An extracellular protease from an extreme thermophile

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 acids on either side of the point of hydrolysis. No esterase activity was detected. Optimal hydrolysis of both casein and albumin occurred at approximately pH 8. Temperature-activity relationships indicated that Caldolysin underwent conformational changes at 50°C and 92°C. Loss of enzyme activity below the latter temperature was thought to be primarily the result of autolysis while above this temperature denaturation occurred. Arrhenius plots showed a break at about 50°C, above which a reduction in activation energy occurred. The Kₘ at 40°C was considerably higher than that at either 65°C or 85°C. Caldolysin is one of the most stable enzymes known. Thermostability was greater than that of most other documented proteases (t ¹/₂ (80° C) = 30 hours; t ¹/₂ (85 °C) = 5-6 hours; t ¹/₂ (90°C) = 1 hour; t ¹/₂ (95°C) = 30 minutes; t ¹/₂ (100°C) = 4 minutes) . In the absence of calcium, thermostability was reduced considerably (t ¹/₂ 80°C) = 8 minutes). Other metal ions provided only a slight thermostabilising effect compared with Ca [Ca» Zn>Sr>Mg>Co>Ba>Cu]. Neither the presence of high ionic concentrations nor the presence of non-ionic solutes had any major effect on thermostability. However, high and low pH values significantly reduced thermostability at 75°C and 90°C. At room temperature in solution, little loss of enzyme activity was noted at pH’s from 7-11 during a 95 day period. Activity half-lives at pH 3.6, 5, and 11.8 were 28 days, more than 60 days, and 4 days, respectively. Little activity loss resulted from storage of the enzyme in the frozen or lyophilised state for a period of six months. Caldolysin was shown to be very stable in the presence of denaturing agents (8M urea, 6M guanidine.HC1, 1% sodium dodecylsulphate) at ambient temperatures. At 75°C, a 50% loss of activity occurred within 59 minutes, 53 minutes, and 320 minutes, respectively. A feature of the Michaelis-Menten kinetics of Caldolysin is marked substrate inhibition (Kₘ (azoalbumin) = 0.028; Kₛ´ (azoalbumin) = 3) which, however, does not occur with small substrates or when the enzyme is immobilised. The enzyme also exhibits an initial activation during incubation at temperatures above 65°C. This effect has been correlated with the ionic strength, the incubation temperature, and the concentration of non-enzyme protein present. The latter causes initial inhibition which is reversed on heating. It is postulated that activation/inhibition and substrate inhibition are related, and are caused by the presence of non-catalytic protein-binding sites in the molecule which are discrete from the active site. Caldolysin was successfully immobilised to glass beads, CM-cellulose, and Sepharose 4B, with enzyme activity retentions of 1%, 31% and 73%, respectively. The Kₘ of Caldolysin was unchanged in the immobilised state, but the pH optimum of Sepharose-caldolysin was reduced by approximately one pH unit. Thermal stability of both apo-and holo-caldolysin-Sepharose was increased by 3-4-fold.