Enhancement of Heat Stability of Alkaline Protease by Chemical Modification

Several chemical modifications of a crystalline alkaline protease (Epolozyme) from Bacillus sp. were attempted to improve its heat stability. 1) Among the reagents tested for chemical modification, hexamethylenediisocyanate (HMD), N-bromosuccinimide (NBS) and iodine enhanced heat stability of the enzyme. 2) The heat stability of the enzyme modified with HMD increased most remarkably, although its enzymatic activity was severely damaged. This enhanced stability was probably due to the cross-linking of the enzyme molecules. 3) The enzyme modified with NBS or I2 retained 70% of its activity after 10 minute incubation at 60°C and pH 10.5, whereas the native enzyme retained only 9% of its activity after the same treatment. 4) When this phenomenon was further investigated in detail, it was found that (i) the optimal pH for the modification reactions was between 7 and 8.5, (ii) the modified enzymes had different UV spectra at the alkaline pH, (iii) heat stability enhanced only in the pH area where the enzyme was active, (iv) the modified enzymes were stable against autolysis and nitration of the enzyme with tetranitromethane (TNM) also increased heat stability. These results strongly suggested that the enhanced heat stability was due to the increased difficulty in splitting of tyrosyl peptide bonds in autolysis because the enzyme having specificity in splitting these bonds was modified at tyrosyl residues by I2 or TNM.