Phthalic acid esters (PAEs) are widely used as plasticizers or additives during the industrial manufacturing of plastic products. PAEs have been detected in both aquatic and terrestrial environments due to their overuse. Exposure of PAEs results in human health concerns and environmental pollution. Diisobutyl phthalate is one of the main plasticizers in PAEs. Cell surface display of recombinant proteins has become a powerful tool for biotechnology applications. In this current study, a carboxylesterase was displayed on the surface of Escherichia coli cells, for use as whole-cell biocatalyst in diisobutyl phthalate biodegradation. A carboxylesterase-encoding gene (carEW) identified from Bacillus sp. K91, was fused to the N-terminal of ice nucleation protein (inpn) anchor from Pseudomonas syringae and gfp gene, and the fused protein was then cloned into pET-28a(+) vector and was expressed in Escherichia coli BL21(DE3) cells. The surface localization of INPN-CarEW/or INPN-CarEW-GFP fusion protein was confirmed by SDS-PAGE, western blot, proteinase accessibility assay, and green fluorescence measurement. The catalytic activity of the constructed E. coli surface-displayed cells was determined. The cell-surface-displayed CarEW displayed optimal temperature of 45 °C and optimal pH of 9.0, using p-NPC2 as substrate. In addition, the whole cell biocatalyst retained ~ 100% and ~ 200% of its original activity per OD600 over a period of 23 days at 45 °C and one month at 4 °C, exhibiting the better stability than free CarEW. Furthermore, approximately 1.5 mg/ml of DiBP was degraded by 10 U of surface-displayed CarEW cells in 120 min. This work provides a promising strategy of cost-efficient biodegradation of diisobutyl phthalate for environmental bioremediation by displaying CarEW on the surface of E. coli cells. This approach might also provide a reference in treatment of other different kinds of environmental pollutants by displaying the enzyme of interest on the cell surface of a harmless microorganism.

中文翻译：

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通过在大肠杆菌表面上功能显示羧酸酯酶降解邻苯二甲酸二异丁酯的全细胞生物催化剂的开发。

邻苯二甲酸酯（PAE）在塑料产品的工业生产过程中被广泛用作增塑剂或添加剂。由于过度使用，在水生和陆地环境中都检测到了PAE。PAE的暴露会导致人类健康问题和环境污染。邻苯二甲酸二异丁酯是PAE中的主要增塑剂之一。重组蛋白的细胞表面展示已成为生物技术应用的强大工具。在这项当前的研究中，羧酸酯酶被展示在大肠杆菌细胞表面，用作邻苯二甲酸二异丁酯生物降解中的全细胞生物催化剂。从芽孢杆菌属中鉴定出的一种编码羧酸酯酶的基因（carEW）。将K91融合到丁香假单胞菌和gfp基因的冰核蛋白（inpn）锚的N端，然后将融合蛋白克隆到pET-28a（+）载体中，并在大肠杆菌BL21（DE3）细胞中表达。通过SDS-PAGE，蛋白质印迹，蛋白酶可及性测定和绿色荧光测量证实了INPN-CarEW /或INPN-CarEW-GFP融合蛋白的表面定位。测定了构建的大肠杆菌表面展示的细胞的催化活性。细胞表面显示的CarEW使用p-NPC2作为底物，显示的最佳温度为45°C，最佳pH为9.0。此外，全细胞生物催化剂在45°C下23天和4°C下1个月内，每个OD600的原始活性分别保持〜100％和〜200％，表现出比游离CarEW更好的稳定性。此外，在120分钟内10 U的表面展示CarEW细胞降解了约1.5 mg / ml的DiBP。这项工作通过在大肠杆菌细胞表面上展示CarEW，为环境生物修复提供了一种经济有效的邻苯二甲酸二异丁酯生物降解的策略。通过在无害微生物的细胞表面展示目的酶，该方法还可为处理其他不同种类的环境污染物提供参考。