| Pulsed electric field(PEF) is an emerging non-thermal treatment technology that applies short pulses of high voltage across a food product placed between two electrodes. Given its high intensity, short time, small temperature rise and low power consumption, PEF has been widely received in food sterilization, enzyme inactivation, bioactive compounds extraction, maintaining food freshness, etc. and become one of research hotspots in food research and development worldwide. Interestingly, some enzyme activity is enhanced by treating with low-intensive PEF during enzyme inactivation. Although enhancements in enzyme activities have been previously investigated and reported, these studies mostly focused on the impact of PEF on the enzyme activation. The conformation and relevant mechanism have not yet been clearly clarified. To explore an efficient, safe, and speedy application of pulsed electric field(PEF) technology for enzymatic modification, effects of PEF treatment on the enzymatic activities, properties, kinetic parameters, thermostability and structure of three widely used glycosidase–α-amylase, glucoamylase and pectinase were investigated. The main conclusions of this study are as follows:(1) The maximum enzymatic activity of α-amylase, glucoamylase and pectinase was obtained under 15 k V/cm, 100 mL ? min-1; 15 k V/cm, 80 mL ? min-1 and 12 k V/cm, 80 mL ? min-1 PEF treatment, in which the enzymatic activity increased by 22.13 ± 1.14%, 20.74 ± 0.71% and 21.89 ± 1.67% respectively compared with control. Suggesting that activation of enzymes treated by low-intensity PEF was possible and activation effects of PEF on these three enzymes were α-amylase > pectinase > glucoamylase. This activation effects of α-amylase, glucoamylase and pectinase could last for 18 h, 23 h and 19 h respectively when they refrigerated at 4°C, and the storage stability of these enzymes were glucoamylase > pectinase > α-amylase.(2) PEF treatment extended the optimum temperature range of α-amylase, glucoamylase and pectinase from 55-60℃, 65-70℃, 50-55℃ to 50-60℃, 60-70℃, 45-55℃ respectively. Besides, the catalytic efficiency of α-amylase treated by PEF at 65℃ and 70℃ were significantly higher than those of untreated, while glucoamylase and pectinase behaved significantly higher catalyst efficiency during the temperature range of 40-60℃ and 30-45℃ respectively. However, it barely exerted any effect on the optimum pH and optimum pH range, the optimum pH of treated and untreated α-amylase, glucoamylase and pectinase were 6.0, 4.5 and 3.5; and the optimum pH range were 5.5-6.5, 3.5-5.0 and 3.5-4.0 respectively.(3) Experimatic kinetics results demonstrated that α-amylase, glucoamylase and pectinase treated by PEF showed a decrease in Km, whereas an increase of Vmax, kcat were observed. Indicating that the affinity between enzyme and their substrates were increased by PEF treatment. The catalytic efficiency increased by the rank of α-amylase > pectinase > glucoamylase.(4) k value(activation rate constant) of α-amylase, glucoamylase and pectinase under PEF treatment were significantly lower than control while t1/2(the half-life time) were significantly higher. Indicating that enzymatic activation occured with a lower rate compared with control, especially for α-amylase. Results also showed significantly decrease in Ea(activation energy), ΔS(entropy) and ΔH(enthalpy) after PEF treatment, whereas ΔG(free energy) were higher than those of untreated. The remarkable reduction in Ea value by PEF treatment demonstrated that the hydrolysis reaction catalyzed by these enzymes could occur more easily. The thermalstability of these enzymes enhanced at certain temperature, and stability enhancement of PEF were α-amylase > pectinase > glucoamylase.(5) SDS-PAGE results showed that polyacrylamide gel electrophoretic band of α-amylase, glucoamylase and pectinase remained unchanged after PEF treatment. Likewise, enzymic molecular weight remained unchanged and no new subunit was found.(6) It can be observed from CD spectra that α-helix of α-amylase, glucoamylase, pectinase increased by 34.76%, 25.86% and 10.28% respectively while random coil content decreased by 12.04%, 8.24% and 17.8% after PEF treatment. Suggesting that PEF treatment made enzymes exhibit more regularity. The degree of ordering was pectinase > α-amylase > glucoamylase according to the decreases in random coil content. Secondary structure of these enzymes changed.(7) Intrinsic fluorescence analysis showed that PEF processing enhanced fluorescence absorption of these enzyme, and fluorescence intensity increased by the order of α-amylase > glucoamylase > pectinase. Indicating that PEF treatment increased the number of amino acid residues, especially that of tryptophan on α-amylase, glucoamylase and pectinase surface, and therefore, the fluorescence increased. These changes in structure describe above have positive effects on enhancing α-amylase, glucoamylase and pectinase activities and properties. |
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