BioEngineering Research GroupJ. Chem. Technol. Biotechnol., 81, 1678-1684 (2006)
Almeida, C. F., Calado, C. R. C., Bernardino, S. A., Cabral, J. M. S., Fonseca, L. P.
Expanded bed adsorption (EBA) was used to recover, concentrate and purify Fusarium solani pisi cutinase secreted by a recombinant Saccharomyces cerevisiae strain directly from whole fermentation culture. A Flow Injection Analysis (FIA) system for monitoring Fusarium solani pisi cutinase based on microencapsulation of p-nitrophenylbutyrate (p-NPB) in a micellar system (sodium cholate, tetrahydrofuran, phosphate buffer) was developed for monitoring this target enzyme at the outlet tube of Expanded Bed Adsorption (EBA) column. Slight different yeast cultivation conditions during cutinase production may influence fermentation performance which affects directly the cutinase adsorption during the loading step and consequently EBA process efficiency. This effect can be especially relevant when it is necessary to stop the application of feedstock to EBA column when the outlet concentration (A) of the desired product is lower than 5% of the feed concentration (Ao). Excellent correlations between the FIA system and the off-line analytical method for monitoring cutinase activity during the different EBA steps were obtained. Additionally, the blocking/fouling of sample injector and tubes of the FIA system initially observed were eliminated due to the excellent surfactant properties of sodium cholate contained in phosphate buffer and used to dilute the enzyme samples. This FIA system showed to be a powerful analytical tool for monitoring cutinase activity almost in real time (45 – 60s) maximizing enzyme adsorption while minimizing product loss and consequently maximizing product recovery yield.
Keywords: on-line monitoring; control; expanded bed adsorption - EBA; cutinase; S. cerevisiae; micellar FIA system
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