Characterization of alginate lyase activity on liquid, gelled, and complexed states of alginate.
A study of alginate lyase was carried out to determine if this enzyme could be used to remove alginate present in the core of alginate/poly-L-lysine (AG/PLL) microcapsules in order to maximize cell growth and colonization. A complete kinetic study was undertaken, which indicated an optimal activity of the enzyme at pH 7-8, 50 degrees C, in the presence of Ca2+. The buffer, not the ionic strength, influenced the alginate degradation rate. Alginate lyase was also shown to be active on gelled forms of alginate, as well as on the AG/PLL complex constituting the membrane of microcapsules. Batch cultures of CHO cells in the presence of alginate showed a decrease of the growth rate by a factor of 2, although the main metabolic flux rates were not modified. The addition of alginate lyase to cell culture medium increased the doubling time 5-7-fold and decreased the protein production rate, although cell viability was not affected. The addition of enzyme to medium containing alginate did not improve growth conditions. This suggests that alginate lyase is probably not suitable for hydrolysis of microcapsules in the presence of cells, in order to achieve high cell density and high productivity. However, the high activity may be useful for releasing cells from alginate beads or AG/PLL microcapsules.
Breguet
Véronique
V
Laboratory of Biological and Chemical Engineering, Ecole Polytechnique Fédérale de Lausanne, Switzerland.
von Stockar
Urs
U
Marison
Ian W
IW
D000464:Alginates / Q000737:chemistry*; D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry*; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation; D004800:Enzymes, Immobilized; D005782:Gels / Q000737:chemistry*; D020723:Glucuronic Acid / Q000737:chemistry; D006603:Hexuronic Acids / Q000737:chemistry; D008422:Materials Testing; D044367:Phase Transition; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000378:metabolism*
D000464:Alginates; D001672:Biocompatible Materials; D004800:Enzymes, Immobilized; D005782:Gels; D006603:Hexuronic Acids; D020723:Glucuronic Acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase