Staining method for determination of the penetration of immobilized enzyme into a porous support

Carleysmith, S. W.; Eames, Malcolm; Lilly, M. D.

doi:10.1002/bit.260220505

Cited by 56 publications

(14 citation statements)

References 3 publications

(1 reference statement)

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“…If immobilization is reversible (0 # 0), Eo = EZ, and the internal profiles of enzyme in the pore and on the pore surfaces will be spatially uniform and at equilibrium (9)…”

Section: And Incomplete Loading Occurs If V/$ >mentioning

confidence: 99%

Modeling enzyme immobilization in porous solid supports

Duong

Clark

Bailey

1982

Biotech & Bioengineering

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Enzymes are often immobilized on the internal surfaces of porous solid by immersing enzyme-free particles in a well mixed solution of enzyme. The ensuing impregnation process involves coupled transient mass transfer and surface attachment of enzyme. A mathematical model is employed to explore the influences of process parameters on the amount of enzyme loaded and the distribution of immobilized enzyme within the support particles. Nonuniform loading of the support occurs under some conditions. This is significant since the distribution of enzyme within the support particle influences the overall activity and stability of the immobilized enzyme catalyst. The model developed here may also be used to describe removal of reversibly immobilized enzyme during washing or utilization of the immobilized enzyme catalyst.

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“…If immobilization is reversible (0 # 0), Eo = EZ, and the internal profiles of enzyme in the pore and on the pore surfaces will be spatially uniform and at equilibrium (9)…”

Section: And Incomplete Loading Occurs If V/$ >mentioning

confidence: 99%

Modeling enzyme immobilization in porous solid supports

Duong

Clark

Bailey

1982

Biotech & Bioengineering

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“…Carlaysmith et al 21 measured the penetration The details of the analytical procedure have rate of proteins into a porous support by staining been given elsewhere. 16 support beads and splitting to expose the crosssection.…”

Section: Site Of Immobilizationmentioning

confidence: 99%

Application of porous microspheres prepared by SPG (Shirasu porous glass) emulsification as immobilizing carriers of glucoamylase (GluA)

Omi

Kaneko

Nakayama

et al. 1997

J. Appl. Polym. Sci.

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Fairly uniform spheres of crosslinked polystyrene (PS) and polymethyl methacrylate (PMMA), prepared by a particular emulsification process using SPG (Shirasu Porous Glass) membranes and subsequent suspension polymerization, were applied for immobilizing carriers of Glucoamylase (GluA). A mixture of monomers, solvents, and oil-soluble initiator was allowed to permeate through the micropores of SPG, suspended in an aqueous solution of poly(vinyl alcohol), and polymerized while retaining the narrow size distribution during polymerization. A small amount of acrylic acid or glycidyl methacrylate (GMA) was incorporated for the immobilization of GluA via covalent bonding. Although GluA has been regarded as being difficult to retain its activity after the immobilization process, a porous structure of the carriers definitely favored the immobilization, and a maximum 55% relative activity (RA) was obtained by the physical adsorption to PMMA spheres. The reaction of epoxide in GMA with 6aminocaproic acid provided a spacer arm for the carboxyl group. An improvement of activity was expected by the incorporation of the spacer arms; however, barely noticeable activity was observed for PMMA carriers either by the physical adsorption or by the covalent bonding. A slight improvement was observed for PS carriers with spacers compared to the carriers without them. The diffusion process of oligosaccharides in the porous carriers seemed to retard the rate of hydrolysis in the case of largest carriers, 60 mm PS-DVB-AA spheres. The activity of immobilized GluA was retained during a long storage period of more than 150 days, some of them even increasing gradually, while the activity of native GluA dropped to zero after 100 days.

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“…On the other hand, we also wanted to quantitatively measure the enzyme distribution in the immobilized derivatives in order to rationally design new improved biocatalysts. Staining with different colored dyes (such as Ponceau S and naphthol blue black) has been used to study enzyme distribution in immobilized biocatalysts (Carleysmith et al 1980;Ampon 1992). Other methods require tagging of the enzyme (e.g.…”

Section: Introductionmentioning

confidence: 99%

Kinetic and microstructural characterization of immobilized penicillin acylase fromStreptomyces lavendulaeon Sepabeads EC-EP

Hormigo

Mata

Castillón

et al. 2009

Biocatalysis and Biotransformation

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Recombinant penicillin acylase from Streptomyces lavendulae was covalently bound to epoxy-activated Sepabeads EC-EP303 † . Optimization of the immobilization process led to a homogeneous distribution of the enzyme on the support surface avoiding the attachment of enzyme aggregates, as shown by confocal electron microscopy. The optimal immobilized biocatalyst had a specific enzymatic activity of 26.2 IU g wet carrier (1 in the hydrolysis of penicillin V at pH 8.0 and 408C. This biocatalyst showed the highest activity at pH 8.5 and 658C, 1.5 pH units lower and 58C higher than its soluble counterpart. Substrate specificity of the derivative also showed its ability to efficiently hydrolyze other natural aliphatic penicillins such as penicillins K, F and dihydroF. The immobilized enzyme was highly stable at 408C and pH 8.0 (t 1/2 0625 h vs. t 1/2 0397 h for the soluble enzyme), and it could be recycled for at least 30 consecutive batch reactions without loss of catalytic activity.

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Staining method for determination of the penetration of immobilized enzyme into a porous support

Abstract: SummaryThe penetration of bovine serum albumin and penicillin acylase into Amberlite XAD7 beads was determined by staining split beads. The rates of penetration were measured and correlated with a theoretically derived equation.

Cited by 56 publications

References 3 publications

Modeling enzyme immobilization in porous solid supports

Modeling enzyme immobilization in porous solid supports

Application of porous microspheres prepared by SPG (Shirasu porous glass) emulsification as immobilizing carriers of glucoamylase (GluA)

Kinetic and microstructural characterization of immobilized penicillin acylase fromStreptomyces lavendulaeon Sepabeads EC-EP

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