title: Purification and chemical characterisation of a cell wall-associated β-galactosidase from mature sweet cherry (Prunus avium L.) fruit.
abstract: Using four different chromatographic steps, β-galactosidase was purified from the ripe fruit of sweet cherry to apparent electrophoretic homogeneity with approximately 131-fold purification. The Prunus avium β-galactosidase showed an apparent molecular mass of about 100 kDa and consisted of four different active polypeptides with pIs of about 7.9, 7.4, 6.9 and 6.4 as estimated by native IEF and β-galactosidase-activity staining. The active polypeptides were individually excised from the gel and subjected to SDS-PAGE. Each of the four native enzymes showing β-galactosidase activity was composed of two polypeptides with an estimated mass of 54 and 33 kDa. Both of these polypeptides were subjected to N-terminal amino acid sequence analysis. The 54 kDa polypeptide of sweet cherry β-galactosidase showed a 43% identity with the 44 kDa subunit of persimmon and apple β-galactosidases and the 48 kDa subunit of carambola galactosidase I. The sweet cherry β-galactosidase exhibited a strict specificity towards p-nitrophenyl β-D-galactopyranoside, a pH optimum of 4.0 and K(m) and V(max) values of 0.42 mM and 4.12 mmol min(-1) mg(-1) of protein respectively with this substrate. The enzyme was also active towards complex glycans. Taken together the results of this study prompted a role for this class of enzymes on sweet cherry fruit ripening and softening.
authors.affiliation: Institute of Sciences of Food Production, C.N.R. Unit of Lecce, via Monteroni, 73100 Lecce, Italy. carmela.gerardi@ispa.cnr.it
authors.forename: Carmela
authors.initials: C
authors.lastname: Gerardi
authors.forename: Federica
authors.initials: F
authors.lastname: Blando
authors.forename: Angelo
authors.initials: A
authors.lastname: Santino
mesh_terms: D000595:Amino Acid Sequence; D002473:Cell Wall / Q000201:enzymology* / Q000378:metabolism; D029788:Diospyros / Q000201:enzymology; D005638:Fruit / Q000201:enzymology* / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D027845:Malus / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009598:Nitrophenylgalactosides / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D021122:Protein Subunits; D027861:Prunus / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D010455:Peptides; D010940:Plant Proteins; D011134:Polysaccharides; D021122:Protein Subunits; D009598:Nitrophenylgalactosides; D001616:beta-Galactosidase