| L-Asparaginase (E.C.3.5.1.1), one kind of effective drugs using in the treatment of acute lymphoblastic leukemia (ALL), may lead to severe side effects such as allergic reactions as well as anaphylactic shocks in the therapeutic application. It concerns how to solve these problems and improve the enzyme performance in clinical research. In this paper, we chemically modified L-Asparaginase with silk fibroins, a kind of natural high molecular material with stability and non-toxicity. We focusd on exploring the best optimization of reaction conditions for modification, purified modified products preliminarily. Physical and chemical properties of modified enzyme were analyzed at last. Main contents and results of the research are as follows:After extracted from cocoon, silk fibroins modifiers were prepared with further separation using gel filtration (Tricorn 10×600, Sephcryl S-300 HR) chromatography. Silk fibroins were separated homogenized in the molecular level.We studied the effects of reaction condition, such as molecular ratio, pH value, reaction temperature and time by orthogonal design experiment. Meanwhile, single factor design experiment on the concentration of crosslinking agent was studied as well. The results showed that the optimized reaction condition was found to be phosphate buffer pH 7.0, molecular ratio of silk fibroin to L-asparaginase was 5:1, reaction time 8 h and reaction temperature 4℃. Under this optimized reaction condition, the recovery of modified L-asparaginase was 66.58%, the rate of modifiedε- amino was 57.88%.Silk fibroin-L-asparaginase conjugations were purified with two consecutive anion-exchange (XK 16×20 Q Sepharose FF) and gel filtration (Tricorn 10×600,Sephcryl S-300 HR) chromatography. These two steps removed most of excess free silk fibroins, crosslinking agent and some other impurity.Finally, we studied physical and chemical properties of modified enzyme. The results showed that, Km of native L-asparaginase was 6.665 times as the value of enzyme after modified. The affinity of modified enzyme to substrate L-asparagine elevated. The optimal reaction temperature and pH value of modified enzyme was 70℃and 7.0 respectively. Thermo and pH stability of L-asparaginase after modified with silk fibroins increased. Meanwhile, the resistance to trypsin digestion of modified enzyme had been improved. |
