Development of a whole-cell biocatalyst for diisobutyl phthalate degradation by functional display of a carboxylesterase on the surface of Escherichia coli
Development of a whole-cell biocatalyst for diisobutyl phthalate degradation by functional display of a carboxylesterase on the surface of Escherichia coli
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DOI：
10.1186/s12934-020-01373-6
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发表时间：
2020-05-29
影响因子：
6.4
通讯作者：
Huang, Zunxi
中科院分区：
文献类型：
--
作者：
Ding, Junmei;Zhou, Yang;Huang, Zunxi
背景：邻苯二甲酸酯（PAEs）在塑料制品的工业生产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邻苯二甲酸酯。邻苯二甲酸酯的暴露导致人类健康问题和环境污染。邻苯二甲酸二异丁酯是PAEs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有力工具。在本研究中，在大肠杆菌细胞表面展示羧酸酯酶，用作邻苯二甲酸二异丁酯生物降解的全细胞生物催化剂。将芽孢杆菌K91的羧酸酯酶编码基因（carEW）融合到假单胞菌的冰核蛋白（inpn）锚和gfp基因的N端，将融合蛋白克隆到pET-28 a（+）载体中，在大肠杆菌BL 21（DE 3）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定证实INPN-CarEW/或INPN-CarEW-GFP融合蛋白的表面定位。构建的E.大肠杆菌表面展示细胞。细胞表面展示的CarEW显示最适温度为45 ℃，最适pH为9.0，使用p-NPC 2作为底物。此外，全细胞生物催化剂在45 ° C下23天和4 ° C下1个月的时间内保持其每OD 600的原始活性的100%和200%，表现出比游离CarEW更好的稳定性。此外，约1.5 mg/ml的邻苯二甲酸二异丁酯降解10 U的表面展示的CarEW细胞在120 min。结论：这项工作提供了一个有前途的战略成本效益的邻苯二甲酸二异丁酯的生物修复的表面展示CarEW的E。大肠杆菌细胞。该方法也可为在无害微生物细胞表面展示目的酶处理其他不同类型的环境污染物提供参考。
Background: 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.Results: 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 degrees C and optimal pH of 9.0, using p-NPC2 as substrate. In addition, the whole cell biocatalyst retained similar to 100% and similar to 200% of its original activity per-OD600 over a period of 23 days at 45 degrees C and one month at 4 degrees 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.Conclusions: 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.