Hydrogel‐Immobilized Supercharged Proteins

The remarkable catalytic potential of enzymes in chemical synthesis, environmental bioremediation, and medical therapeutics is limited by their longevity and stability. Immobilization of enzymes on solid supports is demonstrated to improve the stability of biocatalysts but often relies on multiple chemical steps for covalent attachment and is limited by the physical properties of the various supports. Here, production of enzyme: hydrogel complexes is described via engineering of a cationic supercharged phosphotriesterase. These enzyme: hydrogel complexes are remarkably robust displaying no loss of catalytic activity after 80 d of use and up to 105 turnovers when used in a flow reactor at catalyst loadings as low as 0.0008 mol%. In addition, exceptional resilience to organic solvents is observed. The use of enzyme: hydrogel complexes is likely to be of value in a diverse range of applications such as enantioselective continuous‐flow chemistry, detoxification of poisons, and the formation of functionalized biomaterials.

[1]  C. Barrow,et al.  Large and Small Assembly: Combining Functional Macromolecules with Small Peptides to Control the Morphology of Skeletal Muscle Progenitor Cells. , 2018, Biomacromolecules.

[2]  Vera D. Jäger,et al.  Catalytically-active inclusion bodies-Carrier-free protein immobilizates for application in biotechnology and biomedicine. , 2017, Journal of biotechnology.

[3]  Benjamin T. Porebski,et al.  The role of protein dynamics in the evolution of new enzyme function. , 2016, Nature chemical biology.

[4]  Lukasz P. Kozlowski,et al.  Proteome-pI: proteome isoelectric point database , 2016, Nucleic Acids Res..

[5]  L. Kozlowski.  IPC – Isoelectric Point Calculator , 2016, Biology Direct.

[6]  J. Schneider,et al.  Protein release from highly charged peptide hydrogel networks. , 2016, Journal of materials chemistry. B.

[7]  D. Nisbet,et al.  Tailoring minimalist self-assembling peptides for localized viral vector gene delivery , 2016, Nano Research.

[8]  H. Maynard,et al.  Trehalose hydrogels for stabilization of enzymes to heat. , 2015, Polymer chemistry.

[9]  J. Oakeshott,et al.  Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron , 2015, Applied and Environmental Microbiology.

[10]  J. Keith Joung,et al.  Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo , 2014, Nature Biotechnology.

[11]  Conor C. Horgan,et al.  In vivo assessment of grafted cortical neural progenitor cells and host response to functionalized self-assembling peptide hydrogels and the implications for tissue repair. , 2014, Journal of materials chemistry. B.

[12]  P. Kofinas,et al.  Enzymatic activity preservation and protection through entrapment within degradable hydrogels , 2013, Biotechnology and bioengineering.

[13]  A. Patil,et al.  Enhanced catalytic activity in organic solvents using molecularly dispersed haemoglobin-polymer surfactant constructs. , 2013, Chemical communications.

[14]  Roger A Sheldon,et al.  Enzyme immobilisation in biocatalysis: why, what and how. , 2013, Chemical Society reviews.

[15]  Philip R. Evans,et al.  How good are my data and what is the resolution? , 2013, Acta crystallographica. Section D, Biological crystallography.

[16]  Klavs F. Jensen,et al.  The role of flow in green chemistry and engineering , 2013 .

[17]  A. Cardona,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[18]  Piotr Oleskowicz-Popiel,et al.  The challenge of enzyme cost in the production of lignocellulosic biofuels. , 2012, Biotechnology and bioengineering.

[19]  David R Nisbet,et al.  Self-Assembled Peptides: Characterisation and In Vivo Response , 2012, Biointerphases.

[20]  Dietmar W. Hutmacher,et al.  Design and Fabrication of Tubular Scaffolds via Direct Writing in a Melt Electrospinning Mode , 2012, Biointerphases.

[21]  Ángel Berenguer-Murcia,et al.  Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance , 2011 .

[22]  P. Hartley,et al.  The in vivo performance of an enzyme-assisted self-assembled peptide/protein hydrogel. , 2011, Biomaterials.

[23]  D. Ollis,et al.  The organophosphate-degrading enzyme from Agrobacterium radiobacter displays mechanistic flexibility for catalysis. , 2010, The Biochemical journal.

[24]  E. Ribeiro,et al.  Immobilization of Aspergillus oryzae β-galactosidase in ion exchange resins by combined ionic-binding method and cross-linking , 2010 .

[25]  I. Wheeldon,et al.  Catalytic biomaterials: engineering organophosphate hydrolase to form self-assembling enzymatic hydrogels. , 2010, Protein engineering, design & selection : PEDS.

[26]  David R. Liu,et al.  Potent Delivery of Functional Proteins into Mammalian Cells in Vitro and in Vivo Using a Supercharged Protein , 2010, ACS chemical biology.

[27]  Vincent B. Chen,et al.  PHENIX: a comprehensive Python-based system for macromolecular structure solution , 2010, Acta crystallographica. Section D, Biological crystallography.

[28]  Dan S. Tawfik,et al.  Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase , 2009, Proceedings of the National Academy of Sciences.

[29]  J. Shaw,et al.  Biocatalysis for the production of carbohydrate esters. , 2009, New biotechnology.

[30]  D. G. Gibson,et al.  Enzymatic assembly of DNA molecules up to several hundred kilobases , 2009, Nature Methods.

[31]  David R. Liu,et al.  Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins , 2009, Proceedings of the National Academy of Sciences.

