Enzyme immobilization on tritylagarose

Cashion, P.; Javed, Ali; Harrison, Dolores L.; J, Seeley; Lentini, V.; Sathe, Ganesh M.

doi:10.1002/bit.260240212

Cited by 21 publications

(3 citation statements)

References 49 publications

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“…The immobilization of phosphotriesterase was accomplished by a procedure similar to that of Cashion et al 9 To a slurry of trityl agarose in 125 mM CHES, pH 9.0, was added a predetermined amount of enzyme activity. After gently swirling for at least 10 min, the mixture was poured into a glass column reactor.…”

Section: Preparation Of the Immobilized Phosphotriesterasementioning

confidence: 99%

Detoxification of organophosphate pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta

Caldwell

Raushel

1991

Biotech & Bioengineering

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A purified phosphotriesterase was successfully immobilized onto trityl agarose in a fixed bed reactor. A total of up to 9200 units of enzyme activity was immobilized onto 2.0 mL of trityl agarose (65 micromol trityl groups/mL agarose), where one unit is the amount of enzyme required to catalyze the hydrolysis of one micromole of paraoxon in one min. The immobilized enzyme was shown to behave chemically and kinetically similar to the free enzyme when paraoxon was utilized as a substrate. Several organophosphate pesticides, methyl parathion, ethyl parathion, diazinon, and coumaphos were also hydrolyzed by the immobilized phosphotriesterase. However, all substrates exhibited an affinity for the trityl agarose matrix. For increased solubility and reduction in the affinity of these pesticides for the trityl agarose matrix, methanol/water mixtures were utilized. The effect of methanol was not deleterious when concentrations of less than 20% were present. However, higher concentrations resulted in elution of enzyme from the reactor. With a 10-unit reactor, a 1.0 mM paraoxon solution was hydrolyzed completely at a flow rate of 45 mL/h. Kinetic parameters were measured with a 0.1-unit reactor with paraoxon as a substrate at a flow rate of 22 mL/h. The apparent K(m) for the immobilized enzyme was 3-4 times greater than the K(m) (0.1 mM) for the soluble enzyme. Immobilization limited the maximum rate of substrate hydrolysis to 40% of the value observed for the soluble enzyme. The pH-rate profiles of the soluble and immobilized enzymes were very similar. The immobilization of phosphotriesterase onto trityl agarose provides an effective method esterase onto trityl agarose provides an effective method for hydrolyzing and thus detoxifyuing organophosphate pesticides and mammalian acetylcholinesterase inhinbitors.

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Section: Preparation Of the Immobilized Phosphotriesterasementioning

confidence: 99%

Detoxification of organophosphate pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta

Caldwell

Raushel

1991

Biotech & Bioengineering

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“…The enzyme can be reused by its irreversible immobilization enzyme onto the supports by covalent bonding, but the regeneration of the prepared reactor is limited when the activity of the bound enzyme is damaged or destroyed [13]. Metal-ion chelated immobilization of the enzyme is quite different from conventional approaches, and the enzyme is bound to the support media based on the Lewis acid-base interaction through the divalent cation chelators such as iminodiacetic acid (IDA), which is chemically bound onto the matrix.…”

Section: Introductionmentioning

confidence: 99%

Immobilized metal‐ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ ionization‐time of flight‐mass spectrometry

Guo

Lei

et al. 2003

Electrophoresis

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Immobilized metal-ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ ionization-time of flight-mass spectrometryPeptide mass mapping analysis, utilizing a regenerable enzyme microreactor with metal-ion chelated adsorption of enzyme, combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was developed. Different procedures from the conventional approaches were adopted to immobilize the chelator onto the silica supports, that is, the metal chelating agent of iminodiacetic acid (IDA) was reacted with glycidoxypropyltrimethoxysilane (GLYMO) before its immobilization onto the inner wall of the fused-silica capillary pretreated with NH 4 HF 2 . The metal ion of copper and subsequently enzyme was specifically adsorbed onto the surface to form the immobilized enzyme capillary microreactor, which was combined with MALDI-TOF-MS to apply for the mass mapping analysis of nL amounts of protein samples. The results revealed that the peptide mapping could routinely be generated from 0.5 pmol protein sample in 15 min at 507C, even 20 fmol cytochrome c could be well digested and detected.

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“…Tritylation of cellulose beads was performed as described by Cashion et al 14 Reaction mixtures (10 cm3) were mixed vigorously using a rotating homogenizer type MPW-309 at 2000rev min-' for 1Omin at 25°C before addition oftheenzyme. Lipase reaction was performed in well-shaken flasks at 28°C.…”

Section: Matrix Modificationmentioning

confidence: 99%

Factors influencing the activity and thermostability of immobilized porcine pancreatic lipase

Kéry

Haplová

Tihlárik

et al. 1990

J of Chemical Tech & Biotech

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Lipase from porcine pancreas was immobilized on cellulose beads having various degrees of hydrophobicity, by covalent linking and by hydrophobic adsorption. Lipolytic activity was measured in heterogeneous organicaqueous systems of various hydrophobicities using olive oil as a substrate. The main factors influencing lipase activity were hydrophobicity of the reaction mixture and of the carrier. Carriers with increased hydrophobicity enhanced lipase activity more than less hydrophobic ones. Lipase immobilized covalently on cellulose beads was less active than that adsorbed onto tritylcellulose but was considerably more thermostable.

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Enzyme immobilization on tritylagarose

Cited by 21 publications

References 49 publications

Detoxification of organophosphate pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta

Detoxification of organophosphate pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta

Immobilized metal‐ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ ionization‐time of flight‐mass spectrometry

Factors influencing the activity and thermostability of immobilized porcine pancreatic lipase

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