Spacer effects on enzymatic activity of papain immobilized onto porous chitosan beads

Itoyama, Koki; Tanibe, Hiroaki; Hayashi, Toshio; Ikada, Yoshito

doi:10.1016/0142-9612(94)90258-5

Cited by 29 publications

(21 citation statements)

References 14 publications

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“…Spacer arm constituted by aliphatic chains of six‐carbon atoms has been used to move away‐immobilized glucoamylase from the support. The attachment of six‐carbon atoms hydrophobic spacer‐arm on the poly(HEMA‐EGDMA) microspheres surface could prevent undesirable side interaction between large enzyme molecule and support 7, 22. In this way, more areas of the immobilized glucoamylase could become accessible to its large substrate soluble dextrin.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Immobilization of glucoamylase on the plain and on the spacer arm‐attached poly(HEMA‐EGDMA) microspheres

Arıca

Yavuz

Deni̇zli̇

2001

J of Applied Polymer Sci

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Immobilization glucoamylase onto plain and a six-carbon spacer arm (i.e., hexamethylene diamine, HMDA) attached poly(2-hydroxyethylmethacrylate-ethyleneglycol dimethacrylate) [poly(HEMA-EGDMA] microspheres was studied. The microspheres were prepared by suspension polymerization and the spacer arm was attached covalently by the reaction of carbonyl groups of poly(HEMA-EGDMA). Glucoamylase was then covalently immobilized either on the plain of microspheres via CNBr activation or on the spacer arm-attached microspheres via CNBr activation and/or using carbodiimide (CDI) as a coupling agent. Incorporation of the spacer arm resulted an increase in the apparent activity of the immobilized enzyme with respect to enzyme immobilized on the plain of the microspheres. The activity yield of the immobilized glucoamylase on the spacer armattached poly(HEMA-EGDMA) microspheres was 63% for CDI coupling and 82% for CNBr coupling. This was 44% for the enzyme, which was immobilized on the plain of the unmodified poly(HEMA-EGDMA) microspheres via CNBr coupling. The K m values for the immobilized glucoamylase preparations (on the spacer arm-attached microspheres) via CDI coupling 0.9% dextrin (w/v) and CNBr coupling 0.6% dextrin (w/v) were higher than that of the free enzyme 0.2% dextrin (w/v).The temperature profiles were broader for both immobilized preparations than that of the free enzyme. The operational inactivation rate constants (k iop ) of immobilized enzymes were found to be 1.42 ϫ 10 Ϫ5 min Ϫ1 for CNBr coupled and 3.23 ϫ 10 Ϫ5 min Ϫ1 for CDI coupled glucoamylase.

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Section: Resultsmentioning

confidence: 99%

“…In general, the effect of changes in temperature on the rates of enzyme‐catalyzed reactions does not provide much information on the mechanism of biocatalysts. However, these effects can be important in indicating structural changes in enzyme 3, 7, 22, 23…”

Section: Resultsmentioning

confidence: 99%

Immobilization of glucoamylase on the plain and on the spacer arm‐attached poly(HEMA‐EGDMA) microspheres

Arıca

Yavuz

Deni̇zli̇

2001

J of Applied Polymer Sci

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“…Several reports have described the significance of peptide density on cellular responses [29][30][31], and it is well-documented that the distance between biomolecule and substrate has a significant effect on its activity [32][33][34]. In the current work, the objective was to investigate effects of spacer length on accessibility and activity following controlled immobilization PTH .…”

Section: Discussionmentioning

confidence: 95%

“…The longest (C10) spacer resulted in the largest intracellular content of cAMP. In comparison, Itoyama et al [34] reported that bioactivity of the immobilized enzyme papain peaked with a spacer between four and six carbons or increased monotonically through eight carbons. Whereas PTH(1-34) either randomly immobilized via any available amino group or simply adsorbed on PLGA stimulated the MC3T3-E1 cells, with immobilization being marginally better, they were significantly less effective than peptide bound via the C10 molecule.…”

Section: Discussionmentioning

confidence: 96%

The use of N-terminal immobilization of PTH(1–34) on PLGA to enhance bioactivity

Sharon

Puleo

2008

Biomaterials

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The objective of this work was to control the orientation of bioactive molecules immobilized on a biodegradable substrate to improve their accessibility for binding to cell surface receptors and, therefore, to increase bioactivity. The osteotropic peptide, parathyroid hormone (1-34) (PTH(1-34)), was used to demonstrate the approach. To this end, the intrinsic N-terminal serine residue was oxidized to create an aldehyde group that specifically bound to hydrazide-derivatized poly(lactide-co-glycolide) under neutral conditions to form a hydrazone bond. Use of dihydrazide spacers significantly increased the amount of peptide immobilized compared to simple adsorption or direct, random attachment. In probing accessibility of immobilized PTH(1-34), attachment using longer dihydrazide spacers enhanced binding of an antibody against an epitope in the N-terminal region of the peptide. The longest spacer also increased binding of a C-terminal antibody. Furthermore, substrates with peptide tethered via spacers stimulated intracellular synthesis of cAMP, with activity increasing with dihydrazide length. PTH(1-34) immobilized using the longest spacer was significantly more effective than both random binding and adsorption. Site-directed binding of bioactive peptides to surfaces presents biomolecules for binding with cells so as to enhance interaction with receptors, and therefore the approach may be useful for obtaining preferred localized tissue responses.

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“…1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 Immobilization of the enzyme papain using the two-step mechanism. Papain is a cysteineprotease from C. papaya and was selected for its potential applications in biotechnology [34][35][36] and biomedicine, i.e. as wound debriding formulations used for the treatment of diabetic ulcers or infected wounds 24,37 .…”

Section: Reaction Of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (Ementioning

confidence: 99%

TEMPO-Oxidized Nanofibrillated Cellulose as a High Density Carrier for Bioactive Molecules

et al. 2015

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Controlled and efficient immobilization of specific biomolecules is a key technology to introduce new, favorable functions to materials suitable for biomedical applications. Here, we describe an innovative and efficient, two-step methodology for the stable immobilization of various biomolecules, including small peptides and enzymes onto TEMPO oxidized nanofibrillated cellulose (TO-NFC). The introduction of carboxylate groups to NFC by TEMPO oxidation provided a high surface density of negative charges able to drive the adsorption of biomolecules and take part in covalent cross-linking reactions with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC) and glutaraldehyde (Ga) chemistry. Up to 0.27 μmol of different biomolecules per mg of TO-NFC could be reversibly immobilized by electrostatic interaction. An additional chemical cross-linking step prevented desorption of more than 80% of these molecules. Using the cysteine-protease papain as model, a highly active papain-TO-NFC conjugate was achieved. Once papain was immobilized, 40% of the initial enzymatic activity was retained, with an increase in kcat from 213 to >700 s(-1) for the covalently immobilized enzymes. The methodology presented in this work expands the range of application for TO-NFC in the biomedical field by enabling well-defined hybrid biomaterials with a high density of functionalization.

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Spacer effects on enzymatic activity of papain immobilized onto porous chitosan beads

Cited by 29 publications

References 14 publications

Immobilization of glucoamylase on the plain and on the spacer arm‐attached poly(HEMA‐EGDMA) microspheres

Immobilization of glucoamylase on the plain and on the spacer arm‐attached poly(HEMA‐EGDMA) microspheres

The use of N-terminal immobilization of PTH(1–34) on PLGA to enhance bioactivity

TEMPO-Oxidized Nanofibrillated Cellulose as a High Density Carrier for Bioactive Molecules

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