RESUMEN
In this study, a novel α-amylase was cloned from Bifidobacterium longum and named BiLA. The enzyme exhibited optimal activity at 20 °C and a pH value of 5.0. Kinetic analysis using various carbohydrate substrates revealed that BiLA had the highest k(cat/)K(m) value for amylose. Interestingly, analysis of the enzymatic reaction products demonstrated that BiLA specifically catalyzed the hydrolysis of oligosaccharides and starches up to G5 from the nonreducing ends. To determine whether BiLA can be used to generate slowly digestible starch (SDS), starch was treated with BiLA, and the kinetic parameters were analyzed using porcine pancreatic α-amylase (PPA) and amyloglucosidase (AMG). Compared to normal starch, BiLA-treated starch showed lower k(cat)/K(m) values with PPA and AMG, suggesting that BiLA is a potential candidate for the production of SDS.
Asunto(s)
Bifidobacterium/enzimología , alfa-Amilasas/metabolismo , Amilosa/análisis , Amilosa/metabolismo , Animales , Electroforesis en Gel de Poliacrilamida , Glucano 1,4-alfa-Glucosidasa/metabolismo , Hidrólisis , Cinética , alfa-Amilasas Pancreáticas/metabolismo , Almidón/química , Porcinos , alfa-Amilasas/genéticaRESUMEN
A novel maltose (G2)-forming α-amylase from Lactobacillus plantarum subsp. plantarum ST-III was expressed in Escherichia coli and characterized. Analysis of conserved amino acid sequence alignments showed that L. plantarum maltose-producing α-amylase (LpMA) belongs to glycoside hydrolase family 13. The recombinant enzyme (LpMA) was a novel G2-producing α-amylase. The properties of purified LpMA were investigated following enzyme purification. LpMA exhibited optimal activity at 30 °C and pH 3.0. It produced only G2 from the hydrolysis of various substrates, including maltotriose (G3), maltopentaose (G5), maltosyl ß-cyclodextrin (G2-ß-CD), amylose, amylopectin, and starch. However, LpMA was unable to hydrolyze cyclodextrins. Reaction pattern analysis using 4-nitrophenyl-α-d-maltopentaoside (pNPG5) demonstrated that LpMA hydrolyzed pNPG5 from the nonreducing end, indicating that LpMA is an exotype α-amylase. Kinetic analysis revealed that LpMA had the highest catalytic efficiency (kcat/Km ratio) toward G2-ß-CD. Compared with ß-amylase, a well-known G2-producing enzyme, LpMA produced G2 more efficiently from liquefied corn starch due to its ability to hydrolyze G3.
Asunto(s)
Lactobacillus plantarum/enzimología , Maltosa/metabolismo , Ciclodextrinas/metabolismo , Escherichia coli/genética , Hidrólisis , Cinética , Maltosa/análogos & derivados , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Trisacáridos/metabolismo , alfa-Amilasas/química , alfa-Amilasas/genética , alfa-Amilasas/metabolismoRESUMEN
Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) mainly produces cyclodextrins (CDs) using linear maltooligosaccharides. We performed site-directed saturation mutagenesis on the +1 substrate-binding residue, H233, of CGTase from alkalophilic Bacillus sp. I-5 to prepare specific-length oligosaccharides. The obtained mutant CGTase, H233Y, primarily produced maltoheptaose (G7) using ß-CD via a hydrolysis reaction. A kinetic study of H233Y showed that the kcat/Km value of ß-CD was 7-fold greater than that of G7, which accounts for the accumulation of G7 during the H233Y enzyme reaction. A structure comparison of CGTases with H233Y modeling suggests that the substitution of H233Y may alter the position of the +1 subsite and slow the further hydrolysis of G7 after the ring-opening reaction.
Asunto(s)
Glucanos/metabolismo , Glucosiltransferasas/química , Glucosiltransferasas/metabolismo , Mutagénesis Sitio-Dirigida/métodos , Secuencia de Aminoácidos , Bacillus/enzimología , Bacillus/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Glucosiltransferasas/genética , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Fe65 is characterized as an adaptor precursor (APP) through its PID2 element, as well as with the other members of the APP protein family. With the serum- and glucocorticoid-induced kinase 1 (SGK1) substrate specificity information, we found that the putative site of phosphorylation in Fe65 by SGK1 is present on its Ser(566) residue in (560)CRVRFLSFLA(569)(X60469). Thus, we demonstrated that Fe65 and the fluorescein-labeled Fe65 peptide FITC-(560)CRVRFLSFLA(569) are phosphorylated in vitro by SGK1. Phosphorylation of the Ser(566) residue was also demonstrated using a Ser566 phospho-specific antibody. The phospho Fe65 was found mainly in the nucleus, while Fe65 S556A mutant was localized primarily to the cytoplasm. Therefore, these data suggest that SGK1 phosphorylates the Ser(566) residue of Fe65 and that this phosphorylation promotes the migration of Fe65 to the nucleus of the cell.
