{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/21731"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/21731","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"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$\\sp\\circ$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.","abstract_html":"In this study the enzyme <span class=\"etd-inline-math\">&alpha;</span>-galactosidase (<span class=\"etd-inline-math\">&alpha;</span>-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 <span class=\"etd-inline-math\">&mu;</span>moles/min/mg protein were calculated for pNP-<span class=\"etd-inline-math\">&alpha;</span>-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. <span class=\"etd-inline-math\">&alpha;</span>-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-<span class=\"etd-inline-math\">&alpha;</span>-G) had a protective effect against inhibition by iodoacetamide and NEM. Based on $\\rm K\\sb{i}$ values against the substrate pNP-<span class=\"etd-inline-math\">&alpha;</span>-G, the order of substrate affinity is: raffinose $&gt;$ melibiose $&gt;$ stachyose. Galactose caused competitive product inhibition with a $\\rm K\\sb{i}$ of 6 mM.","abstract_has_math":true,"creators":["Montelongo, Jose Luis"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Food Science and Human Nutrition","degree_department":null,"school":null,"contributors":["Blaschek, Hans-Peter M."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2011,"date_issued":"2011-05-07T13:17:26Z","date_published":"2011-05-07T13:17:26Z","updated_at":"2026-07-22T22:25:18Z","subjects":["Agriculture, Food Science and Technology","Biology, Microbiology"],"languages":["eng"],"rights":["Copyright 1995 Montelongo, Jose Luis"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI9522153","(UMI)AAI9522153"],"render_values":[{"text":"AAI9522153","href":null,"code":true},{"text":"(UMI)AAI9522153","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/21731","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Blaschek, Hans-Peter M."]},{"key":"dc:creator","label":"Author","values":["Montelongo, Jose Luis"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2011-05-07T13:17:26Z","10000-01-01","1995"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Food Science and Human Nutrition"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Agriculture, Food Science and Technology","Biology, Microbiology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 1995 Montelongo, Jose Luis"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI9522153","(UMI)AAI9522153","http://hdl.handle.net/2142/21731"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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.","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: 1995","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) 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 permission","ETDs are only available to UIUC Users without author permission","U of I Only"]},{"key":"dc:title","label":"Title","values":["Alpha-galactosidase from Lactobacillus salivarius: Isolation, purification, and biochemical characterization"]}]}],"canonical_facts":{"dc:contributor":["Blaschek, Hans-Peter M."],"dc:creator":["Montelongo, Jose Luis"],"dc:date":["2011-05-07T13:17:26Z","10000-01-01","1995"],"dc:description":["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.","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: 1995","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) 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 permission","ETDs are only available to UIUC Users without author permission","U of I Only"],"dc:identifier":["AAI9522153","(UMI)AAI9522153","http://hdl.handle.net/2142/21731"],"dc:language":["eng"],"dc:rights":["Copyright 1995 Montelongo, Jose Luis"],"dc:subject":["Agriculture, Food Science and Technology","Biology, Microbiology"],"dc:title":["Alpha-galactosidase from Lactobacillus salivarius: Isolation, purification, and biochemical characterization"],"dc:type":["text"],"thesis:degree_discipline":["Food Science and Human Nutrition"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:25:18Z"}