Protein Engineering Of A L-aspartate-?-decarboxylase And Its Application In The ?-alanine Production

?-alanine is the only?-type amino acid existing in nature.As an important pharmaceutical intermediate,?-alanine has a wide range of applications in various fields,such as of medicine,food,and chemical industries.Compared with the conventional chemical-sysnthesis routes for?-alanine,the biological-synthesis routes for?-alanine are more attractive due to their mild process conditions,high specifity and less environmental pollution.Thus,biological-synthesis routes for?-alanine have significant industrial prospects and research value.L-aspartate-?-decarboxylase?EC 4.1.1.11,PanD?is a kind of enzyme capable of catalyzing decarboxylase of L-aspartate to produce?-alanine.However,PanD has relatively low activity,and the pyruvyl-dependent PanD inactivates due to its inherent mechanism.These limitations significantly inhibit applications of this enzyme.Therefore,it is essential to develop an alternative PanD with a high activity and stability in the further study.In this dissertation,an gene encoding a decarboxylase from Tribolium castaneum,named TcPanD,was expressed in the E.coli BL21?DE3?and used for protein engineering of TcPanD to improve its acitivity and stability.Our results were listed as follows:1.After the first round of random mutatation for TcPanD,a total of 3500 mutants were secreened,and the mutant 16-H5 and 18-G6,with the enzyme activity was 1.61and 1.43 times higher than that of the wild TcPanD enzyme,and the mutant sites were R98H,K305E and I451V respectively.Then,the site-directed saturation mutation,as the second round of mutations based on the random mutation was performed,and the mutant TcPanD-R38H/K305S with the enzyme activity 2.45 times higher than that of the wild TcPanD.With the homology modeling of TcPanD,the mutant amino was lacated at the surface position of protein.Through the auto docking analysis,the mutant site R98 with high activity was far from the substrate L-aspartate,which may improve the structural stability through a certain force.The mutant site K305 near L-aspartate may improve the catalytic activity by enhancing the hydrophilicity to binding the substrate.2.The enzymatic properties of the mutant TcPanD-R38H/K305S and the wild TcPanD were further studied.The optimal concentration of the coenzyme pyridoxal phosphate added to the reaction system for the activity of mutant TcPanD-R38H/K305S was of 0.04%?m/v?,The optimum optimal temperature of for the mutant enzyme TcPanD-R38H/K305S was 55°C,and the optimum pH of for the mutant enzyme and the original wild TcPanD enzyme was were 7.0.With the increase of pH,as pH increased from 7.0 to 11.0,the enzyme activity of TcPanD dropped sharply under alkaline conditions than TcPanD-R38H/K305S.The effects of various metal ions on activities of the TcPanD mutant TcPanD-R38H/K305S and wild enzyme.Under low concentration?1 mM?metal ion conditions,Na⁺,Ni²⁺,Co²⁺,K⁺and Ca²⁺have a certain activation effect on the mutant enzyme TcPanD-R38H/K305S,Fe²⁺,Cu²⁺,Zn²⁺,Ba²⁺have obvious inhibition effect.Under high concentration?5 mM?metal ion conditions,only Ni²⁺and Na⁺has obvious activation effect.The K_m values of TcPanD-R38H/K305S for ketoprenalactone was 1.226 mM,the V_max of the reaction was7.05 U/mg,the catalytic number was 7.633 s^-1 and the catalytic rate was 6.226 s^-1·mM^-1.The K_m values of TcPanD for ketoprenalactone was 1.353 mM,the V_max of the reaction was 2.88 U/mg,the catalytic number was 3.116 s^-1 and the catalytic rate was 2.303s^-1·mM^-1.The mutant TcPanD-R38H/K305S showed better substrate affinity and higher catalytic activity than TcPanD.3.The mutant enzyme TcPanD-R38H/K305S freeze-dried powder as a catalyst was prepared by vacuum freeze-drying technology.The effects of different catalytic conditions on?-alanine production was measured.Its optimum temperature and pH were37°C and 7.0,respectively.The amount of pyridoxal phosphate was 10?L/mL,and the substrate concentration was 50 g/L.Under optimal catalytic conditions,the yield of?-alanine can be up to 10.89 g/L.Finally,the mutant enzyme was induced by 5 L fermenter.The collected cells were subjected to whole-cell catalytic reaction in a reactor.At 37 ^oC,pH 7.0 with a shake of 600 rpm,and 267 g of bottom.L-aspartate was added by fed-batch.After 10 h of conversion,the?-alanine was finally obtained 170.53 g/L in a yield of 95.45%.