共生细菌SB_1降解聚乙烯醇的研究Ⅱ．共生细菌SB_1聚乙烯醇氧化酶的产生、纯

DEGRADATION oF PVA BY SYMBIOTIC BACTERIA SB_1Ⅱ.STUDY ON PRODUCTION,PUKRIFICATION AND PROPERTISE OF PVA OXIDASE OF SYMBIOTIC BACTERIA SB_1

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Ｈ＿２Ｏ＿２，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，Ｐｂ￣（２＋）和水杨酸可使ＰＶＡ氧化酶活力完全丧失。而Ｂａ￣（２＋）与ＮａＮ＿３则对酶活力有较强的激活作用。

The production and purification of PVA oxidase by symbiotic bacteria SB_1,as well asproperties of the enzyme were studied. When symbiotic bacteria SB_1 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 SB_1 strain by ammonium sulfate fractionation(0.4- 0. 5 saturation), colume chromatography on DEAE- Sephadex A_(25_ and 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 O_2,theproduction of H_2O_2 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, Pb￣(2+) and salicylate inactivated the enzyme compltely, and Ba￣(2+)andNaN_3 activated the enzyme.