臺灣博碩士論文加值系統

新興汙染物為『新認定及尚未認定』或『未受法規規範』的化學物汙染物，對於人體健康以及生態環境具有風險性。新興汙染物可分為：『藥品及個人護理物品(pharmaceuticals and personal care products，PPCPs)』，『內分泌干擾物質(endocrine disrupting chmemicals，EDCs)』，『工業化學物質』，『生物可分解之合成化學物質』。四環黴素(tetracycline)為新興汙染物之一，屬於藥品及個人護理用品，廣泛用於人類與家畜的疾病治療，為人和家禽共用抗生素，一般污水系統無法有效去污水中的四環黴素，且在地表水中檢測出四環黴素。傳統汙染物的降解，大多使用細菌降解汙染物，然而用細菌降解抗生素是有風險的。白腐真菌(white root fungi)能產生木質素分解酵素，分解大部分的木質素和化合物，此木質素分解酵素系統也已被研究多年，並且廣泛運用在難分解的化合物。本研究利用白腐真菌Perenniporia tephropora TFRI707和實驗室分離株Cerrena unicolor A8所產生漆氧化酶(laccase)和錳離子過氧化酶(manganese Peroxidase)對四環黴素進行降解。首先找出培養基最佳條件，得知分離株A8和TFRI707在1 mM錳離子的GPCSL中能產生最多的漆氧化酶和錳離子過氧化酶。分離株A8及TFRI707在GPL和GPCSL培養基中降解100 ppm四環黴素，結果顯示四環黴素能被降解，且不同的培養基降解速度兩者相同；在培養第8天時加入四環黴素的降解速度，比培養第0天加入四環黴素的降解速度快。將分離株A8和TFRI707進行高溫滅菌，再令其降解四環黴素，結果顯示四環黴素的濃度並未下降，可知兩者的菌絲不會吸附四環黴素。使用無菌水將分離株A8和TFRI707菌絲上的培養基洗去後，置入四環黴素中進行降解，結果顯示兩者的菌絲在沒有培養基的環境下降解四環黴素的速度較慢。在GPL和GPCSL中，分別培養分離株A8和TFRI707 8天後，萃取出培養液中的酵素，並利用其酵素降解四環黴素，效果不佳，四環黴素依然能抑制E. coli 11848的生長。使用錳離子過氧化酶標準品及漆氧化酶標準品分別降解四環黴素，結果顯示錳離子過氧化酶在Sodium tartrate緩衝溶液中能降解四環黴素，而漆氧化酶在1-hydroxybenzotriazole (HBT) 緩衝液中，能在一小時內完全降解四環黴素。以上結果得知，分離株A8和TFRI707可產生漆氧化酶和錳離子過氧化酶並降解四環黴素，在沒有培養基時菌絲體也能降解四環黴素，且其菌絲不會吸附四環黴素。分離株A8和TFRI707培養基萃取酵素、漆氧化酶和錳離子過氧化酶可以降解四環黴素，但必須使用適當的培養基以及正確的介質(mediator)，方能更有效得降解四環黴素。

