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oai:www.ideals.illinois.edu:2142/19366 2023-07-10 col_2142_5131 col_2142_16338 com_2142_5130 com_2142_16337 com_2142_14793 com_2142_8903

Salyers, Abigail A. Smith, Karen Ann 2011-05-07T12:05:17Z 2011-05-07T12:05:17Z 10000-01-01 1991 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 AAI9136738 (UMI)AAI9136738 http://hdl.handle.net/2142/19366 eng Copyright 1991 Smith, Karen Ann Biology, Microbiology Enzymes involved in utilization of pullulan by Bacteroides thetaiotaomicron text Microbiology Microbiology University of Illinois at Urbana-Champaign Dissertation Ph.D.