Studies on PVA Pectin cryogels containing crosslinked enzyme aggregates of keratinase

MARTINAZ, YANINA; CAVELLO, IVANA; CAVALITTO, SEBASTIÁN FERNANDO; ILLANES, ANDRES; CASTRO, GUILLERMO

COLLOIDS AND SURFACES B-BIOINTERFACES

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2014 vol. 117 p. 284 - 289

0927-7765

Polyvinyl alcohol-pectin (PVA-P) films containing enrofloxacin and keratinase were developed to treat wounds and scars produced by burns and skin injuries. 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. KCLEA 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 X-ray scattering (SAXS) analysis showed that K-CLEA bound to pectin rather than to PVA in the PVA-P matrix.