Studies on PVA pectin cryogels containing crosslinked enzyme aggregates of keratinase

https://doi.org/10.1016/j.colsurfb.2014.02.049Get rights and content

Highlights

  • Keratinase CLEA prevents enzyme inactivation against enzyme denaturants like solvents, high temperature and autolysis.

  • Immobilization of CLEAs into PVA-P cryogel retarded the release in comparison with free enzyme.

  • The control of K-CLEA release is based on the pectin present in the PVA film.

  • K-CLEAs keep 51% of their activity after 2 months at 37 °C.

  • K-CLEA activity was 1.4, 1.7 and 6.6 times higher in acetone, n-hexane and isobutanol than the soluble enzyme.

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 1 h 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.

Introduction

As previously reported, keratinase isolated from Paecilomyces lilacinus strain LPSC#876 was immobilized into polyvinyl alcohol-pectin (PVA-P) cryogel showing a controlled release profile without loss of enzymatic activity [1]. The use of PVA-P cryogel in wound healing, however, exposes the biocatalyst to harsh environmental conditions at the interface and over the skin wound area. The keratinase activity can be influenced by changes of CO2 partial pressure, autolysis–proteolysis, ionic strength, temperature and pH gradients, and desolvation–solvation processes. Partial pressure of CO2 (pCO2) is enhanced in a wound microenvironment because it is insoluble at hyperoxial conditions during the healing process. This phenomena has been observed in animals were ambient pCO2 was increased up to 5% producing respiratory acidosis. The damaged tissue tends to metabolically compensate this effect by alkalosis response reducing collagen deposition [2]. Also, autolysis is a common inactivation phenomenon reported for many proteases [3]. Either combined or individual factors can be a serious drawback during shelf storage of therapeutic devices containing keratinase. Therefore, the main challenge is to keep the enzyme active inside the cryogel, at interfacial conditions and in the wound microenvironment.

Crosslinked enzyme aggregates (CLEAs) are a promising alternative to enhance protein structural stability and to extend the half-life of the biocatalyst even at harsh operational conditions. CLEAs can be produced by a simple method of enzyme precipitation under non-denaturing conditions followed by cross linking with bifunctional reagents, such as glutaraldehyde [4]. Enzyme precipitation can be produced by the addition of many compounds such as salts, organic solvents, non-ionic polymers or acids [5]. In addition, hyperactivation has been observed in the synthesis of CLEAs of some enzymes, such as lipase and penicillin acylase [6], [7].

In order to study enzyme stability at harsh environmental conditions such as interface and solvation changes, the use of organic solvents was proposed as a model system. Solvents partition coefficient (Log P) values regards to solvent's ability to replace water molecules bound to enzymes. Also, dielectric constant (ɛ) values of the different solvents used was taken into account to study the replacement of water molecules bound to the enzyme and how this phenomenon affects enzyme stability [8].

The aim of the present work is to develop and study CLEAs of keratinase (K-CLEAs) from crude extracts of P. lilacinus LPSC#876. Effects of polar and non-polar solvents, kinetic constants, temperature and ionic strength are compared between soluble keratinase and K-CLEA. K-CLEAs were analyzed by electron microscopy. Inclusion of K-CLEAs into PVA and PVA-P cryogels was analyzed by small angle X-ray scattering spectrometry (SAXS). Finally, K-CLEA controlled release was monitored under simulated skin conditions.

Section snippets

Materials

Dimethyl sulfoxide (DMSO), ethanol and isobutanol were of analytical grade and provided by Biopack (Buenos Aires, Argentina). Acetone and buffers were provided from Anedra S.A. (Buenos Aires, Argentina). Azocasein, bovine serum albumin (BSA, fraction V), polyvinyl alcohol (PVA, MWav = 13–23 kDa, 98–99% hydrolyzed) and serine protease (from Streptomyces griseus) were purchased from Sigma–Aldrich (Buenos Aires, Argentina). Pectin of 55% methoxylation degree was kindly supplied by CPKelco (Buenos

Optimization of CLEA production

Keratinase with specific activity of 18.35 EU/mg was precipitated with increasing concentrations of acetone. Full enzyme precipitation was detected in 75% (v/v) acetone without loss of keratinase activity (Fig. 1 of additional material). Keratinase activity was fully recovered up to 90% (v/v) acetone after solvent evaporation and redissolution in buffer (Fig. 2 of additional material). However, the recovery of keratinase was only 60% when acetone concentrations were higher than 90%. Over 90%

Conclusions

The development of CLEAs for keratinase has many advantages such as higher enzyme stability compared to soluble keratinase under harsh environmental conditions, e.g. in the presence of organic solvents, high temperatures and ionic strength, and prolonged storage. In addition, the procedure for the production of K-CLEAs developed is simple, reproducible and scalable.

Immobilization of CLEA in PVA and PVA-P cryogels was tested in kinetic release experiments. In the presence of pectin, K-CLEA

Acknowledgments

The present work was supported by Argentinian grants from CONICET (National Council for Science and Technology, PIP 0214), The National Agency of Scientific and Technological Promotion (ANPCyT) and UNLP (National University of La Plata, 11/X522, 11/545 and PRH 5.2). We want to thank Mr. Erick Araya Garmendia (Escuela de IngenieríaBioquímica, Universidad Católica de Valparaíso, Chile) for his technical help on CLEA synthesis, to Dr. Mateus Cardoso (National Laboratory of Synchrotron Light,

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