From Data Mining of Chitinophaga sp. Genome to Enzyme Discovery of a Hyperthermophilic Metallocarboxypeptidase.

For several centuries, microorganisms and enzymes have been used for many different applications. Although many enzymes with industrial applications have already been reported, different screening technologies, methods and approaches are constantly being developed in order to allow the identification of enzymes with even more interesting applications. In our work, we have performed data mining on the Chitinophaga sp. genome, a gram-negative bacterium isolated from a bacterial consortium of sugarcane bagasse isolated from an ethanol plant. The analysis of 8 Mb allowed the identification of the chtcp gene, previously annotated as putative Cht4039. The corresponding codified enzyme, denominated as ChtCP, showed the HEXXH conserved motif of family M32 from thermostable carboxypeptidases. After expression in E. coli, the recombinant enzyme was characterized biochemically. ChtCP showed the highest activity versus benziloxicarbonil Ala-Trp at pH 7.5, suggesting a preference for hydrophobic substrates. Surprisingly, the highest activity of ChtCP observed was between 55 °C and 75 °C, and 62% activity was still displayed at 100 °C. We observed that Ca2+, Ba2+, Mn2+ and Mg2+ ions had a positive effect on the activity of ChtCP, and an increase of 30 °C in the melting temperature was observed in the presence of Co2+. These features together with the structure of ChtCP at 1.2 Å highlight the relevance of ChtCP for further biotechnological applications.

Investigating host-microbiome interactions by droplet based microfluidics.

BACKGROUND: Despite the importance of the mucosal interface between microbiota and the host in gut homeostasis, little is known about the mechanisms of bacterial gut colonization, involving foraging for glycans produced by epithelial cells. The slow pace of progress toward understanding the underlying molecular mechanisms is largely due to the lack of efficient discovery tools, especially those targeting the uncultured fraction of the microbiota. RESULTS: Here, we introduce an ultra-high-throughput metagenomic approach based on droplet microfluidics, to screen fosmid libraries. Thousands of bacterial genomes can be covered in 1 h of work, with less than ten micrograms of substrate. Applied to the screening of the mucosal microbiota for ß-N-acetylgalactosaminidase activity, this approach allowed the identification of pathways involved in the degradation of human gangliosides and milk oligosaccharides, the structural homologs of intestinal mucin glycans. These pathways, whose prevalence is associated with inflammatory bowel diseases, could be the result of horizontal gene transfers with Bacteroides species. Such pathways represent novel targets to study the microbiota-host interactions in the context of inflammatory bowel diseases, in which the integrity of the mucosal barrier is impaired. CONCLUSION: By compartmentalizing experiments inside microfluidic droplets, this method speeds up and miniaturizes by several orders of magnitude the screening process compared to conventional approaches, to capture entire metabolic pathways from metagenomic libraries. The method is compatible with all types of (meta)genomic libraries, and employs a commercially available flow cytometer instead of a custom-made sorting system to detect intracellular or extracellular enzyme activities. This versatile and generic workflow will accelerate experimental exploration campaigns in functional metagenomics and holobiomics studies, to further decipher host-microbiota relationships. Video Abstract.

Halotolerant aminopeptidase M29 from Mesorhizobium SEMIA 3007 with biotechnological potential and its impact on biofilm synthesis.

The aminopeptidase gene from Mesorhizobium SEMIA3007 was cloned and overexpressed in Escherichia coli. The enzyme called MesoAmp exhibited optimum activity at pH 8.5 and 45 °C and was strongly activated by Co2+ and Mn2+. Under these reaction conditions, the enzyme displayed Km and kcat values of 0.2364 ± 0.018 mM and 712.1 ± 88.12 s-1, respectively. Additionally, the enzyme showed remarkable stability in organic solvents and was active at high concentrations of NaCl, suggesting that the enzyme might be suitable for use in biotechnology. MesoAmp is responsible for 40% of the organism's aminopeptidase activity. However, the enzyme's absence does not affect bacterial growth in synthetic broth, although it interfered with biofilm synthesis and osmoregulation. To the best of our knowledge, this report describes the first detailed characterization of aminopeptidase from Mesorhizobium and suggests its importance in biofilm formation and osmotic stress tolerance. In summary, this work lays the foundation for potential biotechnological applications and/or the development of environmentally friendly technologies and describes the first solvent- and halo-tolerant aminopeptidases identified from the Mesorhizobium genus and its importance in bacterial metabolism.

