Improving the catalytic properties of immobilized Lecitase via physical coating with ionic polymers.
Lecitase Ultra has been immobilized on cyanogen bromide agarose (via covalent attachment) and on octyl agarose (via physical adsorption on the hydrophobic support by interfacial activation). Both immobilized preparations have been incubated in dextran sulfate (DS) or polyethylenimine (PEI) solutions to coat the enzyme surface. Then, the activity versus different substrates and under different experimental conditions was evaluated. The PEI coating generally produced a significant increase in enzyme activity, in some cases even by more than a 30-fold factor (using the octyl-Lecitase at pH 5 in the hydrolysis of methyl phenyl acetate). In opposition, the DS coating usually produced some negative effects on the enzyme activity. The rate of irreversible inhibition of the covalent preparation using diethyl p-nitrophenylphosphate did not increase after PEI coating suggesting that the increase in Lecitase activity is not a consequence of the stabilization of the open form of Lecitase. Moreover, the coating greatly increased the stability of the immobilized Lecitase, for example using DS and the covalent preparation, the half-life was increased by a 30-fold factor in 30% acetonitrile. The stabilizing effect was not found in all cases, in certain cases even a certain destabilization is found (e.g., octyl-Lecitase-DS at pH 7). Thus, the effects of the ionic polymer coating strongly depend on the substrate, experimental conditions and immobilization technique employed.
Instituto de Catálisis-CSIC, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain; Departamento de Engenharia Química, Universidade Federal Do Ceará, Campus Do Pici, CEP 60455-760, Fortaleza, CE, Brazil.
Jose C S
JC
dos Santos
Instituto de Catálisis-CSIC, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain.
Cristina
C
Garcia-Galan
Biotechnology, Bioprocess and Biocatalysis Group, Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Av. Bento Gonçalves, 9500, P.O. Box 15090, ZC 91501-970 Porto Alegre, RS, Brazil.
Rafael C
RC
Rodrigues
Departamento de Engenharia Química, Universidade Federal Do Ceará, Campus Do Pici, CEP 60455-760, Fortaleza, CE, Brazil.
Hosiberto Batista
HB
de Sant' Ana
Departamento de Engenharia Química, Universidade Federal Do Ceará, Campus Do Pici, CEP 60455-760, Fortaleza, CE, Brazil.
Luciana R B
LR
Gonçalves
Instituto de Catálisis-CSIC, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain. Electronic address: rfl@icp.csic.es.
Roberto
R
Fernandez-Lafuente
D001709:Biotechnology; D002384:Catalysis; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D003488:Cyanogen Bromide; D016264:Dextran Sulfate; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D054466:Phospholipases A1 / Q000737:chemistry* / Q000378:metabolism*; D011094:Polyethyleneimine; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012685:Sepharose
D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D011094:Polyethyleneimine; D012685:Sepharose; D016264:Dextran Sulfate; D054466:Phospholipases A1; D003488:Cyanogen Bromide
Enzyme hyperactivation; Enzyme stabilization; Interfacial activation; Lecitase Ultra; Polyethylenimine; Sulfate dextran