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Abstract Background The use of lignocellulosic materials for second generation ethanol production would give several advantages such as minimizing the conflict between land use for food and fuel production, providing less expensive raw materials than conventional agricultural feedstock, allowing lower greenhouse gas emissions than those of first generation ethanol. However, cellulosic biofuels are not produced at a competitive level yet, mainly because of the high production costs of the cellulolytic enzymes. Therefore, this study was aimed at discovering new cellulolytic microorganisms and enzymes. Results Different bacteria isolated from raw composting materials obtained from vegetable processing industry wastes were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. Four strains belonging to the actinomycetes group were selected on the basis of their phenotypic traits and cellulolytic activity on solid medium containing carboxymethylcellulose. The strain showing the highest cellulolytic activity was identified by 16S rRNA sequencing as belonging to Streptomyces genus and it was designated as Streptomyces sp. strain G12. Investigating the enzymes responsible for cellulase activity produced by Streptomyces G12 by proteomic analyses, two endoglucanases were identified. Gene coding for one of these enzymes, named CelStrep, was cloned and sequenced. Molecular analysis showed that the celstrep gene has an open reading frame encoding a protein of 379 amino acid residues, including a signal peptide of 37 amino acid residues. Comparison of deduced aminoacidic sequence to the other cellulases indicated that the enzyme CelStrep can be classified as a family 12 glycoside hydrolase. Heterologous recombinant expression of CelStrep was carried out in Escherichia coli, and the active recombinant enzyme was purified from culture supernatant and characterized. It catalyzes the hydrolysis of carboxymethylcellulose following a Michaelis–Menten kinetics with a KM of 9.13 mg/ml and a vmax of 3469 μM min-1. The enzyme exhibits a half life of around 24 h and 96 h at 60°C and 50°C, respectively and shows a retention of around 80% of activity after 96 h at 40°C. Conclusions In this manuscript, we describe the isolation of a new cellulolytic strain, Streptomyces sp. G12, from industrial waste based compost, the identification of the enzymes putatively responsible for its cellulolytic activity, the cloning and the recombinant expression of the gene coding for the Streptomyces sp. G12 cellulase CelStrep, that was characterized showing to exhibit a relevant thermoresistance increasing its potential for cellulose conversion.

With the aim to reach the maximum recovery of bulk and specialty bioproducts while minimizing waste generation, a multi-product biorefinery for ethanol and lactic acid production from the biomass of cyanobacterium Arthrospira platensis was investigated. Therefore, the residual biomass resulting from different pretreatments consisting of supercritical fluid extraction (SF) and microwave assisted extraction with non-polar (MN) and polar solvents (MP), previously applied on A. platensis to extract bioactive metabolites, was further valorized. In particular, it was used as a substrate for fermentation with Saccharomyces cerevisiae LPB-287 and Lactobacillus acidophilus ATCC 43121 to produce bioethanol (BE) and lactic acid (LA), respectively. The maximum concentrations achieved were 3.02 ± 0.07 g/L of BE by the MN process at 120 rpm 30 °C, and 9.67 ± 0.05 g/L of LA by the SF process at 120 rpm 37 °C. An economic analysis of BE and LA production was carried out to elucidate the impact of fermentation scale, fermenter costs, production titer, fermentation time and cyanobacterial biomass production cost. The results indicated that the critical variables are fermenter scale, equipment cost, and product titer; time process was analyzed but was not critical. As scale increased, costs tended to stabilize, but also more product was generated, which causes production costs per unit of product to sharply decrease. The median value of production cost was US$ 1.27 and US$ 0.39, for BE and LA, respectively, supporting the concept of cyanobacterium biomass being used for fermentation and subsequent extraction to obtain ethanol and lactic acid as end products from A. platensis., (© 2021. The Author(s).)

