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

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\circC, and highest activity was attained at 50\sp\circC. $\alpha$-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.Made available in DSpace on 2011-05-07T13:17:26Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9522153.pdf: 6858379 bytes, checksum: 3a746992077bdd33c2b9a2f7611e9f5c (MD5) Previous issue date: 1995Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding ([email protected]) on 2011-05-07T14:52:49Z Item is restricted indefinitely.Restriction data tranferred 2014-07-01T11:24:22-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permissionETDs are only available to UIUC Users without author permissionU of I Onl