Purification and characterization of extra- and intracellular β-fructofuranosidase from Saccharomyces cerevisiae growing on Eichhornia crassipes leaf extract

Purification and characterization of extra- and intracellular beta-fructofuranosidase produced by Saccharomyces cerevisiae NRRL Y-12632 cultivated on water hyacinth leaf extract are reported. Purification of both extra- and intracellular enzymes was done with different purification techniques giving specific activities (4638.38 and 6900 U/mg protein) with 26 and 41.4 purification fold for extra- and intracellular enzymes respectively. The pure enzymes gave 2 bands for each by SDS-PAGE with molecular weights (42, 36 KDa) and (40, 34 KDa) for extra- and intracellular enzymes respectively, while the molecular weights determined by gel filtration were 69 and 79.4 KDa respectively. Both pure extra- and intracellular enzymes showed maximum activities at pH 5, while the optimum temperatures were 30 and 60 degrees C for extra- and intracellular enzymes respectively. Both enzymes were able to hydrolyze sucrose, raffinose and partially inulin while melizitose, maltose and trehalose were not hydrolyzed by both enzymes. The K-m values of extra- and intracellular enzymes were 0.15 and 0.075 M respectively. Both enzymes were completely inhibited by Hg2+ ion and only intracellular enzyme was completely inhibited by Mg2+ ion. Pure extra- and intracellular enzymes were found to contain 14 and 15 amino acids respectively. End product analysis of the pure enzymes by HPLC revealed that both enzymes possessed transfructosylating activities in addition to their hydrolytic activities.