Cloning, heterologous expression and characterization of a thermostable esterase from Bacillus sp. HJ14 for diethyl-phthalate degradation

Abstract:

[Objective] A thermostable esterase EstZ1 from Bacillus sp. HJ14 able to degrade diethyl-phthalate (DEP) was heterologously expressed in Escherichia coli BL21(DE3) and characterized.[Methods] Full-length EstZ1 was obtained based on specific amplification and genome sequencing, and amino acid sequence of EstZ1 was analyzed. EstZ1 was expressed in Escherichia coli BL21(DE3) using the pEASY-E2 expression system. EstZ1 was purified to electrophoretic homogeneity by Ni²⁺-NTA metal chelating affinity chromatography, and the enzyme was characterized. The degradation products from DEP were detected by high-pressure liquid chromatography and electrospray ionization mass spectrometry.[Results] The 903 bp full-length EstZ1 encoded 300 amino acid residues (EstZ1:33.84 kDa). EstZ1 showed the highest identity of 98% with hormone-sensitive lipase (HSL)-like family in NCBI databases. The optimal temperature and pH was 50℃ and 9.0, respectively, with p-NP butyrate as the best substrate. Meanwhile, it was stable between 40 and 70℃, pH 7.0 to 9.5. Most of metal ions, chemical agents had little impact. DEP could partially be degraded by EstZ1 to its corresponding monoalkyl and alcohol.[Conclusion] Our findings may serve as reference for phthalate esters degradation.

Key words:thermostable esterase;heterologous expression;enzyme characterizations;DEP degradation