Comparing the effect of immobilization methods on the activity of lipase biocatalysts in ester hydrolysis

The activity of various lipases was compared, in both free and immobilized forms, using the kinetics of the hydrolysis reaction of p-nitrophenyl butyrate, which was followed with in situ UV/Vis diode array spectrophotometry. Several enzymes were used to catalyze the reaction, namely Candida antarctica lipase B and Fusarium solani pisi cutinase wildtype and three single-mutation variants. The enzymes were tested in three different forms: free, immobilized as cross-linked aggregates and supported on zeolite NaY. A simple kinetic model was used to allow a quantitative comparison of the behavior of the different catalysts. It was concluded that although immobilization reduces the activity of the enzyme, the zeolite offers a much higher specific activity when compared to the cross-linked aggregates, thus supplying a heterogeneous catalyst with promising catalytic properties.

[1]  M. Aires-Barros,et al.  Cutinase: from molecular level to bioprocess development. , 1999, Biotechnology and bioengineering.

[2]  A. Svendsen,et al.  Lipase protein engineering. , 2000, Biochimica et biophysica acta.

[3]  J. Cabral,et al.  Effect of the immobilization support on the hydrolytic activity of a cutinase from Fusarium solani pisi , 1997 .

[4]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[5]  Abdul Hameed,et al.  Industrial applications of microbial lipases , 2006 .

[6]  J. Cabral,et al.  Stability of a recombinant cutinase immobilized on zeolites , 2002 .

[7]  Uwe T Bornscheuer,et al.  Immobilizing enzymes: how to create more suitable biocatalysts. , 2003, Angewandte Chemie.

[8]  J. Cabral,et al.  Interaction of an anionic surfactant with a recombinant cutinase from Fusarium solani pisi: a spectroscopic study , 2003 .

[9]  T. Ema,et al.  Rational control of enantioselectivity of lipase by site-directed mutagenesis based on the mechanism. , 2005, Chemical communications.

[10]  Roberto Fernandez-Lafuente,et al.  Improvement of enzyme activity, stability and selectivity via immobilization techniques , 2007 .

[11]  U C Banerjee,et al.  Production, purification, characterization, and applications of lipases. , 2001, Biotechnology advances.

[12]  Ipsita Roy,et al.  Enzymes in organic media , 2004 .

[13]  V. Gotor‐Fernández,et al.  Lipases: Useful biocatalysts for the preparation of pharmaceuticals , 2006 .

[14]  W. Windsor,et al.  Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution. , 2003, Protein engineering.

[15]  J. Cabral,et al.  Zeolites as supports for enzymatic hydrolysis reactions. Comparative study of several zeolites , 1996 .

[16]  R. A. Sheldon,et al.  Cross-linked enzyme aggregates (CLEAs): A novel and versatile method for enzyme immobilization (a review) , 2005 .

[17]  A. Klibanov.  Improving enzymes by using them in organic solvents , 2001, Nature.

[18]  A. Illanes,et al.  Production of cephalexin in organic medium at high substrate concentrations with CLEA of penicillin acylase and PGA-450 , 2007 .

[19]  J. Cabral,et al.  Application of factorial design to the study of an alcoholysis transformation promoted by cutinase immobilized on NaY zeolite and Accurel PA6 , 2004 .

[20]  A. Ghanem.  Trends in lipase-catalyzed asymmetric access to enantiomerically pure/enriched compounds , 2007 .

[21]  Ying Mei,et al.  Imaging the distribution and secondary structure of immobilized enzymes using infrared microspectroscopy. , 2003, Biomacromolecules.

[22]  Karl-Erich Jaeger,et al.  Lipases for biotechnology. , 2002, Current opinion in biotechnology.

[23]  R. A. Sheldon,et al.  Cross-linked enzyme aggregates with enhanced activity: application to lipases , 2002, Biotechnology Letters.

[24]  Roger A. Sheldon,et al.  Synthesis of enantiomerically pure (S)-mandelic acid using an oxynitrilase–nitrilase bienzymatic cascade: a nitrilase surprisingly shows nitrile hydratase activity , 2006 .

[25]  A. P. Serro,et al.  Effects of ionic surfactants used in reversed micelles on cutinase activity and stability. , 2000, Biochimica et biophysica acta.