Candida rugosa Lipase (CRL) shows a very low alkaline stability that comprises its immobilization on glyoxyl-agarose, which requires pH above 10. In this way, an adaptation from the original method was used; an enzyme solution at pH 7 was slowly added at a suspension of glyoxyl-agarose prepared in bicarbonate buffer, pH 10. This change of protocol was enough for allowing the preparation of derivatives actives of CRL on glyoxyl-agarose and verifying the effect of this modified procedure on the properties of the immobilized enzyme. The effect of the additives Triton-X-100 and polyethylene glycol (PEG) on the enzymatic activity recovery and immobilized enzyme stability was evaluated.


The glyoxyl-agarose support was prepared by etherification of 6% agarose beads with glycidol and further oxidation with sodium periodate. CRL was immobilized covalently on glyoxyl-agarose support in the absence and presence of 1% (w/v) Triton-X-100 or 5 g L-1 polyethylene glycol (PEG). The lipolysis activity of the free and immobilized enzyme was determined at 37ºC and pH 7.0, using p-nitrophenyl palmitate (p-NPP) as substrate. Profiles of temperature-activity (37-65ºC, pH 7.0) and pH-activity (6.0-9.5, 37ºC) were evaluated as well as thermal (45ºC and pH 8.0) and operational (15 min batches of p-NPP hydrolysis at 50ºC and pH 8.0) stabilities of free and immobilized CRL.


Using a single modification of the original protocol, the CRL poorly stable under alkaline conditions could be immobilized on glyoxyl-agarose in its active conformation (recovered activity varying from 10.3 to 30.4%). Besides, the presence of a detergent (Triton-X-100) and an enzyme stabilizer (PEG) contributed to the preparation of more active and more stable biocatalysts, respectively. CRL immobilized on glyoxyl-agarose in the presence of PEG was around 5 times more stable than the free CRL and around 3 times more stable than the CRL immobilized on glyoxyl-agarose in absence of PEG. The higher stability of the CRL-glyoxyl derivative prepared in the presence of PEG allowed its reuse in four successive 15 min-batches of p-nitrophenyl palmitate hydrolysis at 50ºC and pH 8.0.


The technique of immobilizing enzymes covalently on glyoxyl-agarose showed promising results for Candida rugosa lipase (CRL). The derivatives prepared in the presence of the additives retained two to three times more activity than those prepared in the absence of additives. The enzyme immobilized in presence of PEG was about three times more stable than the enzyme immobilized in absence of this additive. Maximum catalytic activity of the immobilized CRL (in absence of additives) was observed in a temperature 10ºC above that for the free enzyme and the pH of the maximum activity was maintained in the range 6.5-7.5 for free and immobilized CRL.

Keywords: Triton X-100, PEG, Stabilization, Candida rugosa Lipase, Immobilization, Glyoxyl-Agarose.
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