The Study On Expression,Purification And Characterization Of Pyruvate Decarboxylase From Escherichia Coli And Sedoheptulose-1,7-bisphosphatase From Rice

The enzyme sedoheptulose-1,7-bisphosphatase(SBPase) is located in the chloro plast stroma in higher plants. As a key regulator in the regenerative phase of the photosynthetic carbon reduction cycle (Calvin cycle), SBPase converts sedoheptul ose-1,7-bisphosphate to sedoheptulose-7-phosphate and Phosphate. Studying on fun ction and structure of Ri-SBPase contribute to increase Ri-SBPase activity which stimulate photosynthesis and growth in transgenic rice plants and enhance the rice production.Pyruvate decarboxylase (PDHc E1) converts pyruvate to hydroxy-ethylidene-T hDP, which is the first step and only irreversible step in the reaction that pyruva te dehydrogenase multienzyme complex converts pyruvate to acetyl-CoA.Therefore, PDHc E1activity is influencing the overall enzymatic reaction directly.Taking it as a potential target, we may develop novel and efficient fungicides.In this thesis, the study mainly covers two aspects:(1) Soluble, active recombinant SBPase and PDHc E1with a MBP tag were obtained using the plasmid pMal-C2x-Ri-SBPase and pMAL-c2X-ace with the E. coli host strain TB1. Purification of recombinant proteins were performed by MBP affinity chromatography and gel filtration chromatography. Expressing and purifying recombinant proteins by the system,6mg PDHc E1and0.8mg SBPase were obtained in1L cell culture.(2) Measuring the effect of pH, temperature, cofactors and substrate, enzyme kinetic of E. Coli PDHc E1and Rice SBPase have been studied. Michaelis constant Km for substrate and maximum enzymatic reaction rate Vm of E. Coli PDHc E1was42.1μM and4.0U/mg, respectively. Meanwhile, Michaelis constant Km for substrate and maximum enzymatic reaction rate Vm of Rice SBPase was5.26mM and0.15U/mg, respectively. Moreover, the research showed that the effect of MBP tag to enzyme kinetic of E. Coli PDHc E1and Rice SBPase could be neglected.