Immobilization of lipases on hydrophobic supports involves the open form of the enzyme

• Published in: Enzyme and microbial technology Vol. 71; pp. 53 - 57
• Main Authors: Manoel, Evelin A., dos Santos, José C.S., Freire, Denise M.G., Rueda, Nazzoly, Fernandez-Lafuente, Roberto
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2015
• Subjects: agarose; Ascomycota - enzymology; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; bromides; Burkholderia cepacia; Burkholderia cepacia - enzymology; carboxylic ester hydrolases; cyanogen; Cyanogen Bromide; Detergents; Enzymes, Immobilized - antagonists & inhibitors; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Fungal Proteins - antagonists & inhibitors; Fungal Proteins - chemistry; Fungal Proteins - metabolism; Hydrophobic and Hydrophilic Interactions; hydrophobicity; Interfacial activation; Ionic strength; Irreversible inhibition; Kinetics; Lipase - antagonists & inhibitors; Lipase - chemistry; Lipase - metabolism; Lipase immobilization; Octyl-agarose; Osmolar Concentration; Protein Conformation; Sepharose - analogs & derivatives; sodium phosphate; Thermomyces lanuginosus; Octyl-agarose; Interfacial activation; Detergents; Ionic strength; Irreversible inhibition; Lipase immobilization
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2015.02.001

•Lipase immobilization rate on octyl supports is reduced at high ionic strength.•Octyl-lipase is more rapidly inhibited by D-pNPP than covalent preparations.•Octyl-lipase inhibition by D-pNPP did not depend on ionic strength.•Octyl-lipase activity is less depended on ionic strength.•Lipase activity is not only depended on catalytic Ser exposition. The lipases from Thermomyces lanuginosus and Pseudomonas cepacia have been immobilized on octyl and cyanogen bromide (CNBr) agarose beads. The immobilization on octyl-agarose is slowed with increasing ionic strength, while the immobilization on CNBr is not significantly affected by the ionic strength. The inhibition of the immobilized preparations with diethyl p-nitrophenylphosphate (D-pNPP) was analyzed. The inhibition was more rapid using octyl-lipase preparations than using covalent preparations, and the covalent preparations were much more sensitive to the reaction medium. The addition of detergent increased the inhibition rate of the covalent preparation while an increase on the ionic strength produced a slowdown of the inhibition rate by D-pNPP for both lipases. The effect of the medium on the activity versus fully soluble substrate (methyl mandelate) was in the same direction. The octyl preparations presented a slight decrease in activity when comparing the results using different concentrations of sodium phosphate buffer (between 0.025 and 1M), while the CNBr preparations suffered drastic drops in its activity at high ionic strength. The results confirm that the lipases immobilized on octyl agarose presented their open form stabilized while the covalent preparation maintains a closing/opening equilibrium that may be modulated by altering the medium.