Identification and characterization of a new true lipase isolated through metagenomic approach

Abstract

Background: Metagenomics, the application of molecular genomics to consortia of non-cultivated microbes, has the potential to have a substantial impact on the search for novel industrial enzymes such as esterases (carboxyl ester hydrolases, EC 3.1.1.1) and lipases (triacylglycerol lipases, EC 3.1.1.3). In the current work, a novel lipase gene was identified from a fosmid metagenomic library constructed with the "prokaryotic-enriched" DNA from a fat-contaminated soil collected from a wastewater treatment plant.

Results: In preliminary screening on agar containing 1% tributyrin, 2661 of the approximately 500,000 clones in the metagenomic library showed activity. Of these, 127 showed activity on agar containing 1% tricaprylin, while 32 were shown to be true lipase producers through screening on agar containing 1% triolein. The clone with the largest halo was further characterized. Its lipase gene showed 72% identity to a putative lipase of Yersinia enterocolitica subsp. palearctica Y11. The lipase, named LipC12, belongs to family I.1 of bacterial lipases, has a chaperone-independent folding, does not possess disulfide bridges and is calcium ion dependent. It is stable from pH 6 to 11 and has activity from pH 4.5 to 10, with higher activities at alkaline pH values. LipC12 is stable up to 3.7 M NaCl and from 20 to 50°C, with maximum activity at 30°C over a 1 h incubation. The pure enzyme has specific activities of 1722 U/mg and 1767 U/mg against olive oil and pig fat, respectively. Moreover, it is highly stable in organic solvents at 15% and 30% (v/v).

Conclusions: The combination of the use of a fat-contaminated soil, enrichment of prokaryotic DNA and a three-step screening strategy led to a high number of lipase-producing clones in the metagenomic library. The most notable properties of the new lipase that was isolated and characterized were a high specific activity against long chain triacylglycerols, activity and stability over a wide range of pH values, good thermal stability and stability in water-miscible organic solvents and at high salt concentrations. These characteristics suggest that this lipase has potential to perform well in biocatalytic processes, such as for hydrolysis and synthesis reactions involving long-chain triacylglycerols and fatty acid esters.

Figure 1

Isolation, purification and identification of LipC12. (a) Identification of a lipolytic clone that formed a hydrolysis halo in the tributyrin plate assay. (b) Hydrolysis halos formed by lipase-producing clones in the triolein plate assay. FosC12, FosE6 and FosH10 showed the largest hydrolysis halos and were chosen for sequencing. (c) SDS-PAGE of fractions of His-tagged LipC12 eluted from the affinity chromatography column. (d) SDS-PAGE and zymogram analyses of LipC12. The lanes correspond to molecular marker (MM), coomassie blue staining (CB) and lipolytic activity of LipC12 on the SDS-PAGE gel, using tricaprylin (TC) and tributyrin (TB) as substrates.

Figure 2

Phylogenetic analysis of LipC12 and closely related proteins. The enzymes most similar to LipC12 are those from Enterobacteria, such as Yersinia sp., Proteus sp. and Arsenophonus sp. LipC12 is a member of subfamily I.1 [42], with closest homology to Yersinia sp. lipases. Except for LipC12, the protein sequences were retrieved from GenBank (NCBI). The phylogenetic tree was generated using MEGA 5 [65]. The scale represents the number of amino acid substitutions per site.

Figure 3

Characteristic residues in multiple sequence alignment between LipC12 and lipases from subfamilies I.1 and I.2. Conserved residues of the active site (as), cysteine residues forming the disulfide bridge (db) and aspartic residues involved in the calcium binding domains (cb) are annotated. Shaded regions indicate similar amino acids in all the aligned sequences. The accession number and the organism name of the aligned sequences are (from top to the bottom): [GenBank:JF417979], LipC12 lipase; [GenBank:CBY26912], lipase from Yersinia enterocolitica subsp. palearctica Y11; [GenBank:CBA71562], lipase from Arsenophonus nasoniae; [GenBank:ACM67042], lipase from Proteus vulgaris; [GenBank:CAC07191], lipase from Pseudomonas fragi; [GenBank:X80800], lipase from Acinetobacter calcoaceticus; [GenBank:D50587], lipase from Pseudomonas aeruginosa; [GenBank:M58494], lipase from Burkholderia cepacia; [GenBank:AF050153], lipase from Pseudomonas luteola; [GenBank:X70354], lipase from Burkholderia glumae. The figure was generated with GeneDoc 2.7 software.

Figure 4

Enzymatic activity measurements obtained using the titrimetric method with triacylglycerols. (a) LipC12 substrate specificity against triacylglycerols. Among long-chain triglycerides, LipC12 showed the highest activities for pig fat and olive oil. (b) Effect of pH on LipC12 activity. LipC12 showed activity above pH 4.5 and it is more active under neutral to alkaline conditions.