Enzymes involved in utilization of pullulan by Bacteroides thetaiotaomicron
Smith, Karen Ann
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Permalink
https://hdl.handle.net/2142/19366
Description
Title
Enzymes involved in utilization of pullulan by Bacteroides thetaiotaomicron
Author(s)
Smith, Karen Ann
Issue Date
1991
Doctoral Committee Chair(s)
Salyers, Abigail A.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Language
eng
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.)
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