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研究论文

DEGRADATION oF PVA BY SYMBIOTIC BACTERIA SB₁Ⅱ.STUDY ON PRODUCTION,PUKRIFICATION AND PROPERTISE OF PVA OXIDASE OF SYMBIOTIC BACTERIA SB₁

摘要：共生细菌SB_1接种在0.3%聚乙烯醇(PVA)培养基中,30℃摇床振荡培养(500mL三角瓶)或发酵罐(1000L)通气培养时,PVA氧化酶产生的高峰在48h或40h.离心收集的上清液采用40%-50%饱和度的硫酸铵分级沉淀DEAE-Sephadex离子交换柱和Cibacron蓝-Sepharose4B亲和柱层析等纯化步骤,PVA氧化酶收得率30%,比活力提高31倍,该酶经凝胶电泳后,用硝基四氮唑蓝进行活性染色,呈现四条谱带,PVA氧化酶与PVA作用,可释放H₂O₂,同时伴随着粘度降低,PVA氧化酶反应的最适pH为7.0,最适温度为40℃,该酶置-5℃贮存较稳定,贮存500天,其活力仍保留近50%,在所测试的金属离子和化学试剂中,Pb²⁺和水杨酸可使PVA氧化酶活力完全丧失。而Ba²⁺与NaN₃则对酶活力有较强的激活作用。

Abstract：The production and purification of PVA oxidase by symbiotic bacteria SB₁,as well asproperties of the enzyme were studied. When symbiotic bacteria SB₁ were cultured in 0.3%PVA medium under contiuous shaking at 30℃,the culmination of the enzyrme productionwas at 48h in flask (500 mL)and 40 h in fermentation jars( 1 ton). PVA oxidase waspurified from the culture supernatant of SB₁ strain by ammonium sulfate fractionation(0.4-0. 5 saturation), colume chromatography on DEAE-Sephadex A₍25and dye ligand chro-matography on the column of cibacron blue-Sepharose 4B with 31 fold purification and 30%recovery. the purified enzyme was applied to columns of 7.5%polyacrylamide gel. Afterelectrophyoresis,the gels were stained with nitro blue tetrazolium for activity and gave fouractivity bands, The enzyme catalyzed the oxidation of PVA with consumption of O₂,theproduction of H₂O₂ and viscosity decrease of the reaction mixture.VPA oxidase was mostactive at pH 7.0 and 40℃. Kept at-5℃, the enzyme was stable,Among metal ions andchmical reagents tested, Pb²⁺ and salicylate inactivated the enzyme compltely, and Ba²⁺andNaN₃ activated the enzyme.