## 4 Discussion
The activity of cold-adapted enzymes at low temperatures depends on the flexibility of the protein structure, which has rendered cold-adapted enzymes susceptible to denaturation by heat at ambient temperatures compared with their mesophilic counterparts. However, EstDR4 exhibited high stability from 10-40 \({}^{\circ}\)C, and retained over 80% residual activity after 1 h of incubation. The half-lives of EstDR4 at 40 \({}^{\circ}\)C and 50 \({}^{\circ}\)C were 10.13 h and 1.29 h, respectively, significantly higher than those reported cold-adapted esterases PLM3, PsyEst, Est684 and MtEst45 [24; 26; 27; 28]. EstDR4 can be classified as an alkaline esterase, not only because its optimum pH was 8, but it also exhibited high stability at pH 8, with over80% residual activity after 1 h at pH 8. In comparison, an esterase from _Basillus circulans_ was maximally active only between pH 8-9 and stable between pH 6-8 [29]. Similarly, Est1 from a compost metagenome exhibited maximum activity at pH 7 and retained more than 95% activity between pH 6-8 [30]. Moreover, EstDR4 better performed hydrolysis in the presence of some detergents like Tween 80 and Triton X-100. The effect of detergent or surfactant on enzyme activity may be due to the interaction between enzyme, detergent and substrate [31]. EstDR4 activation by Tween 80 and Triton X-100 may be attributed to an increased substrate solubility and probability of entering the substrate pocket [32]. However, EstDR4 activity was inhibited by SDS due to the nonspecific binding between SDS and proteins, which results in the formation of complexes and unfolding of proteins [20]. These characteristics demonstrate the potential value of EstDR4 as a low temperature catalytic industrial use, such as processes involving food ingredients, manufacture of thermolabile pharmaceutical products, and cold-wash detergents [33]. Many metal ions reportedly have inhibitory or activation effects on esterases [34]. In this work, EstDR4 activity was substantially inhibited by Cu\({}^{2+}\) and Zn\({}^{2+}\), a similar phenomenon has been observed in AMS8 from antarctic _Pseudomonas_ and EstATII from a Red Sea brine pool metagenomic [8; 35]. Most tested organic solvents in the current study induced inhibitory effects on the catalytic activity of EstDR4. Only DMSO and n-hexane slightly diminished the esterase activity to 99.59% and 75.59% of the initial activity, respectively. Generally, organic solvents affect the catalytic activity of enzymes due to disruption of the balance between organic solvent phase and enzyme surface, disruption of core hydrophobic interactions that lead to a conformational change of the enzyme molecule, and competition for substrate binding [36]. As an example, [PERSON] et al. [8] discovered that AMS8 lipase loses its helix formation due to hydrogen bond formation interference in the presence of ethanol, 2-propanol, and DMSO, causing the loss of lipase activity. Protein engineering may enhance the organic solvent stability of enzymes in three manners: modification to increase the stabilizing interactions of surface residues, modification of residue flexibility, and surface charge alteration. Lipase T6 from _Geobacillus steatorthermophilus_ achieved enhanced stability in methanol by substituting charged surface residues with hydrophobic residues, which led to extensive formation of new hydrogen bonds [37]. [PERSON] et al. [38] confirmed that modulating the flexibility within the hydrophilic solvent-affecting region can enhance the organic solvent stability of _Candida Antarctica_ lipase B. Due to the frequent use of insecticides in agriculture and other environments, the pollution caused by insecticides has been gradually noticed. While the biodegradation of insecticides has been recognized for its advantages of high efficiency and energy saving. Esterase has a potential degradation effect on insecticides [39], which has been confirmed for EstDR4 in this study.