This article reports a comparison of the ease of disruption of gram-positive (Lactobacillus acidophilus) and gram-negative (Pectobacterium carotovorum) bacteria using high-pressure homogenization (HPH). The location factor for both enzymes calculated from enzyme release kinetics together with localization studies identified them as cytoplasmic enzymes. The results showed that release of -galactosidase by HPH of L. acidophilus was more difficult than the release of L-asparaginase from P. carotovorum. It took nine passes at 55.14MPa for maximum release of -galactosidase (0.949 IU/mL) as compared to six passes at 41.35MPa for L-asparaginase (4.653 IU/mL); 1.7 IU of -galactosidase was released as against 11.5 IU of L-asparaginase per MJ of energy during high-pressure homogenization.