Bacterial biocatalysts play a key role in our transition to a bio-based, post-petroleum economy. However, the discovery of new biocatalysts is currently limited by our ability to analyze genomic information and our capacity of functionally screening for desired activities. Here, we present a simple workflow that combines functional metaproteomics and metagenomics, which facilitates the unmediated and direct discovery of biocatalysts in environmental samples. To identify the entirety of lipolytic biocatalysts in a soil sample contaminated with used cooking oil, we detected all proteins active against a fluorogenic substrate in sample’s metaproteome using a 2D-gel zymogram. Enzymes’ primary structures were then deduced by tryptic in-gel digest and mass spectrometry of the active protein spots, searching against a metagenome database created from the same contaminated soil sample. We then expressed one of the novel biocatalysts heterologously in Escherichia coli and obtained proof of lipolytic activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-017-0247-9) contains supplementary material, which is available to authorized users.

A novel gene encoding for a lipolytic enzyme, designated ML-005, was recently identified using a functional metaproteomics approach. We heterologously expressed this protein in Escherichia coli and biochemically characterized it. ML-005 exhibited lipolytic activity toward short-chained substrates with the preferred substrate being p-nitrophenyl-butyrate, suggesting that ML-005 is an esterase. According to homology analysis and site-directed mutagenesis, the catalytic triad of the enzyme was identified as Ser-99, Asp-164, and His-191. Its optimal pH was determined to be at pH 8. Optimal activity was observed at 45°C. It also exhibited temperature, pH and salt tolerance. Residual relative activity after incubating at 50–60°C for 360 min was above 80% of its initial activity. It showed tolerance over a broad range of pH (5–12) and retained most of its initial activity. Furthermore, incubating ML-005 in 1 – 5M NaCl solution had negligible effect on its activity. DTT, EDTA, and ß-mercaptoethanol had no significant effect on ML-005’s activity. However, addition of PMSF led to almost complete inactivation consistent with ML-005 being a serine hydrolase. ML-005 remains stable in the presence of a range of metal ions, but addition of Cu2+ significantly reduces its relative activity. Organic solvents have an inhibitory effect on ML-005, but it retained 21% of activity in 10% methanol. SDS had the most pronounced inhibitory effect on ML-005 among all detergents tested and completely inactivated it. Furthermore, the Vmax of ML-005 was determined to be 59.8 μM/min along with a Km of 137.9 μM. The kcat of ML-005 is 26 s-1 and kcat/Km is 1.88 × 105 M-1 s-1.

Lipolytic enzymes are produced by animals, plants and microorganisms. With their chemo-, regio-, and enantio-specific characteristics, lipolytic enzymes are important biocatalysts useful in several industrial applications. They are widely used in the processing of fats and oils, detergents, food processing, paper and cosmetics production. In this work, we used a new functional metaproteomics approach to screen sediment samples of the Indian Bakreshwar hot spring for novel thermo- and solvent-stable lipolytic enzymes. We were able to identify an enzyme showing favorable characteristics. DS-007 showed high hydrolytic activity with substrates with shorter chain length (<C8) with the maximum activity observed against p-nitrophenyl butyrate (C4). For substrates with a chain length >C10, significantly less hydrolytic activity was observed. A preference for short chain acyl groups is characteristic for esterases, suggesting that DS-007 is an esterase. Consistent with the high temperature at its site of isolation, DS-007 showed a temperature optimum at 55°C and retained 80% activity even after prolonged exposure to temperatures as high as 60°C. The enzyme showed optimum activity at pH 9.5, with more than 50% of its optimum activity between pH 8.0 and pH 9.5. DS-007 also exhibited tolerance toward organic solvents at a concentration of 1% (v/v). One percent of methanol increased the activity of DS-007 by 40% in comparison to the optimum conditions without solvent. In the presence of 10% methanol, DMSO or isopropanol DS-007 still showed around 50% activity. This data indicates that DS-007 is a temperature- and solvent-stable thermophilic enzyme with reasonable activity even at lower temperatures as well as a catalyst that can be used at a broad range of pH values with an optimum in the alkaline range, showing the adaptation to the habitat’s temperature and alkaline pH.

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