RESUMO
Allyl glycidyl ether-ethylene glycol dimethacrylate copolymer with 25 % crosslink density has been functionalized with various amines. The polymer bearing a dibenzyl amino function efficiently removed the soybean trypsin inhibitor (SBTI) and lipoxygenase (LOX) from soymilk. The polymer binds SBTI and LOX efficiently (25-30 mg/g) through a combination of hydrophobic and ionic interactions and denatures them at room temperature by simple contact for 1 h. The bound proteins can be easily recovered almost quantitatively by elution with 1 % (v/v) acetic acid, and the polymer can be recycled at least 10 times without significant changes in binding capacity.
Assuntos
Lipoxigenase/química , Leite de Soja/química , Proteínas de Soja/química , Inibidores da Tripsina/química , Adsorção , Compostos de Epóxi/química , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Lipoxigenase/isolamento & purificação , Lipoxigenase/metabolismo , Metacrilatos/química , Desnaturação Proteica , Proteínas de Soja/isolamento & purificação , Proteínas de Soja/metabolismo , Inibidores da Tripsina/isolamento & purificação , Inibidores da Tripsina/metabolismoRESUMO
Bovine serum albumin (BSA) was adsorbed on allyl glycidyl ether (AGE)-ethylene glycol dimethacrylate (EGDM) copolymer with 25% crosslink density (AGE-25) at pH 8.0 (16% w/w). The amino, thiol and carboxylic acid functional groups available on protein coated surface were utilized for covalent immobilization of glucose oxidase and ß-galactosidase, both independently, and in a step-wise manner on the same matrix, with no more than 10% loss of enzyme activity during immobilization. Glutaraldehyde cross-linking after immobilization provided stable enzyme preparations. The pH-optima of the immobilized enzymes were similar to those for free enzyme but their thermal stability was vastly improved. The co-immobilized enzyme support was used as a biosensor for determination of lactose in milk with excellent reproducibility and reusability.
Assuntos
Técnicas Biossensoriais/métodos , Lactose/análise , Leite/química , Animais , Bovinos , Estabilidade Enzimática , Enzimas Imobilizadas , Compostos de Epóxi , Glucose Oxidase , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Cinética , Metacrilatos , Soroalbumina Bovina , Temperatura , beta-GalactosidaseRESUMO
Allyl glycidyl ether (AGE)-ethylene glycol dimethacrylate (EGDM) copolymer with 25% crosslink density (AGE-25) shows excellent bovine serum albumin (BSA) adsorption (up to 16% (w/w)) at pH 8.0 and the adsorbed BSA is strongly bound. This protein-coated polymer provides a novel matrix with naturally existing functional groups such as thiol, amino, and carboxylic acid that are available for covalent immobilization of functional enzymes. Employing appropriate strategies, trypsin as a model protein was covalently bound to BSA-coated matrix both independently, and in a stepwise manner on the same matrix, with less than 5% loss of enzyme activity during immobilization. Glutaraldehyde crosslinking after immobilization provide stable enzyme preparation with activity of 510 units/g recycled up to six times without loss of enzyme activity. AFM studies reveal that the polymer surface has protein peaks and valleys rather than a uniform monolayer distribution of the protein and the immobilized enzyme preparation can best be described as polymer supported cross-linked enzyme aggregates (CLEAs).