Biodegradation of Endocrine Disrupting Phthalates by Esterase Enzymes

Phthalate esters are a class of endocrine disrupting compounds that are widely used in various industries, and are suspected carcinogens. The two esterase enzymes named esterase B and G were successfully isolated from soil bacterium Sphingobium sp. SM42. These enzymes are responsible for degradation of one of widely used phthalate ester dibutyl phthalate (DBP). In this study, the genes encoding both enzymes were successfully cloned and sequenced. Both recombinant enzymes were biochemically characterized and found to exhibited optimal activity at 28 oC in Tris-HCl buffer pH 8.0, and esteraseB was completely inhibited by Ca²⁺, Co²⁺ and Mn²⁺, whereas esteraseG was sensitive to Cd²⁺, Cu²⁺, Fe²⁺, Ni²⁺ and Zn²⁺, while 1% Triton X detergent significantly improved both enzyme activities. The attempt to express esterase on the cell surface of the recombinant E.coli was made by cell surface-displayed system using various carrier proteins including antigen43 from E.coli, EstA from Pseudomonas aeruginosa, and OprF from Pseudomonas aeruginosa to improve the efficiency of esterase G enzyme.

The recombinant E.coli cell harboring plasmid pETOprF-EstG expressing highest amount of the fusion protein between esteraseG and its OprF partner was detected by western blot analysis, fluorescence microscopy, and flow cytometry. Interestingly, the E.coli cell with surface-displayed and cytoplasmic esterase G demonstrated the highest DBP-degrading activity compared to E.coli cell expressing only cytoplasmic esteraseG. In conclusion, we successfully improve the microbial DBP-degrading efficiency by genetically engineered the esterase to the cell surface. This recombinant E.coli cell with surface-displayed esterase G is a good model and promising tool to remove toxic chemicals from the contaminated sites in Thailand.

แหล่งข้อมูล:

• โครงการวิจัยชื่อ “Biodegradation of Endocrine Disrupting Phthalates by Esterase Enzymes” – ระยะเวลาโครงการ: September 2011- November 2013