PURIFICATION AND EVIDENCE FOR THE INVOLVEMENT OF TRYPTOPHAN RESIDUE AT THE CATALYTIC SITE OF ΒFRUCTOFURANOSIDASE FROM A THERMOTOLERANT YEAST KLUYVEROMYCES MARXIANUS NCYC 2675

Pharmaceutical Science-Biotechnology

Abstract

β Fructofuranosidase from Kluyveromyces. marxianus NCYC 2675 was purified using hydrophobic interaction chromatography. The purified enzyme had specific activity of 2116 µmoles/min/ml. Molecular weight was determined by gel filtration and SDS-PAGE. This was further confirmed by MALDI-TOF. The protein was found to be a non-glycosylated monomer of molecular mass 67 kDa and exhibited a pI of 3.7. Effects of amino acid specific modifying agents were studied to determine the amino acid residue at the catalytic site. NBS and HNBBr, chemical modifiers of tryptophan showed concentration and time dependant linear inactivation of the enzyme. Modification with 60 μM of NBS and 40 mM of HNBBr resulted in 75% and 70% inactivation respectively. Substrate sucrose, and the products glucose and fructose, significantly lowered the extent of inactivation by the tryptophan modifying reagents. The reactions followed pseudo first-order kinetics and the inactivation kinetics indicated the presence of a single tryptophan residue near or at the catalytic site.