Effect of intense pulsed light on the deactivation of lipase: enzyme-deactivation kinetics and tertiary structural changes by fragmentation

Abstract

Intense pulsed light (IPL), a novel non-thermal technology, was applied to investigate the effect on activity and structure of Chromobacterium viscosum lipase (triacylglycerol hydrolase, EC 3.1.1.3) by employing hydrolysis of triacylglycerol in reverse micelle system. After IPL treatment, the lipase deactivation was observed, and the lipase activity was decreased with increase of pulse fluence and exposure time. The half-life of Chromobacterium viscosum lipase at 8.79 mJ/cm2 (37.63 min) was 4.76-fold longer than that at 14.86 mJ/cm2 (7.90 min). In order to determine deactivation constants (k1 and k2), two-step series type deactivation kinetic model was employed. As a result, k1 (9.05 h-1) and k2 (1.64 h-1) at 14.86 mJ/cm2 were 3.04, 3.88-fold higher than those at 8.79 mJ/cm2 (2.98 h-1, 0.42 h-1), respectively. Meanwhile, pulse fluence showed a dominant impact on deactivation, in that higher pulse fluence treatment with short exposure time showed more decreased activity at the same degree of total fluence. Also, no activity reduction phenomenon appeared below 2.66 mJ/cm2, demonstrating that the IPL treatment to the lipase was composed of susceptible phase and resistant phase. Based on the experiments in lipase hydrolysis, structural analysis was conducted in order to elucidate the cause of deactivation. From secondary structure analysis using circular dichroism spectroscopy, the result spectra were not fluctuated significantly. However, changes in its tertiary structure were detected through fluorescence spectroscopy. SDS-PAGE results showed that the amount of intact lipase (32 kDa) was reduced in a time dependent manner and uncertain fragmented peptides were increased below the main protein band. For validation of fragmentation sites in the sequence of lipase, the peptides were analyzed by MALDI-TOF analysis. It was revealed that the lipase was site-selectively fragmented (<5 kDa) by IPL treatment. Furthermore, evidences for peptide bond cleavage was identified using Findpept® tool. From these findings, it is evident that the lipase deactivation was occurred quantitatively in molecular level.