The protein glycosylation is a post-translational modification of crucial importance for its involvement in molecular recognition, protein trafficking, regulation, and inflammation. Indeed, abnormalities in protein glycosylation are correlated with several disease states such as cancer, inflammatory diseases, and congenial disorders. The understanding of cellular mechanisms through the elucidation of glycan composition encourages researchers to find analytical solutions for their detection. Actually, the multiplicity and diversity of glycan structures bond to the proteins, the variations in polarity of the individual saccharide residues, and the poor ionization efficiencies make their detection much trickier than other kinds of biopolymers. An overview of the most prominent techniques based on mass spectrometry (MS) for protein glycosylation (glycoproteomics) studies is here presented. The tricks and pre-treatments of samples are discussed as a crucial step prodromal to the MS analysis to improve the glycan ionization efficiency. Therefore, the different instrumental MS mode is also explored for the qualitative and quantitative analysis of glycopeptides and the glycans structural composition, thus contributing to the elucidation of biological mechanisms., Competing Interests: The authors declare no conflict of interest.

The need to develop competitive and eco-friendly processes in the cosmetic industry leads to the search for new enzymes with improved properties for industrial bioconversions in this sector. In the present study, a complete methodology to generate, express and screen diversity for the type C feruloyl esterase from Fusarium oxysporium FoFaeC was set up in a high-throughput fashion. A library of around 30,000 random mutants of FoFaeC was generated by error prone PCR of fofaec cDNA and expressed in Yarrowia lipolytica. Screening for enzymatic activity towards the substrates 5-bromo-4-chloroindol-3-yl and 4-nitrocatechol-1-yl ferulates allowed the selection of 96 enzyme variants endowed with improved enzymatic activity that were then characterized for thermo- and solvent- tolerance. The five best mutants in terms of higher activity, thermo- and solvent- tolerance were selected for analysis of substrate specificity. Variant L432I was shown to be able to hydrolyze all the tested substrates, except methyl sinapate, with higher activity than wild type FoFaeC towards methyl p-coumarate, methyl ferulate and methyl caffeate. Moreover, the E455D variant was found to maintain completely its hydrolytic activity after two hour incubation at 55 °C, whereas the L284Q/V405I variant showed both higher thermo- and solvent- tolerance than wild type FoFaeC. Small molecule docking simulations were applied to the five novel selected variants in order to examine the binding pattern of substrates used for enzyme characterization of wild type FoFaeC and the evolved variants., (Copyright © 2019. Published by Elsevier B.V.)

Background: Due to its wide range of applications in the food, pharmaceutical and chemical fields, microbial synthesis of succinic acid is receiving growing attention, generating already relevant industrial results, as well as fueling constant research for improvements. In order to develop a sustainable process, a special focus is now set on the exploitation and conversion of lignocellulosic biomasses into platform chemicals., Results: In the present work we used Basfia succiniciproducens BPP7 in separated hydrolysis and fermentation experiments with Arundo donax as starting material. Fed-batch strategies showed a maximal production of about 37 g/L of succinic acid after 43 h of growth and a productivity of 0.9 g/L h on the pilot scale. Global mass balance calculations demonstrated a hydrolysis and fermentation efficiency of about 75%. Moreover, the application of a material flow analysis showed the obtainment of 88.5 and 52 % of succinic acid, per kg of virgin biomass and on the total generated output, respectively., Conclusions: The use of fed-batch strategies for the growth of B. succiniciproducens on A. donax improved the titer and productivity of succinic acid on pre-pilot scale. Process evaluation through material flow analysis showed successful results and predicted a yield of succinic acid of about 30% in a fed-batch process that uses A. donax as only carbon source also in the feed. Preliminary considerations on the possibility to achieve an energetic valorization of the residual solid coming from the fermentation process were also carried out.

Organic and inorganic xenobiotic compounds can affect the potential ecological function of the soil, altering its biodiversity. Therefore, the response of microbial communities to environmental pollution is a critical issue in soil ecology. Here, a high-throughput sequencing approach was used to investigate the indigenous bacterial and fungal community structure as well as the impact of pollutants on their diversity and richness in contaminated and noncontaminated soils of a National Interest Priority Site of Campania Region (Italy) called "Terra dei Fuochi". The microbial populations shifted in the polluted soils via their mechanism of adaptation to contamination, establishing a new balance among prokaryotic and eukaryotic populations. Statistical analyses showed that the indigenous microbial communities were most strongly affected by contamination rather than by site of origin. Overabundant taxa and Actinobacteria were identified as sensitive biomarkers for assessing soil pollution and could provide general information on the health of the environment. This study has important implications for microbial ecology in contaminated environments, increasing our knowledge of the capacity of natural ecosystems to develop microbiota adapted to polluted soil in sites with high agricultural potential and providing a possible approach for modeling pollution indicators for bioremediation purposes.

