Cloning, production, purification and preliminary crystallographic analysis of a glycosidase from the food lactic acid bacterium Lactobacillus plantarum CECT 748(T).
In recent years, the exquisite stereoselectivity and high efficiency of carbohydrate-processing enzymes have been exploited for many biotechnological applications, including flavor enhancement in foods. In particular, much attention has been focused on the use of beta-glucosidases for the enzymatic hydrolysis of flavorless glycoconjugates present in juices and wine beverages for the release aroma volatiles. With the aim to analyze a novel glycosidase with potential applications food industry we have produced and structurally characterized the Bgl glycosidase from the food lactic acid bacterium Lactobacillus plantarum. For that purpose, we have cloned and heterologously expressed the bgl gene (lp_3629) in Escherichia coli. The recombinant protein containing an amino terminal His(6) tag (Bgl) has been produced in a soluble form. Purified recombinant enzyme shows galactosidase activity against 4-nitrophenyl beta-D-galactopyranoside but not glucosidase activity. Analytical size-exclusion gel filtration chromatography reveals that Bgl behaves in solution as a mixture of monomeric and a high-molecular weight assembly. Purified Bgl has been crystallized by the hanging-drop vapor-diffusion method at 18 degrees C. Diffraction data have been collected at ESRF to a resolution of 2.4A. The crystals belong to the space group C2 with unit-cell parameters a=196.7, b=191.7, c=105.9, beta=102.7 degrees. The structure refinement is in progress.
Acebrón
Iván
I
Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain.
Curiel
José A
JA
de Las Rivas
Blanca
B
Muñoz
Rosario
R
Mancheño
José M
JM
D000595:Amino Acid Sequence; D044602:Cellulases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D048191:Lactiplantibacillus plantarum / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D044602:Cellulases