New application of depth filters for the immobilization of Candida antarctica lipase B.

The objective of this study was to use for the first time depth filters, which are usually intended for clarification of cell culture broth, as a direct immobilization support/matrix for industrially relevant enzymes. With this method, it is not only possible to immobilize pure enzymes; it can be also used for capturing recombinant enzymes directly out of culture supernatant. Therefore, the depth filters were coated with different anionic and cationic polymer layers by Layer-by-Layer (LbL) technology. The immobilization behavior of the model enzyme Candida antarctica lipase B (CalB) was examined. Optimal conditions for lipase immobilization were found for anionic surfaces with Poly (allylamin hydrochlorid) (PAH)/Poly (sodium-4-styrene sulfonate) (PSS) coating in 20 mM acetate buffer pH 4. Stability studies showed that immobilized CalB is 1.7-fold more stable when storage is carried out in buffer at 4 °C, compared to storage in buffer at room temperature or storage after drying at 30 °C for 24 h. The calculated half-life period is 108 days until half of the activity was lost. Furthermore, the possibility of direct capture of the CalB either from sonicated culture broth (Escherichia coli) or from cell-free supernatant was tested. Filter blocking prevented the immobilization of lipase from sonicated culture broth, but immobilization from cell-free supernatant could be performed successfully at moderate biomass content (OD600 = 7.0).

Intra-portal injection of 400- microm microcapsules in a large-animal model.

To date, encapsulated grafts have usually been implanted in the peritoneal cavity. This site is, however, not ideal, mainly because of its low blood supply. We have investigated the feasibility of intra-portal injection of (400 microm) microcapsules in the pig. Ten-thousand microcapsules per kilogram body weight were injected into six Large White pigs. Portal pressure, various biological tests, portographies and liver histology were recorded before and at various time points after injection. As a result, portal pressure increased after injection (15+/-2.3 vs 8.7+/-1.7 mmHg) but remained within an acceptable range (<20 mmHg) and returned to normal values at 3 months (8.5+/-3.7 mmHg). During the 3-month follow up, liver function and liver tests remained stable. Portographies showed a homogenous implantation of the capsule, with the portal flow always directed to the liver. At histological examination after 3 months the capsules demonstrated various degrees of fibrosis. We can thus conclude that these results demonstrate that intra-portal injection of microcapsules is feasible in a large-animal model. Hemodynamic, biological and radiological results are similar to those observed in clinical free-islet transplantation.