Isolation and Characterization of Thermostable Alpha-Galactosidases for Application of High-Temperature Processing of Soy Molasses
King, Michael Roy
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https://hdl.handle.net/2142/83723
Description
Title
Isolation and Characterization of Thermostable Alpha-Galactosidases for Application of High-Temperature Processing of Soy Molasses
Author(s)
King, Michael Roy
Issue Date
1997
Doctoral Committee Chair(s)
Bruce M. Chassy
Department of Study
Food Science and Human Nutrition
Discipline
Food Science and Human Nutrition
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
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.
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