Construction And Optimization Of An Immobilized Catalytic System For The Biosynthesis Of 3’-phosphoadenosine-5’-phosphosulfate

3′-Phosphoadenosine-5′-phosphosulfate（PAPS）is a biologically active form of sulfate that participates in sulfur metabolism in organisms,and also serves as a universal active sulfonation donor in the synthesis of sulfonated products.Therefore,the efficient synthesis of PAPS is of great significance for the production of sulfonated products.At present,PAPS is mainly synthesized through in vitro enzymatic synthesis,which is catalyzed by ATP and SO₄^2-synthesized by a bifunctional enzyme through PAPS synthesis.Due to the poor catalytic stability of free enzymes and their ability to catalyze only one round of reactions,their utilization rate is low.Immobilization of enzymes is a commonly used strategy to solve such problems.However,in this study,due to the substrate ATP being easily consumed by intracellular crude enzymes,using traditional enzyme immobilization methods requires the purification of the PAPS synthesis bifunctional enzyme,which will significantly increase its operating cost.Therefore,this study aims to achieve efficient synthesis of PAPS by constructing a immobilized enzyme catalytic system that does not require protein purification,is simple and easy to separate,and can be reused.The main research content is as follows:Firstly,Different auto-assembly proteins were screened and the intracellular auto-assembly immobilization of the dual functional enzyme ASAK for PAPS synthesis was successfully achieved.The C-terminal of the bifunctional enzyme synthesized by PAPS was fused with a rigid PT linker peptide to auto-assembly protein Cip A,and the specific activity of immobilized ASAK reached 427.4 U·g^-1 wet cell.The fusion of green fluorescent protein and Cip A verified the universality of the advantages of fusing Cip A at the C-terminal.Subsequently,the optimal fermentation conditions for immobilized PAPS synthesis of bifunctional enzymes were established:30°C 0.5 m M IPTG,cell concentration OD₆₀₀=0.9 during induction,and culture time 10 hours after induction.The basic enzymatic properties of the immobilized PAPS synthetic bifunctional enzyme were determined:the specific enzyme activity was 37%of that of the free bifunctional enzyme,the optimal catalytic p H was 7.5,the optimal catalytic temperature was 30°C,and the relative activity after storage at 30°C for 24 hours was 73%,which was 29%higher than that of the free bifunctional enzyme.Secondly,screening higher activity ADP phosphorylase to realize the closed regeneration of by-products ADP and PPi into ATP,improve the conversion rate of PAPS,and realize the immobilization of ADP phosphorylase.Firstly,the polyphosphate exonuclease Pa PPX from Pseudomonas aeruginosa was identified to have the activity of ADP phosphorylase using pyrophosphate(8.5 U·mg^-1)and higher activity.Coupled with a bifunctional enzyme synthesized by PAPS,it forms PAPS without the addition of additional phosphate donors,and the substrate ATP conversion rate reaches 72%.Then the ADP phosphorylase was immobilized,At the C-terminal of ADP phosphorylase,the rigid PT linker peptide was used to fuse the auto-assembly protein Cip A,and the specific activity of immobilized Pa PPX reached 950 U·g^-1 wet cells.The enzymatic properties of immobilized ADP phosphorylase and free enzyme were compared and analyzed.The specific enzyme activity was 42%of free ADP phosphorylase,the optimal catalytic p H was 7.5,and the optimal catalytic temperature was 40°C.Compared with free enzyme,the relative activity of immobilized ADP phosphorylase and free enzyme was increased by 10°C,88%and 20%respectively after storage at 30°C for 24 hours.The stability was significantly improved.Finally,the immobilized bifunctional enzyme for PAPS synthesis and ADP phosphorylase catalyzed PAPS synthesis system were optimized,and the co immobilization of bifunctional enzyme in E.coli cells was realized.The catalytic system of immobilized PAPS for synthesizing bifunctional enzyme and immobilized ADP phosphorylase was optimized.The optimal catalytic system was determined as 10 m M ATP,10 m M Mg Cl₂,20 m M Na₂SO₄,and the molar ratio of ASAK-PT-Cip A and Pa PPX-PT-Cip A was 3:1.In order to further simplify the acquisition of two enzymes and improve the catalytic reaction efficiency,the bifunctional enzyme for PAPS synthesis and the bifunctional enzyme for ADP phosphorylase were co immobilized to synthesize PAPS,and then different stabilizers were selected to improve the stability of the immobilized enzyme.It was determined that when 10%glycerol was added to the system,the co-immobilized enzyme still had 82%relative activity after 24 h incubation at30°C.Finally,the number of repeatable catalytic times of co immobilized enzyme was studied.The first conversion rate of co immobilized enzyme for the synthesis of PAPS reached 90%,and after 10 repetitions,the conversion rate still exceeded 50%.