Metagenomic Screening for Lipolytic Genes Reveals an Ecology-Clustered Distribution Pattern.

Lipolytic enzymes are one of the most important enzyme types for application in various industrial processes. Despite the continuously increasing demand, only a small portion of the so far encountered lipolytic enzymes exhibit adequate stability and activities for biotechnological applications. To explore novel and/or extremophilic lipolytic enzymes, microbial consortia in two composts at thermophilic stage were analyzed using function-driven and sequence-based metagenomic approaches. Analysis of community composition by amplicon-based 16S rRNA genes and transcripts, and direct metagenome sequencing revealed that the communities of the compost samples were dominated by members of the phyla Actinobacteria, Proteobacteria, Firmicutes, Bacteroidetes, and Chloroflexi. Function-driven screening of the metagenomic libraries constructed from the two samples yielded 115 unique lipolytic enzymes. The family assignment of these enzymes was conducted by analyzing the phylogenetic relationship and generation of a protein sequence similarity network according to an integrated classification system. The sequence-based screening was performed by using a newly developed database, containing a set of profile Hidden Markov models, highly sensitive and specific for detection of lipolytic enzymes. By comparing the lipolytic enzymes identified through both approaches, we demonstrated that the activity-directed complements sequence-based detection, and vice versa. The sequence-based comparative analysis of lipolytic genes regarding diversity, function and taxonomic origin derived from 175 metagenomes indicated significant differences between habitats. Analysis of the prevalent and distinct microbial groups providing the lipolytic genes revealed characteristic patterns and groups driven by ecological factors. The here presented data suggests that the diversity and distribution of lipolytic genes in metagenomes of various habitats are largely constrained by ecological factors.

A Novel Carboxylesterase Derived from a Compost Metagenome Exhibiting High Stability and Activity towards High Salinity.

Halotolerant lipolytic enzymes have gained growing interest, due to potential applications under harsh conditions, such as hypersalinity and presence of organic solvents. In this study, a lipolytic gene, est56, encoding 287 amino acids was identified by functional screening of a compost metagenome. Subsequently, the gene was heterologously expressed, and the recombinant protein (Est56) was purified and characterized. Est56 is a mesophilic (Topt 50 °C) and moderate alkaliphilic (pHopt 8) enzyme, showing high thermostability at 30 and 40 °C. Strikingly, Est56 is halotolerant as it exhibited high activity and stability in the presence of up to 4 M NaCl or KCl. Est56 also displayed enhanced stability against high temperatures (50 and 60 °C) and urea (2, 4, and 6 M) in the presence of NaCl. In addition, the recently reported halotolerant lipolytic enzymes were summarized. Phylogenetic analysis grouped these enzymes into 13 lipolytic protein families. The majority (45%) including Est56 belonged to family IV. To explore the haloadaptation of halotolerant enzymes, the amino acid composition between halotolerant and halophilic enzymes was statistically compared. The most distinctive feature of halophilic from non-halophilic enzymes are the higher content of acidic residues (Asp and Glu), and a lower content of lysine, aliphatic hydrophobic (Leu, Met and Ile) and polar (Asn) residues. The amino acid composition and 3-D structure analysis suggested that the high content of acidic residues (Asp and Glu, 12.2%) and low content of lysine residues (0.7%), as well as the excess of surface-exposed acidic residues might be responsible for the haloadaptation of Est56.

Biochemical profiles of two thermostable and organic solvent-tolerant esterases derived from a compost metagenome.

Owing to the functional versatility and potential applications in industry, interest in lipolytic enzymes tolerant to organic solvents is increasing. In this study, functional screening of a compost soil metagenome resulted in identification of two lipolytic genes, est1 and est2, encoding 270 and 389 amino acids, respectively. The two genes were heterologously expressed and characterized. Est1 and Est2 are thermostable enzymes with optimal enzyme activities at 80 and 70 °C, respectively. A second-order rotatable design, which allows establishing the relationship between multiple variables with the obtained responses, was used to explore the combined effects of temperature and pH on esterase stability. The response curve indicated that Est1, and particularly Est2, retained high stability within a broad range of temperature and pH values. Furthermore, the effects of organic solvents on Est1 and Est2 activities and stabilities were assessed. Notably, Est2 activity was significantly enhanced (two- to tenfold) in the presence of ethanol, methanol, isopropanol, and 1-propanol over a concentration range between 6 and 30% (v/v). For the short-term stability (2 h of incubation), Est2 exhibited high tolerance against 60% (v/v) of ethanol, methanol, isopropanol, DMSO, and acetone, while Est1 activity resisted these solvents only at lower concentrations (below 30%, v/v). Est2 also displayed high stability towards some water-immiscible organic solvents, such as ethyl acetate, diethyl ether, and toluene. With respect to long-term stability, Est2 retained most of its activity after 26 days of incubation in the presence of 30% (v/v) ethanol, methanol, isopropanol, DMSO, or acetone. All of these features indicate that Est1 and Est2 possess application potential.

A novel, versatile family IV carboxylesterase exhibits high stability and activity in a broad pH spectrum.

OBJECTIVES: To investigate the properties of a novel metagenome-derived member of the hormone-sensitive lipase family of lipolytic enzymes. RESULTS: A forest soil metagenome-derived gene encoding an esterase (Est06) belonging to the hormone-sensitive lipase family of lipolytic enzymes was subcloned, heterologously expressed and characterized. Est06 is a polypeptide of 295 amino acids with a molecular mass of 31 kDa. The deduced protein sequence shares 61% similarity with a hypothetical protein from the marine symbiont Candidatus Entotheonella sp. TSY1. Purified Est06 exhibited high affinity for acyl esters with short-chain fatty acids, and showed optimum activity with p-nitrophenyl valerate (C5). Maximum enzymatic activity was at 50 °C and pH 7. Est06 exhibited high stability at moderate temperatures by retaining all of its catalytic activity below 30 °C over 13 days. Additionally, Est06 displayed high stability between pH 5 and 9. Esterase activity was not inhibited by metal ions or detergents, although organic solvents decreased activity. CONCLUSIONS: The combination of Est06 properties place it among novel biocatalysts that have potential for industrial use including low temperature applications.