美洲黑杨I-69树皮中多酚氧化酶同工酶的提纯和特性

PURIFICATION AND PROPERTIES OF POLYPHENOLOXIDASE ISOZYMES FROM BARK OF POPULUS DELTOIDES I 69

美洲黑杨Ⅰ-69树皮内有两个多酚氧化酶同工酶(ppo A和B)。ppo A和B经硫酸铵沉淀,Sephadex G-100和DEAE-纤维素柱层析分别提纯6.7和5.9倍,聚丙烯酰胺凝胶电泳显示单一的带。ppo A和B的分子量为38900和33000。ppo A含赖氨酸残基最多(86个),含精氨酸残基最少(3个),不含酪氨酸和脯氨酸残基;ppo B含丝氨酸残基最多(37个),含精氨酸残基最少,不含酪氨酸和色氨酸残基。ppo A和B的最适pH为6.4和7.0,最适温度为50℃和45℃。ppo A比B具有较好的热稳定性。在测定的酚类化合物中,ppo A和B对邻苯二酚的亲和性最大,K_m值最小,分别为0.39mM和0.34mM,均属邻苯二酚酶。ppo A和B的活力受1mM的L-半胱氨酸、硫代硫酸钠、硫脲、二乙基二硫代氨基甲酸钠、氯化钠和蔗糖抑制及被0.1mM的氯化钠和蔗糖促进。

Tow polyphenoloxidase isozymes (ppo A and B) in crude extract from populus deltoides 1-69 bark were purified by ammonium sulfate fractionation. Sephadex C- 100 and DEAE-cellulose column chromatography to homogeneity. The data showed that final purification of ppo A and B obtained were 6. 7 and 5. 9 fold. Photographs ofpolyacrylamide dise gels stained with 0. 03 m catechol and 2% coomassie blue R-250 after electronphoresis of ppo A and B preparation showed single band, respectively. Molecular weight of ppo A and B determined by sodium dodecy 1.sulfate polyacryamide get electronphoresis was 38900 and 33000. From the amino acid composition, it was found that ppo A molecular contanied most large amounts of lysine (residue 86), least amounts of arginine (residue 3) and did not contain tyrosine, and proline, ppo B molecular contained largest amounts of serine (residue 37), least amounts of arginine (residue 3) and did not contain tyrosine. The ppo A and B gave pH optima of 6. 4 and 7. 0 and temperature optima of 50 C and 45 C.Thermostability of ppo A was relatively stable as compured with ppo B. Of a number of phenolic compound tested, Km value of ppo A and B for catechol was least (0. 39 and 0. 34 mM), they had largest affinity for catechol. Therefore, ppo A and B was catecholase. In a number of compound tested, 1mM L-systein, sodium thiosulfate, thiourea, sodium dithyldithiocarbamete, sodium chloride and sucrose produced an inhibiting effect on activity of ppo A and ppo B, 0.1mM sodium chloride and sucrose caused promotive effect of activity of ppo B.