Cloning, expression, and characterization of a peculiar choline-binding beta-galactosidase from Streptococcus mitis.
A Streptococcus mitis genomic DNA fragment carrying the SMT1224 gene encoding a putative beta-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 beta-galactosidase showing a specific activity of around 2,500 U/mg of protein, with optimum temperature and pH ranges of 30 to 40 degrees 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 beta-galactosidase having a modular structure with a choline-binding domain, a peculiar property that can also be useful for some biotechnological applications.
Campuzano
Susana
S
Department of Molecular Microbiology, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
Serra
Beatriz
B
Llull
Daniel
D
García
José L
JL
García
Pedro
P
D002794:Choline / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D034361:Streptococcus mitis / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D002794:Choline