The Open Biochemistry Journal

ISSN: 1874-091X ― Volume 13, 2019

Effects of Triton X-100 and PEG on the Catalytic Properties and Thermal Stability of Lipase from Candida Rugosa Free and Immobilized on Glyoxyl-Agarose

Rafael F. Perna1, *, Poliana C. Tiosso2, Letícia M. Sgobi3, Angélica M.S. Vieira3, Marcelo F. Vieira2, Paulo W. Tardioli4, Cleide M.F. Soares5, Gisella M Zanin2
1 Science and Technology Institute, Federal University of Alfenas, Rod. José Aurélio Vilela, Km 533, 11999, 37715-400 Poços de Caldas, MG, Brazil
2 Department of Chemical Engineering, State University of Maringá, Av. Colombo, 5790, 87020-900 Maringá, PR.W, Brazil
3 Department of Food Engineering, State University of Maringá, Av. Colombo, 5790, 87020-900 Maringá, PR.W, Brazil
4 Departmet of Chemical Engineering, Federal University of São Carlos, Rod. Washington Luis, Km 235, 13565-905, São Carlos, SP, Brazil
5 Institute of Technology and Research, Tiradentes University, Av. Murilo Dantas, 300, 49032-490 Aracaju, SE, Brazil



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.

Article Information

Identifiers and Pagination:

Year: 2017
Volume: 11
First Page: 66
Last Page: 76
Publisher Id: TOBIOCJ-11-66
DOI: 10.2174/1874091X01711010066

Article History:

Received Date: 10/01/2017
Revision Received Date: 07/06/2017
Acceptance Date: 23/06/2017
Electronic publication date: 31/07/2017
Collection year: 2017

Article Metrics:

CrossRef Citations:

Total Statistics:

Full-Text HTML Views: 2189
Abstract HTML Views: 1296
PDF Downloads: 635
ePub Downloads: 334
Total Views/Downloads: 4454

Unique Statistics:

Full-Text HTML Views: 1014
Abstract HTML Views: 725
PDF Downloads: 341
ePub Downloads: 175
Total Views/Downloads: 2255
Geographical View

© 2017 Perna et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Science and Technology Institute, Federal University of Alfenas (ICT / UNIFAL-MG), Rod. José Aurélio Vilela, 11999 (BR 267, Km 533), 37715-400. City: Poços de Caldas. State: Minas Gerais, Brazil; Tel: +55 35 3697-4714; E-mails:,


"We have always had a fruitful cooperation and long-term experience of publishing with Bentham Open."

"A number of our original papers have appeared in its different journals, as the Bentham Open Publishers provides sufficient research in the field of science and technologies (belonging to the pharmaceutical, biotechnology, biomedical industries etc). The staff of the Bentham Open always treat our papers very seriously and gently during the rutile paper reviewing process, so far we always felt their kindness and highly cooperative support. Today, Bentham Open is providing a great support to science and research, giving opportunities of high standard publishing as well as promoting and enhancing selected papers around the world through communication and exchange of global scientific education."

Kholmirzo T. Kholmurodov
Leading Scientist
FLNP (Frank Laboratory of Neutron Physics) JINR (Joint Institute of Nuclear Research) Moscow region, Russia

Browse Contents

Webmaster Contact:
Copyright © 2019 Bentham Open