From a metagenomic source to a high-resolution structure of a novel alkaline esterase.

Esterases catalyze the cleavage and formation of ester bonds and are members of the diverse family of α/ß hydrolase fold. They are useful in industries from different sectors, such as food, detergent, fine chemicals, and biofuel production. In a previous work, 30 positive clones for lipolytic activity were identified from a metagenomic library of a microbial consortium specialized in diesel oil degradation. In this study, a putative gene encoding an esterase/lipase, denominated est8, has been cloned and the corresponding protein expressed recombinantly, purified to homogeneity and characterized functional and structurally. We show that the protein codified by est8 gene, denominated Est8, is an alkaline esterase with high catalytic efficiency against p-nitrophenyl acetate and stable in the presence of up to 10% dimethyl sulfoxide. The three-dimensional structure of Est8 was determined at 1.85-Ǻ resolution, allowing the characterization of the substrate-binding pocket and features that rationalize the preference of Est8 for short-chain substrates. In an attempt to increase the size of ligand-binding pocket and enzyme activity against distinct substrates of long chain, we mutated two residues (Met213 and Phe217) that block the substrate channel. A small increase in the reaction velocity for p-nitrophenyl butyrate and p-nitrophenyl valerate hydrolysis was observed. Activity against p-nitrophenyl acetate was reduced. The functional and structural characterization of Est8 is explored in comparison with orthologues.

Can environmental pollution by metals change genetic diversity? Ucides cordatus (Linnaeus, 1763) as a study case in Southeastern Brazilian mangroves.

Industrial areas on estuarine systems are commonly affected by heavy metals, affecting all local biota. Random Amplified Polymorphic DNA (RAPD) was used to evaluate genetic diversity of Ucides cordatus at mangroves in southeastern Brazil (Juréia, J; São Vicente, SV; and Cubatão, C), with distinct pollution levels by metals. The genetic diversity of this species was compared with concentrations of metals (Cd, Pb, Cu, Cr and Hg) in the environment. A pollution gradient was confirmed (SV>C>J), with low levels detected in water, except for mercury in SV. All metals in the sediment samples were below Threshold Effect Level (TEL), without an apparent biological risk to the biota. Genetic distance was very similar between J and C, with SV occurring as an out-group. RAPD was a powerful tool to investigate the effect of metal pollution on genetic diversity of this mangrove crab, and to evaluate the conservation status of the mangrove ecosystem.

Bg10: A Novel Metagenomics Alcohol-Tolerant and Glucose-Stimulated GH1 ß-Glucosidase Suitable for Lactose-Free Milk Preparation.

New ß-glucosidases with product (glucose) or ethanol tolerances are greatly desired to make industrial processes more marketable and efficient. Therefore, this report describes the in silico/vitro characterization of Bg10, a metagenomically derived homodimeric ß-glucosidase that exhibited a Vmax of 10.81 ± 0.43 µM min-1, Kcat of 175.1± 6.91 min-1, and Km of 0.49 ± 0.12 mM at a neutral pH and 37°C when pNP-ß-D-glucopyranoside was used as the substrate, and the enzyme retained greater than 80% activity within the respective pH and temperature ranges of 6.5 to 8.0 and 35 to 40°C. The enzyme was stimulated by its product, glucose; consequently, the Bg10 activity against 50 and 100 mM of glucose were increased by 36.8% and 22%, respectively, while half of the activity was retained at 350 mM. Moreover, the Bg10 was able to hydrolyse 55% (milk sample) and 100% (purified sugar) of the lactose at low (6°C) and optimum (37°C) temperatures, respectively, suggesting the possibility of further optimization of the reaction for lactose-free dairy production. In addition, the enzyme was able to fully hydrolyse 40 mM of cellobiose at one hour and was tolerant to ethanol up to concentrations of 500 mM (86% of activity), while a 1 M concentration still resulted in 41% residual activity, which could be interesting for biofuel production.

Characterization of EST3: a metagenome-derived esterase with suitable properties for biotechnological applications.

