本文選題：炔苯酰草胺 + 生物降解��； 參考：《南京農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：炔苯酰草胺是1969年美國羅門哈斯公司(RohmHaas)開發(fā)的一種芽后處理的內(nèi)吸傳導(dǎo)型選擇性苯甲酰胺類除草劑,適用作物為苜蓿、大豆、豌豆、萵苣、棉花、向日葵等,對(duì)一年生、多年生雜草和某些闊葉雜草有很好的抑制和鏟除效果。因?yàn)槿脖锦２莅烦莼钚愿哂謱?duì)哺乳動(dòng)物低毒,所以被廣泛使用。但是過度、頻繁的使用炔苯酰草胺,引起了一些環(huán)境問題。因此,尋求能夠把炔苯酰草胺殘留從環(huán)境中消除的有效策略是很有必要的。生物降解被認(rèn)為是消除環(huán)境中污染物的主要因素。目前已經(jīng)分離到了許多能夠降解有機(jī)污染物的微生物,但是關(guān)于炔苯酰草胺的降解菌的研究未見報(bào)道.本研究從長期受農(nóng)藥污染的土壤中分離出能夠快速降炔苯酰草胺的菌株W1,根據(jù)表型特性、生理生化特征和16SrDNA系統(tǒng)發(fā)育分析,將其鑒定為睪丸酮叢毛單胞菌(Comamonas testoteroni)。菌株W1最適生長溫度和pH分別30℃和7.0,為好氧性生長,當(dāng)NaCl濃度小于2%時(shí),菌體生長良好。鑒定了菌株W1代謝炔苯酰草胺的中間產(chǎn)物為3,5-二氯苯甲酸,即該菌株降解炔苯酰草胺是依靠斷裂酰胺鍵來實(shí)現(xiàn)的,而且它能進(jìn)一步降解3,5--二氯苯甲酸。菌株W1其降解炔苯酰草胺的最適溫度和pH分別為30 ℃和7.0。在該條件下,5%接種量的菌株W1在18 h內(nèi)可將80 mg L~(-1)的炔苯酰草胺降解99%以上。但是該菌株不能降解包括敵稗、敵草隆、異丙隆、滅草隆、甲草胺、乙草胺、丙草胺、丁草胺在內(nèi)的酰胺類除草劑。由于3,5-二氯苯甲酸的水溶性比炔苯酰草胺的水溶性高,所以當(dāng)炔苯酰草胺被轉(zhuǎn)化成3,5二氯苯甲酸時(shí),在含有300 mg L~(-1)炔苯酰草胺的LB平板上會(huì)形成透明的水解圈。以此作為篩選標(biāo)記,從W1的基因組文庫中克隆出一個(gè)新的酰胺水解酶的基因camH,該基因大小為1452 bp,編碼483個(gè)氨基酸,G+C含量為49.30%。camH基因序列的Blast比對(duì)顯示,在氨基酸水平上與推測的酰胺酶最高相似性為41%�；盍ξ稽c(diǎn)分析發(fā)現(xiàn)CamH具有酰胺酶特征家族的保守的Lys-Ser-Ser催化中心。因此確定CamH是屬于酰胺酶特征家族的一個(gè)新成員。成功構(gòu)建了該基因的表達(dá)載體pET29a-camH;camH可以在E.coliBL21(DE3)中表達(dá),重組酶CamH經(jīng)Ni-NAT柱純化后獲得純酶。炔苯酰草胺水解酶CamH的最適反應(yīng)溫度為30 ℃,最適反應(yīng)pH值為8.0,具有較好的pH穩(wěn)定性和熱穩(wěn)定性。CamH以炔苯酰草胺為底物時(shí)的K_m和V_(max)分別為0.889mM和0.161mMmin~(-1)。
[Abstract]:Phenylethyl oxalamide is a selective benzoyl amine herbicide developed by Rohm Haas. in 1969. It is suitable for alfalfa, soybean, pea, lettuce, cotton, sunflower and so on.Perennial weeds and some broad-leaved weeds have good inhibition and eradication effects.It is widely used because of its high herbicidal activity and low toxicity to mammals.However, excessive and frequent use of phenylethyl oxalamide has caused some environmental problems.Therefore, it is necessary to search for an effective strategy to remove acetyloxalicamide residues from the environment.Biodegradation is considered to be a major factor in the elimination of pollutants in the environment.Many microorganisms have been isolated which can degrade organic pollutants, but there is no report on the biodegradation of acetyloxalicalamine.In this study, a strain W1 was isolated from soil contaminated by pesticide for a long time. According to phenotypic characteristics, physiological and biochemical characteristics and 16SrDNA phylogeny, the strain W1 was identified as Comamonas testoteroniae.The optimum growth temperature and pH of strain W1 were 30 鈩，

本文編號(hào)：1753769