{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/19366"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/19366","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Enzymes involved in utilization of pullulan by Bacteroides thetaiotaomicron","abstract":"A pullulanase I gene from Bacteroides thetaiotaomicron has been cloned. To determine whether the cloned pullulanase gene was essential for pullulan utilization, I used directed insertional mutagenesis to inactivate the B. thetaiotaomicron pullulanase gene.","abstract_html":"A pullulanase I gene from Bacteroides thetaiotaomicron has been cloned. To determine whether the cloned pullulanase gene was essential for pullulan utilization, I used directed insertional mutagenesis to inactivate the B. thetaiotaomicron pullulanase gene.","abstract_has_math":false,"creators":["Smith, Karen Ann"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Microbiology","degree_department":null,"school":null,"contributors":["Salyers, Abigail A."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2011,"date_issued":"2011-05-07T12:05:17Z","date_published":"2011-05-07T12:05:17Z","updated_at":"2026-07-22T22:25:12Z","subjects":["Biology, Microbiology"],"languages":["eng"],"rights":["Copyright 1991 Smith, Karen Ann"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI9136738","(UMI)AAI9136738"],"render_values":[{"text":"AAI9136738","href":null,"code":true},{"text":"(UMI)AAI9136738","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/19366","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Salyers, Abigail A."]},{"key":"dc:creator","label":"Author","values":["Smith, Karen Ann"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2011-05-07T12:05:17Z","10000-01-01","1991"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Microbiology"]},{"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":["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 1991 Smith, Karen Ann"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI9136738","(UMI)AAI9136738","http://hdl.handle.net/2142/19366"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["A pullulanase I gene from Bacteroides thetaiotaomicron has been cloned. To determine whether the cloned pullulanase gene was essential for pullulan utilization, I used directed insertional mutagenesis to inactivate the B. thetaiotaomicron pullulanase gene.","The pullulanase I-minus insertional mutant 95-1 was still able to grow on pullulan at a rate similar to that of wild-type B. thetaiotaomicron. Thus there must be a second pullulanase in B. thetaiotaomicron. Characterization of the remaining pullulanase activity present in B. thetaiotaomicron 95-1 has identified an $\\alpha$-(1$\\to$4)-D-glucosidic bond cleaving pullulanase which has tentatively been designated a neopullulanase. The neopullulanase (pullulanase II) cleaves $\\alpha$(1$\\to$4)-D-glucosidic linkages in pullulan to produce panose. The neopullulanase also cleaved $\\alpha$(1$\\to$4)-bonds in amylose and in $\\alpha$(1$\\to$4)-linked oligomers of glucose (maltotriose through maltoheptaose). An $\\alpha$-glucosidase from B. thetaiotaomicron 95-1 was partially purified to a preparation containing three proteins; 80 kDa, 57 kDa, and 50 kDa. Pullulan and amylose were not hydrolyzed by the $\\alpha$-glucosidase. Shorter $\\alpha$(1$\\to$4)-D-glucosidic oligosaccharides (maltose to maltoheptaose) were hydrolyzed to glucose by the $\\alpha$-glucosidase. The $\\alpha$-glucosidase also hydrolyzed $\\alpha$(1$\\to$6)-linked oligosaccharides.","pNJR-6, a Bacteroides suicide vector, was utilized to make directed insertional mutants on either side of the pullulanase I gene in the B. thetaiotaomicron chromosome. Cell extracts from these insertional mutants, from wild type B. thetaiotaomicron, and the pullulanase I disruption mutant, B. thetaiotaomicron 95-1, were assayed for pullulanase specific activity to determine if the insertions had any polar effect on pullulanase expression. Neither of the insertions appeared to exhibit a polar effect. The cloned 4.1 kb HindIII fragment which expressed pullulanase activity at high levels in E. coli, was surveyed for the presence of a Bacteroides promoter. In order to do this, the 4.1 kb HindIII fragment and component DNA segments were cloned into a newly developed Bacteroides fusion vector, pMJF-3. This vector contained the reporter gene $\\beta$-glucuronidase (GUS) which had been shown to express in Bacteroides. pMJF-3, and the resulting GUS fusion