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Studies of [alpha]-galactosidases isolated from Clostridium perfringens and waste lager yeast (Saccharomyces carlsbergensis)

University of British Columbia

This study is divided into two parts. Part 1 describes the partial purification and characterization of a Clostridium perfringens α-galactosidase while part 2 describes the isolation and characterization of α-galactosidase from waste lager yeast (Saccharomyces carlsbergensis, also known as S. uvarum). Of 21 strains of C. perfringens tested, 10 utilized either raffinose or melibiose while 2 utilized both sugars. Spore production in Duncan Strong medium was superior in the presence of raffinose as opposed to starch in 12 of 21 strains. One strain of C. perfringens (M34) yielded 1.2 units of α-galactosidase/g washed cells. This enzyme had an isoelectric point of 5.6 and a pH optimum for hydrolysis of p-nitrophenyl α-D-galactopyranoside (PNPG) of 6.3. Molecular weight of the native protein was 96,000. Monomer molecular weight appeared to be 46,000. Km was 0.20 ±0.02mM PNPG. Heat stability of the enzyme at 45°C decreased as purity increased. This trend was only partially reversed by the addition of 2-mercaptoethanol, NADH, cysteine, and/or bovine serum albumin to the reaction mixture. Waste lager yeast was found to contain 27 Units of α-galactosidase /g yeast cells (dry weight). This activity was resolved into 3 active peaks by DEAE cellulose chromatography. Subsequent gel filtration indicated molecular weights of the α-galactosidase in peaks A, B and C to be 118,000, 95,000, and 65,000 respectively. The isoelectric point of all three forms of the enzyme was 4.4 ±0.1. The enzyme of peak C was shown to have a pH optimum of 4.5. Km values (± standard errors) were 2.54 ±0.32 mM p-nitrophenyl PNPG and 21.1 ±1.8 mM raffinose.

Text

2010-05-11

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http://hdl.handle.net/2429/24581

Food Science

University of British Columbia

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