Metagenomic libraries from diverse environments have been extensive sources of many lipases and esterases; nevertheless, most of these enzymes remain biochemically uncharacterized. We previously built a metagenomic fosmid library from a microbial consortium specialized for diesel oil degradation and tested it for lipolytic activity. In the present study, we identified the PL14.H10 clone that was subcloned and sequenced, which enabled the identification of the EST3 protein. This enzyme exhibited 74 % amino acid identity with the uncharacterized alpha/beta hydrolase from Parvibaculum lavamentivorans [GenBank: WP012110575.1] and was classified into lipolytic enzyme family IV. Biochemical characterization revealed that EST3 presents high activity in a wide range of temperature with highest activity from 41 to 45 °C. Also, this thermostable esterase acts from mild acidic to alkaline conditions with an optimum pH of 6.0. The enzyme exhibited activity against p-nitrophenyl esters of different chain lengths and highest catalytic efficiency against p-nitrophenyl caprylate. The activity of the protein was increased in the presence of 0.5 mM of Mn(+2), Li(+), EDTA, and 1 % of CTAB and exhibited half of the activity in the presence of 10 % methanol and ethanol. Moreover, the homology model of EST3 was built and compared to other esterases, revealing a substrate channel that should fit a wide range of substrates. Taken together, the data presented in this work reveal the unique and interesting characteristics of EST3 that might be explored for further use in biotechnological applications.

Est16, a New Esterase Isolated from a Metagenomic Library of a Microbial Consortium Specializing in Diesel Oil Degradation.

Lipolytic enzymes have attracted attention from a global market because they show enormous biotechnological potential for applications such as detergent production, leather processing, cosmetics production, and use in perfumes and biodiesel. Due to the intense demand for biocatalysts, a metagenomic approach provides methods of identifying new enzymes. In this study, an esterase designated as Est16 was selected from 4224 clones of a fosmid metagenomic library, revealing an 87% amino acid identity with an esterase/lipase (accession number ADM63076.1) from an uncultured bacterium. Phylogenetic studies showed that the enzyme belongs to family V of bacterial lipolytic enzymes and has sequence and structural similarities with an aryl-esterase from Pseudomonas fluorescens and a patented Anti-Kazlauskas lipase (patent number US20050153404). The protein was expressed and purified as a highly soluble, thermally stable enzyme that showed a preference for basic pH. Est16 exhibited activity toward a wide range of substrates and the highest catalytic efficiency against p-nitrophenyl butyrate and p-nitrophenyl valerate. Est16 also showed tolerance to the presence of organic solvents, detergents and metals. Based on molecular modeling, we showed that the large alpha-beta domain is conserved in the patented enzymes but not the substrate pocket. Here, it was demonstrated that a metagenomic approach is suitable for discovering the lipolytic enzyme diversity and that Est16 has the biotechnological potential for use in industrial processes.

Investigation, expression, and molecular modeling of ORF2, a metagenomic lipolytic enzyme.

One clone exhibiting lipolytic activity was selected among 30 positives from a metagenomic library of a microbe consortium specialized in petroleum hydrocarbon degradation. From this clone, a sublibrary was constructed and a metagenome contig was assembled and analyzed using the ORF Finder; thus, it was possible to identify a potential ORF that encodes a lipolytic enzyme, denoted ORF2. This ORF is composed of 1035-bp 345 amino acids and displayed 98 % identity with an alpha/beta hydrolase from Pseudomonas nitroreducens (accession number WP024765380.1). When analyzed against a metagenome database, ORF2 also showed 76 % of sequence identity with a hypothetical protein from a marine metagenome (accession number ECT55726.1). The ProtParam analyses indicated that the recombinant protein ORF2 has a molecular mass approximately 39 kDa, as expected from its amino acid sequence, and based on phylogenetic analysis and molecular modeling, it was possible to suggest that ORF2 is a new member from family V. This enzyme exhibits the catalytic triad and conserved motifs typical from this family, wherein the serine residue is located in the central position of the conserved motif GASMGG. The orf2 gene was cloned in the expression vector pET28a, and the recombinant protein was superexpressed in Escherichia coli BL21(DE3) cells. The lipolytic activity of protein bands presented in a SDS-PAGE gel was confirmed by zymogram analyses, indicating ORF2 activity. These discoveries raise the possibility of employing this protein in biotechnological applications, such as bioremediation.