Study On Purification Of Trypsin And Validation Synthesis Of Phosphoryl Dipeptide In Reversed Micelles

Reversed micellar extraction of biological macromolecule, especially proteins, has a promising profit, for the mild operational condition , high extraction yield, low cost and solvent recycling. The dissertation focuses on the research of extraction and purification of trypsin in raw pancreatin, probing into the STM image of trypsin in AOT-isooctane reversed micelles and validation synthesis of AcPheBPA (BPA:Butyl Phosphonic acid) catalysed by α-chymotrypsin.The STM image shows that the conformation of trypsin in reversed micelles is changed and in a partly active state, for increased curly degree, compacted dimensional configuration and enhanced rigidity in contrast with that in water. The conjugated degree of π-π bonds in trypsin is weakened so that ultraviolet and fluorescent spectra are blue-shifted, owing to the rigid state in reversed micelles.Firstly, pH and ionic strength were discussed with biological purified trypsin. ① The relation of pH and extraction rate is fitted no-linear curve . To 0.1mol/L AOT-isooctane, , ; To 0.05mol/L AOT, , ; To 0.01mol/L AOT, , ; ② when pH is in [5.2, 4.8], extraction rate decreases with pH because H+ competes with trypsin for combining to AOT polar terminal; when pH is in [5.3, 10.0], extraction rate increases with difference of pI-pH for more impetus; When AOT concentration is increased, the effect of pH on extraction rate drops but inactivation rate rises. ③ The optimum AOT concentration is 0.05 mol/L, and the optimum pH for extraction is 5.2~5.3 and 9.9~10.0 for reextraction. ④ The effect of different cations on extraction is Ca2+purified under the optimum condition and the results show that total extraction rate is 22.7%, trypsin specific activity is 3.1 times of that raw material, protein of trypsin is 3.6 times of that, and inactivation rate of trypsin is 13.9%. ④ The recycled reversed micelles were reused and the results show total extraction rate is 19.8% and trypsin specific activity is 2.8 times. ⑤ With AOT/Span-40 mixed reversed micelles, total extraction rate is 16.5% and trypsin specific activity is 2.9 times, which are 6.2% and 6.5% lower than sole AOT reversed micellar system respectively.Reversed micelles can solubilize hydrophile and lipophile substrates. And it was found that proteolytic enzyme in reversed micelles can act as synthetase. Phosphoryl Dipeptide was synthesized by AcPheOEt and (1-Aminobutyl)Phosphonic acid in AOT-isooctane reversed micelles at 40℃ and pH 9.4, which is catalysed by α-chymotrypsin. The amino group combined with OPA was examined by fluorescence, and fluorescent intensity is decreased after synthesis for 30 or 60 min. So the synthesis of AcPheBPA is validated.