title: First co-expression of a lipase and its specific foldase obtained by metagenomics.
abstract: BACKGROUND
  Metagenomics is a useful tool in the search for new lipases that might have characteristics that make them suitable for application in biocatalysis. This paper reports the cloning, co-expression, purification and characterization of a new lipase, denominated LipG9, and its specific foldase, LifG9, from a metagenomic library derived from a fat-contaminated soil.
  
  
  RESULTS
  Within the metagenomic library, the gene lipg9 was cloned jointly with the gene of the foldase, lifg9. LipG9 and LifG9 have 96% and 84% identity, respectively, with the corresponding proteins of Aeromonas veronii B565. LipG9 and LifG9 were co-expressed, both in N-truncated form, in Escherichia coli BL21(DE3), using the vectors pET28a(+) and pT7-7, respectively, and then purified by affinity chromatography using a Ni(2+) column (HiTrap Chelating HP). The purified enzyme eluted from the column complexed with its foldase. The molecular masses of the N-truncated proteins were 32 kDa for LipG9, including the N-terminal His-tag with 6 residues, and 23 kDa for LifG9, which did not have a His-tag. The biochemical and kinetic characteristics of the purified lipase-foldase preparation were investigated. This preparation was active and stable over a wide range of pH values (6.5-9.5) and temperatures (10-40°C), with the highest specific activity, of 1500 U mg(-1), being obtained at pH 7.5 at 30°C. It also had high specific activities against tributyrin, tricaprylin and triolein, with values of 1852, 1566 and 817 U mg(-1), respectively. A phylogenetic analysis placed LipG9 in the lipase subfamily I.1. A comparison of the sequence of LipG9 with those of other bacterial lipases in the Protein Data Bank showed that LipG9 contains not only the classic catalytic triad (Ser(103), Asp(250), His(272)), with the catalytic Ser occurring within a conserved pentapeptide, Gly-His-Ser-His-Gly, but also a conserved disulfide bridge and a conserved calcium binding site. The homology-modeled structure presents a canonical α/β hydrolase folding type I.
  
  
  CONCLUSIONS
  This paper is the first to report the successful co-expression of a lipase and its associated foldase from a metagenomic library. The high activity and stability of Lip-LifG9 suggest that it has a good potential for use in biocatalysis.
authors.affiliation: Departamento de Química, Universidade Federal do Paraná, Cx. P. 19081 Centro Politécnico, Curitiba, 81531-980, Paraná, Brazil. viviane.martini@ifpr.edu.br.|Instituto Federal do Paraná - Campus Irati, Rua Pedro Koppe, 100, Irati, 84500-000, Paraná, Brazil. viviane.martini@ifpr.edu.br.
authors.forename: Viviane Paula
authors.initials: VP
authors.lastname: Martini
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Cx. P. 19046, Centro Politécnico, Curitiba, 81531-980, Paraná, Brazil. arnaldoglogauer@yahoo.com.br.|Agência Tecpar de Inovação, Instituto de Tecnologia do Paraná - Tecpar, Curitiba, 81350-010, Paraná, Brazil. arnaldoglogauer@yahoo.com.br.
authors.forename: Arnaldo
authors.initials: A
authors.lastname: Glogauer
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Cx. P. 19046, Centro Politécnico, Curitiba, 81531-980, Paraná, Brazil. marcelomuller@ufpr.br.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Müller-Santos
authors.affiliation: Departamento de Química, Universidade Estadual de Ponta Grossa, Av. Carlos Cavalcanti, 4748, Ponta Grossa, 84070-900, Paraná, Brazil. iulek@uepg.br.
authors.forename: Jorge
authors.initials: J
authors.lastname: Iulek
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Cx. P. 19046, Centro Politécnico, Curitiba, 81531-980, Paraná, Brazil. souzaem@ufpr.br.
authors.forename: Emanuel Maltempi
authors.initials: EM
authors.lastname: de Souza
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Cx. P. 19046, Centro Politécnico, Curitiba, 81531-980, Paraná, Brazil. davidmitchell@ufpr.br.
authors.forename: David Alexander
authors.initials: DA
authors.lastname: Mitchell
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Cx. P. 19046, Centro Politécnico, Curitiba, 81531-980, Paraná, Brazil. fpedrosa@ufpr.br.
authors.forename: Fabio Oliveira
authors.initials: FO
authors.lastname: Pedrosa
authors.affiliation: Departamento de Química, Universidade Federal do Paraná, Cx. P. 19081 Centro Politécnico, Curitiba, 81531-980, Paraná, Brazil. nkrieger@ufpr.br.
authors.forename: Nadia
authors.initials: N
authors.lastname: Krieger
mesh_terms: D000333:Aeromonas / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D008049:Lipase; D054892:Metagenome; D056186:Metagenomics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008049:Lipase