Improving Properties of a Novel β-Galactosidase from Lactobacillus plantarum by Covalent Immobilization.
A novel β-galactosidase from Lactobacillus plantarum (LPG) was over-expressed in E. coli and purified via a single chromatographic step by using lowly activated IMAC (immobilized metal for affinity chromatography) supports. The pure enzyme exhibited a high hydrolytic activity of 491 IU/mL towards o-nitrophenyl β-D-galactopyranoside. This value was conserved in the presence of different divalent cations and was quite resistant to the inhibition effects of different carbohydrates. The pure multimeric enzyme was stabilized by multipoint and multisubunit covalent attachment on glyoxyl-agarose. The glyoxyl-LPG immobilized preparation was over 20-fold more stable than the soluble enzyme or the one-point CNBr-LPG immobilized preparation at 50 °C. This β-galactosidase was successfully used in the hydrolysis of lactose and lactulose and formation of different oligosaccharides was detected. High production of galacto-oligosaccharides (35%) and oligosaccharides derived from lactulose (30%) was found and, for the first time, a new oligosaccharide derived from lactulose, tentatively identified as 3'-galactosyl lactulose, has been described.
Departamento de Biotecnología y Microbiología de Alimentos, Instituto de Investigación en Ciencias de la Alimentación CIAL (CSIC-UAM), Campus de la Universidad Autónoma de Madrid, Nicolás Cabrera 9, 28049 Madrid, Spain. r.benavente@csic.es.
Rocio
R
Benavente
Departamento de Biotecnología y Microbiología de Alimentos, Instituto de Investigación en Ciencias de la Alimentación CIAL (CSIC-UAM), Campus de la Universidad Autónoma de Madrid, Nicolás Cabrera 9, 28049 Madrid, Spain. b.pessela@csic.es.
Benevides C
BC
Pessela
Grupo de Biotecnología Bacteriana, Instituto de Ciencia y Tecnología de Alimentos y Nutrición, (ICTAN-CSIC), Juan de la Cierva 3, 28006 Madrid, Spain. jcuriel@ifi.csic.es.
Jose Antonio
JA
Curiel
Grupo de Biotecnología Bacteriana, Instituto de Ciencia y Tecnología de Alimentos y Nutrición, (ICTAN-CSIC), Juan de la Cierva 3, 28006 Madrid, Spain. blanca.r@ifi.csic.es.
Blanca
B
de las Rivas
Grupo de Biotecnología Bacteriana, Instituto de Ciencia y Tecnología de Alimentos y Nutrición, (ICTAN-CSIC), Juan de la Cierva 3, 28006 Madrid, Spain. rmunoz@ictan.csic.es.
Rosario
R
Muñoz
Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica (ICP-CSIC), Marie Curie 2, Cantoblanco, 28049 Madrid, Spain. jmguisan@icp.csic.es.
Jose Manuel
JM
Guisán
Grupo de Cristalografía y Biología Estructural, Instituto de Química Física Rocasolano (IQFR-CSIC), Serrano 119, 28006 Madrid, Spain. xjosemi@iqfr.csic.es.
Jose M
JM
Mancheño
CBQF-Centro de Biotecnologia e Química Fina, Escola Superior de Biotecnologia, Centro Regional do Porto da Universidade Católica Portuguesa, Rua Dr. António Bernardino Almeida, 4200-072 Porto, Portugal. acardelle@porto.ucp.pt.
Alejandra
A
Cardelle-Cobas
Departamento de Bioactividad y Análisis de Alimentos, Instituto de Investigación en Ciencias de la Alimentación CIAL (CSIC-UAM), Campus de la Universidad Autónoma de Madrid, Nicolás Cabrera 9, 28049 Madrid, Spain. ana.ruiz@csic.es.
Ana I
AI
Ruiz-Matute
Departamento de Bioactividad y Análisis de Alimentos, Instituto de Investigación en Ciencias de la Alimentación CIAL (CSIC-UAM), Campus de la Universidad Autónoma de Madrid, Nicolás Cabrera 9, 28049 Madrid, Spain. nieves.corzo@csic.es.
Nieves
N
Corzo
D004800:Enzymes, Immobilized / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D006038:Glyoxylates / Q000378:metabolism; D006868:Hydrolysis; D048191:Lactiplantibacillus plantarum / Q000378:metabolism*; D007785:Lactose / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D012685:Sepharose / Q000378:metabolism; D013696:Temperature; D001616:beta-Galactosidase / Q000378:metabolism*
D004800:Enzymes, Immobilized; D006038:Glyoxylates; D009844:Oligosaccharides; C070399:glyoxyl agarose; D012685:Sepharose; D001616:beta-Galactosidase; D007785:Lactose; D005690:Galactose