chemical modification of cassava linamarase

Abstract

Chemical modifications of linamarase from cassava petiole were carried out under mild conditions. Loss of β-glucosidase (linamarase) and β-fucosidase activities was observed when modifications were made on carboxyl, tryptophan, histidine, tyrosine and arginine residues. However, modification of Iysine only affected the B-glucosidase (linamarase) but not β-fucosidase activity. The loss of both activities due to the modifications of carboxyl, tryptophan and histidine residues was reduced in the presence of a competitive inhibitor, δ-gluconolactone. However, δ-gluconolactone has no effect on the loss of linamarase activity due to the modification of Iysine, arginine or tyrosine. In addition, δ-gluconolactone decreased the loss of β-fucosidase activity due to tyrosine modification. However, it showed no reduction on the loss of β-fucosidase activity after dialysis. These data suggested that carboxyl (aspartic and glutamic acids), tryptophan and histidine residues were located at the active site of cassava linamarase. By non-denaturing polyacrylamide gel electrophoresis, effects of the chemical modifications on the enzyme structure were studied. The modification of carboxyl groups gave a smear of slow moving bands suggesting that the surface negative charges were reduced by the modification. Modification of tryptophan did not change the ladder-like pattern of bands in the gel. However, the modifications of histidine, tyrosine, Iysine and arginine induced more net negative charges or structural changes to expose more negative charges on the enzyme and hence the enzyme bands that moved faster. The effects of chemical modifications on the molecular size of linamarase were investigated using a Superose 6 column and FPLC. After modification of carboxyl, tryptophan, histidine, tyrosine, Iysine or arginine residues, the molecular size of the enzyme remained in the same range as that of unmodified enzyme, MW = 440,000-2,000,000. However, after Iysine modification, the large molecular aggregates were not found, only the aggregate of 440,000 was present.