[32]  George M Whitesides,et al.  Lysine acetylation can generate highly charged enzymes with increased resistance toward irreversible inactivation , 2008, Protein science : a publication of the Protein Society.

[33]  Erdan Gu,et al.  A Novel Fiber Optic Biosensor for the Determination of Adrenaline Based on Immobilized Laccase Catalysis , 2008 .

[34]  Alica Vikartovská,et al.  A crosslinked inclusion body process for sialic acid synthesis. , 2008, Journal of biotechnology.

[35]  François Stricher,et al.  How Protein Stability and New Functions Trade Off , 2008, PLoS Comput. Biol..

[36]  David R. Liu,et al.  Supercharging proteins can impart unusual resilience. , 2007, Journal of the American Chemical Society.

[37]  P. Wang,et al.  Bioenergy Beads: A Tool for Regeneration of ATP/NTP in Biocatalytic Synthesis , 2006, Artificial cells, blood substitutes, and immobilization biotechnology.

[38]  D. Ollis,et al.  The structure of an enzyme-product complex reveals the critical role of a terminal hydroxide nucleophile in the bacterial phosphotriesterase mechanism. , 2005, Biochimica et biophysica acta.

[39]  Jun Huang,et al.  Immobilization of Pycnoporus sanguineus laccase on magnetic chitosan microspheres , 2005 .

[40]  François Stricher,et al.  The FoldX web server: an online force field , 2005, Nucleic Acids Res..

[41]  Adam Godzik,et al.  FATCAT: a web server for flexible structure comparison and structure similarity searching , 2004, Nucleic Acids Res..

[42]  Rajesh R Naik,et al.  Enzyme immobilization in a biomimetic silica support , 2004, Nature Biotechnology.

[43]  H. Schoemaker,et al.  Dispelling the Myths--Biocatalysis in Industrial Synthesis , 2003, Science.

[44]  B. Greb-Markiewicz,et al.  Immobilization of wood-rotting fungi laccases on modified cellulose and acrylic carriers , 2002 .

[45]  B. Davison,et al.  Enzyme stabilization by covalent binding in nanoporous sol-gel glass for nonaqueous biocatalysis. , 2001, Biotechnology and bioengineering.

[46]  F. Raushel,et al.  Structural determinants of the substrate and stereochemical specificity of phosphotriesterase. , 2001, Biochemistry.

[47]  Chi-Huey Wong,et al.  Enzymes for chemical synthesis , 2001, Nature.

[48]  T. Kajino,et al.  Catalytic Activity in Organic Solvents and Stability of Immobilized Enzymes Depend on the Pore Size and Surface Characteristics of Mesoporous Silica , 2000 .

[49]  Song,et al.  The preparation and characterization of an immobilized l-glutamic decarboxylase and its application for determination of l-glutamic acid. , 2000, Enzyme and microbial technology.

[50]  S. Hober,et al.  Charge engineering of a protein domain to allow efficient ion-exchange recovery. , 2000, Protein engineering.

[51]  Susan E. Brown,et al.  Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance. , 2000, Biochemistry.

[52]  R. Fernández-Lafuente,et al.  Immobilization of lipases by selective adsorption on hydrophobic supports. , 1998, Chemistry and physics of lipids.

[53]  A. Vagin,et al.  MOLREP: an Automated Program for Molecular Replacement , 1997 .

[54]  V. Uversky,et al.  Conformational transitions provoked by organic solvents in beta-lactoglobulin: can a molten globule like intermediate be induced by the decrease in dielectric constant? , 1997, Folding & design.

[55]  P. Vrabel,et al.  Analysis of the mechanism and kinetics of thermal inactivation of enzymes: Critical assessment of isothermal inactivation experiments , 1996 .

[56]  V. V. Mozhaev.  Mechanism-based strategies for protein thermostabilization. , 1993, Trends in biotechnology.

[57]  J. Dordick,et al.  Organic solvents strip water off enzymes , 1992, Biotechnology and bioengineering.

[58]  Frances H. Arnold,et al.  Enzyme Engineering for Nonaqueous Solvents: Random Mutagenesis to Enhance Activity of Subtilisin E in Polar Organic Media , 1991, Bio/Technology.

[59]  F. Raushel,et al.  Purification and properties of the phosphotriesterase from Pseudomonas diminuta. , 1989, The Journal of biological chemistry.

[60]  K. Jaeger,et al.  Fusion of a Coiled‐Coil Domain Facilitates the High‐Level Production of Catalytically Active Enzyme Inclusion Bodies , 2016 .

[61]  John H. Grate,et al.  A green-by-design biocatalytic process for atorvastatin intermediate , 2010 .

[62]  Rein V Ulijn,et al.  Enzyme-assisted self-assembly under thermodynamic control. , 2009, Nature nanotechnology.

[63]  I. Sokolov,et al.  Enzyme-functionalized mesoporous silica for bioanalytical applications , 2009, Analytical and bioanalytical chemistry.

[64]  BMC Biochemistry BioMed Central Research article Benchmarking pK a prediction , 2006 .

[65]  Dan S. Tawfik,et al.  Directed evolution of phosphotriesterase from Pseudomonas diminuta for heterologous expression in Escherichia coli results in stabilization of the metal-free state. , 2005, Protein engineering, design & selection : PEDS.

[66]  Ramanathan Sowdhamini,et al.  BMC Bioinformatics BioMed Central Database , 2004 .

[67]  Karen N. Allen,et al.  research papers Acta Crystallographica Section D Biological , 2003 .

[68]  M. Westlake,et al.  The Council of the European Union , 1995 .