百菌清水解脱氯酶（Chd）的枯草芽孢杆菌分泌表达、发酵优化及应用

发布时间：2018-04-08 16:07

  本文选题：百菌清水解脱氯酶(Chd)　切入点：分泌表达　出处：《南京农业大学》2015年硕士论文

【摘要】：百菌清(四氯间苯二腈)是一种广谱氯代苯腈类杀菌剂,在农业生产中已使用30年之久,是世界上使用最广泛的杀真菌剂之一,在环境中较稳定、残效期长,且具有明显的蓄积毒性。百菌清的广泛使用及其难降解的特性使得其在温室大棚气体、蔬菜和水果中都可被检测到,其给人类健康和食品安全造成的严重威胁引起了广泛关注。百菌清污染的生物降解被认为是一种有效、安全可靠和无二次污染的方法,具有广泛应用前景。本实验室前期分离到一株高效降解百菌清的菌株Pseudomonas sp.CTN-3,其编码的新颖百菌清水解脱氯酶(Chd)无需其他辅因子就能将百菌清水解为毒性变小、水溶性变大的羟基百菌清。枯草芽孢杆菌由于其非致病性、分泌蛋白能力强和良好的发酵基础应用非常广泛,是一些重要工业酶制剂的生产菌株。本论文将利用含P43启动子功能片段和NprB基因信号肽编码序列,将chd基因整合到大肠杆菌-枯草芽孢杆菌穿梭载体PP43NMK中构建分泌表达载体pP43Chd,将其转化至枯草芽孢杆菌WB800的感受态中,成功实现chd基因在枯草芽孢杆菌WB800中的高效胞外分泌表达,解决了 Chd胞内表达而后续分离纯化难度高的难题。重组枯草杆菌WB800(pP43Chd)经液态发酵优化(包括单因素实验、Placket-Burman 实验和 Box-Behnken Design 实验),发酵产 Chd 酶活提高了 1.13 倍,达到14.50 U/1。获得最佳培养基配方为:蛋白胨30 g/l、葡萄糖50 g/1、酵母提取物39 g/1、磷酸氢二钾3 g/1、氯化钠3 g/1、硫酸镁3 g/l。最佳培养条件为:接种量10%、pH7.0、30ml/250ml的装液量、培养温度37℃、摇床转速175rpm、培养12h后加入20 mg/1的百菌清,发酵培养24 h后测酶活。重组枯草杆菌WB800(pP43Chd)经固态发酵优化后,发酵产Chd酶活提高了 2.82倍,达到85.60 U/kg干基质,获得固态发酵最佳培养基和培养条件为:小麦秆粉和麸皮各 2g、稻谷壳 1g,K2HPO4 0.4‰、MgSO40.2‰、蛋白胨5%、D-木糖 5%,含水量为60%,接种量为20%,固体基质颗粒大小2.96 mm、堆料高度为3.3 cm、堆料密度为0.047g/cm3及孔隙度为84.86%时,37‰发酵36h。用最优液态发酵条件下发酵生产的Chd粗酶液对代表不同植物器官的六种蔬菜进行百菌清残留降解实验。结果表明Chd粗酶液对西红柿中百菌清的降解效果最好,百菌清的降解率接近100%,其次是莴苣和生菜,百菌清降解率分别为82%和67%左右,对蘑菇、花菜和胡萝卜中百菌清的降解率处于40%-55%之间。与此同时,酶促反应3 h后,从各种蔬菜洗脱到酶液中的百菌清也基本能完全被Chd降解,酶解反应2 h后残余Chd酶活还维持起始酶活的76%-86%之间。实验结果表明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残留,在解决瓜果蔬菜中百菌清农药残留超标污染具有一定的应用前景。
[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，

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