Screening,Rational Modification Of Polyphosphate Kinases And The Application In Glutathione Synthesis

Polyphosphate kinases（PPK）have been involved in many biocatalytic reactions to provide energy for ATP-depleting reactions due to their cheap and readily available substrate polyphosphate.In this study,in order to obtain PPK enzyme with higher enzyme activity,PPK enzymes were screened by high-throughput extracellular and intracellular screening,and PPK enzymes were modified by rational design mutation.Based on the mutant enzyme,an ATP regeneration system was constructed,and the mutant enzyme PPK was coupled to produce glutathione.The ATP regeneration ability was enhanced to increase glutathione yield and reduce catalytic cost,and the production system was optimized to achieve efficient glutathione production.The main results are as follows:（1）According to PPK classification and phylogenetic tree analysis,seven polyphosphate kinases from different sources were cloned and expressed in Escherichia coli,and the effect of the length and concentration of poly P phosphate chain of substrate on PPKs enzyme activity was studied.The results showed that the seven PPK enzymes had the best catalytic capacity for poly P₆,and the higher the concentration of phosphate,the stronger the inhibition of PPK activity.The highest specific enzyme activity of Ch PPK from Cytophaga hutchinsonii was 30.26±2.08U·mg^-1,which was the best biocatalysis from the seven PPK enzymes using poly P₆ to regenerate ATP.（2）The Ch PPK mutant library was constructed by error-prone PCR.Combined with the intracellular and extracellular high-throughput screening method and site-directed mutagenesis,the double mutant Ch PPK_D82N-K103E was finally screened,and its relative enzyme activity increased to 430.2%,showing the highest catalytic capacity.Molecular docking revealed that the key residues D82 and K103 were located at the entrance of the poly P₆ channel and ADP channel,respectively,and formed ion interaction with the phosphate group of the substrate.After mutation,N82 and E103 still enlarged the substrate channel cavity to improve the catalytic activity of Ch PPK.By RMSD and RMSF analysis,it was found that the mutations of D82N and K103E improved the molecular structural flexibility,which promoted the interaction between the substrate（poly P₆ and ADP）and active pocket.（3）In the EB13 whole-cell catalytic system,coupling with mutant enzyme Ch PPK_D82N-K103E and Gsh AB,can convert 50 m M substrate into 14.2±1.2 m M glutathione with adding 5m M ATP at 40 h.It can achieve 91.6%of the effect of EB11（the Gsh AB enzyme was expressed）whole cell catalytic system（100 m M ATP）,and 59.6%higher than the catalytic yield of Ch PPK coupled with Gsh AB.After optimizing the buffer,bacterial volume,feeding time and substrate concentration of the catalytic system,the EB13 strain could convert 50 m M substrate into34.1±2.6 m M glutathione at 40 h and the conversion rate of the substrate cysteine was 68.2%.However,when the substrate concentration was 100 m M,the highest glutathione yield was51.5±1.3 m M at 40 h,and the conversion rate of the substrate cysteine was 51.5%.（4）In order to further improve the conversion rate of glutathione production,strains EB11,EB12 and EB13 were used to catalyze glutathione production in the form of cell-free lysate after cell fragmentation.By coupling Ch PPK_D82N-K103E with Gsh AB and adding 5 m M ATP,an efficient ATP regeneration efficiency could be obtained.Within 8 h,50 m M substrate could be converted into 27.1±1.9 m M glutathione,which was 51.4%higher than the catalytic yield of Ch PPK and Gsh AB.After optimizing the buffer,bacterial volume,feeding time and substrate concentration of the catalytic system,the EB13 strain could convert 50 m M substrate into46.2±1.4 m M(14.2±0.4 g·L^-1)glutathione at 8 h and the highest conversion rate of the substrate cysteine was 92.4%.When the substrate concentration was 100 m M,the highest glutathione yield was 67.5±1.7 m M(20.7±0.5 g·L^-1)at 8 h,and the conversion rate of the substrate cysteine was 67.5%.