Study On Purification And Characterisation Of Peroxidase From Honeysuckle

Honeysuckle is one kind of food and medicine homologous food that is widely used for food processing.Fresh honeysuckle is prone to browning during processing that leads to loss of functional ingredients and appearance deterioration.Enzymatic browning is an important reason for the deterioration of honeysuckle quality.Studies had shown that peroxidase could be involved in enzymatic browning reactions.Based on extracting and purification of honeysuckle peroxidse by three phase partitioning and Ion exchange chromatography.This paper studied enzymatic properties and inhibitory effects of some kinds of inhibitors,which provided a theoretical basis for controlling enzymatic browning and clarifying the mechanism of enzymatic browning metabolism.The main conclusions are as follows:Based on single factor experiment and orthogonal experiment design,The optimum extraction conditions were as follows: the extraction time was 1 h,the ratio of material to liquid was 1: 7,the buffer pH was 6,under which the the specific activity of honeysuckle peroxidase was 173.27U/ mg.The factors affecting the extraction efficiency of honeysuckle peroxidase were the pH of the buffer,the extraction time,the ratio of material to liquid.The results of variance analysis showed that the effect of buffer pH on the extraction efficiency was significant.Three phase partitioning was used to extract and purify peroxidase from Lonicera Japonica Thunb.The optimal conditions were obtained as follows: pH 5.6,ammonium sulfate concentration(w/v)39.49%,ratio of t-butanol / crude extract(v/v),1.38.Under this condition,the activity recovery was 87.64% and purification factor was 5.849,and a specific activity of 1021.6U/mg was obtained.92% pigment was removed by using three phase partitioning.DEAE 52-cellulose ion exchange chromatography was used for purification of peroxidase from Lonicera Japonica Thunb prepared preliminarily by three-phase partitioning.Two kinds of peroxides,HSP?(honeysuckle peroxidase?)and HSP ?(honeysuckle peroxidase ?),were purified,the specific activity were3312.3U/mg and 564.8U/mg respectively.Studies on the enzymatic properties of honeysuckle peroxidase showed that optimum temperature of this enzyme was 30?,and its Optimum pH was 5.Thermal stability of honeysuckle peroxidase was higher between 10?-40?,and pH stability was higher between4-7.When the concentration of H2O2 was 1 mmol / L,the rate of enzymatic reaction was stable.And when the concentration of guaiacol was 96 mmol /L,the rate of enzymatic reaction became stable.The kinetic analysis of the reaction of honeysuckle peroxidase showed that the two-substrate enzymatic reaction of honeysuckle peroxidase was a ping-pong mechanism.The Km value of the enzyme to guaiacol is 8.12mmol/L and the Vmax is 1.71mmol/L·min.The Km of H2O2 was0.822mmol/L and the Vmax was 1.38 mmol/L·min.Ca2+,Cu2+,Zn2+ could activate honeysuckle peroxidase activity,Mg2+,Mn2+,citric acid,ascorbic acid,L-cysteine,sodium sulphite and sodium metabisulphite showed inhibitory effect.Inhibitory effects and inhibitory kinetics studies of L-cysteine,citric acid,sodium metabisulfite,SDS on honeysuckle peroxidase showed that,the order of inhibitory effect was sodium metabisulfite,L-cysteine,citric acid,SDS.Inhibition type of citric acid,sodium metabisulfite on honeysuckle peroxidase was irreversible inhibition.Inhibition type of L-cysteine and SDS on honeysuckle peroxidase was reversible inhibition.And L-cysteine was a competitive inhibitor that the inhibition constant KI was 0.053mmol/L.And SDS was a non-competitive inhibitor that the inhibition constant KI was 13.4mmol/L and KIS was 11.5mmol/L.