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Purification and Characterization of a Novel β-Galactosidase From the Thermoacidophile Alicyclobacillus vulcanalis

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Abstract

Thermoacidophiles are microorganisms capable of optimum growth under a combination of high temperature and low pH. These microorganisms are a rich source of thermo- and acid- active/stable glycosyl hydrolases. Such enzymes could find use as novel biocatalysts in industrial processes, as operation at elevated temperature can increase substrate solubility, decrease viscosity, and reduce the risk of microbial contamination. We report the purification and characterization of an intracellular β-galactosidase from the thermoacidophile Alicyclobacillus vulcanalis DSM 16176. The enzyme was purified 110-fold, with a 5.89% yield. Denatured (83.7 kDa) and native (179 kDa) molecular masses were determined by SDS-PAGE and gel filtration, respectively, and suggest the enzyme functions as a homodimer. LC-MS/MS analysis confirmed identity, and bioinformatic analysis indicates the enzyme to be a member of the glycosyl hydrolase family 42 (GH42). Highest activity was measured at 70 °C and pH 6.0. The Km on the substrates ONPG and lactose were 5 and 258 mM, respectively. This enzyme is thermostable, retaining 76, 50, and 42% relative activity after 30, 60, and 120 min, respectively, at 70 °C. This property could lend its use to high-temperature industrial processes requiring a thermo-active β-galactosidase.

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This work has been funded by the Irish Research Council under the Embark Initiative.

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  1. Department of Chemical Sciences, School of Natural Sciences, Bernal Institute, University of Limerick, Limerick, Ireland

    Jayne Murphy, Michael P. Ryan & Gary Walsh

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Murphy, J., Ryan, M.P. & Walsh, G. Purification and Characterization of a Novel β-Galactosidase From the Thermoacidophile Alicyclobacillus vulcanalis. Appl Biochem Biotechnol 191, 1190–1206 (2020). https://doi.org/10.1007/s12010-020-03233-w

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