- مبلغ: ۸۶,۰۰۰ تومان
- مبلغ: ۹۱,۰۰۰ تومان
The precipitation of endoglucanase from Aspergillus niger with synthetic and natural electrically charged polymers -poly vinyl sulfonate (PVS) and chitosan (CHS)- was characterized and applied to a simple method of purification of an enzymatic extract obtained from fungal culture under solid-state fermentation (SSF). The kinetics of complex formation was determined. The results of the kinetic profile obtained for CHS and PVS indicated an exothermic mechanism for the formation of the non-soluble complex. CHS exhibited a marked stabilizing effect on endoglucanase. The enzyme precipitated successfully with both polymers. The precipitation method applied to commercial endoglucanase and the fungal extract showed similar patterns with high purification factors. The recovery of the activity in the re-dissolved precipitate from the fungal extract was close to 40% at pH 5.3 using PVS (1% w/w) as precipitating agent and the purification factor was near 9. The purification factor of endoglucanase in the precipitate of the enzymatic extract from SSF with CHS (0.05% w/v) was around 7. These parameters make this precipitation method appropriate to be included in the last stages of a downstream process, with advantages such as simplicity, scalability and ability to concentrate and stabilize the enzyme.
In this work, the conditions of non-soluble complex formation between commercial endoglucanase and PVS and CHS were determined. Solubility diagrams for both complexes were slightly dependent on polymer concentration. The increase in enzymatic activity observed after precipitation with PVS and CHS allowed us to conclude about the stabilizing effect of both polymers; however, CHS presented a more marked effect. Precipitation curves made at different ionic strengths demonstrated that the interactions between the enzyme and the two polymers are predominantly electrostatic. The kinetics of the precipitate formation showed that both complexes (PVS-enzyme and CHS-enzyme) are favored at temperatures below 20 °C, the formation of the PVS-enzyme complex being faster (10 min) than that of CHS-enzyme (40 min). High values of purification factors were obtained when applying the methodology on a commercial endoglucanase. The same trend was observed when the precipitation was carried out on a fungal extract. However, the fungal extract presented higher purification factors (close to 9 fold). These performance parameters make this precipitation method appropriate to be included in the last stages of a downstream process. It is a simple, economical and scalable methodology that uses low quantities of non-toxic and biodegradable polymers such as CHS. Additionally, a concentration and stabilization of the enzyme can be achieved, which represents an advantage over chromatographic methods. If the inclusion of this method in earlier stages were desirable, the recoveries could be enhanced by the re-precipitation of the enzyme by an extra addition of polymer to the supernatant.