Expression,Purification,Characterization,and Crystal Structure Of Two Esterases From Marine Bacteria

As a treasure house of biological enzyme resources,the ocean contains a large number of new enzyme resources with excellent enzyme properties that are yet to be developed and utilized.As an important biocatalyst,esterase can catalyze the hydrolysis,synthesis,and transesterification of esters.It is widely used in food,medicine,chemical industry,as well as energy,sewage treatment and other industrial production fields.Esterase from microorganisms has become the main source of esterase in industrial production because of its high yield,low cost,stable properties,low toxicity of by-products,easy separation,and purification.In this thesis,the enzymatic properties and crystal structure of two novel marine bacterial esterases Erj5 and Pos7 were studied,hoping to provide data support and theoretical basis for the industrial application of marine esterase and the study of esterase catalytic mechanism.Firstly,phylogenetic analysis and multi-sequence alignment analysis of amino acid sequences of esterases Erj5 and Pos7 were carried out,and the results showed that they both belonged to the Family IV esterase.A large number of soluble recombinant proteins were obtained by using E.coli prokaryotic expression system.After affinity purification and molecular sieve purification,esterases Erj5 and Pos7 with a molecular weight of 33 kDa were obtained.Secondly,the enzymatic properties of esterases Erj5 and Pos7 were characterized by the aspects of optimum reaction conditions,thermal stability and additive tolerance.The results showed that the optimum catalytic substrate of esterase Erj5 was p-nitrophenol butyrate,and its optimum reaction conditions are 45? and pH 7.0.The esterase Erj5 showed excellent tolerance to organic solvents,and 1%Triton X-100 promoted its catalytic activity.The esterase Pos7 is an alkaline esterase,and its most suitable catalytic substrate is p-nitrophenol caprylate,which has the highest catalytic activity at 40? and pH 9.5.Esterase Pos7 is also a pH broad-spectrum esterase with pH 5.0 to 11.0.When pH is 11.0,Pos7 still retains about 80%activity.Meanwhile,Pos7 showed excellent tolerance to metal ions,and had strong tolerance to Sr2+,Ba2+,Cd2+,Co2+and Mn2+.The Ca2+and Mg2+could promote the catalytic activity of Pos7 to a certain extent.In addition,Pos7 also showed excellent thermal stability and NaCl tolerance.Finally,the crystal structure method was used to study the protein structure of the esterases Erj5 and Pos7.Through crystal screening and optimization,X-ray diffraction data collection and processing,the complete three-dimensional structures of esterases Erj5 and Pos7 were successfully obtained with resolutions of 2.4 A and 2.3 A,respectively.In this thesis,the enzymatic properties of Family IV esterases Erj5 and Pos7 from marine bacteria were studied,and the results showed that Erj5 and Pos7 had excellent enzymatic properties and were good candidates for industrial application,especially for Pos7 because it's suitable for ester catalytic reaction under strong base conditions.The successful analysis of the three-dimensional crystal structure of esterases Erj5 and Pos7 laid a theoretical foundation for the study of esterase catalytic mechanism and directional modification of esterase.