title: 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.
authors.affiliation: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, SY23 3DA, UK.
authors.forename: Florence
authors.initials: F
authors.lastname: Privé
authors.forename: C Jamie
authors.initials: CJ
authors.lastname: Newbold
authors.forename: Naheed N
authors.initials: NN
authors.lastname: Kaderbhai
authors.forename: Susan G
authors.initials: SG
authors.lastname: Girdwood
authors.forename: Olga V
authors.initials: OV
authors.lastname: Golyshina
authors.forename: Peter N
authors.initials: PN
authors.lastname: Golyshin
authors.forename: Nigel D
authors.initials: ND
authors.lastname: Scollan
authors.forename: Sharon A
authors.initials: SA
authors.lastname: Huws
mesh_terms: D000818:Animals; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D054892:Metagenome; D008969:Molecular Sequence Data; D012417:Rumen / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D013696:Temperature; D014169:Transformation, Bacterial; D014280:Triglycerides / Q000378:metabolism
chemical_list: D014280:Triglycerides; D004950:Esterases; C005830:tributyrin