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First co-expression of a lipase and its specific foldase obtained by metagenomics.

Martini VP, Glogauer A, Müller-Santos M, Iulek J, de Souza EM, Mitchell DA, Pedrosa FO, Krieger N - Microb. Cell Fact. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Química, Universidade Federal do Paraná, Cx. P. 19081 Centro Politécnico, Curitiba, 81531-980, Paraná, Brazil. viviane.martini@ifpr.edu.br.

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.

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Related in: MedlinePlus

Effect of temperature on Lip-LifG9 activity and stability. In both studies, activity was determined by the titrimetric method with tricaprylin as the substrate, while in all incubations and assays the pH was 7.5. For the activity study, relative activities are plotted, with 100% corresponding to the activity at 30°C. For the stability study, Lip-LifG9 was incubated for 1.5 h before determination of the residual activity and 100% corresponds to the activity after incubation at 30°C. The assays were done in triplicate. The error bars represent the standard error of the mean.
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Fig6: Effect of temperature on Lip-LifG9 activity and stability. In both studies, activity was determined by the titrimetric method with tricaprylin as the substrate, while in all incubations and assays the pH was 7.5. For the activity study, relative activities are plotted, with 100% corresponding to the activity at 30°C. For the stability study, Lip-LifG9 was incubated for 1.5 h before determination of the residual activity and 100% corresponds to the activity after incubation at 30°C. The assays were done in triplicate. The error bars represent the standard error of the mean.

Mentions: The highest specific activity of Lip-LifG9 for the hydrolysis of tricaprylin was obtained at 30°C (1566 ± 101 U mg−1). At 50°C, its activity was only half this value and at 60°C it was only 30% of this value (Figure 6). In stability studies involving previous incubation of Lip-LifG9 for 1.5 h at 10-40°C without substrate, residual activities of around 90% or better were obtained. At the higher temperatures of 50°C and 60°C, the residual activities were around 70% and 30%, respectively (Figure 6).Figure 6


First co-expression of a lipase and its specific foldase obtained by metagenomics.

Martini VP, Glogauer A, Müller-Santos M, Iulek J, de Souza EM, Mitchell DA, Pedrosa FO, Krieger N - Microb. Cell Fact. (2014)

Effect of temperature on Lip-LifG9 activity and stability. In both studies, activity was determined by the titrimetric method with tricaprylin as the substrate, while in all incubations and assays the pH was 7.5. For the activity study, relative activities are plotted, with 100% corresponding to the activity at 30°C. For the stability study, Lip-LifG9 was incubated for 1.5 h before determination of the residual activity and 100% corresponds to the activity after incubation at 30°C. The assays were done in triplicate. The error bars represent the standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4305245&req=5

Fig6: Effect of temperature on Lip-LifG9 activity and stability. In both studies, activity was determined by the titrimetric method with tricaprylin as the substrate, while in all incubations and assays the pH was 7.5. For the activity study, relative activities are plotted, with 100% corresponding to the activity at 30°C. For the stability study, Lip-LifG9 was incubated for 1.5 h before determination of the residual activity and 100% corresponds to the activity after incubation at 30°C. The assays were done in triplicate. The error bars represent the standard error of the mean.
Mentions: The highest specific activity of Lip-LifG9 for the hydrolysis of tricaprylin was obtained at 30°C (1566 ± 101 U mg−1). At 50°C, its activity was only half this value and at 60°C it was only 30% of this value (Figure 6). In stability studies involving previous incubation of Lip-LifG9 for 1.5 h at 10-40°C without substrate, residual activities of around 90% or better were obtained. At the higher temperatures of 50°C and 60°C, the residual activities were around 70% and 30%, respectively (Figure 6).Figure 6

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Química, Universidade Federal do Paraná, Cx. P. 19081 Centro Politécnico, Curitiba, 81531-980, Paraná, Brazil. viviane.martini@ifpr.edu.br.

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.

Show MeSH
Related in: MedlinePlus