ABSTRACT
A newly isolated actinomycete belonging to Saccharothrix sp. was found to produce a unique enzyme catalyzing D-amino acid transfer. The enzyme, which was tentatively named D-amino acid transferase, was purified 2600-fold to electrophoretic homogeneity and the molecular mass was 41 kDa. The enzyme was D-configuration specific and recognized aromatic D-amino acid esters to form oligo D-amino acid esters. D-Phenylalanine ester was favored as substrate over other D-amino acid esters. The optimum conditions for oligo D-phenylalanine ester formation by D-amino acid transferase were pH 7.0 and 40 degrees C. The enzyme was inhibited by DAN, EPNP and DFP.
Subject(s)
Actinomycetales/enzymology , Acyltransferases/metabolism , Aminoacyltransferases , Bacterial Proteins , Acyltransferases/antagonists & inhibitors , Acyltransferases/isolation & purification , Enzyme Inhibitors/pharmacology , Isomerism , Phenylalanine/analogs & derivatives , Phenylalanine/metabolism , Substrate SpecificityABSTRACT
Streptomyces limosus was selected because it secreted a novel protease that catalyzed the synthetic reaction forming Pro-Pro-Pro from Pro-Pro. The protease was purified to an electrophoretically homogeneous state and an activity of more than about 20,000-fold that of the culture broth. The molecular mass of the enzyme was estimated to be 50 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme was most active in alkaline pH for the synthetic reaction producing Pro-Pro-Pro from Pro-Pro, although for the hydrolytic reaction forming proline it was most active in neutral pH. The enzyme was inhibited by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and diazoacetyl-DL-norleucine methyl ester (DAN). It can be considered that this enzyme belongs to the class of aspartic proteases. The substrate specificity indicates that this enzyme has a strong affinity for proline as a N-terminal amino acid of peptides.
Subject(s)
Endopeptidases/isolation & purification , Streptomyces/enzymology , Amino Acid Sequence , Ammonium Sulfate , Chromatography, DEAE-Cellulose , Chromatography, Gel , Dipeptides/metabolism , Electrophoresis, Polyacrylamide Gel , Endopeptidases/biosynthesis , Endopeptidases/chemistry , Hydrogen-Ion Concentration , Mass Spectrometry , Molecular Sequence Data , Molecular Weight , Protein Hydrolysates/chemistry , Spectrophotometry, Ultraviolet , Substrate SpecificityABSTRACT
A biotin derivative having a carbodiimide function (fig.1 Carbo-biotin) was synthesized and reacted with DNAs. This compound was used to label biotin to DNAs, by reaction of carbodiimide group and nucleotides as they form stable adducts. Our experiments showed that it is able to bind to both single and double stranded DNAs. The DNA oligomer labeled with this reagent was shown to hybridize to complementary DNA chain. From our results, this compound is expected to be a useful biotinylating reagent for non-radioactive detection.
Subject(s)
Biotin/analogs & derivatives , Carbodiimides , Binding Sites , Biotin/chemical synthesis , Biotin/chemistry , Carbodiimides/chemical synthesis , Carbodiimides/chemistry , DNA/chemistry , Indicators and Reagents/chemical synthesis , Indicators and Reagents/chemistry , Molecular StructureABSTRACT
A transfer reaction catalyzed by an exo-beta-1,4-galactanase from Bacillus subtilis was studied. The enzyme had a broad acceptor specificity and transferred galactobiosyl residues to acceptors such as various alcohols, including hydroxy benzenes and saccharides. Transfer products of glycerol formed by the enzyme were compared with those formed by Escherichia coli beta-galactosidase and by Penicillium citrinum endo-galactanase. E. coli enzyme transferred 90% of galactose residues to the primary hydroxyl groups of glycerol and P. citrinum endo-enzyme transferred 80% of saccharide residues to the secondary hydroxyl group. The B. subtilis exo-galactanase was less specific than the other two enzymes and formed two products (1-DG and 2-DG) with a 2-DG/1-DG ratio of about 2. The structures of the saccharides were examined by 13C-nuclear magnetic resonance analysis and by enzymatic hydrolysis. 1-DG and 2-DG were elucidated to be O-beta-D-galactosyl-(1----4)-O-beta-D-galactosyl-(1----1)-glycerol and O-beta-D-galactosyl-(1----4)-O-beta-D-galactosyl-(1----2)-glycerol, respectively. The efficiency of the transfer reaction was measured at various concentrations of glycerol using galactotriose as a donor. About 40-75% of galactobiosyl residues were transferred at an acceptor concentration range of 20-100 mg/ml.
Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , Glycoside Hydrolases , beta-Galactosidase/metabolism , Carbohydrate Sequence , Glycerol/metabolism , Molecular Sequence Data , Substrate SpecificityABSTRACT
An arabinogalactan 4-beta-D-galactanohydrolase was purified to a homogeneous state from the culture filtrate of a strain of Bacillus subtilis. The enzyme have a molecular mass of 36 kDa and an isoelectric point of pH 7.9. The enzyme is most active at around pH 6.5-7 and at 60 degrees C, and is stable between pH 6-10 and below 55 degrees C. Hg2+ and Cu2+ inhibit the activity. The enzyme hydrolyze soybean arabinogalactan which contains beta-1,4-galactosidic linkages in its main chain structure, but not other polysaccharides with beta-1,3-galactosidic linkages. The hydrolysis products from soybean arabinogalactan are predominantly galactobiose with a small amount of galactotetraose. The enzyme is an exo-enzyme and the ability to transfer galactobiose to other galactobiose molecules is indicated by the formation of galactotetraose.