The enzymatic extracellular mixtures of two new microorganisms - Streptomyces flavogriseus AE64X and AE63X - isolated from Eucalyptus camaldulensis and Populus nigra and producing cellulase and xylanase, were characterized and applied to hydrolysis of pretreated Arundo donax, Populus nigra and Panicum virgatum (10% w/v) replacing the commercial enzymes Accelerase 1500 and Accelerase XY (5.4 and 145 U/g of pretreated biomass, respectively). It is worth of noting that the newly developed extracellular enzymatic mixtures, without any purification step and at the same dosage, presented saccharification yields that are higher (86% for S. flavogriseus AE64X) than those of commercial enzymes (81%). Moreover, these enzymatic mixes allowed us to hydrolyse both cellulose and xylan within the different lignocellulose biomasses substituting both the cellulase and xylanase of commercial source. The produced sugars were also fermentable by Basfia succiniciproducens BPP7 into succinic acid with high yield., (Copyright © 2018. Published by Elsevier Ltd.)

The chemical syntheses currently employed for industrial purposes, including in the manufacture of cosmetics, present limitations such as unwanted side reactions and the need for harsh chemical reaction conditions. In order to overcome these drawbacks, novel enzymes are developed to catalyze the targeted bioconversions. In the present study, a methodology for the construction and the automated screening of evolved variants library of a Type B feruloyl esterase from Myceliophthora thermophila (MtFae1a) was developed and applied to generation of 30,000 mutants and their screening for selecting the variants with higher activity than the wild-type enzyme. The library was generated by error-prone PCR of mtfae1a cDNA and expressed in Saccharomyces cerevisiae. Screening for extracellular enzymatic activity towards 4-nitrocatechol-1-yl ferulate, a new substrate developed ad hoc for high-throughput assays of feruloyl esterases, led to the selection of 30 improved enzyme variants. The best four variants and the wild-type MtFae1a were investigated in docking experiments with hydroxycinnamic acid esters using a model of 3D structure of MtFae1a. These variants were also used as biocatalysts in transesterification reactions leading to different target products in detergentless microemulsions and showed enhanced synthetic activities, although the screening strategy had been based on improved hydrolytic activity.

The type C feruloyl esterase FoFaeC from Fusarium oxysporum is a newly discovered enzyme with high potential for use in the hydrolysis of lignocellulosic biomass but it shows low activity towards sinapates. In this work, small molecule docking simulations were employed in order to identify important residues for the binding of the four model methyl esters of hydroxycinnamic acids, methyl ferulate/caffeate/sinapate/p-coumarate, to the predicted structure of FoFaeC. Subsequently rational redesign was applied to the enzyme' active site in order to improve its specificity towards methyl sinapate. A double mutation (F230H/T202V) was considered to provide hydrophobic environment for stabilization of the methoxy substitution on sinapate and a larger binding pocket. Five mutant clones and the wild type were produced in Pichia pastoris and biochemically characterized. All clones showed improved activity, substrate affinity, catalytic efficiency and turnover rate compared to the wild type against methyl sinapate, with clone P13 showing a 5-fold improvement in catalytic efficiency. Although the affinity of all mutant clones was improved against the four model substrates, the catalytic efficiency and turnover rate decreased for the substrates containing a hydroxyl substitution., Competing Interests: The authors have declared that no competing interests exist.

