Cloning and biochemical characterization of a novel lipolytic gene from activated sludge metagenome, and its gene product

Abstract

In this study, a putative esterase, designated EstMY, was isolated from an activated sludge metagenomic library. The lipolytic gene was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The gene estMY contained a 1,083 bp open reading frame (ORF) encoding a polypeptide of 360 amino acids with a molecular mass of 38 kDa. Sequence analysis indicated that it showed 71% and 52% amino acid identity to esterase/lipase from marine metagenome (ACL67845) and Burkholderia ubonensis Bu (ZP_02382719), respectively; and several conserved regions were identified, including the putative active site, GDSAG, a catalytic triad (Ser203, Asp301, and His327) and a HGGG conserved motif (starting from His133). The EstMY was determined to hydrolyse p-nitrophenyl (NP) esters of fatty acids with short chain lengths (≤C8). This EstMY exhibited the highest activity at 35°C and pH 8.5 respectively, by hydrolysis of p-NP caprylate. It also exhibited the same level of activity over wide temperature and pH spectra and in the presence of metal ions or detergents. The high level of stability of esterase EstMY with unique substrate specificities makes it highly valuable for downstream biotechnological applications.

First co-expression of a lipase and its specific foldase obtained by metagenomics

“…The fact that Lip-LifG9 has a high specific activity against this long chain substrate makes it a true metagenomic lipase [ 73 ],[ 74 ]. Of the many hydrolytic enzymes isolated from metagenomics with claimed lipase activity [ 14 ],[ 27 ],[ 29 ],[ 31 ],[ 64 ],[ 75 ]-[ 78 ], only a few are active against long chain triacylglycerols [ 19 ],[ 24 ]. Notably, Lip-LifG9 has a higher specific activity against triolein, another long chain triacylglycerol, than do some other true lipases obtained through metagenomics: Lip-LifG9 gave a specific activity of 817 U mg −1 with this substrate, while RlipE1 and RlipE2, isolated from a cow rumen metagenomic library [ 19 ], gave specific activities of 346 and 232 U mg −1 , respectively.…”

Cloning, Expression and Characteristics of a Novel Alkalistable and Thermostable Xylanase Encoding Gene (Mxyl) Retrieved from Compost-Soil Metagenome

“…Although, the compost soils are in the acidic pH range, an alkalistable and thermostable endoglucanase had been reported from rice straw compost [13]. The culture independent approach has started yielding the useful biocatalysts from the hidden Pandora's Box [14], [15], [16]. A considerable success has also been achieved in obtaining xylanases with diverse attributes by using metagenomic approaches [17], [18], [19], [20], [21].…”

Mining the metagenome of activated biomass of an industrial wastewater treatment plant by a novel method

“…The functional clones obtained in primary screening, also demonstrate efficiency of the method. JunGang et al [13] reported, only one lipolytic clone from an activated sludge metagenomic library of size 2.1 Gb, while Roh et al [25] also reported one clone in 1,00,000 clones. Ranjan et al [24] reported 13 lipolytic clones from pond water samples.…”