The major industrial use of thermostable and detergent compatible proteinases is in laundry detergent formulations. ' Detergents such as Era Plus@ (Procter and Gamble), Tide@ (Colgate Palmolive), and Dynamo@ (Procter and sulfate precipitation followed by separation of proteinase R and T from contaminants and each other by ion exchange chromatography.Gamble) contain proteolytic enzymes, the majority of which are produced by the members of the genus Bacillus. Although subtilisins have been the enzymes of choice for detergent formulations (US patent Nos. 1240058, 374971, 370482, and 4266031, and UK patent No. 13155937), these are not the ideal detergent enzymes due to low thermal stability in the presence of detergents and short halflife on the shelf. Recently, modified subtilisins have been generated with greater thermal stability by means of site directed mutagenesis te~hnique.~-~ In this report, the laundry detergent compatibility of two novel enzymes6.' from the fungus Tritirachium album Limber is described. The amino acid sequences of these two proteinases already contain some of the changes recently engineered into subtilisins. These two proteinases, termed as proteinase R and T, were extremely stable in the presence of detergents at high temperature. We found the thermal and detergent stabilities of proteinase R to be very similar to those of proteinase K which were much higher than that of subtilisin BPN'. On the other hand, proteinase T demonstrated superior stability to that of proteinase R and K. An earlier report of this work has been published as a meeting abstract.*
MATERIALS AND METHODS
Determination of Proteinases ActivityN-Succinyl-alanyl-alanyl-prolyl phenyl alanyl nitroanilide' was used as the substrate to monitor the production of proteinases during liquid culture while azoalbumin and azocasein were used as substrates in stability studies." To a final volume of 500 pL, 20 p L of azocasein or azoalbumin (a 5% solution in 0.W Tris HCl, pH 7.5, 1 mM CaCl,), 20 pL of enzyme (1-10 pg) and 460 pL of 50 mM Tris HC1, pH 7.5, were added. Following the incubation for 30 min at 37"C, 500 p L of 10% TCA was added and the samples were kept on ice for 15 min. After centrifugation 800 p L of supernatant was added to a tube containing 200 pL of 1.8N NaOH. The optical density was measured at 420 nm against the proper control.
Stability StudiesProteinases at a concentration of 0.2 mg/mL were incubated at 50°C in the presence of different detergents. After specific intervals 10 p L of the sample were removed to determine the residual activity at 37°C using azocasein as the substrate in excess (see above).
RESULTS AND DISCUSSION
Purification of ProteinasesThe physical properties of the proteinases have been described e l~e w h e r e .~.~ Briefly, the molecular weights of proteinase R and T were 30,000 and 33,000 daltons, respectively. The isoelectric point of proteinase R was 9.8 while that of proteinase T was 4.5. The pH optimum of the proteinase R was between 7.0 and 10.0 while proteinase T was maximally active b...