plasmids; pKS30-1, pKS30-2, pKS32-14, pKS33-7, pKS34-7, and pKS35-8 were mobilized into B. thetaiotaomicron by conjugation and their GUS specific activity determined. There was a Bacteroides promoter present on the fragment which appeared to not be regulated and was expressed at a low level. (Abstract shortened with permission of author.)","Made available in DSpace on 2011-05-07T12:05:17Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9136738.pdf: 3437714 bytes, checksum: 9eb8d39d60a541b96c0d6d53c61f8f7e (MD5) Previous issue date: 1991","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:36:28Z Item is restricted indefinitely.","Restriction data tranferred 2014-07-01T11:14:42-05:00 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":["Enzymes involved in utilization of pullulan by Bacteroides thetaiotaomicron"]}]}],"canonical_facts":{"dc:contributor":["Salyers, Abigail A."],"dc:creator":["Smith, Karen Ann"],"dc:date":["2011-05-07T12:05:17Z","10000-01-01","1991"],"dc:description":["A pullulanase I gene from Bacteroides thetaiotaomicron has been cloned. To determine whether the cloned pullulanase gene was essential for pullulan utilization, I used directed insertional mutagenesis to inactivate the B. thetaiotaomicron pullulanase gene.","The pullulanase I-minus insertional mutant 95-1 was still able to grow on pullulan at a rate similar to that of wild-type B. thetaiotaomicron. Thus there must be a second pullulanase in B. thetaiotaomicron. Characterization of the remaining pullulanase activity present in B. thetaiotaomicron 95-1 has identified an $\\alpha$-(1$\\to$4)-D-glucosidic bond cleaving pullulanase which has tentatively been designated a neopullulanase. The neopullulanase (pullulanase II) cleaves $\\alpha$(1$\\to$4)-D-glucosidic linkages in pullulan to produce panose. The neopullulanase also cleaved $\\alpha$(1$\\to$4)-bonds in amylose and in $\\alpha$(1$\\to$4)-linked oligomers of glucose (maltotriose through maltoheptaose). An $\\alpha$-glucosidase from B. thetaiotaomicron 95-1 was partially purified to a preparation containing three proteins; 80 kDa, 57 kDa, and 50 kDa. Pullulan and amylose were not hydrolyzed by the $\\alpha$-glucosidase. Shorter $\\alpha$(1$\\to$4)-D-glucosidic oligosaccharides (maltose to maltoheptaose) were hydrolyzed to glucose by the $\\alpha$-glucosidase. The $\\alpha$-glucosidase also hydrolyzed $\\alpha$(1$\\to$6)-linked oligosaccharides.","pNJR-6, a Bacteroides suicide vector, was utilized to make directed insertional mutants on either side of the pullulanase I gene in the B. thetaiotaomicron chromosome. Cell extracts from these insertional mutants, from wild type B. thetaiotaomicron, and the pullulanase I disruption mutant, B. thetaiotaomicron 95-1, were assayed for pullulanase specific activity to determine if the insertions had any polar effect on pullulanase expression. Neither of the insertions appeared to exhibit a polar effect. The cloned 4.1 kb HindIII fragment which expressed pullulanase activity at high levels in E. coli, was surveyed for the presence of a Bacteroides promoter. In order to do this, the 4.1 kb HindIII fragment and component DNA segments were cloned into a newly developed Bacteroides fusion vector, pMJF-3. This vector contained the reporter gene $\\beta$-glucuronidase (GUS) which had been shown to express in Bacteroides. pMJF-3, and the resulting GUS fusion plasmids; pKS30-1, pKS30-2, pKS32-14, pKS33-7, pKS34-7, and pKS35-8 were mobilized into B. thetaiotaomicron by conjugation and their GUS specific activity determined. There was a Bacteroides promoter present on the fragment which appeared to not be regulated and was expressed at a low level. (Abstract shortened with permission of author.)","Made available in DSpace on 2011-05-07T12:05:17Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9136738.pdf: 3437714 bytes, checksum: 9eb8d39d60a541b96c0d6d53c61f8f7e (MD5) Previous issue date: 1991","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:36:28Z Item is restricted indefinitely.","Restriction data tranferred 2014-07-01T11:14:42-05:00 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":["AAI9136738","(UMI)AAI9136738","http://hdl.handle.net/2142/19366"],"dc:language":["eng"],"dc:rights":["Copyright 1991 Smith, Karen Ann"],"dc:subject":["Biology, Microbiology"],"dc:title":["Enzymes involved in utilization of pullulan by Bacteroides thetaiotaomicron"],"dc:type":["text"],"thesis:degree_discipline":["Microbiology"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:25:12Z"}