Study On The Extraction Of Aspartate Aminotransferase By Aqueous Two Phase System And It's Character Of Aspartate Aminotransferase

Aspartic aminotransferase is the key enzyme in product of L-Phe with the use of phenyl pyruvic acid and L-Asp. The method above which is developed by our lab has arrived at a small scale industrialized production. But the stability of the reaction and the accumulation of the product and by-product can't cater for the requirement of low cost because of the use of mobiled cell.It's necessary for us to research the pure enzyme solution systemic which is acquired by some simple method. Also we can provide the base data for the large-scale industrialized production.In this dissertation, we chose the aspartic aminotransferase from E.coli as an example to study different cell disintegrative method and choose the best one. We use ATPE system to separate the aspartic aminotransferase from the crude enzyme solution, determine the best system and coefficients, study on the enzyme property and the factors which can influent the capability of enzyme reaction.Chemical cell disintegration, high-pressure homogenization and ultrasonication are studied and the last one is choosed as the best method in view of A value, relative enzyme activity and the volume of one-time disposal. We also study on some factors that can influent the ultrasonication results and get the best coefficients: one-time disposed volume is 100mL, the time of prosess of cell disintegration is 20minutes, the power of cell disintegration is 700W.The ability of aqueous two phase system on extraction and separation of aspartate acid amino transferase has been studied. The PEG/Salt system has been systemic researched. One aqueous two phase system was found fine to extract aspartate acid amino transferase .Also many factors affecting the ability of phase separation and extaction has been researched. It is found that the best extraction system is 18%PEG1000/ 20%Na2HPO4, and in this condition, the partition coefficient is 2.36 and the purification coefficient is 2.94.In the processing of reaction, conditions such as temperature, pH, coenzyme 5'-PLP and metal ions had effects on this reaction, conditions of the reaction were chosen as followed: temperature 45℃, pH range 8.5~9.0, metal ions such as Mg2+, Mn2+could accelerate the reaction, while other ions such as Cu2+ had some degree of inhibitive effects. It is proved by experiment that a little existing of analog of phenyl pyruvic acid has little influence to the creation of L-Phe. The various enzyme in reaction system influent L-Phe and pyruvic acid, also it can influent the phenyl pyruvic acid and aspartic aminotransferase in some degree. Inorganic salt can restrain the action of aspartic aminotransferase, so it is necessary to reduce the concentration of inorganic salt.