Effects of ultrasound field on the catalytic kinetics of stem bromelain

Abstract

Previous researches have revealed that the catalytic ability of a protease appeared to be the best if it is in its native state as presented in the plant cells of origin. However, when the protease is extracted and applied to industrial uses, its proteolytic activity is much lower than that in the plant cells. This may be explained by two major aspects. First, the conformation of the enzyme including active site may be altered during extraction and hence the kinetic parameters are changed. Second, the environment for the optimum activity of the enzyme may be disturbed by the changes in type of substrates, pH and existence of other chemical substances. The purpose of this research is to study the effects of ultrasound field on the conformation and the catalytic activity of a typical protease, the stem bromelain (SB). The objectives include (1) to ascertain the influence of ultrasound field on the conformation and the catalytic efficiency of SB; (2) to study the effective mechanisms of ultrasound field based on the experimental findings; (3) to propose a hypothesis model for incorporating the effect of ultrasound into the enzyme kinetics. The experimental results show that the SB activity was first promoted at low ultrasound power (amplitude setting < 5%) and then gradually decreased at a higher power level (amplitude setting > 20%). When the treatment time was less than 5min, the effect of ultrasound on SB activities were similar for the power level at amplitude settings of 30% and 40%. It was also found that at amplitude setting of 5% for 5min, the maximum catalytic activity of SB was about 25% higher than that without treatment, while its activity reduced by about 40% at amplitude setting of 40%. At low ultrasound power, acoustic microstreaming was favored that facilitates the diffusion of substrate to or product away from the active site of the enzyme. It also enhances the 'lock and key' mechanism during enzyme and substrate binding. Thus, the increase in the catalytic efficiency was double at amplitude range of 5% to 10%. At higher ultrasound power, vigorous cavitation was created in the sonicated medium that caused enzyme denaturation resulting in a 50% decrease in the catalytic efficiency at amplitude setting of 40%. The analytic results indicated that ultrasound induced only a minor conformational change of SB without varying its secondary structures. The major change was caused by the interaction between exposed aromatic residues, which were originally buried in the inner zone of the protein, and their surrounding environments. That altered only the tertiary structure. Moreover, ultrasound also reduced the surface hydrophobicity of SB. These effects were more severe at high ultrasound amplitude (40%). As a result, the activity and catalytic efficiency was dramatically decreased. A hypothesis model for incorporating the effect of ultrasound into the enzyme kinetics was proposed by introducing the concept of activation power. This kinetic model suggested that the activation energy for the catalytic reaction could be replaced by the activation power and treatment time. A critical power zone should exist for a given system. Ultrasound activation and inactivation may occur before and after this zone respectively.