Purification Of Aspartate Aminotransferase And Researches Of Characters

Aspartate aminotransferase catalyzes synthetic reaction and degraded reaction of aromatic amino acid In this dissertation,aspartate aminotransferase is abstracted and purified from E.coli,then the enzyme characters and kinetic characters were also investigated. We use a series of steps to abstract and purify aspartate aminotransferase,such as fractional precipitation of(NH4)2S04,hydrophobic interastion chromatography, ion— exchange chromatography,specific activity is enhanced 84.1 64 times,and the yield of enzyme is 1 1.24%.It is determinated that molecular mass of subunit of AspAT is about 43000Da by SDS— PAGE Using purified AspAT,we study on enzyme characters,and the optimum temperature is 50。C,the optimum pH is 8.5.Because this reaction is transfer reaction of di— substrate and di— product,we only use Michadlis-Menten equation to study kinetic character approximtly.It is determined that apparent Km is 1 6.705mmol·L-1。 L ,and apparent Vmax is 0.46639 mmol·L-1·min-1.Transferamination mechanism of transaminase is ping-pang reaction mechanism,SO we study on two— step transamination using ping-pang reaction mechanism.The Km of the first step is 8.28 mmol·L-1,and the Km of the second step is 14.677 mmol ·L-1.So the second step reaction is the rate— limiting reaction.The rate— limiting step is that AspAT with phosphoryridoxamine is transferred to AspAT with phosphopyridoxal.We deduce the enzyme solution added phosphoryridoxamine can more facilitate the genesis of production in the beginning of reaction.It is verified that the yield catalyzed by AspAT added phosphoryridoxamine enhance 6.667%after 10min,after 120min,the yield enhance only 3.397%. Using polyacrylamide as embed material,there are five factors in the procedure of immobilized cell:the concentration of gel,cross— link,the concentration of persulamine,the concentration of TEMED,the concentration of thallus.So we use orthogonal experiments of five factors and four levels to determine the optimum condition of immobilized cell.The optimum condition is:the concentration of gel 6is 15%,cross-Iink is 5%,the Concentration of persulamine is 400 u L the concentration 0f TEMED is 60 μL,the concentration of thallus is 2mL.The approach Michaelis constant of immobilized cell is 10.90109mmo1·L-1 ,Vmax is 0.0618l1 mmol min .The approach Michaelis constant Of free ceIl is 8·7848mm, Vmax is O.352mmol·min.Comparing Km and vmmax enzyme,free ceIl and immobilized cell,it is demonstrated that Km of enzyme is higher than that of free ceIl;and Vmax of free cell is higher than that of immobilized celI.ImmobiIiZed cell affect mass transfer,and the effect of inner diffusion affect the process of substrate entering cell to react.So Km of immobilized cell is higher than that 0f free...