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Motari FINAL 4 28 23 with cert.pdf (1.23 MB)

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Biochemical Characterization of β-galactosidase from Enterobacter sp. YSU

Motari, Fred Ondabu
http://orcid.org/0009-0004-3260-9977
http://rave.ohiolink.edu/etdc/view?acc_num=ysu1682955639568043

Abstract Details

2023, Master of Science in Chemistry, Youngstown State University, Department of Biological Sciences and Chemistry.
The enzyme β-galactosidase plays a role in the hydrolysis of lactose to galactose and glucose. Depending on the source, β-galactosidases can also carry out transglycosylation. This research was aimed at the biochemical characterization of β-galactosidase from Enterobacter sp. YSU. The enzyme is within the family of glycoside hydrolases. The Enterobacter sp. YSU β-galactosidase was overexpressed in E. coli. Subsequently, it was isolated using ammonium sulfate precipitation and a Q-Sepharose ion-exchange column. The single polypeptide chain protein contains 1056 amino acids with a molecular weight of 120 kDa. An in-gel activity test using 4-methylumbelliferyl-β-D-galactopyranoside established that the protein is active in its dimeric form. Dissociation of β-galactosidase into monomers in the presence of detergents like SDS results in the loss of enzymatic activity. The enzyme shows its optimal activity at pH 7.4 and a temperature of 40 °C. It has a limited substrate specificity of o-nitrophenyl-β-D-galactopyranoside (o-NPGal) and lactose. The catalytic parameters of the enzyme for o-NPGal were determined: KM is 0.3 mM, and kcat is 146 s-1. With respect to lactose, KM is 22 mM, and kcat is 3.86×103 min-1. Galactose competitively suppresses β-galactosidase activity, whereas glucose uncompetitively inhibits the enzyme. The enzymatic activity of β-galactosidase was affected by the presence of Mg2+ but not other divalent ions like calcium, zinc, or copper. Dimethyl sulfoxide caused a notable decrease in the activity of β-galactosidase while 2-mercaptoethanol had no effect on the activity of the enzyme. The β-galactosidase from Enterobacter sp. YSU showed a similar KM for lactose with most β-galactosidases isolated from other organisms but a higher kcat, and, therefore, there is a need to explore its applications in the hydrolysis of lactose.
Nina Stourman, PhD (Advisor)
Michael Serra, PhD (Committee Member)
Jonathan Caguiat, PhD (Committee Member)
80 p.
Biochemistry; Biology; Biomedical Research; Chemistry; Food Science; Health Sciences; Molecules
Biochemical characterisation of beta-galactosidase enzyme from Enterobacter species YSU; o-NPGal; Galactose as a competitive inhibitor; Glucose as an uncompetitive inhibitor; Galactooligosaccharides; Galactosylation; hydrolysis of lactose

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Citations

  • Motari, F. O. (2023). Biochemical Characterization of β-galactosidase from Enterobacter sp. YSU [Master's thesis, Youngstown State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1682955639568043

    APA Style (7th edition)

  • Motari, Fred. Biochemical Characterization of β-galactosidase from Enterobacter sp. YSU. 2023. Youngstown State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ysu1682955639568043.

    MLA Style (8th edition)

  • Motari, Fred. "Biochemical Characterization of β-galactosidase from Enterobacter sp. YSU." Master's thesis, Youngstown State University, 2023. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1682955639568043

    Chicago Manual of Style (17th edition)

ysu1682955639568043
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© 2023, all rights reserved.
This open access ETD is published by Youngstown State University and OhioLINK.