The Influence Of High Pressure Processing On The Catalytic Mechanism Of Myrosinase From White Mustard Seeds

Myrosinase has great value to be researched since it can catalyze glucosinolates glycosidase into isothiocyanates which has anti-cancer effects. This study focused on the myrosinase in white mustard seeds, and its enzymic characteristics were studied. Enzyme activity, reaction kinetics and enzymes conformation have been studied to analyse the effects of high pressure processing on myrosinase catalytic reaction. Meanwhile, the effects of pressure and hydroxyl alcohol on thermal stability and reaction proceeding of myrosinase were studied. The aim was to make up the research gaps of the effects of high pressure processing(HPP) on myrosinase and expand the application of myrosinase in food processing.1. The myrosinase was extracted and purificated from white mustard seeds by ammonium sulfate precipitation, dialysis and Con A affinity column chromatography.The results showed that the most active component was that extracted from 0-20% ammonium sulfate saturation and the activity was 7.8 U/mg, so this component was used in the subsequent enzymic characteristics research.2. Molecular weight of myrosinase was determined by SDS-Page, and enzyme activity was determined by the method of glucose kits in combination with high performance liquid chromatography(HPLC) to research its enzymic characteristics, The results showed that the molecular weight of myrosinase from white mustard seeds was about 75 k Da, the optimum p H was 7 and the optimum temperature was 55 °C; Na Cl could inhibite myrosinase activity; Vc could activate enzyme effectively, the enzyme activity in 0.1 mmol/L Vc was 4.8 times higher than that of no Vc added; Enzyme concentration and enzyme activity did not have a linear positive correlation; the Km and maximum reaction rate Vmax under the atmospheric pressure(0.1 MPa)were 1.265 mmol/L and 0.150 mmol/min, respectively.3. The effects of high pressure processing(HPP) on myrosinase catalytic reaction by different factors were studied. These factors included stress intensity, treatment time, p H, buffer and enzyme stabilizers(hydroxyl alcohol). The results were as follows:(1)From 200 to 600 MPa, myrosinase enzyme activity reduced wih pressure rising, and enzyme activity under 600 MPa was 0.53 times lower than that of 0.1 MPa.(2)The effects of different conditions on enzyme activity under different p H and buffer were analyzed. Under 200 MPa, the highest enzyme activity was showed at p H 6, but the activity was greatly influenced by buffer type; Ammonium acetate buffer was an appropriate buffer for catalytic reaction under HPP.(3)At the same time, The enzyme activity under different pressure holding time were analyzed. It was found that increasing the holding time(from 1 min extended to 20 min) had less effect on the enzyme activity, ANOVA analysis showed that the holding time had no statistical difference between the influence of enzyme activity.(4)The effects of enzyme stabilizers hydroxyl alcohol(glycerol and sorbitol) on thermal stability of myrosinase were studied. It was found that 200 MPa treatment reduced the thermal stability of myrosinase, but hydroxyl alcohol under certain conditions could have effective protection. In the case of adding 0.1 mol/L glycerin, glycerol had certain protective effect on myrosinase under high temperature(>65 °C), improved the thermal stability of myrosinase; The thermal stability of myrosinase could be improved by adding 3 mol/L sorbitol. The effects of hydroxyl alcohol on reaction proceeding of myrosinase were studied. It was found that the myrosinase catalytic reaction process could be promoted by adding glycerol but not observably under 0.1 MPa; The myrosinase catalytic reaction process could be promoted by adding sorbitol, and apparent promoting effect showed under high pressure.(5)The kinetic parameters under different HPP were calculated. HPP could improve the affinity ability between myrosinase and substrate. Compared with normal pressure, Km increased firstly and then decreased from 100 to 600 MPa but lower than the value of normal pressure. With the pressure increasing, the maximum reaction rate Vmax showed a downward trend. And reaction activation volume In 0.1 to 600 Mpa at room temperature(25 °C) conditions was calculated, the activation volume △V≠= 4.46 cm3/mol, which indicated that the enzyme catalytic reaction speed could be reduced by improving pressure.4.The effects of HPP on activity of purified myrosinase enzyme have been studied. Circular Dichroism(CD) and Fluorescence spectrum were used to study the effects of HPP on the conformation of purified myrosinase. The results indicated that after high pressure processing, the enzyme activity of purified myrosinase was improved, and disulfide bond content was also increased. HPP could change the secondary structure, the α-helix contents raised and β-sheet contents decreased as the pressure increased. Fluorescence spectrum of myrosinase indicated that HPP could change the tertiary structure, make more hydrophobic Trp expose to polar environment and enhance the enzyme fluid fluorescence intensity.