With the aim to develop biocatalysts for enhanced hydrolysis of (hemi)cellulose into monosaccharides, random diversity by directed evolution was introduced in the gene coding for the endo-β-1,4-glucanase from Streptomyces sp. G12 which had been recombinantly expressed in Escherichia coli and named rCelStrep. The main objectives were therefore to set up a complete strategy for creation and automated screening of rCelStrep evolved direct mutants and to apply it to generate and screen a library of 10,000 random mutants to select the most active variants. The diversity was introduced in the gene by error-prone polymerase chain reaction. A primary qualitative screening on solid plates containing carboxymethylcellulose as the substrate allowed selecting 2200 active clones that were then subjected to a secondary quantitative screening towards AZO-CMC for the selection of 76 improved variants that were cultured in flasks and characterized. Five rCelStrep mutants exhibiting the highest hydrolytic activities than the wild-type enzyme were further characterized and applied to the bioconversion of the pretreated Arundo donax lignocellulosic biomass. It is worth of noting that one of the five tested mutants exhibited a 30% improvement in bioconversion yields compared to the wild-type enzyme, despite the absence of the carbohydrate binding module domain in this variant. Homology models of the three-dimensional structures of the catalytic and binding modules of rCelStrep were obtained and localization of mutations on these models allowed us to speculate on the structure-function relationships of the mutants.

Feruloyl esterases (FAEs) are a diverse group of enzymes that specifically catalyze the hydrolysis of ester bonds between a hydroxycinnamic (e.g. ferulic) acid and plant poly- or oligosaccharides. FAEs as auxiliary enzymes significantly assist xylanolytic and pectinolytic enzymes in gaining access to their site of action during biomass saccharification for biofuel and biochemical production. A limited number of FAEs have been functionally characterized compared to over 1000 putative fungal FAEs that were recently predicted by similarity-based genome mining, which divided phylogenetically into different subfamilies (SFs). In this study, 27 putative and six characterized FAEs from both ascomycete and basidiomycete fungi were selected and heterologously expressed in Pichia pastoris and the recombinant proteins biochemically characterized to validate the previous genome mining and phylogenetical grouping and to expand the information on activity of fungal FAEs. As a result, 20 enzymes were shown to possess FAE activity, being active towards pNP-ferulate and/or methyl hydroxycinnamate substrates, and covering 11 subfamilies. Most of the new FAEs showed activities comparable to those of previously characterized fungal FAEs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A novel fungal species able to synthesize enzymes with potential synergistic actions in lignocellulose conversion was isolated from the biomass of Arundo donax during biodegradation under natural conditions in the Gussone Park of the Royal Palace of Portici (Naples, Italy). In this work, this species was subjected to morphological and phylogenetic analyses. Sequencing of its genome was performed, resulting in 28 scaffolds that were assembled into 27.05 Mb containing 9744 predicted genes, among which 396 belong to carbohydrate-active enzyme (CAZyme)-encoding genes. Here we describe and illustrate this previously unknown species, which was named Talaromyces borbonicus, by a polyphasic approach combining phenotypic, physiological, and sequence data.

4-O-Methyl-d-glucuronic acid (MeGlcA) is a side-residue of glucuronoarabinoxylan and can form ester linkages to lignin, contributing significantly to the strength and rigidity of the plant cell wall. Glucuronoyl esterases (4-O-methyl-glucuronoyl methylesterases, GEs) can cleave this ester bond, and therefore may play a significant role as auxiliary enzymes in biomass saccharification for the production of biofuels and biochemicals. GEs belong to a relatively new family of carbohydrate esterases (CE15) in the CAZy database (www.cazy.org), and so far around ten fungal GEs have been characterized. To explore additional GE enzymes, we used a genome mining strategy. BLAST analysis with characterized GEs against approximately 250 publicly accessible fungal genomes identified more than 150 putative fungal GEs, which were classified into eight phylogenetic sub-groups. To validate the genome mining strategy, 21 selected GEs from both ascomycete and basidiomycete fungi were heterologously produced in Pichia pastoris. Of these enzymes, 18 were active against benzyl d-glucuronate demonstrating the suitability of our genome mining strategy for enzyme discovery., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Here we present the draft genome sequence of the fungus Talaromyces adpressus A-T1C-84X (=CBS 142503). This strain was isolated from lignocellulosic biomass of Arundo donax during biodegradation under natural conditions in the Gussone Park of the Royal Palace of Portici, Naples, Italy., (Copyright © 2018 Cerullo et al.)

After publication of the original article, authors found that there has been a minor mistake in the units of kcat and kcat/Km in Table 2. The units should be 10 3 min -1 g -1 FAE for kcat and mM -1 min -1 g -1 FAE for kcat/Km. This correction does not affect any conclusions drawn within the article.

