本文關(guān)鍵詞： 漆酶 雙孢菇 催化氧化 兒茶素 對乙酰氨基酚 固定化　出處：《南京理工大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：漆酶(EC 1.10.3.2)是一種含銅的多酚氧化酶,廣泛存在于植物、細菌、高等真菌和昆蟲中,在食品工業(yè)、造紙工業(yè)、有機合成、工業(yè)染料和醫(yī)療等領(lǐng)域被廣泛應(yīng)用。漆酶在環(huán)境保護方面的利用也受到了人們的關(guān)注。本研究從雙孢菇子實體中提取漆酶,并以2'-聯(lián)氨-雙-3-乙基苯并噻唑啉-6-磺酸(ABTS)為底物研究漆酶的基本酶學(xué)性質(zhì)。雙孢菇子實體經(jīng)勻漿、過濾、硫酸銨沉淀、透析和陰離子交換層析,獲得的漆酶比活為56.58 U/mg蛋白質(zhì),回收率為3.9%,純化倍數(shù)為2.8。當(dāng)漆酶以ABTS為底物時,測得的酶反應(yīng)最適pH為2.4,最適溫度為65℃。雙孢菇漆酶在30℃和pH 2.4條件下穩(wěn)定性較好。0.05 mol/L的Cu2+對漆酶有激活作用,而Fe2+顯著抑制漆酶活性。在最適條件下,雙孢菇漆酶對ABTS的Km值為2.94 mmol/L,最大反應(yīng)速率Vmax為0.20 mmol/(L·min)。以雙孢菇漆酶為生物催化劑,探討了 pH分別對酶催化兒茶素和對乙酰氨基酚氧化的影響,并對比了其催化兒茶素和對乙酰氨基酚及兩者混合物氧化的效果。在30℃條件下,漆酶對兒茶素和對乙酰氨基酚的最適pH分別為3.0和2.2。在30℃,pH3.0條件下,加入1U/mL漆酶,反應(yīng)96h后5mmol/L兒茶素殘留為3%;在同等條件下,用漆酶處理等摩爾濃度的對乙酰氨基酚,其殘留量為21%。若反應(yīng)體系同時含有兒茶素和對乙酰氨基酚,漆酶優(yōu)先催化兒茶素氧化。本研究以納米SiO_2顆粒為固定化載體,確定了雙孢菇漆酶的固定化條件和酶負載量,并研究了固定化漆酶的酶學(xué)性質(zhì)。0.04g納米SiO_2顆粒加入4mL無水乙醇和80μL3-氨丙基三乙氧基硅烷(APTS),在30℃反應(yīng)2 h后得到表面氨基化的納米SiO_2顆粒;以戊二醛為交聯(lián)劑,在氨基化納米SiO_2懸浮液中加入雙孢菇漆酶,4℃下過夜反應(yīng),得到固定化漆酶。當(dāng)納米SiO_2顆粒的負載量為6U/g時,固定化收率最高。固定化漆酶的最適pH和最適溫度分別為2.2和65℃。在最適條件下,固定化漆酶對ABTS的Km為0.88 mmol/L,Vmax為8.32×10-3 mmol/(L·min);在30℃,pH2.2條件下,固定化酶的穩(wěn)定性最好,固定化漆酶耐酸性比游離酶強,可重復(fù)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，

本文編號：1507152