【作者】 刘孝龙；

【导师】 刘玉焕；

【作者基本信息】 中山大学 ， 生物技术， 2010， 硕士

【摘要】 拟除虫菊酯类农药具有杀虫活性高、杀虫方式多样和用量少等优点,广泛应用于人们的生产和生活中,占整个农药市场的20%以上。但研究发现,其对环境和非目标生物并非完全安全,菊酯类农药的残留问题越来越受到重视。目前,对拟除虫菊酯类农药残留的处理方法主要有物理法、化学法和生物法,其中生物法尤其是生物酶类,在处理拟除虫菊酯类农药的残留时,具有操作简单、安全高效、应用范围广且无二次污染等优点,因而,在污染环境的修复中具有广阔的应用前景。本研究采用大肠杆菌表达系统,诱导表达来源于海底泥宏基因组文库的新型菊酯类农药降解酯酶基因est825。通过表达条件优化和酶学性质研究,在30℃、1.0 mM IPTG条件下诱导8 h,该重组酯酶（Est825）得到高效可溶表达,表达量为200 mg/L;SDS-PAGE电泳分析Est825的分子量为34.1 kDa（其中还有4 kDa的融合标签）;最适反应温度和pH值分别为40℃和6.5;70℃保温2 h后,仍保留50%的酶活性,表明Est825具有较好的热稳定性;1 mM Al³⁺以及1% （w/v） Triton X-100和Tween 80对Est825具有不同程度的激活作用;37℃反应1 h,Est825对氯氟氰菊酯、氯氰菊酯、氰戊菊酯和溴氰菊酯的降解率分别达到80.66%、84.95%、81.90%和76.75%,表明其具有广泛的底物特异性和较好的菊酯类农药降解能力。为进一步提高Est825对菊酯类农药的降解能力,本研究利用易错PCR对est825进行体外定向进化,获得突变酶EstM46。研究突变酶的酶学性质,并与野生酶比较,其酶活性提高1.5倍;最适反应温度提高至45℃;70℃保温2 h后保留约80%的酶活性,热稳定性得到了进一步提高;在相同的条件下,EstM46对上述四种菊酯类农药的降解率分别提升至92.21%、99.75%、93.21%和89.48%,表明突变酶对菊酯类农药的降解能力更强,在处理菊酯类农药残留方面具有良好的应用潜力。更多还原

【Abstract】 Pyrethroids were widely used in our daily life, accounting for more than 20% of the world pesticide market because of its bio-efficiency and safety to human beings. As pyrethroids were not completely safe for the environment, it took more and more attention to deal with the problem of residual amount. So far, the methods used to eliminate the pyrethroids were physical, chemical and biological processes. Among these methods, the biological processes, especially pyrethroids degradated by the biological enzyme, showed potential industrial application due to its environmental safety and no secondary pollution. Over the past two decades, most of the pyrethroid hydrolases were isolated from the cultural microorganisms.In this study, a novel pyrethroids hydrolase gene （est825） originating from a marine mud metagenomic library was ligated into pET-28a（+） and transformed into Escherichia coli BL21（DE3）. The expression product reached to 200 mg/L after induced for 8 h at 30°C with 1.0 mM IPTG. SDS-PAGE analysis revealed that the molecular mass of recombinant Est825 was 30.1 kDa. The optimum pH and temperature of Est825 were 6.5 and 40℃, respectively. It kept more than 50% of its activity after incubated for 2 h at 70℃. The enzyme activity was increased by the presence of 1mM Al³⁺, 1% Triton X-100 and 1% Tween 80. Fortunately, Est825 could degradate cyhalothrin, cypermethrin, sumicidin and deltamethrin at 37℃for 1h with the degradation rates of 80.66%、84.95%、81.90% and 76.75%, respectively.In order to improve the ability of Est825 for pyrethroids degradation, est825 was motified by directed evolution. Random mutations were introduced through error-prone PCR （epPCR）. A mutant enzyme EstM46 was obtained and its characterization was researched. Compared with Est825, the activity of EstM46 was increased by 1.5 times and its optimum temperature was increased to 45℃. EstM46 showed more thermal stability by remaining 80% of its activity after incubated at 70℃for 2 h. About 92.21% cyhalothrin, 99.75% cypermethrin, 93.21% sumicidin and 89.48% deltamethrin were hydrolyzed by EstM46 at 37℃for 1 h.The results showed that a new marine mud-oriented pyrethroid-hydrolyzing esterase Est825 degraded a variety of pyrethroids with broader substrate specificities and higher activity made it an ideal candidate for situ bioremediation where pyrethroids caused environmental contamination problems. Moreover, the degradation activity was further enhanced by epPCR, which promote its potential applications in dealing with residual pyrethroid pesticides.更多还原