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BioMed Research International
Volume 2015, Article ID 250532, 8 pages
Research Article

Improving the Thermostability and Optimal Temperature of a Lipase from the Hyperthermophilic Archaeon Pyrococcus furiosus by Covalent Immobilization

1Programa de Pós-Graduação em Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Avenida Athos da Silveira Ramos 149, Block A, 5th Floor, Room 541, 21941-909 Rio de Janeiro, RJ, Brazil
2Departamento de Biocatálisis, Instituto de Catálisis (CSIC), Campus UAM, Cantoblanco, 28049 Madrid, Spain
3Departamento de Engenharia Bioquímica, Escola de Química, Universidade Federal do Rio de Janeiro, Avenida Athos da Silveira Ramos 149, Block E, 2nd Floor, Room 203, 21949-909 Rio de Janeiro, RJ, Brazil

Received 12 December 2014; Revised 3 February 2015; Accepted 4 February 2015

Academic Editor: Zheng Guo

Copyright © 2015 Roberta V. Branco et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


A recombinant thermostable lipase (Pf2001Δ60) from the hyperthermophilic Archaeon Pyrococcus furiosus (PFUL) was immobilized by hydrophobic interaction on octyl-agarose (octyl PFUL) and by covalent bond on aldehyde activated-agarose in the presence of DTT at pH = 7.0 (one-point covalent attachment) (glyoxyl-DTT PFUL) and on glyoxyl-agarose at pH 10.2 (multipoint covalent attachment) (glyoxyl PFUL). The enzyme’s properties, such as optimal temperature and pH, thermostability, and selectivity, were improved by covalent immobilization. The highest enzyme stability at 70°C for 48 h incubation was achieved for glyoxyl PFUL (around 82% of residual activity), whereas glyoxyl-DTT PFUL maintained around 69% activity, followed by octyl PFUL (27% remaining activity). Immobilization on glyoxyl-agarose improved the optimal temperature to 90°C, while the optimal temperature of octyl PFUL was 70°C. Also, very significant changes in activity with different substrates were found. In general, the covalent bond derivatives were more active than octyl PFUL. The E value also depended substantially on the derivative and the conditions used. It was observed that the reaction of glyoxyl-DTT PFUL using methyl mandelate as a substrate at pH 7 presented the best results for enantioselectivity and enantiomeric excess (ee (%) = 91).