Screening, Characterization And Preliminary Purification Of Oxalate Oxidase From Plants

Oxalate oxidase(OXO,EC 1.2.3.4),which catalyzing the decomposition of oxalate to hydrogen peroxide and carbon dioxide,is potentail to quantify and scavenge oxalate in animal body thus to ameliorate the related diseases. With the aim to enlarge the resource of OXO,and to study the characteristics of OXO as related to medical usage,88 plant species from 45 families were used as raw material to screen Oxalate oxidase(OXO).The result showed that OXO was widely existed in plants,but their activities differed among plant species,and varied in different seasons or at different growth stages even in same species.Ten plant species were chosen as candidates to study their characteristics. The OXO from Lycianthes biflora(Lour.)Bitt showed optimal temperature at 55℃and pH at 3.0,and was stable at temperature range 30～60℃and pH range 2.0～4.0;The OXO from Baphicanthus cusia(Nees.)Bremek showed optimal temperature at 50℃and pH at 3.5,and was stable at temperature range 30～60℃and pH range 2.0～2.5;The OXO from Ardisia japonica (Thunb.)Blune showed optimal temperature at 45℃and pH at 3.0,and was stable at temperature range 45～60℃and pH range 2.0～2.5;The OXO from Kakanchoe diagremontiana Hamet et Perril showed optimal temperature at 45℃and pH at 3.5,and was stable at temperature range 30～45℃and pH range 2.0～2.5;The OXO from Cynanchum stauntonii(Decne.)Schltr.ex Levl showed optimal temperature at 50℃and pH at 3.5,and was stable at temperature range 40～65℃and pH range 2.0～2.5;The OXO from Sarcandra glabra(Thunb.)Nakai showed optimal temperature at 45℃and pH at 3.0, and was stable at temperature range 30～80℃and pH range 2.0～2.5;The OXO from Rhinacanthus nasutus(Linn.)Kurz showed optimal temperature at 35℃and pH at 3.0,and was stable at temperature range 30～55℃and pH range 2.5～3.0;The OXO from Glycyrriza Uralensis Fisch.G.Glabra L showed optimal temperature at 35℃and pH at 3.0,and was stable at temperature range 30～65℃and pH range 2.0～4.0;The OXO from Patrinia villosa (Thunb.)Juss showed optimal temperature at 35℃and pH at 4.0,and was stable at temperature range 45～60℃and pH range 3.5～4.5;The OXO from Echinacea purpurea Moench showed optimal temperature at 40℃and pH at 3.0,and was stable at temperature range 30～80℃and pH range 2.0～3.0.The OXOs all retained more than 27%of their maximal activities when heated at 80℃for 10 min.The OXOs were not inhibited significantly by Cl~-(10mmol/L)except that from Sarcandra glabra(Thunb.)Nakai,and not hydrolysised by pepsin except those from Baphicanthus cusia(Nees.) Bremek.,Cynanchum stauntonii(Decne.)Schltr.ex Levl,Sarcandra glabra (Thunb.)Nakai and Glycyriza Uralensis Fisch.Glabra L.The OXOs from Ardisia japonica(Thunb.)Blune,Rhinacanthus nasutus Linn and Sarcandra glabra(Thunb.)Nakai were inhibited significantly by SDS,while those from Lycianthes biflora(Lour.)Bitt,Baphicanthus cusia(Nees.)Bremek, Kakanchoe diagremontiana Hamet et Perril,Cynanchum stauntonii(Decne.) Schltr.ex Levl,Echinacea purpurea Moench,Patrinia villosa(Thunb.)Juss and Glycyriza Uralensis Fisch.Glabra L were not affected by SDS.The preliminary purification indicated that the activities of OXO from Lycianthes biflora(Lour.)Bitt and Sarcandra glabra(Thunb.)Nakai were increased by 2.29 and 5.59 fold,respectively,by ammonium sulfate fractionation and thermal treatment.Sephacryl S-200 High resolution chromatography and DEAE-Sepharose Fast Flow chromatography had no effect on purification of the OXO from Lycianthes biflora(Lour.)Bitt,and DEAE-Sepharose Fast Flow chromatography had no effect on purification of the OXO from Sarcandra glabra(Thunb.)Nakai.Through ammonium sulfate fractionation,thermal treatment and DEAE-Sepharose Fast Flow chromatography,OXO from Echinacea purpurea Moench was purified to 12.32 fold to a final activity of 0.764 U/mg protein.