Dataset Information

Modification of PEGylated enzyme with glutaraldehyde can enhance stability while avoiding intermolecular crosslinking.

ABSTRACT: We demonstrate an enzyme stabilization approach whereby a model enzyme is PEGylated, followed by controlled chemical modification with glutaraldehyde. Using this stabilization strategy, size increases and aggregation due to intermolecular crosslinking are avoided. Immediately following synthesis, the PEGylated enzyme with and without glutaraldehyde modification possessed specific activities of 372.9 ± 20.68 U/mg and 373.9 ± 15.14 U/mg, respectively (vs. 317.7 ± 19.31 U/mg for the native enzyme). The glutaraldehyde-modified PEGylated enzyme retains 73% original activity after 4 weeks at 37 °C (vs. 2% retention for control).

SUBMITTER: Ritter DW

PROVIDER: S-EPMC4452374 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Modification of PEGylated enzyme with glutaraldehyde can enhance stability while avoiding intermolecular crosslinking.

Ritter D W DW Newton J M JM McShane M J MJ

RSC advances 20140617 53

We demonstrate an enzyme stabilization approach whereby a model enzyme is PEGylated, followed by controlled chemical modification with glutaraldehyde. Using this stabilization strategy, size increases and aggregation due to intermolecular crosslinking are avoided. Immediately following synthesis, the PEGylated enzyme with and without glutaraldehyde modification possessed specific activities of 372.9 ± 20.68 U/mg and 373.9 ± 15.14 U/mg, respectively (<i>vs.</i> 317.7 ± 19.31 U/mg for the native e ...[more]

PMID: 26052433

Similar Datasets

Project description:In this study, six-arm star-shaped poly(N-vinylcaprolactam) (PNVCL) polymers prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization were subjected to aminolysis reaction using hexylamine. Chemically crosslinked gels or highly end-functionalized star polymers can be obtained depending mainly on the type of solvent used during the transformation of the RAFT functional group. An increase in the viscosity of the solution was observed when the aminolysis was carried out in THF. In contrast, when the reaction was conducted in dichloromethane, chain-end thiol (PNVCL)6 star polymers could be obtained. Moreover, when purified (PNVCL-SH)6 star polymers are in contact with THF, the gelation occurs in just a few minutes, with an obvious increase in viscosity, to form physical gels that become chemically crosslinked gels after 12 h. Interestingly, when purified (PNVCL-SH)6 star polymers were stirred in distilled water, even at high aqueous solution concentration (40 mg/mL), there was no increase in the viscosity or gelation, and no evident gels were observed. The analysis of the hydrodynamic diameter (Dh) by dynamic light scattering (DLS) did not detect quantifiable change even after 4 days of stirring in water. On the other hand, the thiol groups in the (PNVCL-SH)6 star polymers were easily transformed into trithiocarbonate groups by addition of CS2 followed by benzyl bromide as demonstrated by UV-Vis spectroscopical analysis and GPC. After the modification, the (PNVCL)6 star polymers exhibit an intense yellow color typical of the absorption band of trithiocarbonate group at 308 nm. To further demonstrate the highly effective new trithiocarbonate end-functionality, the PNVCL polymers were successfully chain extended with N-isopropylacrylamide (NIPAM) to form six-arm star-shaped PNIPAM-b-PNVCL block copolymers. Moreover, the terminal thiol end-functionality in the (PNVCL-SH)6 star polymers was linked via disulfide bond formation to l-cysteine to further demonstrate its reactivity. Zeta potential analysis shows the pH-responsive behavior of these star polymers due to l-cysteine end-functionalization. By this using methodology and properly selecting the solvent, various environment-sensitive star polymers with different end-groups could be easily accessible.

Project description:Common commercial porcine acellular dermal matrix (PADM) products take the form of a thin membrane. Given its dense structure, delaying vascularization after implantation remains an issue to be solved. In addition, overlaying multiple sheets to address deep wounds and large tissue defects that are difficult to repair by self-tissues could hinder tissue ingrowth, angiogenesis, and integration. Here, we creatively prepared PADM microparticles through a homogenizing treatment and crosslinked them to ADM sponges by thermal crosslinking (VT-ADM) and thermal-glutaraldehyde crosslinking (GA-ADM). The resulting VT-ADM was thicker than GA-ADM, and both maintained the natural dermal matrix microstructure and thermal stability. The porosity of GA-ADM (mean 82%) was lower than that of VT-ADM (mean 90.2%), but the mechanical strength and hydrophilicity were significantly higher. The two types of ADM sponges showed no obvious difference in cell adhesion and proliferation without cytotoxicity. Furthermore, the human adipose stem cells were co-cultured with ADM sponges which promoted proliferation, tube formation, and migration of endothelial cells, and the GA-ADM group exhibited better migration behavior. There were no markable differences among expressions of pro-angiogenesis genes, including vascular endothelial growth factor, insulin-like growth factor-1, and epidermal growth factor. In a nude mouse model, the VT-ADM and GA-ADM pre-cultured with human adipose stem cells for 1 week in advance were implanted subcutaneously. The VT-ADM and the GA-ADM showed great histocompatibility without local redness, swelling, or necrosis. The vascular density of the local skin flap above the material was visualized using indocyanine green and showed no statistical difference between the two groups. The collagen tissue deposition in the pores and vessel formation within the sponges increased with time. Although VT-ADM had a higher degradation rate in vivo, the integrity of the two scaffolds was preserved. Collectively, the VT-ADM and the GA-ADM retained a natural matrix structure and presented biocompatibility. Thus, the above-mentioned two crosslinking methods for ADM sponges are safe and practicable. The novel ADM sponges with good physicochemical and biological properties are no longer limited to membrane tissue regeneration but could also realize structure remodeling where they act as scaffolds for a soft tissue filler and three-dimensional reconstruction of the tissue with strength requirements.

Dataset Information

Modification of PEGylated enzyme with glutaraldehyde can enhance stability while avoiding intermolecular crosslinking.

Publications

Modification of PEGylated enzyme with glutaraldehyde can enhance stability while avoiding intermolecular crosslinking.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets