Study On DAHP Synthase Gene Of Microbulbifer Sp. A4B-17 Strain

4-Hydroxybenzoate(4HBA)is a widely used raw material for organic synthesis.Liquid crystal products and other fields.Chemical synthesis from petroleum components can cause environmental pollution;the use of 4HBA synthesized in plants is esterified;the production of 4HBA from recombinant E.coli has production problems.The laboratory collected and isolated more than 20 strains of Microbulbifer from the marine environment,of which the A4B-17 strain had the highest amount of synthetic 4HBA.We completed the genome sequencing and annotation of the A4B-17 strain,and through the in-depth study of the strain we could overcome the drawbacks of previous production methods.In this study,we conducte an in-depth study of a reaction in the synthetic p-hydroxybenzoic acid pathway of A4B-17 strain.There are three genes encoding DHAP synthase in the genome of A4B-17 strain,and the gene numbers are GM002069,GM001679,GM001835.In this study,the properties of the enzymes are studied after high-level expression of these three genes in E.coli.With pCold IV vector,GM002069 optimal reaction conditions for pH 7.5,time 120 minutes,the amount of enzyme 20?L,temperature 41?,the substrate reduced by 97%.GM001679 optimum reaction conditions pH 7.5,time 120 minutes,enzyme amount 20?L,temperature 42?,substrate reduction 99%.GM001835 optimal reaction conditions pH 7.5,time 120 minutes,enzyme amount 30?L,temperature 41?,substrate reduction 97%.The gene is cloned using pET-21a(+)vector.The optimal enzyme activity condition of GM002069 is reaction temperature 38?,time 2 minutes,enzyme amount 20?L,pH 7.5,and the substrate consumed 98.4% of the initial substrate amount.The optimal enzymatic activity conditions of GM001679 are reaction temperature 37?,time 2 minutes,enzyme amount 20?L,pH 7.5,and the substrate consumed 97.7% of the initial substrate amount.It is concluded that the use of this vector to link the target gene clone and the enzymatic activity assay result resulted in a higher enzyme activity.The utilization of the substrate is higher and the yield of the product is correspondingly higher.