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

Affiliation

Department of Agricultural Biotechnology, Seoul National University, Seoul, 08826, Republic of Korea; Center for Food and Bioconvergence, Seoul National University, Seoul, 08826, Republic of Korea; Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea. Electronic address: [Email]

Abstract

The effect of intense pulsed light (IPL) irradiation on Chromobacterium viscosum lipase was investigated with a primary focus on catalytic activity and molecular structure. During IPL irradiation, lipase activity decreased significantly with increasing pulse fluence (Fp) and exposure time (te). IPL-induced deactivation kinetics were further elucidated based on a two-step series-type deactivation model (constant deactivation rate k1 >k2). Fp was found to be the dominant variable affecting the degree of lipase deactivation, and residual activity was not associated with increasing te below a certain Fp energy density (2.66 mJ/cm2), implying a critical threshold for IPL-induced deactivation of lipase. From the results of fluorescence spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), we determined that IPL-induced deactivation was caused by fragmentation, leading to lipase tertiary structural changes. Furthermore, the results of FindPept analysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) indicated that the internal sensitive bonds of lipase were cleaved preferentially by IPL, such that IPL irradiation induced site-sensitive fragmentation and peptide bond cleavage.

Keywords

Chromobacterium viscosum lipase,Deactivation kinetics,Intense pulsed light,Peptide bond cleavage,Site-sensitive fragmentation,