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A protease-producing bacterium was isolated from slaughterhouse waste samples, Hyderabad, India. It was related to Bacillus cereus on the basis of 16S rRNA gene sequencing and biochemical properties. The protease was purified to homogeneity using ammonium sulfate precipitation, and ion exchange chromatography with a fold purification of 1.8 and a recovery of 49%. The enzyme had a relative molecular weight of 28kDa, pH and temperature optima for this protease were 10 and 60 degrees C. The activity was stable between a pH range of 7.0 and 12.0. The activity was inhibited by EDTA and enhanced (four-fold) by Cu(2+) ions indicating the presence of metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents and anionic surfactants. The enzyme also showed stability in the presence of organic solvents.

In many age-related and neurological diseases, formerly native proteins aggregate via formation of a partially unfolded intermediate. γS-crystallin is a highly stable structural protein of the eye lens. In the mouse Opj cataract, a non-conservative F9S mutation in the N-terminal domain core of γS allows adoption of a native fold but renders the protein susceptible to temperature and concentration-dependent aggregation, including fibril formation. Hydrogen/Deuterium exchange and denaturant unfolding studies on this mutant (Opj) have suggested the existence of a partially unfolded intermediate in its aggregation pathway. Here we have used NMR and fluorescence spectroscopy to obtain evidence for this intermediate. In 3.5 M urea Opj forms a stable and partially unfolded entity, characterized by an unstructured N-terminal domain and a largely intact C-terminal domain. Under physiologically relevant conditions, Carr-Purcell-Meiboom-Gill (CPMG) T2-relaxation dispersion experiments showed that the N-terminal domain residues were in conformational exchange with a loosely structured intermediate with a population of 1-2%, which increased with temperature. This provides direct evidence for a model in which proteins of native fold may explore an intermediate state with an increased propensity for formation of aggregates, such as fibrils. For the crystallins, this also shows how inherited sequence variants or environmentally induced modifications can destabilize a well-folded protein allowing the formation of intermediates able to act as nucleation sites for aggregation and the accumulation of light scattering centers in the cataractous lens.

BACKGROUND: Response surface methodology is used to build a predictive model of the combined effects of independent variables (pH, salt concentration starch and casein). The model was validated in a laboratory-scale bioreactor for extracellular protease production from a newly isolated Serratia rubidaea.