本文選題：百菌清水解脫氯酶(Chd)　切入點(diǎn)：分泌表達(dá)　出處：《南京農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：百菌清(四氯間苯二腈)是一種廣譜氯代苯腈類殺菌劑,在農(nóng)業(yè)生產(chǎn)中已使用30年之久,是世界上使用最廣泛的殺真菌劑之一,在環(huán)境中較穩(wěn)定、殘效期長(zhǎng),且具有明顯的蓄積毒性。百菌清的廣泛使用及其難降解的特性使得其在溫室大棚氣體、蔬菜和水果中都可被檢測(cè)到,其給人類健康和食品安全造成的嚴(yán)重威脅引起了廣泛關(guān)注。百菌清污染的生物降解被認(rèn)為是一種有效、安全可靠和無二次污染的方法,具有廣泛應(yīng)用前景。本實(shí)驗(yàn)室前期分離到一株高效降解百菌清的菌株P(guān)seudomonas sp.CTN-3,其編碼的新穎百菌清水解脫氯酶(Chd)無需其他輔因子就能將百菌清水解為毒性變小、水溶性變大的羥基百菌清。枯草芽孢桿菌由于其非致病性、分泌蛋白能力強(qiáng)和良好的發(fā)酵基礎(chǔ)應(yīng)用非常廣泛,是一些重要工業(yè)酶制劑的生產(chǎn)菌株。本論文將利用含P43啟動(dòng)子功能片段和NprB基因信號(hào)肽編碼序列,將chd基因整合到大腸桿菌-枯草芽孢桿菌穿梭載體PP43NMK中構(gòu)建分泌表達(dá)載體pP43Chd,將其轉(zhuǎn)化至枯草芽孢桿菌WB800的感受態(tài)中,成功實(shí)現(xiàn)chd基因在枯草芽孢桿菌WB800中的高效胞外分泌表達(dá),解決了 Chd胞內(nèi)表達(dá)而后續(xù)分離純化難度高的難題。重組枯草桿菌WB800(pP43Chd)經(jīng)液態(tài)發(fā)酵優(yōu)化(包括單因素實(shí)驗(yàn)、Placket-Burman 實(shí)驗(yàn)和 Box-Behnken Design 實(shí)驗(yàn)),發(fā)酵產(chǎn) Chd 酶活提高了 1.13 倍,達(dá)到14.50 U/1。獲得最佳培養(yǎng)基配方為:蛋白胨30 g/l、葡萄糖50 g/1、酵母提取物39 g/1、磷酸氫二鉀3 g/1、氯化鈉3 g/1、硫酸鎂3 g/l。最佳培養(yǎng)條件為:接種量10%、pH7.0、30ml/250ml的裝液量、培養(yǎng)溫度37℃、搖床轉(zhuǎn)速175rpm、培養(yǎng)12h后加入20 mg/1的百菌清,發(fā)酵培養(yǎng)24 h后測(cè)酶活。重組枯草桿菌WB800(pP43Chd)經(jīng)固態(tài)發(fā)酵優(yōu)化后,發(fā)酵產(chǎn)Chd酶活提高了 2.82倍,達(dá)到85.60 U/kg干基質(zhì),獲得固態(tài)發(fā)酵最佳培養(yǎng)基和培養(yǎng)條件為:小麥稈粉和麩皮各 2g、稻谷殼 1g,K2HPO4 0.4‰、MgSO40.2‰、蛋白胨5%、D-木糖 5%,含水量為60%,接種量為20%,固體基質(zhì)顆粒大小2.96 mm、堆料高度為3.3 cm、堆料密度為0.047g/cm3及孔隙度為84.86%時(shí),37‰發(fā)酵36h。用最優(yōu)液態(tài)發(fā)酵條件下發(fā)酵生產(chǎn)的Chd粗酶液對(duì)代表不同植物器官的六種蔬菜進(jìn)行百菌清殘留降解實(shí)驗(yàn)。結(jié)果表明Chd粗酶液對(duì)西紅柿中百菌清的降解效果最好,百菌清的降解率接近100%,其次是萵苣和生菜,百菌清降解率分別為82%和67%左右,對(duì)蘑菇、花菜和胡蘿卜中百菌清的降解率處于40%-55%之間。與此同時(shí),酶促反應(yīng)3 h后,從各種蔬菜洗脫到酶液中的百菌清也基本能完全被Chd降解,酶解反應(yīng)2 h后殘余Chd酶活還維持起始酶活的76%-86%之間。實(shí)驗(yàn)結(jié)果表明Chd粗酶液能有效去除百菌清污染的瓜果蔬菜表面的百菌清殘留,在解決瓜果蔬菜中百菌清農(nóng)藥殘留超標(biāo)污染具有一定的應(yīng)用前景。
[Abstract]:Chlorothalonil (tetrachloro-isophthalonitrile) is a broad-spectrum chlorobenzonitrile fungicides, which have been used in agricultural production for 30 years. It is one of the most widely used fungicides in the world.And has obvious accumulative toxicity.The widespread use of chlorothalonil and its non-degradable properties make it detectable in greenhouse gases vegetables and fruits. The serious threat to human health and food safety caused by chlorothalonil has attracted wide attention.Biodegradation of chlorothalonil is considered to be an effective, safe and reliable method without secondary pollution.A highly efficient chlorothalonil degrading strain Pseudomonas sp. CTN-3 was isolated in our laboratory, which encodes a novel chlorothalonil water dechlorination enzyme (Chd), which can hydrolyze chlorothalonil into hydroxy chlorothalonil which is less toxic and more water-soluble without other cofactors.Bacillus subtilis (Bacillus subtilis) is widely used for its non-pathogenicity, strong secretory protein ability and good fermentation foundation. It is a production strain of some important industrial enzyme preparations.In this paper, we will use the P43 promoter functional fragment and NprB gene signal peptide coding sequence.The chd gene was integrated into the Escherichia coli-Bacillus subtilis shuttle vector PP43NMK to construct the secretory expression vector pP43Chd. the expression of chd gene was successfully realized in the highly efficient extracellular secretion of Bacillus subtilis WB800 by transforming it into the competent state of Bacillus subtilis WB800.The problem of Chd expression in cell and the difficulty of further separation and purification was solved.The recombinant Bacillus subtilis WB800pP43Chd was optimized by liquid fermentation (including single factor experiment Placket-Burman test and Box-Behnken Design experiment). The activity of Chd produced by fermentation increased by 1.13 times to 14.50 U / 1.The optimum medium was as follows: peptone 30 g / l, glucose 50 g / 1, yeast extract 39 g / 1, potassium hydrogen phosphate 3 g / 1, sodium chloride 3 g / 1, magnesium sulfate 3 g / l.After the fermentation of recombinant Bacillus subtilis WB800pP43Chd, the enzyme activity of Chd was increased by 2.82 times to 85.60 U/kg dry substrate. The optimum medium and culture conditions were obtained as follows: wheat straw powder and bran 2 g each, and rice husk 1 g K2HPO 4 0.4 鈥，

本文編號(hào)：1722338