Isolation and Screening of Bacillus Cereus from Soil, Followed By Media Optimization, Pilot Scale Production and Partial Purification of Protease Enzymes

Proteases are a unique class of enzymes, since they are of immense physiological as well as commercial importance. They possess both degradative and synthetic properties. Microbes are a gold mine of proteases and represents the preferred sources of enzymes in view of their rapid growth, limited space required for cultivation, and ready accessibility to genetic manipulation. Purification of protease to homogeneity is a perquisite for studying their mechanism of action. The catalytic site of protease is flanked on one or both sides by specificity sub sites, each able to accommodate the side chain of a single amino acid residue from the substrate. Serine protease usually follow a two step reaction for the hydrolysis in which a covalently linked enzyme peptide intermediate is formed with the loss of the amino acid or peptide fragment. This acylation step is followed by a deacylation process that occurs by a nucleophillic attack on the intermediate by water, resulting in hydrolysis of the peptide. Serine endopeptidases can be classified into 3 groups based mainly on their primary substrates preference: (i) trypsin like, which cleave after positively charged residues; (ii) elastase like, which cleave after small hydrophobic residues. Aspartic endopeptidases depend on the aspartic acid residue for their catalytic activity. Crystallographic studies have shown that the enzymes of the pepsin family are bilobed molecules with the active site cleft located between the lobes and each lobe contributing one of the pair of aspartic acid residues that is essential for the catalytic activity. The mechanism of action involves general acid base catalysis with lytic water molecule that directly participates in the reaction. These enzymes depend on the presence of bound divalent cations and can be inactivated by the dialysis or the addition of chelating agents. Most of the metallo protease are enzymes containing the His-Glu-Xaa-His(HEXXH) motif, which has been shown by X-ray crystallography to form a part of the site for binding of the metal, usually zinc. Cysteine protease catalyses the hydrolysis of a carboxylic acid derivates through a double displacement pathway involving general acid base formation and hydrolysis of an acyl-thiol intermediate. The mechanism of action of cysteine protease is thus very similar to that of serine protease. Studies of the mechanism of action of protease have revealed that they exhibit different types of mechanism based on their active-site configuration.