title: Digestion of protein substrates by subtilisin: immobilization changes the pattern of products.
abstract: Subtilisin BPN' (Bacillus protease strain N') was immobilized on glass-bead carriers of controlled pore size by the glutaraldehyde method. The Vmax and Km values of the synthetic substrate were similar for immobilized and free enzymes. However, the hydrolytic patterns of immobilized and free enzymes toward casein and carboxymethylated lysozyme were different. The free enzyme rapidly hydrolyzed the substrate in the early stage of the reaction to produce peptides of various sizes. The immobilized enzyme, however, slowly digested the casein and lysozyme during digestion; even in the late stage of digestion the original substrates were present in the reaction mixture. The peptide size produced by immobilized enzyme depended on the pore size of the carrier; enzyme immobilized on glass of smaller pore size produced smaller peptide products. These phenomena found with our system of immobilized protease and a protein substrate can be explained by a multiple attack mechanism, in which the substrate that has been forced to enter the matrix is attacked many times by the protease to be completely hydrolyzed, because the substrate and the intermediate-sized product are trapped inside the matrix under reduced diffusion movement. To explain the effective digestion that forms amino acids, we have proposed that a multiple type of attack is responsible for the intracellular protein degradation that takes place in cellular organelles in which hydrolytic enzymes are entrapped.
mesh_terms: D002364:Caseins / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005898:Glass; D006868:Hydrolysis; D007700:Kinetics; D009113:Muramidase / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D013381:Subtilisins / Q000378:metabolism*
chemical_list: D002364:Caseins; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D009113:Muramidase; D013381:Subtilisins