Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome

Abstract

Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78% following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.

Fig. 1

Neighbour-joining analysis of lip1 to lip14 and lipolytic proteins from different families. The scale indicates the number of substitution events. The numbers associated with the branches refer to the bootstrap values (confidence limits) resulting from 1000 replicate resamplings. Roman numerals correspond to the lipolytic families as defined by Arpigny and Jaeger (1999)

Fig. 2

The effect of pH on the activity of lipases isolated from the rumen metagenome of cattle. The pH assays were carried out using ρ-nitrophenyl caprate (C10) as the substrate for lip4 and lip13ss, ρ-nitrophenyl caproate (C6) for lip6 and ρ-nitrophenyl caprylate (C8) for pl1 and pl2ss, at a constant temperature of 39 °C in a wide-range pH buffer set at the indicated pH values

Fig. 3

The effect of temperature on the activity of lipases isolated from the rumen metagenome of cattle. The temperature assays were carried out using ρ-nitrophenyl caprate (C10) as the substrate for lip4 and lip13ss, ρ-nitrophenyl caproate (C6) for lip6 and ρ-nitrophenyl caprylate (C8) for pl1 and pl2ss, in a wide-range pH buffer with pH being 6.5 for all assays