Investigation on the stability and regeneration of penicillin G acylase immobilized in immobilized metal affinity membrane

Autor: Cheng-Ju Yao, 姚呈儒
Rok vydání: 2007
Druh dokumentu: 學位論文 ; thesis
Popis: 95
The regenerated cellulose-based membrane (RC membrane) was employed to construct the immobilized metal affinity membrane (IMAM) for purification and immobilization of penicillin G acylase (PGA). This research is constituted of two parts. The first part was the preparation of IMAM on the RC membrane and the purifications of the PGA by using IMAM. For the preparation of IMAM, factors such as chelator density on the surface, epoxy functional group, NaOH concentration and elution solutions were investigated. The epichlorohydrin (EPI) reaction was enhanced as increasing NaOH concentration. However, the RC membrane was deteriorated when the NaOH concertation was higher than 1.6M. The optimal reaction conditions were concluded as follows: for one RC membrane, 5ml EPI and 20ml 1.4M NaOH was used and the reaction conditions were 24℃, 150rpm for 14h. After that, the membrane was immersed in 25ml of 1M IDA (dissolved in 1M sodium carbonate) and allowed to react at 24℃ for 12h. The amount of Cu2+ chelated on IMAM under this condition was 75μmol/disc and the adsorbed PGA was 1.8 IU/disc. By applying this conditions to the flow experiments in a cartridge, a 21.31-times of purification in specific activity with 97.1﹪recovery for PGA purification was obtained. Meanwhile, the activity of immobilized PGA could be retained for more than 40 days under 16-times repeated use at 37℃. The second part was to discuss the regeneration procedures of immobilized enzyme membrane. The regeneration was conducted by immersing the immobilized enzyme membrane in the strip buffer (300mM NaCl, 100mM EDTA, 20mM Sodium phosphate buffer, pH8)for 30 min to elute Cu2+ ion on the membrane. Then the membrane was immersed in 0.5M HCl and 0.5M NaOH respectively for 10 min to wash out the residual reactants, hydrophobic protein, etc. If was found that even under regeneration 5 times, the activity of the immobilized PGA membrane still could be maintained above 99%. This means a repeated use of the PGA immobilized on the membrane is feasible with a marked stability on the immobilized PGA activity.
Databáze: Networked Digital Library of Theses & Dissertations
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