Gene Cloning,Expression And Application Of Nitrilases

To estimate a country's comprehensive national strength,the scale and power of chemical industry is an important indicator.After years'efforts,China's fine chemical industry has formed a considerable scale in the chemical industry and the output of some products has been considered to be top of the world.However,environmental pollution is the biggest challenge to domestic fine chemical industry.Developing economic at the cost of destroying the environment is not a long-term solution,green chemical production and sustainable development must be adopted.Nitrilase has important applications in organic synthesis—the preparation of a host of intermediates(organic acids).Preparation of organic acids using nitrilases has unparalleled advantages against many chemical methods with high efficiency,high selectivity,mild reaction conditions,less environmental pollution and optical purity production,in line with the direction of developing atom economy and green chemistry.Biological production of iminodiacetic acid is not only in accord with green chemistry and sustainable development requirements,but also is one important pathway of synthesizing glyphosate herbicide.This paper focus on reengineered genetically protein to produce iminodiacetic acid.Through homology modeling,molecular docking,a method to identify the type of nitrilases has been established.After structure and enzymatic properties research on the screened nitrilases,an nitrilase with a higher conversion of iminodiacetonitrile has been well prepared and reengineered;one of the mutants has the potential for industrial production.Firstly,nine nitrilase sequences with a large diversity in amino acid sequence were selected from the NCBI database.After condon optimication using E.coli as target host.These gene sequences were synthesized and cloned into the vector pET28b for expression.After purification by affinity chromatograph,the purity of target protein met electrophoresis homogeneity.In addition,a rapid method of determining sets of nitrilases has been preliminarily established by docking analysis and then the key amino acids responsible for catalytic activity have been recognized by comparing the nitrilase structures of different kinds.Considering substrate specificity,we selected adiponitrile as the co-substrate,and the enzymatic properties study for the nine nitrilases were based on it.As a result,the nitrilase activity of AcfN?KpN?GpN underwent little change between 40-60 ?,which suggests they are highly potential for industry applications.When the temperature is above 70 ?,all nitrilases lose hydrolyze activity basically.The catalytic optimum pH for most nitrilase was alkalescent between pH 7.0 and 8.0.When the pH<3.0,or>10.0,the activity of almost all nitrilases dropped dramatically,which will provide a reference value for scaling up our post-experimental design.In the investigation of effects of Metal Ions on nitrilases,all the vitality of activities is less than or equal to 10%of control with the presence of Ag+ and Hg2+,which demonstrated that sulfhydryl groups play an important role in nitrilase catalysis.Additionally,Ca2+?Mg2+?Zn2+ slightly improved nitrilase activity and little inhibition was induced by EDTA,indicating that the nitrilases do not need metal ions involved in the catalysis.Finally,the nitrilase AcfN was selected to study its catalytic reaction process from iminodiacetonitrile to iminodiacetic acid.After mutating some important amino acids,one mutant with a high conversion(>95%)was ultimately obtained and the possible catalytic mechanism was analyzed,which also verifies the results from structural analysis.