Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase

Abstract

A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli. Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350 Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria, Bacteroidetes, Acidobateria, Firmicutes, Verrucomicrobia, Chloroflexi, Spirochaetes, Thermotogae, Armatimonadetes, and Planctomycetes. Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct α/β-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new α-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p-nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p-nitrophenol-α-(l)-arabinopyranose with a high specific activity of about 2.7 kU/mg protein. Most of the α/β-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40 min of incubation at 90 °C. Furthermore, enzymes were active in organic solvents (e.g., >30 % methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6873-x) contains supplementary material, which is available to authorized users.

Cellulases from Thermophiles Found by Metagenomics

“…The use of metagenomics, the study of whole communities’ genomes, has proven to be a useful tool for the discovery of novel cellulases, both in the functional and the sequence-based approaches [ 10 , 11 ]. Several studies had found cellulases in a wide variety of natural thermophilic environments, such as hydrothermal vents [ 56 , 57 ], continental geothermal pools and hotsprings [ 58 , 59 ], and man-made environments like vermicompost [ 60 ], compost [ 37 , 61 , 62 ], and biogas digesters [ 63 ]. Nevertheless, high-temperature acting enzymes have also been found by metagenomics on moderate-temperature samples like soils [ 40 , 64 , 65 ] and aquatic environments [ 66 ], and in microorganisms associated with animals like microbial communities in rabbit cecum [ 67 ], ruminants rumen [ 36 , 68 , 69 ], earthworm casts [ 70 ], and thermite guts [ 71 , 72 ].…”

Biodiversity for biocatalysis: A review of the α/β-hydrolase fold superfamily of esterases-lipases discovered in metagenomes

“…Using (R,S)-1-phenylethyl acetate, Im-LipG9 (KM023399) showed excellent enantio-selectivity for the R-isomer (E > 200), giving an ee of higher than 95% for the products at 49% conversion. Using a racemic mixture, the capacity to hydrolyze preferentially (S)-racemic ofloxacin butyl ester with an ee of 70.3% was found using an esterase ( (KP861227), LIPESV12_26 (KP861228) from hydrothermal vent sediments of the Levante Bay (Placido et al 2015) were also found to be enantio-selective, to different degrees and preferences, for at least eight chiral esters, including methyl-(±)-mandelate, methyl-(±)-lactate, (±)-menthylacetate, (±)-neomenthyl acetate, (±)-glycidyl 4-nitrobenzoate, (±)-pantolactone, and (±)-methyl -3-hydroxybutyrate.…”

“…Hydrolysis of aryl esters such as methyl phenyl-acetate and phenyl acetate have also been from hydrothermal vent sediments (Placido et al 2015), and 5 cold-active and salt-resistant esterases (JX133669, JX133670, JX133672-JX133675) from marine metagenomes (Tchigvintsev et al 2015). These studies demonstrated that esterases/lipases discovered in metagenomes show reactivity and promiscuity levels that are superior to those of the homologous hydrolases reported to date.…”

A-GAME: improving the assembly of pooled functional metagenomics sequence data

“…Coverage depth is typically high and levels of sequence redundancy within relatively short (typically <45kbp) inserts are expected, in general, to be low. Indeed, costs associated with sequencing can be further reduced by sequencing libraries of pooled inserts [ 15 – 17 ]. Lam et al used pooled and individually sequenced inserts to provide a detailed evaluation of this approach [ 16 ], demonstrating that many inserts could be completely or almost completely assembled in one or two fragments and assigned to their clones of origin through Sanger sequencing of the original insert ends (end-tag sequencing).…”