Emerging contaminants are either new or unrecognized chemical that pose risks on human health and ecological environment. It can be divided into four groups: "Pharmaceuticals and personal care products, PPCPs", "Endocrine disrupting chemicals, EDCs" "industrial chemicals "and “Biosynthesis chemicals". Tetracycline is one of the emerging contaminants and belongs to PPCPs. Tetracycline is widely used in the treatment of humans and animals. General sewage system cannot effectively remove tetracycline; also, tetracycline can be detected in most surface water. Traditionally, bacteria are used to break down pollutants, but there is a risk that it might create super bacterial resist with antibiotics. White rot fungi can break down most of the lignin and polycyclic compounds by lignin-degrading enzyme. Lignin-degrading enzyme has been studied for many years. It is widely used in the degradation of compounds which are difficult to degrade. In this study, we used Perenniporia tephropora TFRI707 and laboratory isolates Cerrena unicolor A8 to produce laccase and manganese peroxidase degrade tetracycline. Isolate A8 and TFRI707 could produce highest concentration of enzyme in the 1mM Mn2+ GPCSL medium. Isolate A8 and TFRI707 could degrade 100ppm tetracycline in the GPCSL and GPL, and their degradation rates in these two different mediums were the same. After high-temperature sterilization, Isolate A8 and TFRI707 could not degrade tetracycline in the GPL. This result confirmed that their mycelium do not adsorb tetracycline. After washing mycelium of Isolate A8 and TFRI707 by sterile water, their mycelium was put into tetracycline and executed degradation. The results showed that the degradation rates of both mycelium were slower without medium. Enzymes extracted from the culture medium of GPL and GPCSL had poor degradation ability and tetracycline still could inhibit growth of E.coli 11848. Using manganese peroxidase standard (SIGMA) and laccase standard (SIGMA) to degrade tetracycline, we found that manganese peroxidase could degrade tetracycline in sodium tartrate buffer, and laccase could degrade in 1-hydroxybenzotriazole (HBT) buffer in 1 hour. According to the results above, C. unicolor A8 and P. tephropora TFRI707 could degrade tetracycline, and their mycelium do not absorb tetracycline. Enzymes from mediums, laccase and manganese peroxidase also could degrade tetracycline when appropriate medium and correct mediator is involved. That is, it could be more effective to degrade tetracycline.

中文摘要........................................ I
Abstract....................................... IV
目 錄....................................... VI
表 目 錄...................................... X
圖 目 錄...................................... XI
前 言 ....................................... 1
一、 新興汙染物............................... 1
二、 四環黴素................................. 3
三、 細菌分解抗生素及抗藥性..................... 5
四、 白腐真菌................................. 7
五、 白腐真菌之胞外木質素分解酵素系統............ 9
六、 漆氧化酶的催化與特性...................... 10
七、 錳離子過氧化酶的催化與特性................. 12
八、 白腐真菌降解抗生素 ........................ 14
實 驗 目 的................................... 17
材 料 方 法................................... 18
一、 菌株來源與保存............................ 18
二、 培養基成分............................... 18
三、 菌種保存................................. 19
四、 漆氧化酶酵素活性測量....................... 19
五、 錳離子過氧化酶酵素活性測量................. 20
六、 四環黴素的偵測............................ 22
七、 抗菌能力的測試............................ 23
八、 漆氧化酶產量和錳離子過氧化酶之初步篩選....... 24
九、 培養基條件最佳化.......................... 24
十、 分離株A8和TFRI707於培養基中降解四環黴素..... 25
十一、 分離株A8和TFRI707菌絲吸附四環黴素.......... 25
十二、 分離株A8和TFRI707的菌絲降解四環黴素........ 26
十三、 萃取培養基中的酵素........................ 27
十四、 培養基萃取酵素降解四環黴素................. 27
十五、 漆氧化酶降解四環黴素...................... 28
十六、 錳離子過氧化酶降解四環黴素................. 29
十七、 酵素活性測量方法的確認..................... 30
結 果..................................... 31
一、 漆氧化酶產量和錳離子過氧化酶之初步篩選....... 31
二、 培養基條件最佳化.......................... 31
三、 四環黴素濃度的測量 ......................... 33
四、 分離株A8和TFRI707於培養基中降解四環黴素..... 33
五、 分離株A8和TFRI707吸附四環黴素.............. 43
六、 分離株A8和TFRI707的菌絲降解四環黴素........ 45
七、 培養液的酵素降解四環黴素................... 48
八、 漆氧化酶降解四環黴素...................... 50
九、 錳離子過氧化酶降解四環黴素................. 51
十、 酵素活性測量法的準確性.................... 53
討 論.................................... 54
結 論.................................... 67
參 考 文 獻................................... 69

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