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Title:Enzymes involved in utilization of pullulan by Bacteroides thetaiotaomicron
Author(s):Smith, Karen Ann
Doctoral Committee Chair(s):Salyers, Abigail A.
Department / Program:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Microbiology
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.
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.)
Issue Date:1991
Rights Information:Copyright 1991 Smith, Karen Ann
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9136738
OCLC Identifier:(UMI)AAI9136738

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