One of the major ecological concerns associated with biodiesel production is the generation of waste/crude glycerol during the trans-esterification process. Purification of this crude glycerol is not economically viable. In this context, the development of an efficient and economically viable strategy would be biotransformation reactions converting the biodiesel derived crude glycerol into value added chemicals. Hence the process ensures the sustainability and waste management in biodiesel industry, paving a path to integrated biorefineries. This review addresses a waste to wealth approach for utilization of crude glycerol in the production of value added chemicals, current trends, challenges, future perspectives, metabolic approaches and the genetic tools developed for the improved synthesis over wild type microorganisms were described., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Water hyacinth a fresh water aquatic plant is considered as a noxious weed in many parts of the world since it grows very fast and depletes nutrients and oxygen from water bodies adversely affecting the growth of both plants and animals. Hence conversion of this problematic weed to value added chemicals and fuels helps in the self-sustainability especially for developing countries. The present review discusses the various value added products and fuels which can be produced from water hyacinth, the recent research and developmental activities on the bioconversion of water hyacinth for the production of fuels and value added products as well as its possibilities and challenges in commercialization., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Five feruloyl esterases (FAEs; EC 3.1.1.73), FaeA1, FaeA2, FaeB1, and FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464, were tested for their ability to catalyze the transesterification of vinyl ferulate (VFA) with prenol in detergentless microemulsions. Reaction conditions were optimized investigating parameters such as the medium composition, the substrate concentration, the enzyme load, the pH, the temperature, and agitation. FaeB2 offered the highest transesterification yield (71.5 ± 0.2%) after 24 h of incubation at 30 °C using 60 mM VFA, 1 M prenol, and 0.02 mg FAE/mL in a mixture comprising of 53.4:43.4:3.2 v/v/v n-hexane:t-butanol:100 mM MOPS-NaOH, pH 6.0. At these conditions, the competitive side hydrolysis of VFA was 4.7-fold minimized. The ability of prenyl ferulate (PFA) and its corresponding ferulic acid (FA) to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was significant and similar (IC 50 423.39 μM for PFA, 329.9 μM for FA). PFA was not cytotoxic at 0.8-100 μM (IC 50 220.23 μM) and reduced intracellular reactive oxygen species (ROS) in human skin fibroblasts at concentrations ranging between 4 and 20 μM as determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.

In this study, a high-throughput sequencing approach was applied to discover novel biocatalysts for lignocellulose hydrolysis from three dedicated energy crops, Arundo donax, Eucalyptus camaldulensis and Populus nigra, after natural biodegradation. The microbiomes of the three lignocellulosic biomasses were dominated by bacterial species (approximately 90%) with the highest representation by the Streptomyces genus both in the total microbial community composition and in the microbial diversity related to GH families of predicted ORFs. Moreover, the functional clustering of the predicted ORFs showed a prevalence of poorly characterized genes, suggesting these lignocellulosic biomasses are potential sources of as yet unknown genes. 1.2%, 0.6% and 3.4% of the total ORFs detected in A. donax, E. camaldulensis and P. nigra, respectively, were putative Carbohydrate-Active Enzymes (CAZymes). Interestingly, the glycoside hydrolases abundance in P. nigra (1.8%) was higher than that detected in the other biomasses investigated in this study. Moreover, a high percentage of (hemi)cellulases with different activities and accessory enzymes (mannanases, polygalacturonases and feruloyl esterases) was detected, confirming that the three analyzed samples were a reservoir of diversified biocatalysts required for an effective lignocellulose saccharification.

