Characterization of Novel Family IV Esterase and Family I.3 Lipase from an Oil-Polluted Mud Flat Metagenome

Abstract

Two genes encoding lipolytic enzymes were isolated from a metagenomic library constructed from oil-polluted mud flats. An esterase gene, est3K, encoded a protein of 299 amino acids (ca. 32,364 Da). Est3K was a family IV esterase with typical motifs, HGGG, and HGF. Although est3K showed high identity to many genes with no information on their enzymatic properties, Est3K showed the highest identity (36 %) to SBLip5.1 from forest soil metagenome when compared to the enzymes with reported properties. A lipase gene, lip3K, encoded a protein of 616 amino acids (ca. 64,408 Da). Lip3K belonged to family I.3 lipase with a C-terminal secretion signal and showed the highest identity (93 %) to the lipase of Pseudomonas sp. MIS38. The presence of several newly identified conserved motifs in Est3K and Lip3K are suggested. Both Est3K and Lip3K exerted their maximal activity at pH 9.0 and 50 °C. The activity of Lip3K was significantly increased by the presence of 30 % methanol. The ability of the enzymes to retain activities in the presence of methanol and the substrates may offer a merit to the biotechnological applications of the enzymes such as transesterification. The activity and the thermostability of Lip3K were increased by Ca(2+). Est3K and Lip3K preferred p-nitrophenyl butyrate (C4) and octanoate (C8), respectively, as the substrate and acted independently on the substrates with no synergistic effect.

Characterization of an alkaline esterase from an enriched metagenomic library derived from an oil-spill area

“…W130-35 [11]. The large esterases were found in family I.3, likely Lip3K (64 kDa) from the oilpolluted mud flat metagenome [9], or in most of family VII (~55 kDa) [1]. Based on the results, Est7S was a large-sized enzyme with 55 kDa.…”

“…We also have previously reported two novel enzymes, a family IV esterase and a family I. 3 lipase, from an oil-polluted mud flat metagenome [9]. When the sample was enriched with specific substrates, microbial diversity could hugely increase.…”

“…Oil-spilled fine gravels and sands were sampled, as described previously [9]. The sample was enriched by adding 0.5% crude oil into BM medium [2% sea salt, 10 mM phosphate buffer, 0.25 g MgSO 4 ·7H 2 O, 0.02 g CaCl 2 ·2H 2 O, 0.02 g FeSO 4 ·7H 2 O, 2.38 g (NH 4 ) 2 SO 4 per L] and grown three times at 25 ºC for 10 days.…”

Crystal structure of PMGL2 esterase from the hormone-sensitive lipase family with GCSAG motif around the catalytic serine

“…The amino acid sequences of bacterial lipolytic enzymes belonging to the HSL family contain several conserved motifs [13,18,20,52], which participate in the active site formation or are situated nearby. PMGL2 also shares these motifs, including 105 HGGAF 109 , 269 GGRD 272 , 296 FxxGxxHxxF 305 and others ( Figs 1A and 2).…”

“…Several esterases from this family were previously cloned and characterized including enzymes from metagenomic DNA libraries [17]. Common structural features of these esterases include the conserved HGG motif [13,18], which participates in the oxyanion hole formation, motifs GxSxG, GGRD and FxxGxxHxxF [13,[18][19][20], containing active site residues as well as YRMP motif [20,21]. According to the structure of the motif surrounding the catalytic serine residue, members of the HSL family are further divided into two subfamilies with GDSAG and GTSAG variants of this sequence [18,19]; the latter subfamily includes different hydrophilic residues, except for Asp/Glu, in the position before the catalytic serine.…”