Biofunctional membranes: an EPR study of active site structure and stability of papain non-covalently immobilized on the surface of modified poly(ether) sulfone membranes through the avidin-biotin linkage

Bhardwaj, Anita; Lee, Jinbo; Glauner, Kathi; Ganapathi, Sowmya; Bhattacharyya, Dibakar; Butterfield, D. Allan

doi:10.1016/0376-7388(96)00124-x

Cited by 34 publications

(17 citation statements)

References 18 publications

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“…However, the enzyme loading per unit mass of support is usually considerably low. Alternatively, high enzyme loading can be achieved with porous materials such as membranes (40)(41)(42), gel matrices (36,43,44), and porous particles (45,46). Porous materials, however, suffer a much greater diffusional limitation (24,26,32,47).…”

Section: Introductionmentioning

confidence: 99%

Enzyme‐Carrying Polymeric Nanofibers Prepared via Electrospinning for Use as Unique Biocatalysts

Jia

Zhu

Vugrinovich

et al. 2002

Biotechnology Progress

415

213

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Improvement of catalytic efficiency of immobilized enzymes via materials engineering was demonstrated through the preparation of bioactive nanofibers. Bioactive polystyrene (PS) nanofibers with a typical diameter of 120 nm were prepared and examined for catalytic efficiency for biotransformations. The nanofibers were produced by electrospinning functionalized PS, followed by the chemical attachment of a model enzyme, alpha-chymotrypsin. The observed enzyme loading as determined by active site titration was up to 1.4% (wt/wt), corresponding to over 27.4% monolayer coverage of the external surface of nanofibers. The apparent hydrolytic activity of the nanofibrous enzyme in aqueous solutions was over 65% of that of the native enzyme, indicating a high catalytic efficiency as compared to other forms of immobilized enzymes. Furthermore, nanofibrous alpha-chymotrypsin exhibited a much-improved nonaqueous activity that was over 3 orders of magnitude higher than that of its native counterpart suspended in organic solvents including hexane and isooctane. It appeared that the covalent binding also improved the enzyme's stability against structural denaturation, such that the half-life of the nanofibrous enzyme in methanol was 18-fold longer than that of the native enzyme.

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Section: Introductionmentioning

confidence: 99%

Enzyme‐Carrying Polymeric Nanofibers Prepared via Electrospinning for Use as Unique Biocatalysts

Jia

Zhu

Vugrinovich

et al. 2002

Biotechnology Progress

415

213

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“…The reverse reaction of hydrolysis of papain also can be employed in the synthesis of peptides and oligomers based on amino acids [3–5], especially immobilized papain, which has been employed in the enzymic syntheses of peptides and their derivatives in organic solvents [6]. Therefore, great attention has been focused on the investigation of immobilized papain recently [7–11]. In this paper, we report the immobilization of papain with poly( N ‐diethylenediamine)acrylamide fibre carrier, in which glutaraldehyde and casein were used as coupling agent and substrate respectively.…”

Section: Introductionmentioning

confidence: 99%

Papain immobilization on a nitrilon fibre carrier containing primary amine groups

Jia

et al. 2001

Biotech and App Biochem

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A carrier containing primary amine groups was synthesized by the reaction between nitrilon fibre and diethylenetriamine, and papain was immobilized on this kind of carrier by using glutaraldehyde as a coupling agent. The factors involved with the activity recovery of the immobilized papain and the enzymic properties of the resulting immobilized papain were studied in comparison with free papain, for which casein was chosen as a substrate. The results show that the activity recovery of immobilized papain can reach 47.3%, and that the immobilized papain exhibits better environmental adaptability and reusability than free papain.

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“…Most of these cysteine residues are thought to lie within clefts between the US monomers. These thiol functionalities can be rapidly and selectively derivatized by reaction with the thiosulfonate spin label MTS [(l-oxyl-2,2,5,5-tetramethylpyrroline -3-methyl)methanethiosulfonate] (Berliner et al, 1982;Butterfield and Lee, 1994;But-terfIeld et al, l994a;Trad et al, 1995;Bhardwaj et al, 1996;Butterfield, 1996a;Subramaniam et al, 1996). This reaction can be monitored in real time using EPR.…”

mentioning

confidence: 99%

Oxidatively Induced Structural Alteration of Glutamine Synthetase Assessed by Analysis of Spin Label Incorporation Kinetics: Relevance to Alzheimer's Disease

Butterfield

Hensley

Cole

et al. 1997

Journal of Neurochemistry

171

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The activity of the astrocytic enzyme glutamine synthetase (GS) is decreased in the Alzheimer's disease brain, which may have relevance to mechanisms of chronic excitotoxicity. The molecular perturbation(s) that results in GS inactivation is not known, although oxidative lesioning of the enzyme is one likely cause. To assess structural perturbation induced in GS by metal-catalyzed oxidation, a series of spin-labeling studies were undertaken. Ovine GS was oxidized by exposure to iron/hydrogen peroxide and subsequently labeled with the thiol-specific nitroxide probe MTS [(1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate]. The reaction of MTS with cysteine residues within GS was monitored in real time by electron paramagnetic resonance spectrometry. Structural perturbation of GS, manifested as decreased thiol accessibility, was inferred from an apparent decrease in the rate constant for the secondorder reaction of MTS with protein thiols. A subsequent spin-labeling study was undertaken to compare the structural integrity of GS purified and isolated from Alzheimer's disease-afflicted brain (AD-GS) with that of GS isolated from nondemented, age-matched control brain (C-GS). The rate constant for reaction of MTS with AD-GS was markedly decreased relative to that for the reaction of spin label with C-GS. The kinetic data were partially corroborated by spectroscopic data obtained from circular dichroism analysis of control and peroxide-treated ovine GS. In an adjunct experiment, the interaction of GS with a synthetic analogue of the Alzheimer's-associated ßamyloid peptide, known to induce free radical oxidative stress, indicated strong interaction of the enzyme with the peptide as reflected by a decrease in the rate constant for MTS binding to reactive protein thiols.

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Biofunctional membranes: an EPR study of active site structure and stability of papain non-covalently immobilized on the surface of modified poly(ether) sulfone membranes through the avidin-biotin linkage

Cited by 34 publications

References 18 publications

Enzyme‐Carrying Polymeric Nanofibers Prepared via Electrospinning for Use as Unique Biocatalysts

Enzyme‐Carrying Polymeric Nanofibers Prepared via Electrospinning for Use as Unique Biocatalysts

Papain immobilization on a nitrilon fibre carrier containing primary amine groups

Oxidatively Induced Structural Alteration of Glutamine Synthetase Assessed by Analysis of Spin Label Incorporation Kinetics: Relevance to Alzheimer's Disease

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