Intense PEGylation of Enzyme Surfaces: Relevant Stabilizing Effects.
This chapter describes the physicochemical coating of the surface of immobilized enzymes with a dense layer of polyethylene glycol (PEG) to improve enzyme stability. One hypothesis is that a dense, viscous, polar PEG layer around the enzyme would enhance enzyme thermal stability, while still providing access to the active site. PEG groups were attached by using aldehyde-dextran polymers, the dextran polymers are in turn attached to the enzyme surface that have been enriched with excess primary amino groups. The enzymes themselves were initially attached onto porous solids such that they may be separated easily from the reaction mixtures for easy downstream processing and that they may be recycled to reduce the cost of the biocatalyst. The hierarchical modification of enzyme surface with three different sublayers, under chemical design, provided a rational control at several structural levels. Few methods for increasing the number of amino groups on the surface of the enzyme are described: (a) chemical amination of carboxyl residues and (b) coating of the enzyme surface with cationic polymers containing a high percentage of primary amines. Reliable protocols for the PEGylation of four different enzymes are described here. For example, lipases from Thermomyces lanuginosa, Candida antarctica B, and Rhizomucor miehei attached to octyl sepharose and chemically modified via PEGylation are stabilized from 7- to 50-fold when compared to the stability of the corresponding unmodified enzyme. A derivative of endoxylanase from Trichoderma reesei, immobilized by multipoint covalent attachment on glyoxyl agarose, is stabilized by 50-fold. Very likely, the PEG layer generated a dense, high viscosity medium surrounding the enzyme surface and this increase in viscosity around the enzyme microenvironment resists distortion of enzyme structure by heat or other denaturing agents.
Institute of Catalysis, Spanish Research Council, CSIC, Madrid, Spain.
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Moreno-Pérez
Institute of Catalysis, Spanish Research Council, CSIC, Madrid, Spain.
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AH
Orrego
Institute of Catalysis, Spanish Research Council, CSIC, Madrid, Spain.
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Romero-Fernández
Institute of Catalysis, Spanish Research Council, CSIC, Madrid, Spain.
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Trobo-Maseda
Institute of Catalysis, Spanish Research Council, CSIC, Madrid, Spain.
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Martins-DeOliveira
Institute of Catalysis, Spanish Research Council, CSIC, Madrid, Spain.
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Munilla
Institute of Catalysis, Spanish Research Council, CSIC, Madrid, Spain. Electronic address: gflorente@icp.csic.es.
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Fernández-Lorente
Institute of Catalysis, Spanish Research Council, CSIC, Madrid, Spain. Electronic address: jmguisan@icp.csic.es.
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Guisan
D043364:Endo-1,4-beta Xylanases / Q000737:chemistry; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry*; D006031:Glycosylation; D007700:Kinetics; D008049:Lipase / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*
D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D011092:Polyethylene Glycols; D008049:Lipase; C431349:lipase B, Candida antarctica; D043364:Endo-1,4-beta Xylanases
Aldehyde; Amination; Amine; Dextran; Endoxylanase; Immobilized; Lipase; PEG; PEGylation; Primary amine; Stabilization