ABSTRACT
Glucose dehydrogenase (GDH) is of interest for its potential applications in the field of glucose sensors. To improve the performance of glucose sensors, GDH is required to have strict substrate specificity. A novel flavin adenine dinucleotide (FAD)-dependent GDH was isolated from Mucor prainii NISL0103 and its enzymatic properties were characterized. This FAD-dependent GDH (MpGDH) exhibited high specificity toward glucose. High specificity for glucose was also observed even in the presence of saccharides such as maltose, galactose and xylose. The molecular masses of the glycoforms of GDH ranged from 90 to 130 kDa. After deglycosylation, a single 80 kDa band was observed. The gene encoding MpGDH was cloned and expressed in Aspergillus sojae. The apparent kcat and Km values of recombinant enzyme for glucose were found to be 749.7 s(-1) and 28.3 mM, respectively. The results indicated that the characteristics of MpGDH were suitable for assaying blood glucose levels.
Subject(s)
Aspergillus/genetics , Glucose 1-Dehydrogenase/isolation & purification , Glucose 1-Dehydrogenase/metabolism , Glucose/metabolism , Mucor/enzymology , Cloning, Molecular , Flavin-Adenine Dinucleotide/metabolism , Galactose/metabolism , Galactose/pharmacology , Gene Expression , Glucose/pharmacology , Glucose 1-Dehydrogenase/chemistry , Glucose 1-Dehydrogenase/genetics , Glycosylation , Maltose/metabolism , Maltose/pharmacology , Molecular Weight , Substrate Specificity/drug effects , Xylose/metabolism , Xylose/pharmacologyABSTRACT
Depsipeptides are peptide-like polymers consisting of amino acids and hydroxy acids, and are expected to be new functional materials for drug-delivery systems and polymer science. In our previous study, D-alanyl-D-lactate, a type of depsipeptide, was enzymatically synthesized using D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 (TmDdl) by Y207F substitution. Thereafter, in this study, further mutagenesis was introduced, based on structural comparison between TmDdl and a well-characterized D-alanine-D-alanine ligase from Escherichia coli. The S137A/Y207F mutant showed higher D-alanyl-D-lactate and lower D-alanyl-D-alanine synthesizing activity than the Y207F mutant. This suggests that substitution at the S137 residue contributes to product selectivity. Saturated mutagenesis on S137 revealed that the S137G/Y207F mutant showed the highest D-alanyl-D-lactate synthesizing activity. Moreover, the mutant showed broad substrate specificity toward D-amino acid and recognized D-lactate and D,L-isoserine as substrates. On the basis of these characteristics, various depsipeptides can be produced using S137G/Y207F-replaced TmDdl.
Subject(s)
Amino Acid Substitution , Depsipeptides/biosynthesis , Peptide Synthases/chemistry , Peptide Synthases/metabolism , Thermotoga maritima/enzymology , Amino Acid Sequence , Escherichia coli/enzymology , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Peptide Synthases/genetics , Protein Structure, Tertiary , Substrate SpecificityABSTRACT
Cellobiose phosphorylase from Clostridium thermocellum catalyzed the beta-anomer-selective synthesis of alkyl glucosides from cellobiose. Synthesis of alkyl beta-glucoside from inexpensive sucrose using cellobiose phosphorylase and sucrose phosphorylase from Pseudomonas saccharophilia was investigated. By combined use of these two phosphorylases, alkyl beta-glucoside was anomer-selectively synthesized from sucrose and alkyl alcohol.
