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dc.contributor.authorPeek, Keith
dc.contributor.authorDaniel, Roy M.
dc.contributor.authorMonk, Colin R.
dc.contributor.authorParker, Lynne
dc.contributor.authorCoolbear, Tim
dc.date.accessioned2010-08-31T04:35:42Z
dc.date.available2010-08-31T04:35:42Z
dc.date.issued1992
dc.identifier.citationPeek, 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.urihttps://hdl.handle.net/10289/4487
dc.description.abstractThermus 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.isoen
dc.publisherWileyen_NZ
dc.relation.urihttp://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1992.tb17140.x/abstracten_NZ
dc.subjectenzymeen_NZ
dc.subjectRt41Aen_NZ
dc.titlePurification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41Aen_NZ
dc.typeJournal Articleen_NZ
dc.identifier.doi10.1111/j.1432-1033.1992.tb17140.xen_NZ


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