In this paper we combined three chromatographic separation and purification technique such as affinity chromatography, ion exchanger chromatography and hydrophobic interaction chromatography to develope a new technology of stimutaneous extraction of three enzyme from pancreatin. We optimized the technology by studying the methods of purification and assured the technology as: The crude extraction from the dissolution of Pancreatin is directly absorbed on the DEAE gelose fast flow columnEquilibrating buffer is 0.01mol/L NaoAc-HoAc buffer(pH4.5; eluting buffer is 0.2～0.35mol/LNaCl in 0.01mol/LNaoAc-HoAc buffer (pH4.5), and then be eluted by two steps to acquire the peak of kallikrein.The solution which can"t be adsorbed by DEAE gelose fast flow column is adsorbed on affinity chromatographic column Equilibrating buffer is 0.01mol/LTris-HCl buffer(pH7.5, eluting buffer is 0.5mol/LNaCl in 0.01mol/Ltris-HCl buffer(pH7.5)and then be eluted by one step to acquire the peak of trypsin.The solution which can"t be adsorbed by is pretreated with 30%～80%(NH_4)_2SO_4 fractional precipitation, the deposition of the precipitation is dissolved to beabsorbed on phenyl gelose fast flow columnhydrophobic interaction chromatography condition is Equilibrating buffer is lmol/L(NH_4_2SO_4 in 0.01mol/LNaoAc-HoAc buffer(pH4.5), eluting buffer is 0～0.6mol/L(NH_4)_2SO_4 in 0.01mol/LNaoAc-HoAc buffer (pH4.5) and then be eluted by two steps to acquire the peak of chymotrypsin.

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