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Applied and Environmental Microbiology, September 2009, p. 5972-5980, Vol. 75, No. 18
0099-2240/09/$08.00+0     doi:10.1128/AEM.00618-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Cloning, Expression, and Characterization of a Peculiar Choline-Binding β-Galactosidase from Streptococcus mitis{triangledown}

Susana Campuzano,{dagger} Beatriz Serra,{dagger},{ddagger} Daniel Llull, José L. García, and Pedro García*

Department of Molecular Microbiology, Centro de Investigaciones Biológicas, and Ciber de Enfermedades Respiratorias, Madrid, Spain

Received 16 March 2009/ Accepted 18 July 2009

A Streptococcus mitis genomic DNA fragment carrying the SMT1224 gene encoding a putative β-galactosidase was identified, cloned, and expressed in Escherichia coli. This gene encodes a protein 2,411 amino acids long with a predicted molecular mass of 268 kDa. The deduced protein contains an N-terminal signal peptide and a C-terminal choline-binding domain consisting of five consensus repeats, which facilitates the anchoring of the secreted enzyme to the cell wall. The choline-binding capacity of the protein facilitates its purification using DEAE-cellulose affinity chromatography, although its complete purification was achieved by constructing a His-tagged fusion protein. The recombinant protein was characterized as a monomeric β-galactosidase showing a specific activity of around 2,500 U/mg of protein, with optimum temperature and pH ranges of 30 to 40°C and 6.0 to 6.5, respectively. Enzyme activity is not inhibited by glucose, even at 200 mM, and remains highly stable in solution or immobilized at room temperature in the absence of protein stabilizers. In S. mitis, the enzyme was located attached to the cell surface, but a significant activity was also detected in the culture medium. This novel enzyme represents the first β-galactosidase having a modular structure with a choline-binding domain, a peculiar property that can also be useful for some biotechnological applications.

* Corresponding author. Mailing address: Department of Molecular Microbiology, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain. Phone: 34 918373112. Fax: 34 915360432. E-mail: pgarcia{at}cib.csic.es

{triangledown} Published ahead of print on 24 July 2009.

{dagger} S.C. and B.S. contributed equally to this work.

{ddagger} Present address: Grupo de Bio-Geoquímica Ambiental, Laboratorio de FisicoQuímica Ambiental, Instituto de Geografía, Universidad Nacional Autónoma de México, Coyoacán 04510, D.F., Mexico.

Applied and Environmental Microbiology, September 2009, p. 5972-5980, Vol. 75, No. 18
0099-2240/09/$08.00+0     doi:10.1128/AEM.00618-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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