Expression, Enzymatic Properties And Structure Of Esterase E53 From Erythrobacter Longus

Esterases are widespread in plants,animals,and microorganisms,which catalyze hydrolysis of ester bond and participate in various reactions including esterification,interesterification and transesterification.Microbial esterase is one of the most important industrial enzymes,which shows many advantages such as stable reaction conditions,high catalytic activity and product easy separation.These esterases have been widely used in food processing,medicine production,sewage purification,energy utilization,and other fields.The research of microbial esterase is becoming a hot spot of application enzymology.In this paper,an esterase was screened from an Erythrobacteraceae bacterial genome.The esterase was studied throngh heterologous express,isolation and purification,enzymatic properties,crystal structure analysis and construction of mutants and enzymatic activity.The results provide theoretical support for the further study of the catalytic mechanism and the relationship between structure and function of esterase.Firstly,Esterase E53 was heterologous expressed,purified and biochemically characterized,which was screened from the Erythrobacter longus genome.The results showed that the molecular weight of esterase E53 was 33.13 kDa.E53 showed the highest activities at 40°C,pH 9.0,by hydrolysis of p-nitrophenol butyrate.Esterase E53 has good tolerance to some metal ions(such as Mn²⁺,Mg²⁺,Ca²⁺ and Ba²⁺)and most organic solvents?such as isopropanol,ethanol,methanol and acetonitrile?,so it has potential application prospect.Secondly,the high purity protein of Esterase E53 was obtained by heterologous expressed,purified.In order to obtain the single crystal with good shape and crystal form,the crystallization conditions of protein need to be screened and optimized.By collecting the crystal X-ray diffraction data and analyzing and modifying the crystal structure,the three-dimensional structure of esterase E53 were resolved.The three-dimensional structure could be used to further study the catalytic mechanism and industrial application.Finally,the twelve mutants of Esterase E53 were constructed by site-directed mutagenesis.The enzymology properties of esterase mutants were studied.The results showed that the enzymatic activity and catalytic rate of esterase mutants E53-D161 A,E53-S285 G and E53-N288 A were about 1% to about 20% of the wild-type esterase.Therefore,the above three sites of amino acids play an important role in the catalytic reaction of esterase.The activities of enzyme activity and catalytic response of E53-A167 L,E53-F191 Y,E53-V211 A and E53-S216 A were increased more than 50% compared with wild-type esterase.So the above four sites of amino acids have a certain impact in the catalytic reaction of esterase.The activity of the mutant esterase E53-S162 A,E53-D254 A and E53-H284 A were only about one ten thousandth of the wild type,indicating that the active site played a very important role in the catalytic reaction of esterase.In conclusion,the study of the enzymatic properties of esterase E53 showed that it had potential application value.The crystal structure of esterase E53 was well studied for the further study of the catalytic mechanism and the relationship between structure and function of esterase.The construction of esterase E53 mutants and its enzymatic properties have been a good theoretical basis for exploring the amino acid residues.The result of esterase E53 was important to the research of catalytic reaction of esterases and the further construction of engineered enzymes suitable for industrial applications.