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Looking at a single enzyme molecule
UPDATE
DDT: TARGETS Vol. 3, No. 3 June 2004
NEWS
Looking at a single enzyme molecule Vida Foubister, [email protected]
A new class of enzyme catalysts has been created by surrounding single molecules with a porous organic/inorganic structure that significantly enhances their stability [1]. These caged single-enzyme nanoparticles (SENs) maintain good activity and thus hold potential for proteomic analysis and protein therapeutics, among other applications.
Enzyme stabilization Until recently, the potential to harness the specificity and catalytic activity of enzymes for applications outside the cell has been limited by their short life spans. Enzyme modification by genetic engineering or chemical methods typically results in enzymes that retain good activity but still have limited stability [2]. Immobilization onto surfaces or encapsulation in porous solids, in contrast, results in good enzyme stability but often limits the availability of enzyme substrates and, as a result, lowers activity [3]. Those interested in enzyme catalysis have begun approaching the problem in new ways. Altus Biologics Inc. (http://www.altus.com/) created cross-linked enzyme crystals, known as CLECs, with robust activity and long term stability [4]. Manfred T. Reetz, Director of the Max-Planck-Institut für Kohlenforschung (http://www.mpi-muelheim.mpg.de/kofo/ mpikofo_home.html) won the Sigma-Aldrich Reagent of the Year in 1997 for his development of sol-gel entrapped lipases. This immobilization procedure incorporates lipases in hydrophobic organic/inorganic hybrid materials that have increased enzyme activity, long-term stability and other features.
Porous armor Researchers at the US Department of Energy’s Pacific Northwest National Laboratory (http://www.pnl.gov/) have found an innovative way to stabilize enzymes – as single, nanometer-scale particles (Figure 1).
Figure 1. SENs as seen by high resolution transmission electron microscopy image. Image courtesy of J. Kim and J.W. Grate of the Pacific Northwest National Laboratory.
Using both trypsin and α-chymotrypsin, two common protein-splitting enzymes, they have created caged molecules through synthetic modifications of surface amino acids. ‘It’s a multi-step synthesis,’ says Jay Grate, a PNNL Laboratory Fellow. A covalent reaction modifies the enzyme surface and then the molecules are solubilized in an organic solvent.Two polymerizations follow: the first adds vinyl polymers to the enzyme surface and the second, which takes place once the enzymes are extracted into water, cross-links these surface chains to create a basketball net-like structure. SENs demonstrate long-term stability – more than five months at 4ºC – and maintain good enzyme activity [1].‘The kinetic data presented seems to show that, under initial-rate conditions, mass transfer is not limiting,’ says Ben Davis, Lecturer in Organic and Biological Chemistry at the University of Oxford (http://www.ox.ac.uk/). ‘This shows promise and it will be interesting to see the actual synthetic flux levels in true synthetic applications.’
Great potential An advantage to this approach is that the thickness of the net-like structure can be
1741-8372/04/$ – see front matter ©2004 Elsevier Ltd. All rights reserved. PII: S1741-8372(04)02440-5
specifically adapted to enhance the stability and activity of other water-soluble enzymes. ‘We can control these properties by changing the synthetic parameters,’ explains Jungbae Kim, PNNL Senior Research Scientist. Enzymes stabilized as SENs could have applications that range from bioremediation to protein therapeutics.Trypsin, for example, is currently used in proteomic analysis but loses the specificity with which it cleaves proteins over time.‘We have shown that converting free trypsin to single enzyme nanoparticle trypsin not only stabilized the activity so the enzyme last longer, but it also stabilized the selectivity of the activity,’ says Grate. It’s ability to protect enzymes from proteolytic destruction also holds promise in drug delivery.‘The novel architecture [of SENs] may allow their use, like CLECs or PEGylated proteins, in therapies that would benefit from delivery of enzyme activity and I look forward to an assessment of their in vivo stability,’ says Davis. However, more data is need to determine ‘whether this technology is suitable for large scale production,’ adds Ping Wang, Assistant Professor in the Chemical Engineering Department at the University of Akron, Ohio (http://www.uakron.edu/).
References 1 Kim, J. and Grate, J.W. (2003) Single-enzyme nanoparticles armored by a nanometer-scale organic/inorganic network. Nano Lett. 3, 1219–1222 2 DeSantis, G. and Jones, J.B. (1999) Chemical modification of enzymes for enhanced functionality. Curr. Opin. Biotechnol. 10, 324–330 3 Tischer, W. and Wedekind, F. (1999) Immobilized enzymes: methods and applications. Topics Curr. Chem. 200, 95–126 4 Margolin, A.L. and Navia, M.A. (2001) Protein crystals as novel catalytic materials. Angew. Chem. Int. Ed. Engl. 40, 2204–2222
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87
DDT: TARGETS Vol. 3, No. 3 June 2004
NEWS
Looking at a single enzyme molecule Vida Foubister, [email protected]
A new class of enzyme catalysts has been created by surrounding single molecules with a porous organic/inorganic structure that significantly enhances their stability [1]. These caged single-enzyme nanoparticles (SENs) maintain good activity and thus hold potential for proteomic analysis and protein therapeutics, among other applications.
