Doctor of Philosophy, The Ohio State University, 2024, Food Science and Technology

This dissertation investigates the enzymatic parameters of a β-galactosidase derived from the probiotic candidate, Lactobacillus helveticus OSU-PECh-4A (Lb. helveticus), with a focus on its potential for galacto-oligosaccharide (GOS) production. β-galactosidase is a hydrolytic enzyme that catalyzes the breakdown of lactose into its simpler sugar, glucose, and galactose. Simultaneously with lactose hydrolysis and under specific conditions, β-galactosidase can also produce highly demanded prebiotics known as galacto-oligosaccharides (GOS). In this dissertation, a thermostable β-galactosidase from Lb. helveticus has been isolated through diafiltration and size-exclusion chromatography and characterized through enzymatic assay and differential scanning fluorimetry (DSF). The isolated enzyme consists of a heterodimer with a molecular mass of 110 kDa, with a small and large subunit of 36 kDa and 74 kDa, respectively. The Km and Vmax values for lactose hydrolysis were 29.87 ± 1.05 mM and 1.88 ± 0.02 μmol D-glucose released per min per mg of protein, respectively. The enzyme is stable under a wide range of pH and high temperatures in terms of activity (≥ 40 °C). Once characterized, the enzyme capacity to produce GOS was evaluated through a 2x2 factorial design using substrate concentration and temperature as variables. The GOS produced were quantified and identified using high-performance liquid chromatography coupled with a charged aerosol detector (HPLC-CAD) and liquid chromatography with electrospray ionization mass spectroscopy (LC-ESI-MS/MS), respectively. To provide a perspective of the many possible applications of the enzyme, the enzyme's capacity to use different sugar acceptors to produce fucose-containing GOS was also evaluated by comparison. A maximum GOS yield of 12% was obtained at an initial lactose concentration of 200 g/L under 45 °C for 12 h. The GOS mixture consisted of 82% GOS, from which 6'galactosyl-lactose (trisaccharide), lactulose (disaccharides) (open full item for complete abstract)

Committee: Rafael Jiménez-Flores (Advisor); Valente Álvarez (Committee Member); Monica Giusti (Committee Member); Osvaldo Campanella (Committee Member) Subjects: Food Science

Master of Science in Chemistry, Youngstown State University, 2023, 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.

Committee: Nina Stourman PhD (Advisor); Michael Serra PhD (Committee Member); Jonathan Caguiat PhD (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Chemistry; Food Science; Health Sciences; Molecules