First co-expression of a lipase and its specific foldase obtained by metagenomics
BackgroundMetagenomics 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.ResultsWithin 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 Ni2+ 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 (Ser103, Asp250, His272), 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0171-7) contains supplementary material, which is available to authorized users.
[…] The clone with the genes lipg9 and lifg9, for the lipase and foldase, respectively, was previously isolated from a metagenomic DNA library built from a fat-contaminated soil . The plasmid DNA was isolated, by the alkaline lysis method , for template usage for PCR and for DNA sequencing. For the latter, a collection of derivative plasmids containing randomly inserted EZ-Tn5 < KAN-2 > was obtained by using an in vitro transposon insertion reaction with the EZ-Tn5 < KAN-2 > Insertion Kit (Epicentre). These derivative plasmids were then used to generate new clones. Genes from 96 inactive clones were then sequenced with the ABI 377 (Genetic Analyzer, Applied Biosystems/HITACHI, Foster City, CA, USA) and MegaBACE 1000 (GE Healthcare, Uppsala, Sweden) sequencers, from both ends, using DYEnamic ET Dye Terminator Kit (GE Life Sciences). Sequence assembly and editing were performed with the CodonCode Aligner software (CodonCode Corporation, Centerville, MA, USA). The open reading frames (ORFs) were identified with the ORF Finder tool (NCBI) . […]
to analyse the site's operation and effectiveness, to display ads tailored to your interests
and to provide you with relevant promotional messages and other information about products,
events and services of ours or our sponsors and partner companies.
These cookies are needed for the site to work and to be optimized.
These cookies are needed to interact with the social network plugins on this site.
These cookies are used to track visitors across websites.
The intention is to display ads that are relevant and engaging for the users.
These cookies are needed in order to better understand how
this site is used and to improve the user experience.
At omicX, we believe trust is of the utmost importance. Transparency allows trust.
This is why we want you to understand what data we collect and how we use it.