来源于铜绿假单胞菌GF31的拟除虫菊酯降解酶在大肠杆菌细胞的功能性表面展示

Functional display of pyrethroid-degrading enzyme from Pseudomonas aeruginosa GF31 on the surface of Escherichia coli

拟除虫菊酯类农药由于其高疏水性和高分子质量,难以被微生物吸附、摄取和降解,如何提高此类底物的生物可及性是环境生物修复的关键之一.利用细胞表面展示技术将拟除虫菊酯降解酶展示到大肠杆菌细胞表面,构建新型全细胞催化剂,克服拟除虫菊酯类农药的吸附-摄取限制.将丁香假单胞菌冰核蛋白InaK的N-末端作为锚定基序与来自铜绿假单胞菌GF31的拟除虫菊酯降解酶基因融合,转化大肠杆菌Rosetta-gami,构建全细胞催化剂.结果表明在0.05mmol/L的IPTG条件下诱导培养12 h后,该全细胞催化剂的氨基肽酶活性达到0.25 U/OD_(600)/mL,对高效氯氰菊酯农药底物的降解速率达到35.9μmol L^(-1)d^(-1),且对5种常用拟除虫菊酯类农药均具有降解能力.通过细胞的分级分离和免疫荧光分析确定了功能蛋白在细胞膜上的正确位置.该全细胞催化剂经过10次重复反应,残余活性仍能保持在70%以上,具有较好的稳定性.本研究实现了拟除虫菊酯降解酶在大肠杆菌细胞表面上的功能性展示,为高疏水性有机农药的环境修复提供了一种方法.

Pyrethroid pesticides are difficult to adsorb,ingest,and degrade by microorganisms because of their high hydrophobicities and molecular weights.Improving the bioavailability of such substrates is key to environmental bioremediation.A pyrethroid-degrading enzyme was displayed on the cell surface of E.coli using cell surface display technology,and a new whole-cell catalyst was constructed to overcome the adsorption uptake limitation of pyrethroid pesticides.The N-terminal of Pseudomonas syringae ice nucleation protein InaK was used as an anchor motif to fuse with the pyrethroid-degrading enzyme gene from Pseudomonas aeruginosa GF31 and then transformed into Rosetta-gami^(TM)to construct a whole-cell catalyst.After induction and culture for 12 h in the presence of 0.05 mmol/L IPTG,the aminopeptidase activity of the whole cell catalyst was 0.25 U/OD_(600)/mL,and the degradation rate ofβ-cypermethrin pesticide substrate was 35.9μmol L^(-1)d^(-1),and it can degrade five common pyrethroid pesticides.The correct positions of the functional proteins on the cell membrane were determined by cell fractionation and immunofluorescence analysis.After ten repeated reactions,the residual activity of the whole-cell catalyst was maintained at more than 70%and had good stability.This study achieved the functional display of a pyrethroid-degrading enzyme on the surface of Rosetta-gami^(TM)cells and provides a method for the environmental remediation of highly hydrophobic organic pesticides.