Twenty-four Actinobacteria strains, isolated from Arundo donax, Eucalyptus camaldulensis and Populus nigra biomass during natural biodegradation and with potential enzymatic activities specific for the degradation of lignocellulosic materials, were identified by a polyphasic approach. All strains belonged to the genus Streptomyces ( S .) and in particular, the most highly represented species was Streptomyces argenteolus representing 50% of strains, while 8 strains were identified as Streptomyces flavogriseus (synonym S. flavovirens ) and Streptomyces fimicarius (synonyms Streptomyces acrimycini, Streptomyces baarnensis, Streptomyces caviscabies , and Streptomyces flavofuscus ), and the other four strains belonged to the species Streptomyces drozdowiczii, Streptomyces rubrogriseus, Streptomyces albolongus , and Streptomyces ambofaciens . Moreover, all Streptomyces strains, tested for endo and exo-cellulase, cellobiase, xylanase, pectinase, ligninase, peroxidase, and laccase activities using qualitative and semi-quantitative methods on solid growth medium, exhibited multiple enzymatic activities (from three to six). The 24 strains were further screened for endo-cellulase activity in liquid growth medium and the four best endo-cellulase producers ( S. argenteolus AE58P, S. argenteolus AE710A, S. argenteolus AE82P, and S. argenteolus AP51A) were subjected to partial characterization and their enzymatic crude extracts adopted to perform saccharification experiments on A. donax pretreated biomass. The degree of cellulose and xylan hydrolysis was evaluated by determining the kinetics of glucose and xylose release during 72 h incubation at 50°C from the pretreated biomass in the presence of cellulose degrading enzymes (cellulase and β-glucosidase) and xylan related activities (xylanase and β-xylosidase). The experiments were carried out utilizing the endo-cellulase activities from the selected S. argenteolus strains supplemented with commercial β-gucosidase and xylanase preparations from Genencore (Accellerase BG and Accellerase XY). Cellulose and xylan conversion, when conducted using commercial (hemi)cellulases, gave glucose and xylose yields of 30.17 and 68.9%, respectively. The replacement of the cellulolytic preparation from Genencor (Accellerase 1500), with the endo-cellulase from S. argenteolus AE58P resulted in almost 76% of the glucose yield obtained in the presence of the commercial counterpart. Due to the promising results obtained by using the enzymatic crude extracts from S. argenteolus AE58P in the pretreated A. donax saccharification experiments, the proteins putatively responsible for endo-cellulase activity in this strain were identified by proteomics. Several proteins were confidently identified in different Streptomyces spp., eight of which belong to the class of Carbohydrate active enzymes. Overall results highlighted the biotechnological potential of S. argenteolus AE58P being an interesting candidate biocatalyst-producing bacterium for lignocellulose conversion and production of biochemicals and bioenergy.

In the present work the recently isolated strain Basfia succiniciproducens BPP7 was evaluated for the production of succinic acid up to the pilot fermentation scale in separate hydrolysis and fermentation experiments on Arundo donax, a non-food dedicated energy crop. An average concentration of about 17g/L of succinic acid and a yield on consumed sugars of 0.75mol/mol were obtained demonstrating strain potential for further process improvement. Small scale experiments indicated that the concentration of acetic acid in the medium is crucial to improve productivity; on the other hand, interestingly, short-term (24h) adaptation to higher acetic acid concentrations, and strain recovery, were also observed., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The world economy is moving toward the use of renewable and nonedible lignocellulosic biomasses as substitutes for fossil sources in order to decrease the environmental impact of manufacturing processes and overcome the conflict with food production. Enzymatic hydrolysis of the feedstock is a key technology for bio-based chemical production, and the identification of novel, less expensive and more efficient biocatalysts is one of the main challenges. As the genomic era has shown that only a few microorganisms can be cultured under standard laboratory conditions, the extraction and analysis of genetic material directly from environmental samples, termed metagenomics, is a promising way to overcome this bottleneck. Two screening methodologies can be used on metagenomic material: the function-driven approach of expression libraries and sequence-driven analysis based on gene homology. Both techniques have been shown to be useful for the discovery of novel biocatalysts for lignocellulose conversion, and they enabled identification of several (hemi)cellulases and accessory enzymes involved in (hemi)cellulose hydrolysis. This review summarizes the latest progress in metagenomics aimed at discovering new enzymes for lignocellulose saccharification.

