双孢菇漆酶的分离提取及其应用

发布时间：2018-02-13 02:49

  本文关键词： 漆酶 双孢菇 催化氧化 儿茶素 对乙酰氨基酚 固定化　出处：《南京理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：漆酶(EC 1.10.3.2)是一种含铜的多酚氧化酶,广泛存在于植物、细菌、高等真菌和昆虫中,在食品工业、造纸工业、有机合成、工业染料和医疗等领域被广泛应用。漆酶在环境保护方面的利用也受到了人们的关注。本研究从双孢菇子实体中提取漆酶,并以2'-联氨-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)为底物研究漆酶的基本酶学性质。双孢菇子实体经匀浆、过滤、硫酸铵沉淀、透析和阴离子交换层析,获得的漆酶比活为56.58 U/mg蛋白质,回收率为3.9%,纯化倍数为2.8。当漆酶以ABTS为底物时,测得的酶反应最适pH为2.4,最适温度为65℃。双孢菇漆酶在30℃和pH 2.4条件下稳定性较好。0.05 mol/L的Cu2+对漆酶有激活作用,而Fe2+显著抑制漆酶活性。在最适条件下,双孢菇漆酶对ABTS的Km值为2.94 mmol/L,最大反应速率Vmax为0.20 mmol/(L·min)。以双孢菇漆酶为生物催化剂,探讨了 pH分别对酶催化儿茶素和对乙酰氨基酚氧化的影响,并对比了其催化儿茶素和对乙酰氨基酚及两者混合物氧化的效果。在30℃条件下,漆酶对儿茶素和对乙酰氨基酚的最适pH分别为3.0和2.2。在30℃,pH3.0条件下,加入1U/mL漆酶,反应96h后5mmol/L儿茶素残留为3%;在同等条件下,用漆酶处理等摩尔浓度的对乙酰氨基酚,其残留量为21%。若反应体系同时含有儿茶素和对乙酰氨基酚,漆酶优先催化儿茶素氧化。本研究以纳米SiO_2颗粒为固定化载体,确定了双孢菇漆酶的固定化条件和酶负载量,并研究了固定化漆酶的酶学性质。0.04g纳米SiO_2颗粒加入4mL无水乙醇和80μL3-氨丙基三乙氧基硅烷(APTS),在30℃反应2 h后得到表面氨基化的纳米SiO_2颗粒;以戊二醛为交联剂,在氨基化纳米SiO_2悬浮液中加入双孢菇漆酶,4℃下过夜反应,得到固定化漆酶。当纳米SiO_2颗粒的负载量为6U/g时,固定化收率最高。固定化漆酶的最适pH和最适温度分别为2.2和65℃。在最适条件下,固定化漆酶对ABTS的Km为0.88 mmol/L,Vmax为8.32×10-3 mmol/(L·min);在30℃,pH2.2条件下,固定化酶的稳定性最好,固定化漆酶耐酸性比游离酶强,可重复使用5-10次。
[Abstract]:Laccase EC 1.10.3.2) is a copper-containing polyphenol oxidase found widely in plants, bacteria, higher fungi and insects, in the food industry, paper industry, organic synthesis, The use of laccase in environmental protection has attracted much attention. In this study, laccase was extracted from fruiting bodies of Pleurotus bisporus. The basic enzymatic properties of laccase were studied on the substrates of 2ON-HIGH-HYDROXYLAZOLINE-6-ETHYL benzothiazoline-6-sulfonic acid (ABTS). The fruiting bodies of Pleurotus bisporus were homogenized, filtered, precipitated with ammonium sulfate, dialysis and anion exchange chromatography. The specific activity of laccase was 56.58 U / mg protein, the recovery was 3.9 and the purification multiple was 2.8. When laccase was used as substrate, The optimum pH of enzyme reaction was 2.4 and the optimum temperature was 65 鈩，

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