Purification and characterization of phenoloxidase from brine shrimp Artemia sinica  

作　　者：Tingjun Fan Zhao Jing Xianyuan Fan Miaomiao Yu Guojian Jiang 

机构地区：[1]Department of Marine Biology, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China

出　　处：《Acta Biochimica et Biophysica Sinica》2011年第9期722-728,共7页生物化学与生物物理学报（英文版）

摘　　要：Phenoloxidase from Artemia sinica （AsPO） was purified by Superdex 200 gel-Vdtration and Q Sepharose fast flow ionexchange chromatography, and its properties were characterized biochemically and enzymatically by using L-dihydroxyphenylalanine （L-DOPA） as the specific substrate. Results showed that AsPO was isolated as a monomeric protein of 125.5 kDa in molecular mass. The optimal pH value and temperature are 7.0 and 50℃, respectively, for its PO activity. The AsPO had an apparent Km value of 4.2 mM on L-DOPA, and 10.9 mM on catechol, respectively. Oxidase inhibitor on PO activity showed that the AsPO was extremely sensitive to ascorbic acid, sodium sulfite, and citric acid; and was very sensitive to eysteine, benzoic acid, and 1-phenyl-2-thiourea. Combined with its specific enzyme activity on L-DOPA and catechol, it can be concluded that AsPO is most probably a typical catechol-type O-dipheno- loxidase. Its PO activity was also sensitive to metal ions and chelators, and 20 mM DETC-inhibited PO activity was obviously recovered by 15 mM Cu^2＋, indicating that AsPO is most probably a copper-containing metaHoenzyme. All these data about specific substrate, sensitivity to oxidase inhibitor metal ions and chelators indicate that the AsPO has the properties of a catechol-type copper-containing Odiphenoloxidase that functions as a vital humoral factor in host defense via melaninization as in other Crustaceans.Phenoloxidase from Artemia sinica （AsPO） was purified by Superdex 200 gel-Vdtration and Q Sepharose fast flow ionexchange chromatography, and its properties were characterized biochemically and enzymatically by using L-dihydroxyphenylalanine （L-DOPA） as the specific substrate. Results showed that AsPO was isolated as a monomeric protein of 125.5 kDa in molecular mass. The optimal pH value and temperature are 7.0 and 50℃, respectively, for its PO activity. The AsPO had an apparent Km value of 4.2 mM on L-DOPA, and 10.9 mM on catechol, respectively. Oxidase inhibitor on PO activity showed that the AsPO was extremely sensitive to ascorbic acid, sodium sulfite, and citric acid; and was very sensitive to eysteine, benzoic acid, and 1-phenyl-2-thiourea. Combined with its specific enzyme activity on L-DOPA and catechol, it can be concluded that AsPO is most probably a typical catechol-type O-dipheno- loxidase. Its PO activity was also sensitive to metal ions and chelators, and 20 mM DETC-inhibited PO activity was obviously recovered by 15 mM Cu^2＋, indicating that AsPO is most probably a copper-containing metaHoenzyme. All these data about specific substrate, sensitivity to oxidase inhibitor metal ions and chelators indicate that the AsPO has the properties of a catechol-type copper-containing Odiphenoloxidase that functions as a vital humoral factor in host defense via melaninization as in other Crustaceans.

关 键 词：PHENOLOXIDASE Artemia sinica L- dihydroxyphenylalanine O-phenoloxidase METALLOENZYME 

分 类 号：TQ243.12[化学工程—有机化工] TS201.21[轻工技术与工程—食品科学]