Effect Of Dynamic High Pressure Microfluidization On Activity And Conformation Of Polygalacturonase

Dynamic High Pressure Microfluidization(DHPM) is a newly developed technology,which uses the combined forces of shear, cavitation, and ultra-high pressures up to 200 MPa.Polygalacturonase (PG) is a member of pectinase family that acts onα-1 4 linkages of polygalacturonic acid in pectin, a cementing substance in plant cell wall, and causes structural degradation. Its tissue-softening property is commercially exploited in the food industry in the extraction and clarification of fruit juices.This subject was to study the effect of DHPM on the activity and conformation of polygalacturonase mainly,effects of thermal,metal ions and ethanol on PG were observed at the same time by means of activity detection,far-UV circular dichroism(CD),UV absorption and sulphydryl groups detection..The result showed the relationship between activity change and conformation change of PG.The effect of DHPM on the activity and conformation of PG was showed as follows, the optimal temperature of PG is 50℃.DHPM treatment decreased the activity of PG treated under different pressures,the residual activity of PG was in the range 76.5%-94.5%.The analysis of conformation indicated that after microfluidization treatment under different pressure,the content ofβ-sheet reduced and PG structure became looser.In addition, there were some indices that the decrease of PG relative activity was accompanied by the decrease in theβ-sheet content. Tip and Tyr residues,which buried inside the PG molecular, exposed to the solvent, S-S bonds on the surface of PG were ruptured and formed new free SH residues, while the total amount of S-S bonds new formed was more than the amount of ruptured S-S bonds.Effect of combined thermal and DHPM on the activity and conformation of PG was studied,the result was showed as follows, The activity of enzyme incubated in 50℃for 30 min is the maxmium,the activities of PG incubated under other temperature were reducted, there was 39.2% of residual activity of enzyme incubated in 70℃for 30 min.The decrease of PG actvity under DHPM treatment after incubated under different temperature was more than the decrease of PG activity under DHPM treatment.lt demonsrated that the effect of DHPM-temperature treatment is more than DHPM or temperature treatment alone.Temperature profile of PG at the value of 218 nm showed that the denaturation temperature is in the range 60-65℃.The result indicated that PG is sensitiveness to temperature,the high temperature,specially.Some metal ions stimulate the activity of PG,and some inhibit,others had no significance effect on the activity of PG.This subject was to study the effect of Ca2+ Ba2+,Na+,Zn2+,Mn2+,Mg2+ on the activity and conformation of PG.The result showed that, Ca2+,Ba2+,Zn2+,Mg2+ inhibited the activity of PG, Mn2+ stimulate the activity of PG,and Na+ had no effect on the activity of PG.But the mechanism of their different effect on the PG was different.DHPM treatment had no effect on the result,except Mn2+, Mn2+ had no stimulation on the activity of PG after DHPM treatment.All these metal ions had no effect on the temperature profile of PG at the value of 218 nm.The changes of activity and conformation of polygalacturonase in ethanol(20%) were observed.The activity of PG in ethanol(20%) decreased,only 75% of redidual activity of PG leaved.The decreasement increased by the time goes.24 hours later,the decrease amplitude accelerated.However,ethanol(20%) had no conspicuous effect on the far-UV CD spectra of PG.It means that ethanol did not change the secondary structure,it had effect on the tertiary structure to change the activity of PG.Ethanol had no effect on the temperature profile of PG at the value of 218 nm,the denaturation temperature is in the range 60-65℃,which had no changed.