Don A. Cowan

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.

β-Galactosidase from a strain of the anaerobic thermophile, Thermoanaerobacter

An intracellular β-galactosidase was isolated from an extremely thermophilic gram-negative anaerobe (similar to Thermoanaerobacter). This enzyme had an apparent molecular weight of 154,000 (two types of subunits were indicated) and an isoelectric point of 5.0. It was inhibited by p-chloromercuribenzoate, by alkaline earth and transition metal ions, but was activated by alkaline metal salts and by reducing reagents. The enzyme hydrolysed o-nitrophenol-β-d-galactopyranoside optimally at pH 6.0 with an apparent Km of 4.3 mm and a maximal turnover number of 431s−1 (assuming a single active site). The specific activity at 70°C was 99 μmol o-nitrophenol produced min−1 mg−1 protein. Hydrolysis of lactose demonstrated a substrate concentration-dependent Km and Vmax. The enzyme was moderately thermostable, with a half-life of 155 min at 75°C.

A modification for increasing the sensitivity of the casein-agar plate assay: a simple semiquantitative assay for thermophilic and mesophilic proteases

A casein-agar plate assay was used for the quantitative determination of both mesophilic and thermophilic proteases. Because many proteases are thermostable, assay at higher temperatures is possible. The sensitivity of the plate assay increased with temperature, the optimum assay temperature depending on the thermostability of the enzyme (e.g. Thermus protease, 75 degrees C; thermolysin, 65 degrees C; trypsin, 65 degrees C; alpha-chymotrypsin, 45 degrees C). A positive correlation was observed between incubation temperature and the density of the para-casein precipitate, increasing the accuracy of diameter measurement. Using this modified method, thermostable proteases could be assayed at levels well below the limits of detection of other methods (e.g. 40 pg of thermolysin and 300 pg of trypsin detectable at 65 degrees C, a 16-fold increase in the sensitivity for trypsin compared with a conventional plate assay (Fossum, K. (1970) Acta Pathol. Microbiol. Scand. Sect. B 78, 350-361)). The sensitivity of the plate assay could be further increased by the inclusion of some detergents and chaotropic agents in the gel.

Some observations on the inhibition and activation of a thermophilic protease.

The activity of Caldolysin, the extracellular protease from the extreme thermophile Thermus aquaticus strain T351, was reduced in the presence of high protein concentrations. The absence of this effect after enzyme immobilization, or when using chromogenic substrates, suggests that a steric mechanism is involved. The apparent activation of caldolysin under certain conditions was shown to be related to both temperature and the ionic strength of the aqueous environment. The effects on activity, substrate affinity and thermostability of chemical modification with various reagents are also discussed.