Studies on PVA Pectin cryogels containing crosslinked enzyme aggregates of keratinase

MARTINEZ N YANINA; CAVELLO IVANA; CAVALITTO SEBASTIÁN; ILLANES ANDRES; GUILLERMO R. CASTRO

Lille

Congreso; 0th European Symposium on Biochemical Engineering Sciences and 6th International Forum on Industrial Bioprocesses in collaboration with ACS; 2014

Universite of Lille / ACS

Polyvinyl alcohol-pectin (PVA-P) films containing enrofloxacin and keratinase were developed to treat wounds and scars produced by burns and skin injuries[1]. However,in order to prevent enzyme inactivation at the interface between the patch and the scars, crosslinked enzyme aggregates (CLEAs) from a crude extract of keratinase produced by Paecilomyces lilacinus (LPSC#876) were synthesized by precipitation with acetone and crosslinking with glutaraldehyde. Soluble vs. CLEA keratinase (K-CLEA)activities were tested in 59 % (v/v) hydrophobic (isobutanol and n-hexane) and hydrophilic (acetone and dimethylsulfoxide) solvents mixtures. K-CLEA activity was 1.4,1.7 and 6.6 times higher in acetone, n-hexane and isobutanol than the soluble enzyme at 37 ºC after 1 h of incubation, respectively. K-CLEA showed at least 45 % of enzyme residual activity in the 40 to 65ºC range, meanwhile the soluble biocatalyst was fully inactivated at 65 ºC after 1 h incubation. Also, the soluble enzyme was completely inactivated after 12 hours at pH 7.4 and 45 ºC, even though K-CLEA retained full activity. The soluble keratinase was completely inactivated at 37 ºC after storage in buffer solution (pH= 7.4) for two months, meanwhile K-CLEAs kept 51 % of their activity.K-CLEA loaded into polyvinyl alcohol (PVA) and PVA-P cryogels showed six times lower release rate compared to the soluble keratinase at skin pH (5.5). Small angle Xray scattering (SAXS) analysis showed that K-CLEA bound to pectin rather than to PVA in the PVA-P matrix.