Tentative Exploration Of The Effect And Mechanism Of High Hydrostatic Pressure On Yarrowia Lipolytica Lipase Lip2

The application of high hydrostatic pressure(HHP) in food processing has recently attracted more and more attention. Its effect on the enzyme activity has also been taken into account. Yarrowia lipolytica Lipase Lip2(Y. lipolytica lipase) is of high catalytic activity, but so far, its characteristics under HHP have not been studied. Selecting Y. lipolytica lipase as object, this paper studied the effect of pressure, temperature, pH, dwell time and buffers on the lipase activity and conformation after HHP treatment and the effect of HHP on improving the thermal stability of the lipase.Firstly, Y. lipolytica lipase was purified using the strong anion purification column Mono Q 10/100 GL and the gel filtration column HiLoad 16/60 Superdex 200 prep. Checked by SDS-PAGE analysis, the purified lipase has been proved to be of electrophoretic homogeneity.Secondly, the influences of different factors on the lipase activity treated by HHP were studied. Results suggested that the enzyme activity of the lipase was improved by HHP. Optimization by orthogonal test suggested that at optimum conditions of pressure 450 MPa, temperature 45℃, dwell time 10 min, pH 7.5, the enzyme activity increased to 220%. The HHP treatment increased also the stability of the lipase; the maximum activity was observed at 45℃. With the extension of the dwell time, the lipase activity at first increased and then decreased, the highest lipase activity was obtained at dwell time of 10 min. The optimum pH shifted 0.5 towards alkaline direction. After HHP treatment, the enzyme activity decreased with the extension of storage time, then stabilized, because the lipase conformation was partially restored.The enzyme activity of lipase was influenced by environmental factors such as buffers and additives either at atmospheric pressure or at high pressure. Among all the buffers, phosphate buffer was the best. Polyol can stabilize the lipase. The effect was more obvious under atmospheric pressure. Mannitol had the highest stabilizing effect under high pressure: with the dose of 3 mg/mL, the lipase activity increased by 8%. The activity of immobilized lipase treated directly under high pressure declined, but the activity of that treated in phosphate buffer at first increased and then decreased with a maximum increase of 15% at 200 MPa.The conformational changes of the lipase were investigated by SDS-PAGE, circular dichroism(CD), intrinsic fluorescence spectroscopy and Native-PAGE, and their relationship to the activity changes was confirmed. It was found that no polymerization or changes of protein primary structure was induced by HHP. On the other hand, the secondary and tertiary structures were obviously modified by HHP. These conformational changes had a turning point at about 450 MPa, consisting with the evolution of the enzyme activity.The isokineticity diagram for the combined effects of pressure-temperature on thermal denaturation of protein was established. The elliptical shape of the diagram was close to that deduced from thermodynamics, indicating the stabilization of the lipase against thermal denaturation. The study showed that pressure has a remarkable effect on the stabilization of Y. lipolytica lipase.