厚朴过氧化物酶的纯化、性质及清除双酚A特性研究

Purification and Properties of Peroxidase from the Leaves of Magnolia officinalis Rehd.etWils and its Function of Removing Bisphenol A

厚朴为鄂西南道地药材,其药用部位主要为干皮和枝皮,在其漫长生长过程中产生的大量叶片不能实现经济价值;我们前期研究发现厚朴叶片具有较高过氧化物酶活性,而过氧化物酶在酚类物质生物降解途径中能够发挥重要作用。为充分开发厚朴叶的经济价值,进一步提高资源综合利用效率,我们对厚朴叶过氧化物酶进行了纯化并初步研究了其性质;在此基础上,进一步研究了厚朴叶过氧化物酶在降解环境污染物双酚A上的特性。结果显示,经过硫酸铵分级沉淀、DEAE-纤维素离子交换层析,从厚朴叶中分离提纯过氧化物酶,纯化倍数为52.8倍,回收率为14%。该酶的比活力为184140 U/mg蛋白,最适p H为6.0,对H_2O_2的Km值为4.23mmol/L,对愈创木酚的Km值为57.86 mmol/L,最适温度为80℃,热稳定性较好,75℃以下不易失活。其活性受到硫酸亚铁明显抑制。厚朴叶POD对BPA具有良好清除能力,在p H 4～9,温度25～65℃,H_2O_2/BPA浓度之比为0.8条件下,经过3 h,4000 U/L POD对0.2 mmol/L BPA清除率可达95%以上,MLP在酚类物质生物降解途径中具有潜在的重大利用价值。

As the local herbs of southwest of Hubei ,Magnolia officinalis Rehd. etWils is valued for its medicinal proper- ties on the bark. The leaves of Magnolia officinalis are ignored in the perspective of pharmaceutics. It was found that the leaves of Magnolia officinalis have the peroxidase activity in the previous studies. Regarding the compelling effect of per- oxidase in process of phenols biodegradation, the purification and properties of the peroxidase from the leaves of M. offici- nulls were studied to fully develop the value of the leaves and to improve the resource utilization efficiency. Based on that,its function of removing bisphenol A was further investigated. The results showed that, after ammonium sulfate pre- cipitation and DEAE-ccllulose ion-exchange chromatography,the yield of peroxidase purified from the leaves of M. offici- halls was 14% ,and purification fold was 52.8 with specific activity of 184140 U/rag. The optimal pH was 6.0. The Kin value of POD was 4.23 mmol/L on H2O2 ,and 57.86 mmol/L on guaiacol,respectively. The optimal temperature was 80 ℃. The activity of the enzyme could be maintained for a long time below 75 ℃ （good thermal stability）. Its activity was significantly inhibited by ferrous sulfate. M. officinalis leaves peroxidase had good scavenging ability on BPA. Under the conditions of pH 4-9,25-65 ℃ and tl202/BPA 0.8 ,the clearance rate of 0.2 mmol/L BPA derivatives for 4000U / L M. officinalis leaves peroxidase was 95% in 3 hours later. M. offIcinalis leaves peroxidase had convincing future in re- searches of phenols biodegradation.