Monitoring the heat-induced structural changes of alkaline phosphatase by molecular modeling, fluorescence spectroscopy and inactivation kinetics investigations

Abstract

The heat induced conformational changes of calf alkaline phosphatase (ALP) were analyzed using different methods, based on fluorescence spectroscopy, m o l e c u l a r m o d e l i n g a n d i n a c t i v a ti o n s t u d i e s . Experimental studies were conducted in buffer solution in the temperature range between 25 and 70°C. Molecular dynamic (MD) simulation provided details on thermally induced changes in ALP structure, highlighting that heating favored the hydrophobic exposure and important alteration of the catalytic site above 60°C. Additional information to MD data were obtained by using different fluorescence spectroscopy methods, which revealed a complex mechanism of thermal denaturation. Therefore, the emissive properties indicated an unfolding of ALP at temperatures below 60°C, whereas at higher temperatures, the polypeptides chains fold leading to a higher exposure of Trp residues. In order to establish a structure-function relationship, the results were correlated with inactivation studies of ALP in buffer at pH 9.0. The inactivation data were fitted using a first-order kinetic model, resulting in an activation energy value of 207.26±21.68 kJ·mol −1 .

Molecular Dynamics Simulation for Mechanism Elucidation of Food Processing and Safety: State of the Art

“…Other studies involving alkaline phosphate lipase have shown that conformational changes caused by thermal treatment C 2018 Institute of Food Technologists ® Vol. 18, 2019 r Comprehensive Reviews in Food Science and Food Safety 253 were the loss of helix and the exposure of hydrophobic regions, when temperature increased from 25 to 60°C (Dumitrașcu et al, 2015). This study indicated that the unfolding of this enzyme was promoted at temperature above 60°C and that the interaction of the catalytic residues with other residues was changed, with disappearance and reforming of hydrogen bonds at temperatures over 60°C.…”

“…Simulations have shown that the exposure of hydrophobic core and the transition from an ordered to a disordered state are the common phenomena during the thermal denaturation of these proteins. Other studies involving alkaline phosphate lipase have shown that conformational changes caused by thermal treatment were the loss of helix and the exposure of hydrophobic regions, when temperature increased from 25 to 60 °C (Dumitrașcu et al., ). This study indicated that the unfolding of this enzyme was promoted at temperature above 60 °C and that the interaction of the catalytic residues with other residues was changed, with disappearance and reforming of hydrogen bonds at temperatures over 60 °C.…”

Biochemical characterization of digestive membrane‐associated alkaline phosphatase from the velvet bean caterpillar Anticarsia gemmatalis

“…Also, it was observed that enzymatic activity remained stable after 2 hr of incubation at 65°C (Figure c). Alkaline phosphatases with thermal stability have been described before in microorganisms, plant seeds, and bovine samples (Dumitrascu et al, ; Mahesh, Neha, Rajesh, Somashekhar, & Puttiah, ; Vemuri, Sunkara, Arumalla, Nelapati, & Lakkoju, ; Fadiloğlu, Erkmen, & Sekeroğlu, ). We chose to perform all next experiments in pH 9.5 at 37°C.…”

Development of a Continuous Pulsed Electric Field (PEF) Vortex-Flow Chamber for Improved Treatment Homogeneity Based on Hydrodynamic Optimization

“…where r RA is a reaction term considering the reduction of RA by thermal inactivation. The reaction is modeled as a first order kinetic, as described elsewhere (Jaeger et al, 2010;Dumitraşcu et al, 2015), thus…”