Full text
| |
| Author(s): |
Regiane da Silva
Total Authors: 1
|
| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) |
| Defense date: | 2002-08-15 |
| Examining board members: |
Ronaldo Nogueira de Moraes Pitombo;
José Abrahão Neto;
Valdir Augusto Neves
|
| Advisor: | Ronaldo Nogueira de Moraes Pitombo |
| Abstract | |
Bacterial L-asparaginase (L-asparaginase amidohydrolase, E.C. 3.5.1.1) are enzymes of high therapeutic value due to their use in the treatment of lymphocytic acute leukemia. Escherichia coli L-asparaginase is a periplasmic enzyme of high affinity, particularly effective in some kinds of childhood cancer therapies. Several studies have showed that there are specific stabilizing additives preserve the structure and the biological activity of protein molecules (lyoprotectant). However, the protection mechanism for these excipients has not been totally elucidated yet. The aim of this work was investigate the effect of freeze-drying on the enzymatic activity of L-asparaginase using both the purified enzyme as well as intact cells of Escherichia coli. Until recently the way to evaluate the behavior of an addictive one and the behavior of the water in the stabilization of a protein during the freeze-drying consisted of the measure of the activity parameters after the reidratação, even so now modem techniques of the Nuclear Magnetic Resonance of low resolution have been used to understand the behavior of the water in the interactions with proteins. It is Infrared Spectroscopy for Fourier Transformed it presents a great potential in the study of stabilization of proteins during the freeze-drying. The calculation of percentage of activity retention was used to express the studied values of enzymatic activity. These calculations were accomplished for the frozen systems and for the frozen systems and freeze-dryied. The systems were treated in three speeds of different freezing (20°C/min, 5°C/min and 2°C/min), being the freeze-drying for 24 hours. Results are presented on the effect of the freeze-drying in the different systems addictive-enzyme and addictive-cell. The addictive ones were used: sucrose, maltose, lactose, inositol, manitol and trehalose tested in different concentrations (30, 90 and 150mM), to identify which the conditions and the addictive ones that presented a satisfactory cryoprotection. For the systems enzyme-maltose, frozen slowly, the results of activity retention were: 8,67%, 14,02% e 30,80% to 30,90 e 150mM,respectively . The system enzyme-maltose (150mM) frozen quickly and freeze-dryied presented the largest activity retention (111,11 %) and also the largest value of T2 (81 µs) in the referring results NMR. The systems enzyme-trealose in the concentrations of 90 and 150mM they presented retention of activity 89,93% and 79,74%, respectively. (AU) | |