Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail

Background A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product. Results In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45–55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (p NPbetaG) and p NP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g-1 dry biomass, using p NPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg-1 dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g-1 dry biomass in the basis of p NPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions) after 96–120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2) and 2–38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays. Conclusions The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases

The great screen anomaly—a new frontier in product discovery through functional metagenomics

Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product

Vinyl sulfone-activated silica for efficient covalent immobilization of alkaline unstable enzymes: Application to levansucrase for fructooligosaccharide synthesis

[EN] Most methodologies for covalent immobilization of enzymes usually take place at high pH values to enhance the nucleophilicity of protein reactive residues; however, many enzymes inactivate during the immobilization process due to their intrinsic instability at alkaline pH values. Vinyl sulfone (VS)-activated carriers may react with several protein side-chains at neutral pHs. In this work, levansucrase-an alkaline unstable enzyme of technological interest because it forms fructooligosaccharides (FOS) and levan from sucrose-was covalently attached to VS-activated silica at pH 7.0 in a short time (5 h). Theoretical immobilization yields were close to 95% but the apparent activity did not surpass 25%, probably due to random attachment with unproductive orientations and rigidification of the enzyme structure. Due to diffusional hindrance and/or local microenvironmental effects caused by the silica surface, the immobilized levansucrase was unable to produce levan but synthesized a similar amount of FOS than the free enzyme [95 g L in 28 h, with a major contribution of FOS of the β(2 → 1) type]. The VS-activated biocatalysts showed a notable operational stability in batch reactors.Spanish Ministry of Economy and Competitiveness (BIO2013-48779-C4-1-R). We thank the support of COST-Action CM1303 on Systems Biocatalysts. P. S.-M. thanks the Spanish Ministry of Education for FPU Grant (FPU13/01185)

Tailored Enzymatic Synthesis of Chitooligosaccharides with Different Deacetylation Degrees and Their Anti-Inflammatory Activity

By controlled hydrolysis of chitosan or chitin with different enzymes, three types of chitooligosaccharides (COS) with MW between 0.2 and 1.2 kDa were obtained: fully deacetylated (fdCOS), partially acetylated (paCOS), and fully acetylated (faCOS). The chemical composition of the samples was analyzed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and MALDI-TOF mass spectrometry. The synthesized fdCOS was basically formed by GlcN, (GlcN)2, (GlcN)3, and (GlcN)4. On the contrary, faCOS contained mostly GlcNAc, (GlcNAc)2 and (GlcNAc)3, while paCOS corresponded to a mixture of at least 11 oligosaccharides with different proportions of GlcNAc and GlcN. The anti-inflammatory activity of the three COS mixtures was studied by measuring their ability to reduce the level of TNF-α (tumor necrosis factor) in murine macrophages (RAW 264.7) after stimulation with a mixture of lipopolysaccharides (LPS). Only fdCOS and faCOS were able to significantly reduce the production of tumor necrosis factor (TNF)-α at 6 h after stimulation with lipopolysaccharides.Fundación Ramón Areces (XIX Convocatoria de Ayudas a la Investigación en Vida y Ciencias de los Materiales) y el Ministerio de Economía y Competitividad de España (Becas BIO2016-76601-C3-1-R y BIO2016-76601-C3-3-R).El programa Horizonte 2020 de la Unión Europea también financió este trabajo (Blue Growth: Unlocking the potencial of Seas and Oceans; acuerdo de subvención No. 634486; INMARE).EU COST-Action CM1303.3.520 JCR (2019) Q2, 65/159 Chemistry, Physical0.722 SJR (2019) Q2, 51/174 Physical and Theoretical ChemistryNo data IDR 2019UE

Detailed analysis of galactooligosaccharides synthesis with β-galactosidase from Aspergillus oryzae

