Purification and Characterization of a Carboxypeptidase Y from Kluyveromyces fragilis JSB95

¹ Department of Food Science and Agricultural Chemistry, Macdonald Campus of McGill University, Ste. Anne de Bellevue, QC, Canada H9X 3V9

A carboxypeptidase Y was purified to homogeneity from the crude cell extracts of Kluyveromyces fragilis JSB95 grown in yeast, peptone, and dextrose broth using an FPLC® system equipped with ion-exchange and gel filtration columns. The enzyme was purified 216-fold over the crude extract; recovery was 18%. The estimated molecular mass was 56 kDa and consisted of two subunits. The maximum activity of the purified enzyme was obtained at pH 6.0 and 25°C and was strongly inhibited by diisopropylphosphofluoridate and phenylmethylsulfonylfluoride as well as by some metal ions. The Michaelis constant and maximum velocity values for n-benzoyl-L-tyrosine-p-nitroanilide substrate were 5.1 mM and 22.58 µM/min per mg of protein; for Cbz-Phe-Ala, values were 2.98 mM and 22.58 µM per min/mg of protein. Carboxypeptidase Y showed hydrolytic activity against some hydrophobic peptides of the tryptic digests of _s1- and ß-caseins

Key Words: carboxypeptidase Y • Kluyveromyces fragilis • cheese ripening • bitterness removal

Submitted on March 24, 1997
Accepted on October 3, 1997