Preparation and characterization of cross-linked enzyme aggregates (CLEA) of subtilisin for controlled release applications.
Enzyme stabilization is one of the major challenges in the biocatalytic process optimization. Subtilisin was aggregated using ammonium sulphate and polyethylene glycol with surfactants like triton X-100 and tween 20. The resultant aggregates on cross-linking with glutaraldehyde produced insoluble and catalytically active enzyme. The effect of pH, temperature, kinetic parameter, thermal stability and stability in organic solvents were studied. The cross-linked enzyme aggregates (CLEA) exhibited broad pH optima of 9.0 and higher temperature optima of 70 degrees C. Reusability and surface morphology of the CLEA were also studied. CLEA of subtilisin has good stability in nonpolar organic solvents, such as hexane, and cyclohexane and it has high thermal stability up to 60 degrees C and therefore can be used as a catalyst for the biotransformation of compounds which are not soluble in aqueous medium. The CLEAs were entrapped in the hydrogel composite beads of alginate:guar gum (3:1) which were resistant to low pH conditions in the stomach and thus was found to be useful for the oral drug delivery. This process can be used to deliver the protein and peptide drugs which involve high concentrations at the delivery stage, and which usually degrades in the stomach before reaching the jejunum. Application of these pH-sensitive beads for the controlled release of subtilisin in vitro was studied and found to be a feasible strategy.
Sangeetha
K
K
Chemical Science & Technology Division, Regional Research Laboratory (CSIR), Trivandrum 695019, India.
Abraham
T Emilia
TE
D000284:Administration, Oral; D001412:Bacillus subtilis / Q000201:enzymology*; D001711:Biotransformation; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003692:Delayed-Action Preparations; D004337:Drug Carriers / Q000737:chemistry*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism; D005976:Glutaral / Q000737:chemistry; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D009930:Organic Chemicals / Q000737:chemistry; D011485:Protein Binding; D012997:Solvents / Q000737:chemistry; D020860:Subtilisin / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
D003432:Cross-Linking Reagents; D003692:Delayed-Action Preparations; D004337:Drug Carriers; D004800:Enzymes, Immobilized; D009930:Organic Chemicals; D012997:Solvents; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D020860:Subtilisin; D005976:Glutaral