title: Improving the thermostability and optimal temperature of a lipase from the hyperthermophilic archaeon Pyrococcus furiosus by covalent immobilization.
abstract: 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 (E = 22) and enantiomeric excess (ee (%) = 91).
authors.affiliation: Programa 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 ; Departamento de Biocatálisis, Instituto de Catálisis (CSIC), Campus UAM, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Roberta V
authors.initials: RV
authors.lastname: Branco
authors.affiliation: Departamento de Biocatálisis, Instituto de Catálisis (CSIC), Campus UAM, Cantoblanco, 28049 Madrid, Spain ; Departamento 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.
authors.forename: Melissa L E
authors.initials: ML
authors.lastname: Gutarra
authors.affiliation: Departamento de Biocatálisis, Instituto de Catálisis (CSIC), Campus UAM, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Jose M
authors.identifier: 0000-0003-1627-6522
authors.initials: JM
authors.lastname: Guisan
authors.affiliation: Programa 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.
authors.forename: Denise M G
authors.initials: DM
authors.lastname: Freire
authors.affiliation: Programa 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.
authors.forename: Rodrigo V
authors.initials: RV
authors.lastname: Almeida
authors.affiliation: Departamento de Biocatálisis, Instituto de Catálisis (CSIC), Campus UAM, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Palomo
mesh_terms: D001105:Archaea / Q000378:metabolism*; D001426:Bacterial Proteins; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006038:Glyoxylates / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008049:Lipase / Q000737:chemistry*; D020027:Pyrococcus furiosus / Q000378:metabolism*; D012685:Sepharose / Q000737:chemistry; D013237:Stereoisomerism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D006038:Glyoxylates; C070399:glyoxyl agarose; D012685:Sepharose; D008049:Lipase; C428386:thermostable lipase
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