An extracellular protease from an extreme thermophile
type: CreativeWork
description: The major extracellular protease of the Thermus-like organism Thermus T-351, has been isolated by Sephadex SP-C25 ion exchange chromatography, affinity chromatography on CBZ-D-phe-TETA-Sepharose and Sephadex G75 gel filtration chromatography. The purified enzyme, assigned the title “Caldolysin”, was shown to be homogeneous by a variety of electrophoretic techniques. Two other minor extracellular proteolytic enzymes (totally 12% to 25% of initial enzyme activity) were separated during ion exchange chromatography, but have not been studied. The molecular weight and isoelectric point of purified Caldolysin were estimated to be approximately 20,000 Daltons and 8.5, respectively. The presence of 6 disulphide bonds was indicated by the reaction with dithionitro-benzoate. No free sulphydryl groups were detected. When tested against a wide range of inhibitors, significant responses to EDTA, EGTA, and iodoacetic acid, but not o-phenanthroline or p-chloromercuri benzoate, were noted. Together with the presence of lytic activity (Caldolysin lysed a broad range of Gram-negative bacteria but only few Gram-positive organisms), this permitted the classification of Caldolysin as a metal-chelator-sensitive lytic protease. A number of similarities with Myxobacter and Sorangium lytic proteases have been noted. It was shown that calcium was essential for the stability of Caldolysin, but not for its activity. No evidence was found for the presence of an active site-bound metal ion. This is a significant dissimilarity to Thermolysin, which contains a single catalytic zinc atom. Casein, albumin, haemoglobin, collagen, elastin, and fibrin were all hydrolysed to varying degrees by Caldolysin. An apparent minimum size requirement for activity (a minimum of 3 or 4 amino acid residues) precluded the use of amino acid and dipeptide substrate analogues for the measurement of proteolytic activity. A preliminary determination of specificity indicated a preference for small neutral aliphatic amino …
abstract: The major extracellular protease of the Thermus-like organism Thermus T-351, has been isolated by Sephadex SP-C25 ion exchange chromatography, affinity chromatography on CBZ-D-phe-TETA-Sepharose and Sephadex G75 gel filtration chromatography. The purified enzyme, assigned the title “Caldolysin”, was shown to be homogeneous by a variety of electrophoretic techniques. Two other minor extracellular proteolytic enzymes (totally 12% to 25% of initial enzyme activity) were separated during ion exchange chromatography, but have not been studied. The molecular weight and isoelectric point of purified Caldolysin were estimated to be approximately 20,000 Daltons and 8.5, respectively. The presence of 6 disulphide bonds was indicated by the reaction with dithionitro-benzoate. No free sulphydryl groups were detected. When tested against a wide range of inhibitors, significant responses to EDTA, EGTA, and iodoacetic acid, but not o-phenanthroline or p-chloromercuri benzoate, were noted. Together with the presence of lytic activity (Caldolysin lysed a broad range of Gram-negative bacteria but only few Gram-positive organisms), this permitted the classification of Caldolysin as a metal-chelator-sensitive lytic protease. A number of similarities with Myxobacter and Sorangium lytic proteases have been noted. It was shown that calcium was essential for the stability of Caldolysin, but not for its activity. No evidence was found for the presence of an active site-bound metal ion. This is a significant dissimilarity to Thermolysin, which contains a single catalytic zinc atom. Casein, albumin, haemoglobin, collagen, elastin, and fibrin were all hydrolysed to varying degrees by Caldolysin. An apparent minimum size requirement for activity (a minimum of 3 or 4 amino acid residues) precluded the use of amino acid and dipeptide substrate analogues for the measurement of proteolytic activity. A preliminary determination of specificity indicated a preference for small neutral aliphatic amino …
author: Cowan, Don A.
license: https://researchcommons.waikato.ac.nz/bitstreams/5a5a079f-b5cf-4bcc-8313-9413ca85fb4d/download
date published: 1980
date modified: 2026-07-24T05:57:46Z
provider: University Waikato
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: The University of Waikato