Enzyme stabilization Until recently, the potential to harness the specificity and catalytic activity of enzymes for applications outside the cell has been limited by their short life spans. Enzyme modification by genetic engineering or chemical methods typically results in enzymes that retain good activity but still have limited stability [2]. Immobilization onto surfaces or encapsulation in porous solids, in contrast, results in good enzyme stability but often limits the availability of enzyme substrates and, as a result, lowers activity [3]. Those interested in enzyme catalysis have begun approaching the problem in new ways. Altus Biologics Inc. (http://www.altus.com/) created cross-linked enzyme crystals, known as CLECs, with robust activity and long term stability [4]. Manfred T. Reetz, Director of the Max-Planck-Institut für Kohlenforschung (http://www.mpi-muelheim.mpg.de/kofo/ mpikofo_home.html) won the Sigma-Aldrich Reagent of the Year in 1997 for his development of sol-gel entrapped lipases. This immobilization procedure incorporates lipases in hydrophobic organic/inorganic hybrid materials that have increased enzyme activity, long-term stability and other features.
Porous armor Researchers at the US Department of Energy’s Pacific Northwest National Laboratory (http://www.pnl.gov/) have found an innovative way to stabilize enzymes – as single, nanometer-scale particles (Figure 1).
Figure 1. SENs as seen by high resolution transmission electron microscopy image. Image courtesy of J. Kim and J.W. Grate of the Pacific Northwest National Laboratory.
Using both trypsin and α-chymotrypsin, two common protein-splitting enzymes, they have created caged molecules through synthetic modifications of surface amino acids. ‘It’s a multi-step synthesis,’ says Jay Grate, a PNNL Laboratory Fellow. A covalent reaction modifies the enzyme surface and then the molecules are solubilized in an organic solvent.Two polymerizations follow: the first adds vinyl polymers to the enzyme surface and the second, which takes place once the enzymes are extracted into water, cross-links these surface chains to create a basketball net-like structure. SENs demonstrate long-term stability – more than five months at 4ºC – and maintain good enzyme activity [1].‘The kinetic data presented seems to show that, under initial-rate conditions, mass transfer is not limiting,’ says Ben Davis, Lecturer in Organic and Biological Chemistry at the University of Oxford (http://www.ox.ac.uk/). ‘This shows promise and it will be interesting to see the actual synthetic flux levels in true synthetic applications.’
Great potential An advantage to this approach is that the thickness of the net-like structure can be
1741-8372/04/$ – see front matter ©2004 Elsevier Ltd. All rights reserved. PII: S1741-8372(04)02440-5
specifically adapted to enhance the stability and activity of other water-soluble enzymes. ‘We can control these properties by changing the synthetic parameters,’ explains Jungbae Kim, PNNL Senior Research Scientist. Enzymes stabilized as SENs could have applications that range from bioremediation to protein therapeutics.Trypsin, for example, is currently used in proteomic analysis but loses the specificity with which it cleaves proteins over time.‘We have shown that converting free trypsin to single enzyme nanoparticle trypsin not only stabilized the activity so the enzyme last longer, but it also stabilized the selectivity of the activity,’ says Grate. It’s ability to protect enzymes from proteolytic destruction also holds promise in drug delivery.‘The novel architecture [of SENs] may allow their use, like CLECs or PEGylated proteins, in therapies that would benefit from delivery of enzyme activity and I look forward to an assessment of their in vivo stability,’ says Davis. However, more data is need to determine ‘whether this technology is suitable for large scale production,’ adds Ping Wang, Assistant Professor in the Chemical Engineering Department at the University of Akron, Ohio (http://www.uakron.edu/).
References 1 Kim, J. and Grate, J.W. (2003) Single-enzyme nanoparticles armored by a nanometer-scale organic/inorganic network. Nano Lett. 3, 1219–1222 2 DeSantis, G. and Jones, J.B. (1999) Chemical modification of enzymes for enhanced functionality. Curr. Opin. Biotechnol. 10, 324–330 3 Tischer, W. and Wedekind, F. (1999) Immobilized enzymes: methods and applications. Topics Curr. Chem. 200, 95–126 4 Margolin, A.L. and Navia, M.A. (2001) Protein crystals as novel catalytic materials. Angew. Chem. Int. Ed. Engl. 40, 2204–2222
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87