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Title:Isolation and Characterization of Thermostable Alpha-Galactosidases for Application of High-Temperature Processing of Soy Molasses
Author(s):King, Michael Roy
Doctoral Committee Chair(s):Bruce M. Chassy
Department / Program:Food Science and Human Nutrition
Discipline:Food Science and Human Nutrition
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Chemistry, Biochemistry
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.
Issue Date:1997
Type:Text
Language:English
Description:157 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1997.
URI:http://hdl.handle.net/2142/83723
Other Identifier(s):(MiAaPQ)AAI9812655
Date Available in IDEALS:2015-09-25
Date Deposited:1997


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