Caldolase, a chelator-insensitive extracellular serine proteinase from a Thermus spp

Abstract

An extracellular alkaline serine proteinase from Thermus strain ToK3 was isolated and purified to homogeneity by (NH4)2SO4 precipitation followed by ion-exchange chromatography on DEAE-cellulose and QAE-Sephadex, affinity chromatography on N alpha-benzyloxycarbonyl-D-phenylalanyl-triethylenetetraminyl-Sepha rose 4B and gel-filtration chromatography on Sephadex G-75. The purified enzyme had a pI of 8.9 and an Mr determined by gel-permeation chromatography of 25,000. The specific activity was about 37,700 proteolytic units/mg with casein as substrate, and the pH optimum was 9.5. Proteolytic activity was inhibited by low concentrations of di-isopropyl phosphorofluoridate and phenylmethanesulphonyl fluoride, but was unaffected by EDTA, EGTA, o-phenanthroline, N-ethyl-5-phenylisoxazolium-3'-sulphonate, N alpha-p-tosyl-L-phenylalanylchloromethane, N alpha-p-tosyl-L-lysylchloromethane, trypsin inhibitors and pepstatin A. The enzyme contained approx. 10% carbohydrate and four disulphide bonds. No Ca2+, Zn2+ or free thiol groups were detected. It hydrolysed several native and dye-linked proteins and synthetic chromogenic peptides and esters. The enzyme was very thermostable (half-life values were 840 min at 80 degrees C, 45 min at 90 degrees C and 5 min at 100 degrees C). The enzyme was unstable at low ionic strength: after 60 min at 75 degrees C in 0.1 M-Tris/acetate buffer, pH 8, only 20% activity remained, compared with no loss in 0.1 M-Tris/acetate buffer, pH 8, containing 0.4 M-NaCl.

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

“…The poor sedimentation of the ammonium sulphate precipitate resulted in low yields with 70% losses being typical at this stage. Similar losses were also observed at this stage for caldolase [8]. However, the yield of proteinase from the recovered precipitate was good, typically over 70% recovery being obtained (Table 1).…”

“…Five proteinases, all of which are serine-type proteinases, have been characterized from extremely thermophilic Thermus isolates . Two of these enzymes, caldolase [8] (from strain Tok3) and aqualysin I [7] (from T. aquaticus) are chelator-resistant, whilst caldolysin [5] (from strain T351) and aqualysin I1 [9] (from T . aquaticus) are chelatorsensitive.…”

“…The types of ester cleaved by Rt41A proteinase are similar to those cleaved by the Thermus sp. proteinases caldolase [8] and aqualysin I [7], but no direct comparisons can be made due to the lack of kinetic data for these enzymes. Both caldolase and aqualysin I showed most activity towards the alanine ester, for which Rt41A proteinase had the highest k,,,.…”

Isolation and characterisation of two chymotrypsins fromAllocyttus niger(black oreo dory) viscera

“…Substrate inhibition of chymotrypsin by Suc-AAPF-NA has not been previously reported although substrate inhibition is a common feature of proteases when hydrolysing large substrates such as azocasein (e.g., Cowan & Daniel 1982;Saravani et al 1989;Freeman et al 1993). Substrate inhibition of trypsin by small ester substrates was reported by Yoshinaka et al (1984Yoshinaka et al ( ,1985 (Genicot et al 1987).…”

Effects of medium composition on extracellular proteinase stability and yield in batch cultures of a Thermus sp.

“…has been investigated Jones et al 1988;Kanasawud et al 1989) but most studies have concentrated on the properties of the enzymes (Cowan and Daniel 1982;Taguchi et al 1983;Cowan et al 1987;Saravani et al 1989). Thermophilic bacteria investigated to date produce proteinases at levels about an order of magnitude lower than do most mesophiles, and an increase in the level of production is necessary before thermophilic proteinases can beCome competitive as industrial enzymes .rather than speciality enzymes only (Cowan et al 1985).…”