Chemically stabilized trypsin used in dipeptide synthesis

Murphy, Anne N.; Fágáin, Ciarán Ó

doi:10.1002/(sici)1097-0290(19980520)58:4<366::aid-bit3>3.0.co;2-h

Cited by 14 publications

(10 citation statements)

References 31 publications

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“…In many cases, however, low catalytic efficiency and stability of enzymes have been seen as barriers for the development of largescale operations to compete with traditional chemical processes (1)(2)(3)(4)(5)(6). Methodologies such as genetic and protein engineering (7-10), evolution (11,12), solvent engineering (2,(13)(14)(15), and chemical modification (16)(17)(18)(19)(20)(21) are among the most actively investigated strategies to improve the functionality and performance of enzymes for bioprocessing applications.…”

Section: Introductionsupporting

confidence: 89%

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

Jia

Zhu

Vugrinovich

et al. 2002

Biotechnology Progress

414

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: Introductionsupporting

confidence: 89%

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

Jia

Zhu

Vugrinovich

et al. 2002

Biotechnology Progress

414

213

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“…In this regard, chemical modification of solventaccessible reactive side chains has been frequently used to redesign the enzyme stability and activity. Among the surface-located residues, Lys modification has succeeded in stabilizing a number of enzymes, including aminotransferase, a-amylase, trypsin, a-chymotrypsin, and HRP (26)(27)(28)(29). Accordingly, most of the stabilized chemical derivatives of HRP reported to date have involved Lys modifications (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41).…”

Section: Introductionmentioning

confidence: 99%

Structural Stabilization and Functional Improvement of Horseradish Peroxidase upon Modification of Accessible Lysines: Experiments and Simulation

Mogharrab

Ghourchian

Amininasab

2007

Biophysical Journal

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Horseradish peroxidase (HRP) is an important heme enzyme with enormous medical diagnostic, biosensing, and biotechnological applications. Thus, any improvement in the applicability and stability of the enzyme is potentially interesting. We previously reported that covalent attachment of an electron relay (anthraquinone 2-carboxylic acid) to the surface-exposed Lys residues successfully improves electron transfer properties of HRP. Here we investigated structural and functional consequences of this modification, which alters three accessible charged lysines (Lys-174, Lys-232, and Lys-241) to the hydrophobic anthraquinolysine residues. Thermal denaturation and thermoinactivation studies demonstrated that this kind of modification enhances the conformational and operational stability of HRP. The melting temperature increased 3 degrees C and the catalytic efficiency enhanced by 80%. Fluorescence and circular dichroism investigations suggest that the modified HRP benefits from enhanced aromatic packing and more buried hydrophobic patches as compared to the native one. Molecular dynamics simulations showed that modification improves the accessibility of His-42 and the heme prosthetic group to the peroxide and aromatic substrates, respectively. Additionally, the hydrophobic patch, which functions as a binding site or trap for reducing aromatic substrates, is more extended in the modified enzyme. In summary, this modification produces a new derivative of HRP with enhanced electron transfer properties, catalytic efficiency, and stability for biotechnological applications.

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“…However, one attempt to reduce autolysis of immobilized trypsin by reductive methylation of the primary amine groups met with limited success, because reduced autodigestion was accompanied by a decrease in activity . Other modifications to stabilize trypsin in solution included conjugation with methoxypoly(ethylene glycol)s (PEGylation), intramolecular cross-linking, conjugation with beta-cyclodextrin derivatives, transformation of tyrosines into aminotyrosines, and acetylation. − The effect of most of these modifications was assessed with low-molecular-weight substrates. It therefore remains to be seen how they affect the digestion of proteins.…”

Section: Introductionmentioning

confidence: 99%

Chemically Modified, Immobilized Trypsin Reactor with Improved Digestion Efficiency

Freije¹,

Mulder

Werkman

et al. 2005

J. Proteome Res.

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Tryptic digestion followed by identification using mass spectrometry is an important step in many proteomic studies. Here, we describe the preparation of immobilized, acetylated trypsin for enhanced digestion efficacy in integrated protein analysis platforms. Complete digestion of cytochrome c was obtained with two types of modified-trypsin beads with a contact time of only 4 s, while corresponding unmodified-trypsin beads gave only incomplete digestion. The digestion rate of myoglobin, a protein known to be rather resistant to proteolysis, was not altered by acetylating trypsin and required a buffer containing 35% acetonitrile to obtain complete digestion. The use of acetylated-trypsin beads led to fewer interfering tryptic autolysis products, indicating an increased stability of this modified enzyme. Importantly, the modification did not affect trypsin's substrate specificity, as the peptide map of myoglobin was not altered upon acetylation of immobilized trypsin. Kinetic digestion experiments in solution with low-molecular-weight substrates and cytochrome c confirmed the increased catalytic efficiency (lower K(M) and higher k(cat)) and increased resistance to autolysis of trypsin upon acetylation. Enhancement of catalytic efficiency was correlated with the number of acetylations per molecule. The favorable properties of the new chemically modified trypsin reactor should make it a valuable tool in automated protein analysis systems.

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Chemically stabilized trypsin used in dipeptide synthesis

Cited by 14 publications

References 31 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

Structural Stabilization and Functional Improvement of Horseradish Peroxidase upon Modification of Accessible Lysines: Experiments and Simulation

Chemically Modified, Immobilized Trypsin Reactor with Improved Digestion Efficiency

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