dc.contributor.author | Peek, Keith | |
dc.contributor.author | Daniel, Roy M. | |
dc.contributor.author | Monk, Colin R. | |
dc.contributor.author | Parker, Lynne | |
dc.contributor.author | Coolbear, Tim | |
dc.date.accessioned | 2010-08-31T04:35:42Z | |
dc.date.available | 2010-08-31T04:35:42Z | |
dc.date.issued | 1992 | |
dc.identifier.citation | Peek, K., Daniel, R.M., Monk, C., Parker, L. & Coolbear, T. (1992). Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A. European Journal of Biochemistry, 207(3), 1035-1044. | en_NZ |
dc.identifier.uri | https://hdl.handle.net/10289/4487 | |
dc.description.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 cation-exchange 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 CaCl2 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. | en_NZ |
dc.language.iso | en | |
dc.publisher | Wiley | en_NZ |
dc.relation.uri | http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1992.tb17140.x/abstract | en_NZ |
dc.subject | enzyme | en_NZ |
dc.subject | Rt41A | en_NZ |
dc.title | Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A | en_NZ |
dc.type | Journal Article | en_NZ |
dc.identifier.doi | 10.1111/j.1432-1033.1992.tb17140.x | en_NZ |