{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/83723"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/83723","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Isolation and Characterization of Thermostable Alpha-Galactosidases for Application of High-Temperature Processing of Soy Molasses","abstract":"The $\\alpha$-Galactosidases from the novel thermophilic strain KM-THCJ and hyperthermophilic Thermotoga neapolitana were examined for their potential use in processing of soy oligosaccharide waste by-products. The $\\alpha$-galactosidase of strain KM-THCJ has been purified to homogeneity and has a MW of ca. 176 kDa and a subunit MW of ca. 88 kDa occurring as a dimer, as determined by gel filtration (SDS-PAGE ca. 80 kDa). The enzyme exhibits a pH optimum of 8.0 and demonstrates broad pH stability. The optimum temperature of enzyme activity was 77.5$\\sp\\circ$C, with a demonstrated temperature stability up to 70$\\sp\\circ$C. Known sulfhydryl groups inhibitors decrease enzyme activity. Attempts have been made to clone the Strain KM-THCJ $\\alpha$-galactosidase, providing numerous positive results, however, no expressing clone has been obtained. A cosmid library of T. neapolitana has been screened for $\\alpha$-galactosidase expression. One cosmid clone, RU72, was shown to express high levels of $\\alpha$-galactosidase activities. Examination of crude extracts of RU72 demonstrated an $\\alpha$-galactosidase temperature optimum ranging from 93 to 97$\\sp\\circ$C and temperature stability up to 84$\\sp\\circ$C. Restriction enzyme digested cosmid insert was sub-cloned and sequenced, elucidating an $\\alpha$-galactosidase gene. The $\\alpha$-galactosidase gene demonstrated nucleic and amino acid sequence similarities to the $\\alpha$-galactosidases of Thermoanaerobacter ethanolicus, Pediococcus pentosaceus, Streptococcus mutans, and Escherichia coli. The $\\alpha$-galactosidase appears to exist in an operon containing galactose transport and transfer genes. The putative $\\alpha$-galactosidase gene was PCR amplified and inserted into pCR2.1 which expressed a thermostable $\\alpha$-galactosidase. A 61 kDa protein was observed in partially purified extracts, similar to the deduced size of the putative gene. Both bacterial strains produce $\\alpha$-galactosidases which hydrolyze soy $\\alpha$-galactosides. The $\\alpha$-galactosidase from Thermotoga neapolitana, stable at 80$\\sp\\circ$C, meets the criteria required for use in soy processing. The moderately stable $\\alpha$-galactosidase from Strain KM-THCJ may have applications for use in the sucrose industry due to increased activity and stability at moderate alkaline pH.","abstract_html":"The <span class=\"etd-inline-math\">&alpha;</span>-Galactosidases from the novel thermophilic strain KM-THCJ and hyperthermophilic Thermotoga neapolitana were examined for their potential use in processing of soy oligosaccharide waste by-products. The <span class=\"etd-inline-math\">&alpha;</span>-galactosidase of strain KM-THCJ has been purified to homogeneity and has a MW of ca. 176 kDa and a subunit MW of ca. 88 kDa occurring as a dimer, as determined by gel filtration (SDS-PAGE ca. 80 kDa). The enzyme exhibits a pH optimum of 8.0 and demonstrates broad pH stability. The optimum temperature of enzyme activity was 77.5$\\sp\\circ$C, with a demonstrated temperature stability up to 70$\\sp\\circ$C. Known sulfhydryl groups inhibitors decrease enzyme activity. Attempts have been made to clone the Strain KM-THCJ <span class=\"etd-inline-math\">&alpha;</span>-galactosidase, providing numerous positive results, however, no expressing clone has been obtained. A cosmid library of T. neapolitana has been screened for <span class=\"etd-inline-math\">&alpha;</span>-galactosidase expression. One cosmid clone, RU72, was shown to express high levels of <span class=\"etd-inline-math\">&alpha;</span>-galactosidase activities. Examination of crude extracts of RU72 demonstrated an <span class=\"etd-inline-math\">&alpha;</span>-galactosidase temperature optimum ranging from 93 to 97$\\sp\\circ$C and temperature stability up to 