Expression Of Recombiant Carboxypeptidase Y In Pichia Pastoris And Its Application In Kinetically Controlled Solid Phase Enzymatic Peptide Synthesis (SPEPS) Of The Antioxidant Dipeptide Tyr-Ala

The key point of this study is to combine enzymatic and chemical peptide synthesis methods,with optimizing the preparation conditions,in order to develop a new method of peptide synthesis with both advantages.Firstly,the pichia pastoris engineering bacteria was constructed by genetic engineering,and the recombinant carboxypeptidase Y was obtained after fermentation.Secondly,the dipeptide precursors BOC-Tyr-Ala was prepared by enzymatic synthesis in liquid phase,and the enzymaticaly catalytic conditions were optimized for higher yield.The kinetic model of this synthsis reaction was also established.Finally,the model dipeptide Tyr-Ala was synthesized by using solid phase enzymatic peptide synthesis,and the application of enzyme in peptide synthesis was expanded.The conclusions were drawn as follows:?1?The expression of carboxypeptidase Y in P.pastoris GS115.The carboxypeptidase Y gene from A.Oryzae RIB40 was transformed into P.pastoris GS115 using molecular biology technique,and the enzyme activity was 90.379U/mg after purification.The best shaking flask fermentation conditions were obtained by testing different fermentation conditions for P.pastoris GS115-CPY's OD₆₀₀ value and the enzyme activity.The results were as followed:methanol concentration 1.0%,the shaking speed 250 rpm,pH 6.0,temperature 28?,and adding glucose to the concentration of 4 mg/mL.In the end of fermentation,the maximum OD₆₀₀ value was39.65,and the enzyme activity was 89.53U/mL,which was 56.72%and 56.03%higher than the unoptimized experimental results.?2?The synthesis of the antioxidant dipeptide precursor BOC-Tyr-Ala in liquid phase.The antioxidant dipeptide precursor BOC-Tyr-Ala was prepared through the kinetically controlled enzymatic peptide synthesis reaction,catalyzed by the recombinant carboxypeptidase Y?CPY?.t-butyloxycarbonyl-L-tyrosine-methyl ester?BOC-Tyr-Ala?was used as acyl donor and L-alanine?L-Ala?was amino donor in this reaction.The reaction conditions were optimized to be:30oC,pH 9.5,organic phase?methanol?/aqueous phase=1:20,BOC-Tyr-OMe 0.05 mol/L,Ala 0.5 mol/L,and reaction time 12 h.Under these conditions the dipeptide yield reached 49.84%,and then the kinetic model of the synthesis reaction was established as the Michaelis–Menten equation form based on the reaction-time curve and the transpeptidation reaction mechanism.The apparent Michaelis constantK_m^appand the apparent maximum reaction rater_max^appwere calculated as2.9946?10^-2mol/L and2.0406?10^-2mmol/?mL?h?,respectively.?3?The preparetion of antioxidant peptide Tyr-Ala by solid phase enzymatic peptide synthesis.An antioxidant dipeptide Tyr-Ala was produced by the novel solid phase enzymatic peptide synthesis?SPEPS?,catalyzed by recombinant carboxypeptidase Y.77.92%yield of Tyr-Ala with the high product purity of 90.971%was obtained under the optimized conditions?1.8 g of Boc-Tyr?Wang Resin?-OMe,0.8 mol/L of Ala,8 mL/g of liquid/solid ratio,30oC,pH 9.5?.Finally,the antioxidant activity of Tyr-Ala was determined by ABTS method,indicating that the synthesized Tyr-Ala obtained by SPEPS shows a superior antioxidant ability than that of commercial glutathione,which is a common used antioxidant.