Directional Modification Of Escherichia Coli Phytase AppA

Phytase is the enzyme that breaks down phytate into less-phosphorylated myo-inositol derivatives and inorganic phosphates.Phytate(myo-Inositol hexakisphosphate,IP6)is the main storage form of phosphorus in plant seeds and can be able to chelate inorganic ion and protein.As a result,phytate is most commonly regarded as an antinutritional compound.Meanwhile,phosphorus is one of the necessary mineral elements to promote the growth and reproduction of animals.Gastric animals are unable to use phytate phosphorus effectively because they lack the necessary enzymes to breaks down phytate.As an important feed supplement,phytase can solve above problems.Because of the need for treated at high-temperature in the feed pelleting process,how to improve the thermal stability of phytase has become more and more improtant.Phytase mainly plays a role in the digestive system of animals.There are a lot of proteases in the digestive system.Sensitivity to protease is an important factor to restricte the commercial application of phytase.In order to improve the thermalstability and the protease tolerance of Escherichia coli phytase app A,the directional modification was carried out by genetic engineering.It was also helpful to understand the relationship between the structure and function of phytase.The main results were shown as following:1?The effects of N-glycosylation on phytase app AN-glycosylation is an important post-translational modification of proteins.N-glycosylation modification existed in phytase expressed by Pichia pastoris.Based on sequence analysis and structural modeling,results showed that there were three N-glycosylation sites(N139,N204 and N317)in phytase app A,which located in ?-helix,?-sheet and random coil respectively.To study the effects of N-glycosylation on the enzymatic properties of Escherichia coli phytase app A,a series of N-glycosylated phytase mutants were designed and transfected into Pichia pastoris for expression and purification.The enzymatic properties of wild-type phytase app A-WT and N-glycosylated phytase mutants were studied and compared.Results showed that the most beneficial mutant app A-Mut5 had a specific activity of 3187 U/mg with an MW of approximately 60 k Da.Its Km and Vmax were 445 ?mol/L and 3654 U/mg,respectively.The optimal p H of app A-Mut5 was 4.5,which was the same as app A-WT.However,the optimal temperature for app A-Mut5 was 65 oC,5 °C higher than app A-WT.The melting temperature(Tm)of app A-Mut5 was 11 °C higher than app A-WT.After incubating at 75 °C for 15 min,89 % activity of app A-WT was lost;no app A-Mut3 activity was observed.However,app A-Mut5 remained 43 % of activity.Phytase mutant app A-Mut5 was not only had good thermalstability,but also showed a stronger pepsin and trypsin resistance than app A-WT and other mutants.Based on our design,mutant app A-Mut5 contained eight potential N-glycosylation sites.LC-MS/MS analysis was used to characterize the attachment sites of N-linked oligosaccharides in details.The results showed that six sites in app A-Mut5 were N-glycosylated,which were modified by various glycan forms and displayed different N-glycosylation degree at residues N74,N139,N171,N204,N317 and N349.No glycopeptide was detected at residues N258 and N282.In summary,N-glycosylation modification was heterogeneous and played an important role in improving stability of phytase app A.At the same time,the three new sites(N74,N171 and N349)were identified and N-glycosylated by Pichia pastoris,which laid the foundation for the directional modification of phytase.2?Improvement trypsin tolerance of phytase app A by rational designPrevious studies showed that phytase app A had good tolerance to pepsin and it was sensitive to trypsin.In this study,through structure-based rational design,six trypsin cleavage sites(K74,K75,K180,R181,K183 and K363),which could be easily attacked by trypsin,were selected and replaced to improve trypsin tolerance of Escherichia coli phytase app A.Inspection of the three-dimensional structure and computational design via hydrogen bond analysis and optimal site mutations of K74D/K75Q/ K180N/R181N/K183S/K363 N,which strengthened the hydrogen bonding,were performed to generate the mutant app A-M6.Results showed that the purified app A-M6 had a specific activity of 3262 U/mg with molecular weight of approximately 52–55 k Da.Similar to app A-WT,the optimal p H(4.5)and temperature(60 °C)of app A-M6 were unchanged.Compared with app A-WT,app A-M6 showed a significant enhancement in resistance to trypsin and a 3.8 °C increase in melting temperature(Tm).We concluded that mutations in trypsin cleavage sites resulted in decreased enzyme flexibility due to the introduction of hydrogen bonds,increased the enzyme stability against proteolysis and thermal denaturation.3?The obtainement of phytase mutants with good thermalstability and high trypsin tolerance by multipoints combinationIn order to improve the thermalstability and trypsin tolerance of phytase app A at the same time,based on previous results,we designed and compared the enzymatic properties of wild-type phytase app A-WT and phytase mutants app A-M8,app A-M10,app A-M12,app A-M15 and app A-M16,respectively.Through screening,sixteen sites(W46E,Q62 W,A73P,K74 N,K75Q,G76 T,S146E,R159 Y,P173S,K180 N,R181N,K183 S,N204C,Y255 D,Q349N and K365N)were identified as the best combination of mutation sites.The best phytase mutant was app A-M16.The phytase mutant app A-M16 remained more than 40 % activity after being treated at 80 oC for 25 min,while app A-WT was completely inactivated.Phytase mutant app A-M16 not only had good thermalstability,but also its trypsin tolerance was improved.The glycosylation modification of phytase mutant app A-M16 was detected by mass spectrometry.The results of mass spectrometry showed that the three newly introduced sites N74,N171 and N349 were N-glycosylated in phytase mutant app A-M16 exactly.Compared with wild-type phytase app A-WT,the structure of the phytase mutant app A-M16 changed.Due to the introduction of new hydrogen bonds and N-glycosylation modification,the rigidity of the structure was enhanced,and the conformation of phytase mutant app A-M16 was more stable.Thus,the thermalstability of phytase and the resistance of the protease were improved.In this study,a phytase mutant app A-M16 with improved thermalstability and trypsin resistance was successfully obtained by combination mutation and optimal site selection.The mechanism of phytase structural stability was explored.Our study also provided a theoretical basis for directional modification of phytase.The phytase mutant strains generated in this study had good application prospects.