Enzymatic Characterization And Crystal Structure Of HSL Family Esterases From Erythrobacteraceae

Esterase catalyzes the hydrolysis,synthesis and transesterification of esters,and widely found in eukaryotes and prokaryotes.Most of the industrial esterase in food,cosmetic and pharmaceuticals are from bacteria.The bacterial HSL esterase possesses harsh conditions adaptability,broad substrate spectrum and has been used in food flavor preparation and safety detection.The catalytic characteristics of HSL family esterase from different bacterial sources are quite different,therefore,it is very important to study the protein structure in order to explain the relationship between the structure and catalytic characteristics and to guide the transformation of enzyme engineering.In this study,HSL family esterase genes were screened from the genome of the type strains of Erythrobacteraceae which has the ability of alkane dregradation.The enzyme characterization and crystal structure study provided data support for the catalytic mechanism and function of HSL family esterase.Twelve putative HSL family esterases were screened from the genomes of thirteen type strains of Erythrobacteraceae by sequence alignment.Through amino acid analysis,gene cloning,hetero-expression and purification,two research objects Poc14 and Pot7 with high soluble expression and stable properties were identified.The molecular weight of esterases Poc14 and Pot7 are 35.7 kDa and 33.7 kDa.The results of enzymatic characterization showed that esterase Poc14 and Pot7 exhibited the maximum catalytic activity towards p-nitrophenol hexanoate,and the optimum temperature was 55 ?C and pH was 6.0–9.0.Poc14 and Pot7 had strong thermostability and highly resistance to several metal ions,organic solvents and NaCl.Poc14 was more tolerant than Pot7.Moreover,both of them had the ability to degrade the phthalates,indicating that Poc14 and Pot7 can be used for the detection of phthalates.The protein structure of esterase Poc14 and Pot7 was studied.The crystal structure was successfully resolved through protein crystallization,crystal X-ray diffraction data collection and molecular replacement.Finally,the crystal structure of Poc14 with a resolution of 1.8 ? and the crystal structure of Pot7 with a resolution of 2.1 ? were obtained,which provided data support for studying the structure,function and catalytic mechanism of HSL family esterase.In summary,the HSL family esterase Poc14 and Pot7 with excellent enzymatic properties were screened by bioinformatics analysis,and the crystal structure of the two esterases were successfully resolved.It lays a foundation for the study of the catalytic mechanism and function of bacterial HSL family esterase enzyme,and provides alternative enzymes for the food industry.