The synthesis of galactooligosaccharides (GOS) catalyzed by β-galactosidase from Aspergillus oryzae (Enzeco) was studied. Using 400 g/L of lactose and 15 U/mL, maximum GOS yield, measured by HPAEC-PAD, was 26.8% w/w of total carbohydrates, obtained at approximately 70% lactose conversion. No less than 17 carbohydrates were identified; the major transgalactosylation product was 6′-O-β-galactosyl-lactose, representing nearly one-third (in weight) of total GOS. In contrast with previous reports, the presence of at least five disaccharides was detected, which accounted for 40% of the total GOS at the point of maximum GOS concentration (allolactose and 6-galactobiose were the major products). A. oryzae β-galactosidase showed a preference to form β(1→6) bonds, followed by β(1→3) and β(1→4) linkages. Results were compared with those obtained with β-galactosidases from Kluyveromyces lactis and Bacillus circulans. The highest GOS yield and specific productivity were achieved with B. circulans β-galactosidase. The specificity of the linkages formed and distribution of di-, tri-, and higher GOS varied significantly among the three β-galactosidases. © 2013 American Chemical Society.Peer Reviewe

Ants impact the energy reserves of natural enemies through the shared honeydew exploitation

1. Ants, as well as many species of parasitoids and predators, rely on sugar-rich foods such as honeydew to fulfill their energetic needs. Thus, ants and natural enemies may interact through the shared honeydew exploitation. 2. Ant-exclusion experiments were performed in a citrus orchard to test the hypothesis that ants may impact the energy reserves of predators and parasitoids through the competition for honeydew. Through the use of high-performance liquid chromatography (HPLC) the level of ant activity with the energy reserves and feeding history of individual specimens collected in the field during representative days of spring, summer, and autumn were related. 3. Out of 145 Aphytis chrysomphali Mercet parasitoids captured in the field, 65% were classified as sugar-fed and 24.7% as honeydew-fed. In summer, when ant activity peaked, there was a significant negative correlation between the level of ant activity and the total sugar content and honeydew feeding incidence by A. chrysomphali. Out of 47 individuals of the predator Chrysoperla carnea sensu lato (Stephens), captured in the field, 55.3% were classified as sugar-fed. We found a significant negative effect of the level of ant activity on the sugar-feeding incidence by C. carnea in spring. 4. The present study provides evidence that ants can interfere with the energy reserves of natural enemies. This interaction may be widespread in various ecosystems with important consequences for the arthropod community composition and with practical implications for biological control given that absence of sugar feeding is detrimental for the fitness of many species of predators and parasitoids.This work was supported by the project (RTA2010‐00012‐C02‐02) assigned to F. G. M from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Spain and the project (BIO2013‐48779‐C4‐1‐R) from Spanish Ministry of Science and Innovation and COST action CM1303 on Systems Biocatalysis.Peer Reviewe

Sweet-and-salty biocatalysis: Fructooligosaccharides production using Cladosporium cladosporioides in seawater

Production of fructooligosaccharides (FOS) from sucrose was obtained using a bioprocess entirely performed in seawater. The halophilic fungus Cladosporium cladosporioides MUT 5506 was grown in a seawater-based medium and mycelium displayed an optimal activity in seawater at 50–60 °C, being stable up to 60 °C. Under optimized conditions in seawater (50 °C, sucrose 600 g/L, lyophilized mycelium 40 g/L), C. cladosporioides gave a maximum FOS yield of 344 g/L after 72 h with a preponderance of 1F-FOS derivatives (1-kestose 184 g/L, 1-nystose 98 g/L and 1-fructofuranosylnystose 22 g/L), and the noteworthy presence of the non-conventional disaccharide blastose (30 g/L after 144 h). Lyophilized mycelium exhibited good stability in seawater (76% of the initial activity was retained after 15 cycles of reutilization). This proof-of-concept application reports for the first time the production of FOS in a non-conventional medium such as seawater.This work was supported by the European Community FP7-KBBE.2012.3.2-02 project MACUMBA, grant agreement no. 311975.Peer Reviewe