Leonardus Johannes Sofie Marie Mulleners

Structural and Functional Consequences of Engineering the High Alkaline Serine Protease PB92

The ability to remove proteinaceous fabric stains made the alkaline proteases powerful tools in the hands of detergent manufacturers. The application of proteases in detergents requires that the proteases are stable and active in the presence of typical detergent ingredients such as surfactants, builders, bleaching agents, bleach activators, fillers, fabric softeners, various formulation aids, etc. To meet the high alkaline conditions in heavy duty powder detergents, extracellular Bacillus serine proteases have been extensively screened for good detergent stability and a high wash performance at this pH. In such a screening an alkalophilic Bacillus strain, PB92, was isolated1 which produces a serine protease with optimal performance in the pH range 10 to 12. The gene encoding this high-alkaline protease PB92 has been cloned and characterized2. The mature protease has a molecular weight of 26,900 and consists of 269 amino acids. A high sequence homology exists with protease Yab3 (83%) which is also produced by an alkalophilic Bacillus while moderate homology is found with the well known serine proteases from Bacillus amyloliquefaciens (subtilisin BPN’/NOVO, 59%)4,5, B. licheniformis (subtilisin Carlsberg, 60%)6,7, Tritirachium album Limber (proteinase-K, 34%)8,9, and Thermoactinomyces vulgaris (thermitase, 47%)10. In a recent review Siezen et al.11 proposed the name’subtilases’ for the group of serine proteases which share amino acid sequence homology with BPN’/NOVO in order to distinguish them from the (chymo)trypsin related serine proteases.