The Study Of Pilot Scale Preparation Process And Catalytic Domain Study Of Thermophilic Inorganic Pyrophosphatase

The inorganic pyrophosphatase（PPase）is an enzyme that catalyzes the hydrolysis of pyrophosphate（PPi）into two phosphate molecules（Pi）in the presence of magnesium ions.By preventing the precipitation of pyrophosphate with magnesium,this enzyme enhances the rate of in vitro transcription（IVT）reactions and has become an important component of RNA preparation.Large-scale production of RNA for m RNA vaccine preparation and biochemical characterization of RNA often requires the consumption of large amounts of PPase.Additionally,PPase is widely used in experimental and industrial production of applications such as PCR amplification reactions,industrial sugar production,and gene sequencing.Although there is much research on the application of PPase derived from Escherichia coli and yeast,there is relatively little research on the catalytic domain of PPase from thermophilic bacteria.In this study,site-directed mutagenesis was used to modify thermophilic PPase,in order to observe the effects of changes in multiple amino acids in the catalytic domain of the enzyme on its enzymatic properties and activity,to further characterize and identify the catalytic domain of PPase.The study also involved recombinant expression,pilot fermentation,and purification protocols for thermophilic PPase,with the main research objectives outlined below:The wild-type amino acid sequence of the thermostable inorganic pyrophosphatase was obtained from the NCBI protein database,and 12 mutants with different amino acid changes were designed.After codon optimization,the mutants were expressed using an E.coli expression system,and the recombinant proteins were identified by SDS-PAGE and Western blotting,with a molecular weight of approximately 21 k Da.The optimal expression conditions for the recombinant enzyme were established as an induction temperature of 25℃,IPTG concentration of0.3m M,and induction time of 8h.A pilot-scale fermentation process was established using a 5L fermenter with LB medium,and a biomass of 63.93mg/m L was achieved after a 12-hour fermentation process.A large amount of bacterial cells was harvested and subjected to heat treatment at 75℃for 30min to remove the host proteins.Two purification methods were provided for the recombinant thermostable inorganic pyrophosphatase in this study.One method involved column chromatography with a nickel column,Capto Q anion exchange chromatography,and S100 molecular sieve chromatography.The other method involved precipitation with polyethyleneimine（PEI）followed by stepwise elution with high-salt buffer to obtain the protein.Both column chromatography and PEI polymer purification methods achieved good purification results,and the recombinant PPase protein with high purity and low nucleic acid residue was obtained.The activity of the thermophilic inorganic pyrophosphatase is indirectly measured by determining the free Pi concentration in the reaction system using the molybdenum blue colorimetric method.The recombinant enzyme has an optimal reaction temperature of 75℃,an optimal p H of 9,and the optimal divalent metal ion in the reaction process is Mg²⁺.Na F and MDP inhibit enzyme activity,and high concentrations of PPi and Mg²⁺in the reaction process also inhibit enzyme activity.The recombinant thermophilic inorganic pyrophosphatase exhibits extremely high thermal stability,retaining about 80%of its maximum catalytic activity even after incubation at 100℃for 5 hours.Furthermore,incubating the enzyme with an appropriate concentration of Mg²⁺and recombinant PPase can further maintain the enzyme’s thermal stability.The kinetic parameters of the recombinant enzyme for hydrolyzing PPi,K_m and V_max,are 1.008m M and 1.793m M min^-1,respectively.Adding the recombinant enzyme to the PCR amplification system can significantly alleviate the inhibitory effect of excess PPi on PCR amplification.Adding the recombinant thermophilic inorganic pyrophosphatase to the IVT reaction can also increase product yield.The catalytic process of inorganic pyrophosphatase is the result of the synergistic action of key residues in its active center,divalent metal ions,and water molecules.Studies on E.coli PPase have shown that some Asp residues coordinate with four Mg²⁺ions to form a metal catalytic cage.The substrate binds to Lys29,Lys142,and Arg43 through electrostatic interactions,while Asp67 promotes the deprotonation of bridging water molecules.These amino acids constitute a highly conserved metal catalytic cage.Through multiple sequence alignments,evolutionary tree analysis,conservation analysis,and homology modeling,the active center of the thermophilic PPase was determined.Like other Family I PPases,it is composed of 13-17 key amino acids.Based on the electrostatic and functional properties of residues,site-directed mutagenesis and combination mutagenesis were performed on K30,D43,R44,Y56,D66,D71,D103,and Y140,and 12 mutated thermophilic PPases were prepared and characterized.Structural simulations showed that the Y56K,D71N,and D103N mutants lost their catalytic activity completely due to changes in spatial distance and reduced hydrogen bonding,which reduced their affinity for substrates or Mg²⁺.The K30R mutation made it difficult for the catalytic product to dissociate,and the D66N mutation reduced the total negative charge of the active site,reducing its binding ability to divalent metal ions as M1,M2,and PPi.The Y140K mutation increased the distance between the side chain and the substrate,making it difficult to bind.Ultimately,K30R,D66N,and Y140K retained 57%,11%,and 13%of the wild-type enzyme activity,respectively.D43N,D43E,and R44K retained 96%,93%,and97%of the activity,respectively,indicating that changes in this residue had little effect on enzyme activity.The K30R D66N,Y56K D66N,and K30R Y56K D66N mutants,which involved multiple point mutations,also completely lost their enzymatic activity.The Michaelis constants of most mutated enzymes increased,indicating a decrease in affinity for the substrate,and the turnover numbers of the mutants were lower than that of the wild-type,resulting in significantly reduced enzyme activity.This article studied the physical and catalytic functional changes of multiple mutant variants obtained by changing the amino acids of the key sites in the wild-type PPase.Y56,D71,and D103 were determined to play important roles in the enzymatic catalytic domain for the first time,providing strong evidence for designing enzymes with increased activity and stability under different conditions.The results of this study contribute to the understanding of the enzymatic properties of PPase and the relationship between its structure and function.