84$\\sp\\circ$C. Restriction enzyme digested cosmid insert was sub-cloned and sequenced, elucidating an <span class=\"etd-inline-math\">&alpha;</span>-galactosidase gene. The <span class=\"etd-inline-math\">&alpha;</span>-galactosidase gene demonstrated nucleic and amino acid sequence similarities to the <span class=\"etd-inline-math\">&alpha;</span>-galactosidases of Thermoanaerobacter ethanolicus, Pediococcus pentosaceus, Streptococcus mutans, and Escherichia coli. The <span class=\"etd-inline-math\">&alpha;</span>-galactosidase appears to exist in an operon containing galactose transport and transfer genes. The putative <span class=\"etd-inline-math\">&alpha;</span>-galactosidase gene was PCR amplified and inserted into pCR2.1 which expressed a thermostable <span class=\"etd-inline-math\">&alpha;</span>-galactosidase. A 61 kDa protein was observed in partially purified extracts, similar to the deduced size of the putative gene. Both bacterial strains produce <span class=\"etd-inline-math\">&alpha;</span>-galactosidases which hydrolyze soy <span class=\"etd-inline-math\">&alpha;</span>-galactosides. The <span class=\"etd-inline-math\">&alpha;</span>-galactosidase from Thermotoga neapolitana, stable at 80$\\sp\\circ$C, meets the criteria required for use in soy processing. The moderately stable <span class=\"etd-inline-math\">&alpha;</span>-galactosidase from Strain KM-THCJ may have applications for use in the sucrose industry due to increased activity and stability at moderate alkaline pH.","abstract_has_math":true,"creators":["King, Michael Roy"],"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":["Bruce M. Chassy"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-09-25T21:10:12Z","date_published":"2015-09-25T21:10:12Z","updated_at":"2026-07-22T22:26:21Z","subjects":["Chemistry, Biochemistry"],"languages":["eng"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["(MiAaPQ)AAI9812655"],"render_values":[{"text":"(MiAaPQ)AAI9812655","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/83723","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Bruce M. 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The $\\alpha$-galactosidase of strain KM-THCJ has been purified to homogeneity and has a MW of ca. 176 kDa and a subunit MW of ca. 88 kDa occurring as a dimer, as determined by gel filtration (SDS-PAGE ca. 80 kDa). The enzyme exhibits a pH optimum of 8.0 and demonstrates broad pH stability. The optimum temperature of enzyme activity was 77.5$\\sp\\circ$C, with a demonstrated temperature stability up to 70$\\sp\\circ$C. Known sulfhydryl groups inhibitors decrease enzyme activity. Attempts have been made to clone the Strain KM-THCJ $\\alpha$-galactosidase, providing numerous positive results, however, no expressing clone has been obtained. A cosmid library of T. neapolitana has been screened for $\\alpha$-galactosidase expression. One cosmid clone, RU72, was shown to express high levels of $\\alpha$-galactosidase activities. Examination of crude extracts of RU72 demonstrated an $\\alpha$-galactosidase temperature optimum ranging from 93 to 97$\\sp\\circ$C and temperature stability up to 84$\\sp\\circ$C. Restriction enzyme digested cosmid insert was sub-cloned and sequenced, elucidating an $\\alpha$-galactosidase gene. The $\\alpha$-galactosidase gene demonstrated nucleic and amino acid sequence similarities to the $\\alpha$-galactosidases of Thermoanaerobacter ethanolicus, Pediococcus pentosaceus, Streptococcus mutans, and Escherichia coli. The $\\alpha$-galactosidase appears to exist in an operon containing galactose transport and transfer genes. The putative $\\alpha$-galactosidase gene was PCR amplified and inserted into pCR2.1 which expressed a thermostable $\\alpha$-galactosidase. A 61 kDa protein was observed in partially purified extracts, similar to the deduced size of the putative gene. Both bacterial strains produce $\\alpha$-galactosidases which hydrolyze soy $\\alpha$-galactosides. The $\\alpha$-galactosidase from Thermotoga neapolitana, stable at 80$\\sp\\circ$C, meets the criteria required for use in soy processing. The moderately stable $\\alpha$-galactosidase from Strain KM-THCJ may have applications for use in the sucrose industry due to increased activity and stability at moderate alkaline pH.","Made available in DSpace on 2015-09-25T21:10:12Z (GMT). No. of bitstreams: 2 license.txt: 4848 bytes, checksum: 96035ab3f5e1c23cc7138a224ce498bd (MD5) 9812655.pdf: 8451062 bytes, checksum: 593179c46f0260bc7f4f0618c3dc4319 (MD5) Previous issue date: 1997","Embargo set by: Seth Robbins for item 85004 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","U of I Only","157 p.","Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1997."]