Cloning, production, purification and preliminary crystallographic analysis of a glycosidase from the lactic acid bacterium Lactobacillus plantarum CECT 748T

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 b-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 His6 tag (Bgl) has been produced in a soluble form. Purified recombinant enzyme shows galactosidase activity against 4-nitrophenyl b-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 C. Diffraction data have been collected at ESRF to a resolution of 2.4 Å. The crystals belong to the space group C2 with unit-cell parameters a = 196.7, b = 191.7, c = 105.9, b = 102.7 . The structure refinement is in progressJMM thanks the Ministerio de Educación y Ciencia for a research grant (BFU2007-67404/BMC) and ‘‘Factoría de Cristalización” Consolider- Ingenio 2010 in support of his research and the ESRF (Grenoble, France) for provision of synchrotron radiation facilities. This work was also supported by Grants AGL2008-01052 and CSD2007-00063 FUN-C-FOOD (MICINN), RM2008-00002 (INIA), and S-0505/AGR/000153 ALIBIRD (CAM). J.A. Curiel is a recipient of a FPI predoctoral fellowship from the MICINN.Peer reviewe