title: Genetic and biochemical characterization of an oligo-α-1,6-glucosidase from Lactobacillus plantarum.
abstract: Although encoded in the genome of many Lactobacillus spp. strains, α-glucosidases have received little attention compared to other glycosyl hydrolases. In this study, a putative oligosaccharide(oligo)-α-1,6-glucosidase-encoding gene (malL) was identified in the genome of Lactobacillus plantarum LL441. malL coded for 572 amino acid residues with a calculated total molecular mass of 66.31kDa. No predicted signal peptide was observed, suggesting this enzyme to be localized within the cytoplasm of the cell. Homology studies of the deduced amino acid sequence in the area of its active sites classified the enzyme as a member of the α-amylase (AmyAC) superfamily of glycosyl hydrolases (GH), family 13 (GH13), subfamily 31 (GH13_31). malL was cloned in Escherichia coli and the coded enzyme overexpressed as a histidine-tagged protein (MalLHis). It was then purified and characterized. MalLHisprotein showed strong hydrolytic activity towards 4-nitrophenyl-α-d-glucopyranoside (pNP-α-Glu) but not to other pNP-α-d- or pNP-β-d-derivatives. When using pNP-α-Glu as a substrate, MalLHisshowed similar specific activities between pH5.0 and 6.0, and between 20 and 42°C (optimum 30°C). Among the natural carbohydrates assayed, MalLHisshowed specificity towards isomaltose (Vmaxand Kmvalues of 40.64μmolmin-1mg-1and 6.22mM) and much less to isomaltulose (Vmaxand Kmvalues of 168.86μmolmin-1mg-1and 244.52mM). However, under the conditions of the assay, the enzyme showed no transglycosylation activity. Characterization of the entire complement of glycosidases in L. plantarum might reveal how strains of this species could be used in new biotechnological applications or in the development of functional foods.
authors.affiliation: Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA), Consejo Superior de Investigaciones Científicas (CSIC), Paseo Río Linares s/n, 33300 Villaviciosa, Spain.
authors.forename: Susana
authors.initials: S
authors.lastname: Delgado
authors.affiliation: Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA), Consejo Superior de Investigaciones Científicas (CSIC), Paseo Río Linares s/n, 33300 Villaviciosa, Spain.
authors.forename: Ana Belén
authors.initials: AB
authors.lastname: Flórez
authors.affiliation: Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA), Consejo Superior de Investigaciones Científicas (CSIC), Paseo Río Linares s/n, 33300 Villaviciosa, Spain.
authors.forename: Lucía
authors.initials: L
authors.lastname: Guadamuro
authors.affiliation: Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA), Consejo Superior de Investigaciones Científicas (CSIC), Paseo Río Linares s/n, 33300 Villaviciosa, Spain. Electronic address: baltasar.mayo@ipla.csic.es.
authors.forename: Baltasar
authors.initials: B
authors.lastname: Mayo
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005960:Glucosides / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007778:Lactobacillus / Q000235:genetics; D048191:Lactiplantibacillus plantarum / Q000201:enzymology* / Q000235:genetics*; D008320:Maltose / Q000737:chemistry; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D009844:Oligosaccharides / Q000378:metabolism; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013696:Temperature; D000516:alpha-Amylases / Q000378:metabolism; D000520:alpha-Glucosidases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D005960:Glucosides; D009844:Oligosaccharides; C019502:4-nitrophenyl alpha-glucoside; D006639:Histidine; D008320:Maltose; D006026:Glycoside Hydrolases; D000516:alpha-Amylases; D000520:alpha-Glucosidases
keywords: Cloning; Glycosyl hydrolase; Heterologous expression; Lactic acid bacteria; Lactobacillus plantarum; α-Glucosidase