Isolation and amino acid sequence of a glutamic acid specific endopeptidase from Bacillus licheniformis.
An endopeptidase cleaving specifically at the carboxyl side of acidic amino acid residues, preferentially at glutamic acid, has been isolated from a commercial extract obtained by fermentation with Bacillus licheniformis. Using ion-exchange chromatography and affinity chromatography on bacitracin-Sepharose, it was possible, from 100 ml commercial extract, to isolate 100 mg homogeneous enzyme in a yield of 50%. It is the first description of a large-scale isolation of a Glu/Asp-specific enzyme. The preparation was essentially free of contaminating activities. The isolated enzyme consists of one peptide chain of 222 amino acid residues and has a calculated molecular mass of 23,589 Da. The determined amino acid sequence shows similarity to the Glu/Asp-specific enzymes previously isolated from Staphylococcus aureus V8, Actinomyces sp. and Streptomyces thermovulgaris. The substrate preference of the enzyme has been investigated. Although non-specific cleavages were observed after prolonged hydrolysis at high enzyme concentrations the enzyme appears to be essentially specific for Glu-Xaa and Asp-Xaa, with strong preference for the former. The isolated enzyme exhibits a bell-shaped pH/activity profile with an optimum at pH 7.5-8.0. The activity is adversely affected by high ionic strength and beneficially affected by the inclusion of calcium ions in the assay medium. The enzyme is completely inhibited by diisopropylfluorophosphate, suggesting that it is a serine endopeptidase. It is partially inhibited by EDTA.
Carlsberg Laboratory, Department of Chemistry, Copenhagen Valby, Denmark.
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Svendsen
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Breddam
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D002122:Calcium Chloride / Q000494:pharmacology; D002845:Chromatography; D010450:Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000737:chemistry; D013379:Substrate Specificity
D005971:Glutamates; D010446:Peptide Fragments; D018698:Glutamic Acid; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D002122:Calcium Chloride