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Weidner FINAL 8 5 24 with cert.pdf (927.48 KB)

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Characterization of β-galactosidase from Enterobacter sp. YSU, GalB2, and Methods to Purify and Characterize Galacto-oligosaccharides

Weidner, Cody J
http://rave.ohiolink.edu/etdc/view?acc_num=ysu172297260577738

Abstract Details

2024, Master of Science in Chemistry, Youngstown State University, Department of Biological Sciences and Chemistry.
Beta-galactosidases are enzymes that hydrolyze β-glycosidic bonds with galactose at the nonreducing end. These enzymes have been used in the food and pharmaceutical industries for processing waste into environmentally friendly compounds and the synthesis of prebiotics. GalB2 is a novel β-galactosidase from Enterobacter sp. YSU that was expressed in Escherichia coli. The enzyme was isolated with ammonium sulfate precipitation and purified by an anionexchange chromatography. The optimal pH for GalB2 was found to be 7.4. There was no loss in enzymatic activity when GalB2 was incubated at temperatures 37 ºC and 40 ºC, but activity significantly diminished after incubation at temperatures greater than 45 ºC. The window of substrates is narrow with only o-nitrophenyl-β-D-galactopyranoside (o-NPGal) and lactose being evident. The catalytic parameters were determined for both substrates. For the substrate o-NPGal, the Michaelis constant, KM was determined to be 0.18 mM and a catalytic constant, kcat was 44 s-1. With respect to lactose, the KM was found to be 2.5 mM with a kcat of 51 min-1. Glucose inhibited the enzyme in an uncompetitive manner, but galactose demonstrated competitive inhibition of GalB2. The enzyme shows dependence from magnesium ions. Native gel electrophoresis with exposure to fluorogenic substrate 4-methylumbelliferyl-β-D-galactopyranoside indicated that enzyme is active as a dimer. No transglycosylation activity for GalB2 was observed. Transglycosylated products from immobilized commercially available lactase were confirmed using LC/ESI-MS. Attempts to isolate oligosaccharides using a silica-gel column with an acetonitrile:water eluting solvent were unsuccessful.
Nina Stourman, Ph.D. (Advisor)
Michael Serra, Ph.D. (Committee Member)
Jonathan Caguiat, Ph.D. (Committee Member)
63 p.
Biochemistry; Biology; Chemistry; Health Sciences
Enzymology; beta-galactosidase; galacto-oligosaccharides; transglycosylation

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Citations

  • Weidner, C. J. (2024). Characterization of β-galactosidase from Enterobacter sp. YSU, GalB2, and Methods to Purify and Characterize Galacto-oligosaccharides [Master's thesis, Youngstown State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ysu172297260577738

    APA Style (7th edition)

  • Weidner, Cody. Characterization of β-galactosidase from Enterobacter sp. YSU, GalB2, and Methods to Purify and Characterize Galacto-oligosaccharides. 2024. Youngstown State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ysu172297260577738.

    MLA Style (8th edition)

  • Weidner, Cody. "Characterization of β-galactosidase from Enterobacter sp. YSU, GalB2, and Methods to Purify and Characterize Galacto-oligosaccharides." Master's thesis, Youngstown State University, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=ysu172297260577738

    Chicago Manual of Style (17th edition)

ysu172297260577738
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This open access ETD is published by Youngstown State University and OhioLINK.