},{"key":"dc:title","label":"Title","values":["Isolation and Characterization of Thermostable Alpha-Galactosidases for Application of High-Temperature Processing of Soy Molasses"]}]}],"canonical_facts":{"dc:contributor":["Bruce M. Chassy"],"dc:creator":["King, Michael Roy"],"dc:date":["2015-09-25T21:10:12Z","10000-01-01","1997"],"dc:description":["The $\\alpha$-Galactosidases from the novel thermophilic strain KM-THCJ and hyperthermophilic Thermotoga neapolitana were examined for their potential use in processing of soy oligosaccharide waste by-products. The $\\alpha$-galactosidase of strain KM-THCJ has been purified to homogeneity and has a MW of ca. 176 kDa and a subunit MW of ca. 88 kDa occurring as a dimer, as determined by gel filtration (SDS-PAGE ca. 80 kDa). The enzyme exhibits a pH optimum of 8.0 and demonstrates broad pH stability. The optimum temperature of enzyme activity was 77.5$\\sp\\circ$C, with a demonstrated temperature stability up to 70$\\sp\\circ$C. Known sulfhydryl groups inhibitors decrease enzyme activity. Attempts have been made to clone the Strain KM-THCJ $\\alpha$-galactosidase, providing numerous positive results, however, no expressing clone has been obtained. A cosmid library of T. neapolitana has been screened for $\\alpha$-galactosidase expression. One cosmid clone, RU72, was shown to express high levels of $\\alpha$-galactosidase activities. Examination of crude extracts of RU72 demonstrated an $\\alpha$-galactosidase temperature optimum ranging from 93 to 97$\\sp\\circ$C and temperature stability up to 84$\\sp\\circ$C. Restriction enzyme digested cosmid insert was sub-cloned and sequenced, elucidating an $\\alpha$-galactosidase gene. The $\\alpha$-galactosidase gene demonstrated nucleic and amino acid sequence similarities to the $\\alpha$-galactosidases of Thermoanaerobacter ethanolicus, Pediococcus pentosaceus, Streptococcus mutans, and Escherichia coli. The $\\alpha$-galactosidase appears to exist in an operon containing galactose transport and transfer genes. The putative $\\alpha$-galactosidase gene was PCR amplified and inserted into pCR2.1 which expressed a thermostable $\\alpha$-galactosidase. A 61 kDa protein was observed in partially purified extracts, similar to the deduced size of the putative gene. Both bacterial strains produce $\\alpha$-galactosidases which hydrolyze soy $\\alpha$-galactosides. The $\\alpha$-galactosidase from Thermotoga neapolitana, stable at 80$\\sp\\circ$C, meets the criteria required for use in soy processing. The moderately stable $\\alpha$-galactosidase from Strain KM-THCJ may have applications for use in the sucrose industry due to increased activity and stability at moderate alkaline pH.","Made available in DSpace on 2015-09-25T21:10:12Z (GMT). No. of bitstreams: 2 license.txt: 4848 bytes, checksum: 96035ab3f5e1c23cc7138a224ce498bd (MD5) 9812655.pdf: 8451062 bytes, checksum: 593179c46f0260bc7f4f0618c3dc4319 (MD5) Previous issue date: 1997","Embargo set by: Seth Robbins for item 85004 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","U of I Only","157 p.","Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1997."],"dc:identifier":["http://hdl.handle.net/2142/83723","(MiAaPQ)AAI9812655"],"dc:language":["eng"],"dc:subject":["Chemistry, Biochemistry"],"dc:title":["Isolation and Characterization of Thermostable Alpha-Galactosidases for Application of High-Temperature Processing of Soy Molasses"],"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":"2026-07-22T22:26:21Z"}