The Effect of Pectinase on the Bubble Fractionation of Invertase from α-Amylase

Abstract

Fermentation broth normally contains many extracellular enzymes of industrial interest. To separate such enzymes on-line could be useful in reducing the cost of recovery as well as in keeping their yield at a maxi­mum level by minimizing enzyme degradation from broth proteases (either the desired enzymes or the proteases could be removed selec­tively or both removed together and then separated). Several large-scale separation methods are candidates for such on-line recovery such as ultrafiltration, precipitation, and two-phase partitioning. Another promising technique for on-line recovery is adsorptive bubble fractiona­tion, the subject of this study. Bubble fractionation, like ultrafiltration, does not require contaminating additives and can complement ultrafil­tration by preconcentrating the enzymes using the gases normally pre­sent in a fermentation process. A mixture of enzymes in an aqueous bubble solution can, in principle, be separated by adjusting the pH of that solution to the isoelectric point (pI) of each enzyme as long as the enzymes have different pls. The model system investigated here is com­prised of three enzyme separations and the problem is posed as the effect of pectinase (a charged enzyme) on the bubble fractionation of invertase (a relatively hydrophilic enzyme) from α-amylase (a relatively hydrophobic enzyme).

The primary environmental variable studied, therefore, is the pH in the batch bubble fractionation column. Air was used as the carrier gas. This prototype mixture exemplifies an aerobic fungal fermentation process for producing enzymes. The enzyme concentration here is mea­sured as total protein concentration by the Coomassie Blue (Bradford) solution method (1), both as a function of time and column position for each batch run. Since, from a previous study (2), it was found that invertase and α-amylase in a two-enzyme system can be partially separated in favor of one vs the other at two different pHs (pH 5.0 and 9.0) with significant separation ratios, emphasis is placed on the effect of pecti­nase at these pHs. In this study, the addition of pectinase reduced the total separation ratio of the α-amylase-invertase mixture at both pHs.

Author to whom all correspondence and reprint requests should be addressed.