Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A

Abstract

Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25 -10.5) which can be exploited in purification by using cationexchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75% of this activity was retained in the pH range 7.0-10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0-10.0 and maximum activity, in a 10-min assay, was observed at 90°C with 5 mM CaCI, present. No loss of activity was observed after 24 h at 70°C and the half-lives at 80°C and 90°C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60 "C and the half-life at 70 "C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents.Rt41A proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1' amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1' position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1' sites.Generally there is no firm evidence to suggest that an enzyme isolated from an extremely thermophilic organism will have a higher specific activity than its mesophilic counterpart (when assayed at a temperature appropriate to the growth temperature of the producing organism). However, mesophilic proteins tend to denature and become more susceptible to proteolysis at the temperatures at which thermostable enzymes operate optimally. Thus, proteinases from extreme thermophiles have higher specific activities against mesophilic proteins than do most microbial proteinases [l]. The inherent stability of thermophilic proteinases to high temperatures as well as to detergents, organic solvents and chaotropic agents, makes these enzymes potentially useful in a range of biotechnological applications [2, 31.

Characterization of poly(L-lactide)-degrading enzyme produced by thermophilic filamentous bacteria Laceyella sacchari LP175

“…Although EDTA and EGTA are strong inhibitors of metalloproteinase, much of the inhibition of hyperthermophilic serine protease was considered to be due to the loss of activity from enzyme denaturation rather than inhibition of enzyme molecules by removing Ca 2+ ions from the enzyme, leading to the loss both of stabilizing Ca 2+ ions and activity when treated with EDTA and EGTA (Catara et al, 2003;Peek et al, 1992). Furthermore, EDTA is not a good indicator for a metalloproteinase inhibitor because a large number of other enzymes require calcium for activity and EDTA is an excellent calcium chelator (Powers et al, 1986).…”

The use of phenyl-Sepharose for the affinity purification of proteinases

“…The binding of a thermostable extracellular microbial serine proteinase, Rt 41A [5] by a range of alkyl substituted agarose supports was compared to that of phenyl Sepharose.…”

“…The Thermus strain, Rt41A, was grown and the culture supernatant obtained as described previously [5]. The supernatant was clarified with a HFK-131 membrane (Koch Membrane Systems Inc.) and concentrated to 12 litres with a YM10 ultrafiltration membrane (Amicon Corp.).…”

“…(specific activity, 5887 U/mg) was inactivated with PMSF as already described [5] to give a preparation with a specific activity of 687 U/mg, equivalent to an inactivation of 88%. 500 µg and 150 µg of inactivated pepsin and Rt41A respectively were applied to separate 1 ml (0.4 cm x 2.5 cm) columns of phenyl Sepharose equilibrated with either 20 mM Na Formate pH 3.5 (pepsin) or Tes buffer (Rt41A).…”

“…However, we have found that some of the commonly used affinity supports to be less than satisfactory for the purification of a number thermostable proteinases under our investigation; for example, the serine proteinase from the Thermus strain Rt 41A [5] fails to adsorb to either benzamidine agarose or immobilised carboxy-D-phenylalanine and the acid proteinase from the Bacillus strain Wai 21a fails to asdsorb to pepstatin agarose (Prescott, unpublished …”