Alpha-galactosidase from Lactobacillus salivarius: Isolation, purification, and biochemical characterization
Montelongo, Jose Luis
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Permalink
https://hdl.handle.net/2142/21731
Description
Title
Alpha-galactosidase from Lactobacillus salivarius: Isolation, purification, and biochemical characterization
Author(s)
Montelongo, Jose Luis
Issue Date
1995
Doctoral Committee Chair(s)
Blaschek, Hans-Peter M.
Department of Study
Food Science and Human Nutrition
Discipline
Food Science and Human Nutrition
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Agriculture, Food Science and Technology
Biology, Microbiology
Language
eng
Abstract
In this study the enzyme $\alpha$-galactosidase ($\alpha$-D-galactoside-galactohydrolase, EC 3.2.122) has been isolated and characterized from the soluble intracellular fraction of Lactobacillus salivarius. Growth on galactose, raffinose, melibiose and lactose produced the highest levels of intracellular enzyme activity. The levels observed after growth in glucose or sucrose were 10-fold lower than those observed after growth in galactose. A nearly homogeneous 142-fold purified preparation was prepared by (1) ammonium sulfate fractionation, (2) octyl-sepharose chromatography, and (3) Mono Q anion exchange chromatography. The enzyme appeared as a homogeneous 80 kDa protein in 12% SDS-PAGE gels. The $\rm M\sb{r}$ estimated from Superdex G-75 chromatography was also 80 kDa. A $\rm K\sb{m}$ of 0.96 mM and Vmax of 233 $\mu$moles/min/mg protein were calculated for pNP-$\alpha$-G. The enzyme was found to be stable between 20 and 50$\sp\circ$C, and highest activity was attained at 50$\sp\circ$C. $\alpha$-Galactosidase activity was lost rapidly below pH 4.5. Enzyme activity was inhibited by PHMB, HgCl$\sb2,$ and CuSO$\sb4.$ It appeared that a thiol group is required for catalytic activity. The substrate (pNP-$\alpha$-G) had a protective effect against inhibition by iodoacetamide and NEM. Based on $\rm K\sb{i}$ values against the substrate pNP-$\alpha$-G, the order of substrate affinity is: raffinose $>$ melibiose $>$ stachyose. Galactose caused competitive product inhibition with a $\rm K\sb{i}$ of 6 mM.
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