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Triton X-114-aided Extraction and Partial Characterization of β-Galactosidase from Grape Berry Pulp (Vitis vinifera L.)

¹ Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, Campus de Espinardo, E-30071 Murcia, Spain.

Acknowledgments: This work was partially supported by MEC and FEDER (project BIO2007-62510) and by Programa de Ayudas a Grupos de Excelencia de Región de Murcia (project 04541/GERM/06), Plan Regional de Ciencia y Tecnología 2007/2010, Fundación Séneca, Agencia de Ciencia y Tecnología de la Región de Murcia.

The authors are grateful to Encarna Gómez-Plaza (Universidad de Murcia) for her gift of Monastrell grape berries.

^* Corresponding author (email: mpg{at}um.es; tel: 34 968 36 48 37; fax: 34 968 36 41 47)

β-Galactosidase from grape berry pulp was extracted after a 2 hr digestion with the non-ionic detergent Triton X-114. Subsequent phase partitioning eliminated ~75% of the phenolic compounds, enabling the enzyme to be recovered in the aqueous upper phase with an 8-fold increase in its specific activity. The enzyme was kinetically characterized using o-nitrophenyl β-galactopyranoside as substrate and was active at acidic pH with an optimum at pH 4.2, a value at which V_max and K_m were 0.009 µmol/min/mg protein and 1.2 mM, respectively. β-Galactosidase was competitively inhibited by free galactose with K_i of 0.8 mM. The enzyme had an optimum temperature of 65°C, although its thermostability is very low at this temperature.

Key words: β-galactosidase, pulp, Triton X-114, phase partitioning

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				M. Perez-Gilabert, E. Tellez, and F. Garcia-Carmona Extraction and Partial Purification of {beta}-Galactosidase from Grape Berry Skin (Vitis vinifera L.) with Triton X-114 Am. J. Enol. Vitic., March 1, 2010; 61(1): 135 - 139. [Abstract] [Full Text] [PDF]