| Thermozyme have high similarity to homologous mesozymes due to their highly typically similar sequence homology ranging from 40% to 85%, superimposed three-dimensional structure and similar catalytic mechanisms. However, several structural characteristics of hyperthermozyme make it possess high thermostability and optimal activity even above 110℃. Some factors usually impact the thermostability of proteins, such as hydrogen bonds, hydrophobic interaction, ion pairs, electrostatic interaction, disulfide bridges, but only one factor could not be totally responsible for the thermostability of enzyme temperature. Therefore, study of the influence mechanisms of these factors on the structure and thermostability of hyperthermozyms could not only transform the thermostability of enzymes to adapt the harsh environments in industry, but also research the evolution of enzymes by using hyperthermozyme as a model.Hyperthermophilic esterase APE1547 from the thermophilic archaeon Aeropyrum pernix K1, belongs to prolyl oligopeptidase (POP) family. The gene of APE1547 encoded 582 amino acid residues. It possesses the activity of both peptidase and esterase. It contains two domains:β-propeller domain at N-terminal as well asα/βhydrolysis domain at C-tertminal. Three-dimensional structure ofβ-propeller domain is highly conservative, but sequence homology ofβ-propeller domain is low in different enzymes. The main effect ofβ-propeller domain stabilizes holoenzyme.The effect of typical side-chain hydrogen bonds ofβ-propeller domain on the stability of APE1547 was investigated through the mutation of amino acids in different blades ofβ-propeller domain. Three mutants (R292L, Y253L and R160L/D158V) were obtained through the mutation of one of the amino acids in different pairs of blades (Asp52-Arg292 locating between the blades 1 and 7, Thr209-Tyr253 locating between the blades 5 and 6, Arg160-Ile202 and Asp158 -Ile202 locating between the blades 4 and 5 ).Basic properties of the wild and mutated esterases were compared. The optimal temperature of mutated esterase is 92.5℃, 2.5℃lower than that of wild type. The thermostability of wild and mutated esterases was investigated through determining the half-life of denaturation and the free energy of thermal denaturation reaction. Comparing with the half-life of denaturation wild APE1547 (15.26h) , the half-life of denaturation of mutant R292L, mutant Y253L and mutant R160L/D158V decreased 2.36h, 3.02h and 3.65h respectively, which indicated that the deletion of hydrogen bonds declined the thermostability of APE1547 and the decline of mutant R160L/D158V was most obvious.Dynamics analysis showed that Km of mutants had little change comparing with that of wild type, which illustrated that the affinity between esterase and substrate was not almost changed. However, the kcat of mutants was higher than that of wild type, indicating that the catalytic efficiency of APE1547 to substrate increased obviously.The study of the stability of mutants to chemical denaturant (guanidine hydrochloride, Gdn-HCl) showed that the activity of both mutants and wild type lost, following the change of conformation. Although 90% of the activity lost under 2M Gdn-HCl, the conformation was still stable. However, the loss of activity and the change of conformation of mutants decreased were faster than those of wild type. Among the mutants, the decrease of mutant R160L/D158V was the fastest, followed by mutant Y253L, and the speed of mutant R292L wild type. The main reason for this change is that Arg292 is located in the loop of the 7th blade, while Tyr253, Arg160 and Asp158 are located in theβ-sheet of the blades. Removing of the hydrogen bonds in theβ-sheet of the blades weakens the interaction between two blades and looses the structure so that the stability decreased but the activity increased.To further study the effect of hydrogen bonds on the stability of APE1547,β-propeller domain containing hydrogen bonds was cloned and expressed. The thermostability of both mutants and wild type were determined through fluorescence spectrum. The results showed that under thermal denaturation and Gdn-HCl denaturation, the structure ofβ-propeller in wild type was closer and thus more stable, while the structure ofβ-propeller in mutants was looser and that in mutant R160L/D158V was loosest. The results also showed thatβ-propeller domain was sensitive to temperature but resistant to Gdn-HCl.In this thesis, several hydrogen bonds inβ-propeller domain were invested. Three mutants (R292L, Y253L and R160L/D158V) were obtained through amino acid substitution. Analysis of basic enzymatic properties, structure and stability showed that hydrogen bonds were important to stabilize the holoenzyme. Theβ-propeller domain of both mutated and wild APE1547 were cloned and expressed to study and analysis the stability of APE1547. The results showed that hydrogen bonds played an important role on the stability ofβ-propeller domain, which provided theoretical basis for improving thermostability of esterases and transforming mesozyme.
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