Alpha-galactosidase from Lactobacillus salivarius: Isolation, purification, and biochemical characterization

In this study the enzyme α-galactosidase (α-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 μmoles/min/mg protein were calculated for pNP-α-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. α-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-α-G) had a protective effect against inhibition by iodoacetamide and NEM. Based on $\rm K\sb{i}$ values against the substrate pNP-α-G, the order of substrate affinity is: raffinose $>$ melibiose $>$ stachyose. Galactose caused competitive product inhibition with a $\rm K\sb{i}$ of 6 mM.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Food Science and Human Nutrition

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Montelongo, Jose Luis

Contributors dc:contributor

• Blaschek, Hans-Peter M.

• Agriculture, Food Science and Technology
• Biology, Microbiology

Statement dc:rights

• Copyright 1995 Montelongo, Jose Luis

Language dc:language

eng

Identifier

AAI9522153
(UMI)AAI9522153

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/21731