Purification and some properties of an extracellular protease (caldolysin) from an extreme thermophile.
An extracellular metal-chelator-sensitive lytic protease (assigned the trivial name caldolysin) was isolated from a Thermus-like organism, Thermus T-351. Caldolysin was purified by affinity chromatography on Cbz-D-phenylalanine-TETA-Sepharose 4B and by gel filtration. It contained 13% carbohydrate, a single zinc atom, had a molecular weight of approx. 21,000, a pH optimum of 8 (azocasein substrate), and an isoelectric point of about 8.5. It was capable of hydrolysing many soluble and insoluble protein substrates, including collagen and elastin. No esterase activity was detected, and small peptides (less than four amino acids) and low molecular weight chromogenic substrates were not hydrolysed. A specificity for small aliphatic amino acids on either side of the splitting point was indicated. Caldolysin lysed heat-killed Gram-negative bacterial cells, but had little effect on Gram-positive organisms. Caldolysin exhibited a very high degree of thermostability (t 1/2(80 degrees C) approximately 30 h, t 1/2(90 degrees C) = 1 h). The stability (but not activity) was shown to be dependent on the presence of Ca2+ (t 1/2(75 degrees C, 10 mM calcium) greater than 193 h; t 1/2(75 degrees C, no calcium) = 4.8 min). None of the other metal ions tested (Co, Zn, Sr, Mg, Ba and Cu) was as effective as calcium in conferring thermostability of EDTA-treated caldolysin. Caldolysin was stable at room temperature in moderately acid and alkaline (pH 5 to 11) buffers for periods of greater than 90 days. Little loss of enzyme activity was detected after the incubation of caldolysin at 18 degrees C in the presence of 8 M urea, 6 M guanidine hydrochloride or 1% sodium dodecyl sulphate for 24 h. At 75 degrees C, the activity half-life of caldolysin in these denaturing agents was reduced to approx. 1 h, 1 h and more than 5 h, respectively.
D002241:Carbohydrates / Q000032:analysis; D004355:Drug Stability; D010450:Endopeptidases / Q000302:isolation & purification*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008666:Metalloendopeptidases; D008970:Molecular Weight; D013379:Substrate Specificity; D013816:Thermodynamics; D013824:Thermus / Q000201:enzymology*
D002241:Carbohydrates; D010450:Endopeptidases; D008666:Metalloendopeptidases; C035717:caldolysin