The above mutations also caused slight change to the optimum temperature and pH required for enzyme hydrolysis. The optimum temperature for Y45N was reduced to 30 c' compered to 40 c' for wild-type ,while the optimum temperature for the other mutants remained unchanged (data not shown).Asn has a more flexible side chain than the side chain of Tyr, which consists of rigid phenyl group. This increased flexibility would typically cause enzyme instability. On the other hand , the optimum pH for M3751 changed to pH 8.0 compered to pH 7.0 for wild-type (data not shown). The enzyme's isoelectric point (pl) also changed when there were variations in amino acid composition dus to mutation,therefore altering the optimum pH for activity