title: Studies on PVA pectin cryogels containing crosslinked enzyme aggregates of keratinase.
abstract: 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 °C after 1 h of incubation, respectively. K-CLEA showed at least 45% of enzyme residual activity in the 40-65 °C range, meanwhile the soluble biocatalyst was fully inactivated at 65 °C after 1h incubation. Also, the soluble enzyme was completely inactivated after 12 h at pH 7.4 and 45 °C, even though K-CLEA retained full activity. The soluble keratinase was completely inactivated at 37 °C after storage in buffer solution (pH 7.4) for 2 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.
authors.affiliation: Nanobiomaterials Laboratory, Institute of Applied Biotechnology (CINDEFI, UNLP-CONICET-CCT La Plata), School of Sciences, Universidad Nacional de La Plata, Calle 50 # 227, 1900 La Plata, Argentina.
authors.forename: Yanina N
authors.initials: YN
authors.lastname: Martínez
authors.affiliation: Institute of Applied Biotechnology (CINDEFI, UNLP-CONICET-CCT La Plata), School of Sciences, Universidad Nacional de La Plata, Calle 50 # 227, 1900 La Plata, Argentina.
authors.forename: Ivana
authors.initials: I
authors.lastname: Cavello
authors.affiliation: Institute of Applied Biotechnology (CINDEFI, UNLP-CONICET-CCT La Plata), School of Sciences, Universidad Nacional de La Plata, Calle 50 # 227, 1900 La Plata, Argentina.
authors.forename: Sebastián
authors.initials: S
authors.lastname: Cavalitto
authors.affiliation: Escuela de Ingeniería Bioquímica, Universidad Católica de Valparaíso, Avenida Brasil, 2147, Valparaiso, Chile.
authors.forename: Andres
authors.initials: A
authors.lastname: Illanes
authors.affiliation: Nanobiomaterials Laboratory, Institute of Applied Biotechnology (CINDEFI, UNLP-CONICET-CCT La Plata), School of Sciences, Universidad Nacional de La Plata, Calle 50 # 227, 1900 La Plata, Argentina. Electronic address: grcastro@gmail.com.
authors.forename: Guillermo R
authors.initials: GR
authors.lastname: Castro
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry*; D059985:Cryogels / Q000737:chemistry*; D004795:Enzyme Stability; D007700:Kinetics; D009994:Osmolar Concentration; D010143:Paecilomyces / Q000201:enzymology; D010368:Pectins / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D011142:Polyvinyl Alcohol / Q000737:chemistry*; D066329:Protein Aggregates; D053838:Scattering, Small Angle; D012995:Solubility; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D014961:X-Ray Diffraction
chemical_list: D003432:Cross-Linking Reagents; D059985:Cryogels; D066329:Protein Aggregates; D012997:Solvents; D010368:Pectins; D011142:Polyvinyl Alcohol; D010447:Peptide Hydrolases; C035855:keratinase
keywords: Autolysis; CLEA; Controlled release; Cryogel; Keratinase; PVA