ELSEVIER zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Effect of the immobilization support on the hydrolytic activity of a cutinase from zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Fusarium solani pisi A. P. V. Gonsalves, J. M. Lopes, F. Lemos, F. Ram6a Ribeiro, D. M. F. Prazeres, J. M. S. Cabral, and M. R. Aires-Barros zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFE Centro de Engenharia BioMgica e Quimica, Instituto Superior Tknico, Lisboa, Portugal Fusarium solaui pisi zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA recombinant cutinase was immobilized on two support materials (Nay zeolite and polyam- ide-Accurel PA6). These preparations were used to catalyze the hydrolysis of tricaprylin in a nonconventional medium. Water content is a very important factor in relation to enzyme activity in organic media, and the effect of the water in the reaction medium as well as the effect of the organic solvent at different water concentrations on the hydrolytic activity of the immobilized cutinase were investigated. Factors that are also important for the catalytic activity of the enzyme such as immobilization pH, ionic strength, and pH of the reaction medium and temperature were optimized. The selected conditions for further work were pH 9 (the immobilization pH) and 30°C. In order to evaluate cutinase selectivity, the kinetic parameters of the immobilized enzyme were determined with triglycerides containing fatty acids ranging from zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB C, up to C,,. A selectivity to C, was observed with both supports. The thermal stability of the enzyme was also investigated and a particularly good stability was observed with cutinase immobilized on the NaY zeolite with no loss of initial activity after 4.5 days. 0 1997 by Elsevier Science Inc. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Keywords: Cutinase; NaY zeolite; Accurel PA6; immobilization; hydrolysis; water content: organic solvents: enzyme selectivity; thermal stability Introduction Cutinase from Fusarium solani pisi is a lipolytic enzyme that catalyzes the hydrolysis of the water-insoluble biopoly- ester cutin which covers plant surfaces.’ This enzyme is also able to hydrolyze soluble p-nitrophenyl esters and triglyc- erides with a wide variety of chain lengths.* Cutinase is able to hydrolyze soluble esters and triacylglycerols as effi- ciently as esterases and lipases do. The enzyme active site is composed by the usual catalytic triad of lipases (Ser-Asp- His) and located in a crevice between two hydrophobic loops (residues 80-90 and residues 182-189). It is thought that these loops can be involved in both interfacial and substrate binding. 3.4 Its catalytic versatility makes cutinase an interesting enzyme to be applied to biocatalytic reactions in nonconventional media. Several methodologies are known for the use of lipases Address reprint requests to Dr. Maria Raquel Aires-Batros, Centro de Engenharia Biologica e Quimica, Institnto Superior Tecnico, Av. Rovisco Pais, 1096 Lisboa Codex, Portugal Received 18 December 1995; revised 11 March 1996; accepted 20 March 1996 in organic solvents such as microencapsulation in reversed micelles,5,6 entrapment in a polymer gel,’ or covalent im- mobilization on a solid supp~rt.~~~ Because enzymes are insoluble in organic solvents, one practical approach for enzyme immobilization is to immobilize the enzyme on a solid support. The immobilization often increases the thermostability of the enzyme through interaction of the support with the enzyme which leads to a more rigid conformation of the enzyme molecules. The support also entraps water in the microenvironment of the enzyme molecules’o which is es- sential for enzyme stabilization and activity. The enzyme dispersibility in the organic solvent is improved and the immobilized enzyme can be reused and operated in a con- tinuous reactor. In systems containing enzymes immobilized on solid supports and working in organic media, the support has a significant influence on the total enzymatic activity and can also displace the reaction equilibrium (hydrolysis toward synthesis) due to the interaction of the support with the water molecules.” Also, the water content in the medium and the organic solvent are important factors in determining the enzymatic activity. Thus, the choice of a suitable sup- Enzyme and Microbial Technology 20:93-101, 1997 0 1997 by Elsevier Science Inc. 655 Avenbe of the Americas, New York, NY 10010 0141-0229/97/$17.00 PII SOl41-0229(96)00089-0