Discovery and Characterization of a Thermostable Esterase from an Oil Reservoir Metagenome

Abstract

With the aim of identifying novel thermostable esterases, comprehensive sequence databases and cloned fosmid libraries of metagenomes derived from an offshore oil reservoir on the Norwegian Continental Shelf were screened for enzyme candidates using both sequence-and function-based screening. From several candidates identified in both approaches, one enzyme discovered by the functional approach was verified as a novel esterase and subjected to a deeper characterization. The enzyme was successfully over-produced in Escherichia coli and was shown to be thermostable up to 90˚C, with the highest esterase activity on short-chain ester substrates and with tolerance to solvents and metal ions. The fact that the thermostable enzyme was solely found by functional screening of the oil reservoir metagenomes illustrates the importance of this approach as a complement to purely sequence-based screening, in which the enzyme candidate was not detected. In addition, this example indicates the large potential of deep-sub-surface oil reservoir metagenomes as a source of novel, thermostable enzymes of potential relevance for industrial applications.

Recent Advances in Function-Based Metagenomic Screening

“…The enzyme assays for short chain and long chain esterase activity were performed by adding the crude cell extracts to microtiter plates containing nitrophenyl acetate and nitrophenyl palmitate as substrate, respectively. One enzyme showing the highest activity on short chain ester substrate was identified as a novel esterase exhibiting high thermo-stability and high tolerance to metal ions and solvents [50] .…”

“…Mangrove soil E. coli , plasmid[39]Agar plate screeningDNA polymeraseHeterologous complementationCold sensitive mutant strainGlacial ice E. coli , fosmid[40]Agar plate screeningHydrogenaseHeterologous complementationFreshwater enrichmentNot stated E. coli , plasmid[41]Agar plate screeningGenes resistant to toxic elementsPhenotypical detectionpH 1.8Acidic water (Tinto river) E. coli , plasmid[42]Agar plate screeningGenes resistant to toxic elementsPhenotypical detectionDifferent number of antibioticsHuman fecal E. coli , fosmid[43]Agar plate screeningGenes resistant to toxic elementsPhenotypical detectionDifferent number of antibioticsSoil E. coli , plasmid[44]Agar plate screeningGenes resistant to toxic elementsPhenotypical detectionSeveral antibioticsDairy cow manure E. coli , fosmid[45]Agar plate screeningGenes resistant to toxic elementsPhenotypical detectionSeveral antibioticsCheese food matrix E. coli , fosmid[46]Agar plate screeningGenes resistant to toxic elementsPhenotypical detectionChloride saltsSurface water (Mississippi River) E. coli , fosmid[47]Agar plate screeningGenes resistant to toxic elementsHeterologous complementationAcrylateWastewater treatment plant E. coli , cosmid[48]Microtiter plate screeningCellulaseAbsorbance measurementDinitrophenol-cellobiosideSoil, Buffalo rumen, etc. E. coli , fosmid[49]Microtiter plate screeningEsteraseAbsorbance measurementNitrophenyl acetateOil reservoirNot stated, fosmid[50]Microtiter plate screeningPlant polymer decomposing enzymesAbsorbance measurementMultiple substrates…”

Thermophilic Carboxylesterases from Hydrothermal Vents of the Volcanic Island of Ischia Active on Synthetic and Biobased Polymers and Mycotoxins

“…Thus, the metagenomic carboxylesterases from the Ischia hydrothermal vents are the thermophilic enzymes highly active at 70-80 ºC with IS11 and IS12 also showing significant thermostability at temperatures from 60 to 80 ºC. Furthermore, the thermostability of IS11 and IS12 was comparable with, or exceeded the, thermostability of other metagenomic esterases identified in high-temperature environments (40,(57)(58)(59)(60).…”

Metagenomic Exploration of Plastic Degrading Microbes for Biotechnological Application

“…Similarly, identification of other novel enzymes, viz ., alkane hydroxylase, carboxylase, esterases [ 81 ], lipase, tannase [ 82 ], etc . is also carried on from metagenomic libraries of diverse ecology like cold marine to hot spring, antarctic desert to contaminated terrestrial and oil spill, even from the gut microbes of invertebrates, etc [ 83 - 85 ]. Further, heterologous expression of such codon-optimized ‘synthetic’ genes enhances the prospects of new gene identification through functional metagenomics.…”