ELSEVIER zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Covalent immobilization of pure isoenzymes from lipase of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJI Candida rugosa JosC-Maria Moreno, Miguel Arroyo, Maria-Jo& Hernhiz, and Jo&-Vicente Sinisterra zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Department of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, Universidad Complutense, Madrid, Spain Covalent irnmobilizution of pure lipases A and B from Candida rugosa on agarose and silica is described. The immobilization increases the half-life of the biocatalysts (tx. = 5 h) with respect to the native pure lipases (tl,? = 0.28 h). The percentage immobilization of lipases A and B is similar in both supports (33-40%). The remaining activity zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA of the biocatalysts immobilized on agarose (70-75%) is greater than that of the enzymatic derivatives immobilized on SiO, (40-50%). The surjixce area and the hydrophobic/hydrophilic properties of the support control the lipase activiw of these derivatives. The thermal stability of the immobilized lipase A derivatives is greater than that of lipase B derivatives. The nature of the support influences the thermal deactivation profile of the immobilized derivatives. The immobilization in agarose (hydrophilic support) gives biocatalysts that show a greater initial specific reaction rate than the biocatalysts immobilized in Si02 (hydrophobic support) using the hydrolysis of the esters of(R) or(S) 2-chloropropanoic and of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF (R,S) 2-phenylpropanoic acids as the reaction test. The enzymatic derivatives are active for at least 196 h under hydrolysis conditions. The stereospecifi+v of the native and the immobilized enzymes is the same. 0 1997 Elsevier Science Inc. Keywords: Pure isoenzymes; Can&ix rugosa; covalent immobilization; enzymatic stabilization: enantioselective hydrolysis of esters Introduction In general, microbial lipases from culture supernatants have been covalently immobilized without any previous purifi- cation. This is the case for the commercial lipase from Candida rugosa (CCRL) which has not been solubilized following different methodologies. For instance, Yamane et al. ’ used octyl and phenyl Sepharose 4B for lipase adsorp- tion. The hydrolytic activity of the immobilized derivatives obtained by adsorption gradually decreased due to the weak attachment of the enzyme to the support. Lavayre and Baratti’ immobilized lipase on propylated Spherosil beads and yielded lipolytic activity equivalent to only 1% that of the native lipase. The low activity was believed to be due to mass transfer resistance of the oil substrate through the pores of the support. To overcome these problems, some authors have tested different activated supports for covalent Address reprint requests to Dr. J. V. Sinisterro. Universidad Complutense, Faculty of Pharmacy, Dept. Organic and Pharmaceutical Chem. 28040 Madrid. Spain Received 26 June 1996; revised 21 April 1997: accepted 7 May 1997 bonding. Otero et aL3 immobilized lipase from C. rugosa on Sepharose 6B previously activated with 2,3 epoxy-l-propa- nol. These authors obtained 90-100% of the covalent attachment to the matrix and the catalytic efficiency was improved (15532%). Shaw et al.” tried with six different supports; the results varied widely among all the prepared biocatalysts. In previous papers, we have reported the preparation of high thermostable covalent immobilized derivatives of the commercial lipase of C. rugosa. Our research group improved the commercial lipase loading yields employing tosylated agarose (30-35 mg CCRL ml-’ wet gel)” and inorganic supports activated with trichloro- triazine (51-62 mg CCRL ml-’ wet ge1)6 and achieved highly efficient biocatalysts (70-76% of activity with re- spect to the free enzyme) for the hydrolysis of olive oil. Native and immobilized forms of the commercial lipase show high S-enantiopreference in the hydrolysis of the esters of (R,S) 2-phenylpropanoic acid.’ In the present paper and for the first time, two methods have been tested for the covalent bonding of pure lipases A and B from C. rugosa. The enzymatic derivatives were tested for the hydrolysis of pure methyl (R) or (S) 2- Enzyme and Microbial Technology 21:552-558, 1997 0 1997 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010 0141-0229/97/$17.00 PII SOl41-0229(97)00064-l