日本酚氧化酶的分离纯化及其部分生物化学性质研究  

Purification and partial characterization of phenoloxidase from crab Charybdis japonica

作　　者：杨玲玲[1] 樊廷俊[1] 丛日山[1] 汤志宏[1] 孙文杰[1] 刘光兴[1] 孟祥红[1] 朱丽岩[1] 

机构地区：[1]中国海洋大学生命科学与技术学部海洋生物系,山东青岛266003

出　　处：《海洋科学》2008年第2期29-35,共7页Marine Sciences

基　　金：国家863青年基金项目(2001AA649040);教育部重点项目(02132)

摘　　要：利用离子交换层析和凝胶过滤层析等方法,从日本(Charybdis japonica)血淋巴中分离纯化出了酚氧化酶,并以L-二羟苯丙氨酸(L-DOPA)作为特异性底物对其生化性质和酶性质进行了研究。结果表明,酚氧化酶和酚氧化酶原的分子质量分别为64.5 ku和69.5 ku。以L-DOPA为底物对酚氧化酶纯品进行研究发现,其最适pH值为6.0、最适温度为40℃。对底物L-DOPA和儿茶酚的米氏常数Km值分别为3.41和7.97 mmol/L。该酶对亚硫酸钠、苯硫脲极为敏感,对硫脲、苯甲酸非常敏感,表明该酶很可能是一种儿茶酚酶型的酶。此外,EDTA,DETC,Zn2+,Mg2+和Cu2+均能显著抑制该酶活性,且10 mmol/L Cu2+能有效地回复该酶被DETC所抑制的酶活性,表明该酶确为一种Cu-金属酶。Phenoloxidase （PO） from hemolymph of Charybdis japonica was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular weight and enzymatic properties by using L-dihydroxyphenylalanine （L-DOPA） as the specific substrate in this study. It was found that prophenoloxidase （proPO）, isolated as a monomeric protein, had a molecular weight of 69.5 ku, and a 64.5 ku PO molecule was often contained in preparations. The PO showed an optimal pH value of 6.0, and an optimal temperature of 40 ℃, respectively. And an apparent Km value of PO was 3.41 on L-DOPA, and 7. 97 on catechol, respectively. PO activity was extremely sensitive to sodium sulfite and 1-phenyl-2-thiourea, and quite sensitive to thiourea and benzoic acid. Based on its sensitivity to different oxidase inhibitors and a higher affinity on catechol, this PO was thus classified as an o-diphenoloxidase. The PO activity was also strongly inhibited by Zn^2＋ , Mg^2＋ , thylenediaminetetraacetic acid （EDTA） and diethyldithiocarbamate （DETC）. And the DETC-inhibited PO activity can be recovered perfectly by Cu^2＋. Then it can be concluded that Charybdis PO is most probably a metalloenzyme that required Cu^2＋ for its full activity.

关 键 词：日本蟳(Char ybdis ja ponica) 酚氧化酶 L-DOPA(L—dihydroxyphenylalanine) 儿茶酚酶 金属酶 

分 类 号：Q55[生物学—生物化学]