title: Trigger factor assisted folding of the recombinant epoxide hydrolases identified from C. pelagibacter and S. nassauensis.
abstract: Epoxide hydrolases (EHs), are enantioselective enzymes as they catalyze the kinetic resolution of racemic epoxides into the corresponding enantiopure vicinal diols, which are useful precursors in the synthesis of chiral pharmaceutical compounds. Here, we have identified and cloned two putative epoxide hydrolase genes (cpeh and sneh) from marine bacteria, Candidatus pelagibacter ubique and terrestrial bacteria, Stackebrandtia nassauensis, respectively and overexpressed them in pET28a vector in Escherichia coli BL21(DE3). The CPEH protein (42kDa) was found to be overexpressed as inactive inclusion bodies while SNEH protein (40kDa) was found to form soluble aggregates. In this study, the recombinant CPEH was successfully transformed from insoluble aggregates to the soluble and functionally active form, using pCold TF vector, though with low EH activity. To prevent the soluble aggregate formation of SNEH, it was co-expressed with GroEL/ES chaperone and was also fused with trigger factor (TF) chaperone at its N-terminus. The TF chaperone-assisted correct folding of SNEH led to a purified active EH with a specific activity of 3.85μmol/min/mg. The pure enzyme was further used to biocatalyze the hydrolysis of 10mM benzyl glycidyl ether (BGE) and α-methyl styrene oxide (MSO) with an enantiomeric excess of the product (eep) of 86% and 73% in 30 and 15min, respectively. In conclusion, this is the first report about the heterologous expression of epoxide hydrolases using TF as a molecular chaperone in pCold TF expression vector, resulting in remarkable increase in the solubility and activity of the otherwise improperly folded recombinant epoxide hydrolases.
authors.affiliation: Department of Biochemistry, Panjab University, Sector 14, Chandigarh 160 014, India. Electronic address: 27priyasaini@gmail.com.
authors.forename: Priya
authors.initials: P
authors.lastname: Saini
authors.affiliation: Department of Biochemistry, Panjab University, Sector 14, Chandigarh 160 014, India. Electronic address: shahdil.wani@gmail.com.
authors.forename: Shadil Ibrahim
authors.initials: SI
authors.lastname: Wani
authors.affiliation: Department of Biochemistry, Panjab University, Sector 14, Chandigarh 160 014, India. Electronic address: ranjai.pu@gmail.com.
authors.forename: Ranjai
authors.initials: R
authors.lastname: Kumar
authors.affiliation: Department of Biochemistry, Panjab University, Sector 14, Chandigarh 160 014, India. Electronic address: chhabra.ravneet9@gmail.com.
authors.forename: Ravneet
authors.initials: R
authors.lastname: Chhabra
authors.affiliation: Department of Chemistry, Guru Nanak Dev University, Amritsar 143 005, Punjab, India. Electronic address: sschimni@yahoo.com.
authors.forename: Swapandeep Singh
authors.initials: SS
authors.lastname: Chimni
authors.affiliation: Department of Biochemistry, Panjab University, Sector 14, Chandigarh 160 014, India. Electronic address: diptsare@pu.ac.in.
authors.forename: Dipti
authors.initials: D
authors.lastname: Sareen
mesh_terms: D039903:Actinobacteria / Q000235:genetics*; D020561:Alphaproteobacteria / Q000235:genetics*; D055162:Biocatalysis; D003001:Cloning, Molecular; D004851:Epoxide Hydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004852:Epoxy Compounds / Q000737:chemistry; D004926:Escherichia coli; D006868:Hydrolysis; D007700:Kinetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013237:Stereoisomerism
chemical_list: D004852:Epoxy Compounds; D011994:Recombinant Proteins; C020344:glycidyl ethers; D004851:Epoxide Hydrolases
keywords: Candidatus pelagibacter ubique; Chaperones; Epoxide hydrolase; Solubility; Stackebrandtia nassauensis; Trigger factor