A new esterase EstD2 isolated from plant rhizosphere soil metagenome

Abstract

Soil metagenome constitutes a reservoir for discovering novel enzymes from the unculturable microbial diversity. From three plant rhizosphere metagenomic libraries comprising a total of 142,900 members of recombinant plasmids, we obtained 14 recombinant fosmids that exhibited lipolytic activity. A selected recombinant plasmid, pFLP-2, which showed maximum lipolytic activity, was further analyzed. DNA sequence analysis of the subclone in pUC119, pELP-2, revealed an open reading frame of 1,191 bp encoding a 397-amino-acid protein. Purified EstD2 exhibited maximum enzymatic activity towards p-nitrophenyl butyrate, indicating that it is an esterase. Purified EstD2 showed optimal activity at 35 °C and at pH 8.0. The K(m) and K(cat) values were determined to be 79.4 μM and 120.5/s, respectively. The esterase exhibited an increase in enzymatic activity in the presence of 15% butanol and 15% methanol. Phylogenetic analysis revealed that the lipolytic protein EstD2 may be a member of a novel family of lipolytic enzymes. Several hypothetical protein homologs of EstD2 were found in the database. A hypothetical protein from Phenylobacterium zucineum HLK1, a close homolog of EstD2, displayed lipolytic activity when the corresponding gene was expressed in Escherichia coli. Our results suggest that the other hypothetical protein homologs of EstD2 might also be members of this novel family.

Rhodococcus sp. Strain CR-53 LipR, the First Member of a New Bacterial Lipase Family (Family X) Displaying an Unusual Y-Type Oxyanion Hole, Similar to the Candida antarctica Lipase Clan

“…2A), indicating that this kind of active site is a highly conserved region. However, no matches of the conserved motifs existed with respect to the previously described lipase families (26,27,28), suggesting that neither LipR nor the putative lipase sequences clustered with it and used for the alignment belonged to any of the known bacterial lipase families. Moreover, the sequence Tyr-Asp-Ser-Leu ( Fig.…”

“…However, no new bacterial lipase families were described at that time. More recently, new families of bacterial lipases have been proposed by several authors (26,27,28) on the basis of phylogenetic criteria but not on the basis of the presence of conserved blocks of sequence motifs and/or biological function, as was the case for the original bacterial lipase classification (2). Rhodococcus sp.…”

Identification and characterization of novel esterases from a deep-sea sediment metagenome

“…Bacterial lipolytic enzymes have been classified into eight families (I-VIII) according to their amino acid sequences (Arpigny and Jaeger 1999). Recently, using metagenomics, new families of lipolytic enzymes were identified, including LipG, from a Korean tidal flat sediments metagenomic library (Lee et al 2006); EstA, from a South China Sea surface water metagenomic library (Chu et al 2008); LipEH166, from an intertidal flat metagenomic library ); EstD2, from a plant rhizosphere soil metagenomic library (Lee et al 2010); EstGK1 and EstZ3, from a sheep rumen metagenomic library (Bayer et al 2010), and EstF, from a South China Sea marine sediment metagenomic library (Fu et al 2011).…”

Genomics and the Rhizosphere

“…Functional screening of libraries has been employed to identify novel esterases from red pepper rhizospheres. In this study, lipolytic activity was screened by the detection of halos around colonies grown on tributyrin plates (Lee et al, 2010). A novel antimicrobial gene cluster was identified by screening a forest soil metagenome for antimicrobial activity against Saccharmyces cerevisiae (Chung et al, 2008).…”