RESUMO
Cyclomaltodextrinases (CDases) belong to Glycoside Hydrolases (GH) family 13, which show versatile hydrolyzing and/or transglycosylation activity against cyclodextrin (CD), starch, and pullulan. Especially, some CDases have been reported to hydrolyze acarbose, a potent α-glucosidase inhibitor, and transfer the resulting acarviosine-glucose to various acceptors. In this study, a novel CDase (LPCD) gene was cloned from Lactobacillus plantarum WCFS1, which encodes 574 amino acids (64.6 kDa) and shares less than 44% of identities with the known CDase-family enzymes. Recombinant LPCD with C-terminal six-histidines was produced and purified from Escherichia coli. It showed the highest activity on ß-CD at 45°C and pH 5.0, respectively. Gel permeation chromatography analysis revealed that LPCD exists as a dodecameric form (~826 kDa). Its hydrolyzing activity on ß- CD is almost same as that on starch, whereas it can hardly attack pullulan. Most interestingly, LPCD catalyzed the unique modes of action in acarbose hydrolysis to produce maltose and acarviosine, as well as to glucose and acarviosineglucose.
Assuntos
Acarbose/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Lactobacillus plantarum/enzimologia , Lactobacillus plantarum/genética , Sequência de Aminoácidos , Amino Açúcares/metabolismo , Clonagem Molecular , DNA Bacteriano/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Glucanos/metabolismo , Glucose/metabolismo , Glicosídeo Hidrolases/classificação , Concentração de Íons de Hidrogênio , Hidrólise , Lactobacillus plantarum/metabolismo , Maltose/metabolismo , Peso Molecular , Especificidade por SubstratoRESUMO
The roles of conserved amino acid residues (Val329-Ala330- Asn331-Glu332), constituting an extra sugar-binding space (ESBS) of Thermus maltogenic amylase (ThMA), were investigated by combinatorial saturation mutagenesis. Various ThMA mutants were firstly screened on the basis of starch hydrolyzing activity and their enzymatic properties were characterized in detail. Most of the ThMA variants showed remarkable decreases in their hydrolyzing activity, but their specificity against various substrates could be altered by mutagenesis. Unexpectedly, mutant H-16 (Gly-Leu-Val-Tyr) showed almost identical hydrolyzing and transglycosylation activities to wild type, whereas K-33 (Ser-Gly-Asp-Glu) showed an extremely low transglycosylation activity. Interestingly, K-33 produced glucose, maltose, and acarviosine from acarbose, whereas ThMA hydrolyzed acarbose to only glucose and acarviosine-glucose, which proposes that the substrate specificity, or hydrolysis or transglycosylation activity of ThMA can be modulated by combinatorial mutations near the ESBS.
