Research Of Coorperative Production Technology Of Kallikrein, Trypsin And Chymotrypsin

Pancreatin is a common material drug for medical treatment and a biochemistry reagent in industry, it costs low and easy to acquire. There are three important enzymes in pancreatin: kallikrein, trypsin and chymotrypsin, they are all materials for modern biological research and have valuable clinic use. It is huge economically and applicably valuable to extract the three high cost enzymes from pancreatin. But pancreatin is a mixtrure of various enzymes, each enzyme has a low purity and many enzymes have similar molecule structure and physicochemical properties, when extracting one enzyme, it is easy to abandon the otherone as impurity. This is why there are few technoloies of stimutaneous extraction of more than three enzyme from pancreatin and all the technoloies have shortages. By the development of the technique of the separation and purification of enzyme, it is urgent to develop new technologies of stimutaneous extraction to meet the standards of industry.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 column(Equilibrating 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%(NH₄)₂SO₄ fractional precipitation, the deposition of the precipitation is dissolved to beabsorbed on phenyl gelose fast flow column(hydrophobic interaction chromatography condition is Equilibrating buffer is lmol/L(NH₄)₂SO₄ in 0.01mol/LNaoAc-HoAc buffer(pH4.5), eluting buffer is 0～0.6mol/L(NH₄)₂SO₄ in 0.01mol/LNaoAc-HoAc buffer (pH4.5)) and then be eluted by two steps to acquire the peak of chymotrypsin.The production of three enzymes have high specific activity and recovery of activity:the specific activity of kallikrein is 550u/mg, the recovery of kallikrein is 73%, the specific activity of trypsin is 12500 which is 5.5times of pharmacopoeia standard, the recovery of trypsin is 79%, the specific activity of chymotrypsin is 8000 which is 10 times of pharmacopoeia standard, the recovery of chymotrypsin is 80%.Each enzyme showed a few impurity bands in SDS-PAGE.The technology is simple, fast, costs low and has good repetition, it has great potential for industry use.