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

From Data Mining of <i>Chitinophaga</i> 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

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

<div><p>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 <i>in silico</i>/<i>vitro</i> characterization of Bg10, a metagenomically derived homodimeric ß-glucosidase that exhibited a V_max of 10.81 ± 0.43 μM min^-1, K_cat of 175.1± 6.91 min^-1, and K_<i>m</i> of 0.49 ± 0.12 mM at a neutral pH and 37°C when <i>p</i>NP-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.</p></div

“Group I view”: Phylogenetic relationship between Bg10 and other previously characterized GH1 β-glucosidases.

<p>The tree was constructed using the Bayesian model with algorithmic tests to determine the better amino acid substitution matrix (Phangorn at “R” software) and phylogenetic model (Mr Bayes software) using two billion generations (Ngen) to find the better method. The scale bar indicates the number of amino acid substitutions per site. In this view, the focus is on the related group I. The sequence for Bg10 fell in group II. Observation: for a complete tree view, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167932#pone.0167932.s001" target="_blank">S1 Fig</a>; for visualization of other groups, see also Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167932#pone.0167932.g002" target="_blank">2</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167932#pone.0167932.g004" target="_blank">4</a>. Colour code: Firmicutes, dark green; Proteobacteria, pink; Thermotogales, orange; Dictyglomales, light blue; Petrotogales, brow; unculturable, purple. Numbers in white specify the amount of sequences in each collapsed branch.</p

Effects of pH, temperature and different substrates on Bg10 activity.

<p><b>A:</b> Effect of pH on β-glucosidase activity. The reactions were performed at 37°C and at pH values of 3.5 to 10.5 using 1 mM of the <i>p</i>NP-ß-D-glucopyranoside as the substrate. The reagents labelled citrate and phosphate are the sodium salts. <b>B:</b> Effect of temperature on the activity. Enzyme activity was assayed at various temperatures from 4 to 50°C in 20 mM sodium phosphate buffer (pH 7.0). <b>C:</b> Stability of β-glucosidase at the different temperatures from 10°C (control) to 50°C in 20 mM sodium phosphate buffer (pH 7.0) using 1 mM of the <i>p</i>NP-ß-D-glucopyranoside as the substrate. <b>D:</b> Enzymatic assay for Bg10 for different substrates. Enzyme activity was assayed at 37°C in 20 mM sodium phosphate buffer (pH 7.0) using 2 mM of the respective substrates: <i>p</i>NP-ß-D-glucopyranoside (<i>p</i>NP-B-Gl, which was used as the standard substrate for the activity assay), <i>p</i>NP-ß-D-fucopyranoside (<i>p</i>NP-B-F), <i>p</i>NP-ß-D-cellobioside (pNP-B-C), <i>p</i>NP-ß-D-galactopyranoside (<i>p</i>NP-B-Ga) and <i>p</i>NP-α-D-glucopyranoside (<i>p</i>NP-A-Gl). The relative activity levels or V₀ values represent the averages of the means ± SD of triplicate reactions. Small letters in the graphics indicate the significant difference between each condition performed in the experiment, according to ANOVA and Tukey’s test at 5% probability.</p

Effects of various salts on enzyme activity.

<p>Effects of various salts on enzyme activity.</p

Extraction, purification and hydrolytic activity of recombinant Bg10 ß-glucosidase from <i>Escherichia coli</i> BL21(DE3) host cells carrying the pET28a-bg10 vector.

<p><b>A:</b> Electrophoretic profile of Ni-NTA affinity chromatography purification fractions of Bg10 under denatured conditions in an SDS-PAGE gel (10% polyacrylamide). M: molecular weight standards (Thermo Scientific). Lane 1: soluble extract of the induced cells. Lane 2: flow-through fraction from affinity chromatography. Lanes 3 to 5: eluted fractions with 20, 500 and 100 mM imidazole, respectively. <b>B</b>. Zymogram of recombinant Bg10 under non-denaturing conditions. P1: Protein used as molecular weight standard, ‘Bovine Serum Albumin’ (BSA, Sigma, St. Louis, MO, USA). Lane 1: purified protein. Lane 2: ß-1-4-glycosidic activity of purified Bg10 band using <i>p</i>NP-ß-D-glucopyranoside as substrate (Sigma, St. Louis, MO, USA). <b>C:</b> Chromatographic profile of Bg10 by gel filtration using a Superdex 16.600.200 column (GE healthcare) at a flow rate of 0.5 mL/min. The internal image corresponds to the electrophoretic migration profile in an SDS-PAGE gel (10% polyacrylamide) for the analysed samples. M: molecular weight standards (Bio-Rad, Hercules, CA, USA). S: Sample of Bg10 before the gel filtration chromatography assay. Lanes 1 to 7: eluted fractions from gel filtration for the enzyme Bg10. <b>D:</b> Estimation of Bg10 molecular size by gel filtration based on the linear correlation of the relative migration patterns of Bg10 and protein standards versus their log molecular size values. P2-P5: Proteins used as molecular weight standards, corresponding to thyroglobulin bovine, ƴ-globulin, albumin, ribonuclease A and P-aminobenzoic acid, respectively (Protein Standard Mix 15–600 kDa, Sigma, St. Louis, MO, USA).</p

Sequence and structural features of metagenomic ß-glucosidase (Bg10).

<p><b>A:</b> Sequence homology-based prediction of protein folding for the Bg10 homodimeric structure using the tool ‘Automated Mode Modelling’ from the Swiss Model Web-server, which was developed on the basis of a similar template, 1GNX (Bgl3 β-glucosidase) and represents the protein surface, showing one enzyme pocket entrance (circle). The two entrances from each protein chain cannot be visualized together because they are asymmetrically arranged, opening in opposite directions. <b>B:</b> The Bg10 predicted structure is represented by a cartoon view, where each protein chain (ca and cb) exhibits a TIM barrel (α/β) 8 super-secondary organizational motif, which is canonical for GH1 enzymes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167932#pone.0167932.ref039" target="_blank">39</a>]. The chain b pocket entrance is indicated by the red sphere. <b>C:</b> Details of the functional pocket, showing the active site and residues from the -1 to +3 subsites, which were predicted on the basis of amino acid alignment with the experimentally characterized <i>Streptomyces sp</i>. Bgl3 β-glucosidase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167932#pone.0167932.ref034" target="_blank">34</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167932#pone.0167932.ref038" target="_blank">38</a>].</p

“Group V and VI view”: Phylogenetic relationship between Bg10 and other previously characterized GH1 β-glucosidases.

<p>The tree was constructed using the Bayesian model with algorithmic tests to determine the better amino acid substitution matrix (Phangorn at “R” software) and phylogenetic model (Mr Bayes software) using two billion generations (Ngen) to find the better method. The scale bar indicates the number of amino acid substitutions per site. In this view, the focus is on the related groups V and VI; for the complete tree see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167932#pone.0167932.s001" target="_blank">S1 Fig</a> and Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167932#pone.0167932.g001" target="_blank">1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167932#pone.0167932.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167932#pone.0167932.g004" target="_blank">4</a>. The sequence for Bg10 fell in group II. Colour code: Firmicutes, dark green; Actinobacteria, red; Proteobacteria, pink; Thermotogales, orange; Dictyglomales, light blue; unculturable, purple. Numbers in white specify the amount of sequences in each collapsed branch.</p