## 3 Results and Discussion
### Degrading Ability of Purified EstA  
To investigate the ability to degrade different pyrethroids, the substrate specificity of EstA was evaluated with six commonly used pyrethroids at an initial 20 mg/L. As shown in Figure 5, after 2 h of reaction, the degradation of each compound reached to 40.5-100%, indicating that the purified EstA was a broad-spectrum pyrethroid-hydrolyzing enzyme. This may be because the most pyrethroid pesticides possess a similar ester bond in their molecular structure [21]. As pesticide residues are mixtures in the environment, a broad-spectrum pyrethroid-degrading enzyme would have boarder range in practical application. And as each enzyme has its optimal substrate, in this study, EstA showed the highest degrading ability to beta-cypermethrin, which was considered as 100% for its enzyme degrading activity and the activity of enzymatic degradation was 7.9 mg/min. The relative enzyme activity to degrade other pyrethroid was normalized to the highest degradation percentage (the activity to degrade beta-cypermethrin, in this case). Compared to a previous study, the enzyme activity in our case exhibited relatively higher activity compared with the enzyme isolated by [PERSON] et al (3.29 U for degrading 1 nmol beta-cypermethrin, in this case, and 123.34 U for degrading 1 nmol lambda-cyhalothrin, with U indicated as the enzyme dosage employed to degrade 1 ug of beta-cypermethrin per minute) (cypermethrin: 95.7%, fenvalerate: 85.7%, deltamethrin: 70.4%, cyhalothrin: 55.2%, and permethrin: 40.5%) (Figure 5) [36].  
Figure 4: SDS-PAGE analysis of induced protein and its purification protein. (**A**) SDS-PAGE analysis of induced EstA protein at different temperatures. M = protein marker (Code No.: 3450, Takara, Dalian, China); lane 1, 2, and 3 = the full cell protein of _Escherichia coli_ BL21/ pET-32a-_estA_ induced in 30 \({}^{\circ}\)C, 25 \({}^{\circ}\)C, and 20 \({}^{\circ}\)C; lane 4, 5, and 6 = the full cell protein of _Escherichia coli_ BL21/PET-32a (+) induced in 30 \({}^{\circ}\)C, 25 \({}^{\circ}\)C, and 20 \({}^{\circ}\)C; (**B**) M = protein marker; lane 1 = purified EstA; lane 2 = the full cell protein of _Escherichia coli_ BL21/pET-32a-_estA_ expressed at 20 \({}^{\circ}\)C.  
Generally, the function of specifically expressed proteins can be easily affected by different external conditions. For providing more information to facilitate the application of the purified enzyme, the tolerance of EstA under wide temperatures was assessed from 15 \({}^{\circ}\)C to 50 \({}^{\circ}\)C. As shown in Figure 6A, the optimal temperature for EstA catalysis was 35 \({}^{\circ}\)C, and lower or higher temperature could significantly inhibit enzyme activity, since relative activities dropped below 40% at 15 \({}^{\circ}\)C and 50 \({}^{\circ}\)C. Additionally, EstA activity was also determined under various pH values ranging from pH 5.0 to 9.5 (Figure 6B). These results indicated that EstA preferred neutral and alkaline conditions (pH 6.5-9.0) where the enzyme activity of EstA remained above 60%. Maximal enzyme activity was measured at pH 8. The purified protein of EstA was able to exert degrading function over a wide range of pH and temperature. This is a very important feature of an organism to be employed for bioremediation in variable environments. The condition in field is very complex and influenced by many factors, and the parameter in laboratory condition could not be introduced directly to the practical application. Still, the optimal cultivation conditions, preferred enzymatic activity, and biodegradation mechanism illustrated in this study could enrich the available biodegradation resources.  
Figure 5: The relative activity of EstA to various pyrethroid insecticides.  
Figure 6: Effects of temperature and pH on EstA activity. **(A)** Temperature; **(B)** pH.