Lignocellulosic biomass is the most abundant, low-cost, bio-renewable resource that holds enormous importance as alternative source for production of biofuels and other biochemicals that can be utilized as building blocks for production of new materials. Enzymatic hydrolysis is an essential step involved in the bioconversion of lignocellulose to produce fermentable monosaccharides. However, to allow the enzymatic hydrolysis, a pretreatment step is needed in order to remove the lignin barrier and break down the crystalline structure of cellulose. The present manuscript is dedicated to reviewing the most commonly applied "green" pretreatment processes used in bioconversion of lignocellulosic biomasses within the "biorefinery" concept. In this frame, the effects of different pretreatment methods on lignocellulosic biomass are described along with an in-depth discussion on the benefits and drawbacks of each method, including generation of potentially inhibitory compounds for enzymatic hydrolysis, effect on cellulose digestibility, and generation of compounds toxic for the environment, and energy and economic demand., Competing Interests: Compliance with ethical standards Funding This study was funded by grant from the European Union Grant agreement N. 613868. Conflict of interest The authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

The actualization of a circular economy through the use of lignocellulosic wastes as renewable resources can lead to reduce the dependence from fossil-based resources and contribute to a sustainable waste management. The integrated biorefineries, exploiting the overall lignocellulosic waste components to generate fuels, chemicals and energy, are the pillar of the circular economy. The biological treatment is receiving great attention for the biorefinery development since it is considered an eco-friendly alternative to the physico-chemical strategies to increase the biobased product recovery from wastes and improve saccharification and fermentation yields. This paper reviews the last advances in the biological treatments aimed at upgrading lignocellulosic wastes, implementing the biorefinery concept and advocating circular economy., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cosmeceuticals are cosmetic products containing biologically active ingredients purporting to offer a pharmaceutical therapeutic benefit. The active ingredients can be extracted and purified from natural sources (botanicals, herbal extracts, or animals) but can also be obtained biotechnologically by fermentation and cell cultures or by enzymatic synthesis and modification of natural compounds. A cosmeceutical ingredient should possess an attractive property such as anti-oxidant, anti-inflammatory, skin whitening, anti-aging, anti-wrinkling, or photoprotective activity, among others. During the past years, there has been an increased interest on the enzymatic synthesis of bioactive esters and glycosides based on (trans)esterification, (trans)glycosylation, or oxidation reactions. Natural bioactive compounds with exceptional theurapeutic properties and low toxicity may offer a new insight into the design and development of potent and beneficial cosmetics. This review gives an overview of the enzymatic modifications which are performed currently for the synthesis of products with attractive properties for the cosmeceutical industry.

This study reports enzymatic hydrolysis of the biomass of the giant reed (Arundo donax L.) after ammonia fibre expansion (AFEX) pretreatment. In particular, the capacity of the arabinofuranosidase from the fungus Pleurotus ostreatus recombinantly expressed in Pichia pastoris rPoAbf, its evolved mutant rPoAbf F435Y/Y446F and the endo-cellulase from Streptomyces sp. G12 CelStrep recombinantly expressed in Escherichia coli to enhance the hydrolysis of AFEX-treated A. donax was investigated, using the corn stover as reference feedstock. The investigated enzymes were assayed using a mixture of purified cellulases (CBHI, CBHII, EGI and βG), endoxylanases (LX3, LX4) and accessory hemicellulases (LarbF and LβX) as reference enzyme mixture and substituting EGI with rCelStrep and LarbF with rPoAbf or rPoAbf F435Y/Y446F. The use of rPoAbf F435Y/Y446F in the substitution of LarbF led to improvements in sugar conversion, giving a glucan, xylan and arabinan conversion after 72 h of around 62, 63 and 80 %, respectively, similar or higher than those (44, 66 and 55 %) achieved by 72 h hydrolysis with commercial enzymes Novozymes Cellic®, Ctec3 and Htec3. The enzymes rPoAbf, rPoAbf F435Y/Y446F and rCelStrep were also investigated for their effect on hydrolysis of AFEX-pretreated A. donax by addition to commercial enzyme mixture Novozymes Cellic®, Ctec3 and Htec3, and it was shown that the addition of rPoAbf and its evolved mutant rPoAbf F435Y/Y446F enhanced both xylan and arabinan conversions, which achieved 80 % after 6 days of saccharification with rPoAbf F435Y/Y446F.

The lignocellulosic fractions of municipal solid waste (MSW) can be used as renewable resources due to the widespread availability, predictable and low pricing and suitability for most conversion technologies. In particular, after the typical paper recycling loop, the newspaper waste (NW) could be further valorized as feedstock in biorefinering industry since it still contains up to 70 % polysaccharides. In this study, two different physicochemical methods-ammonia fiber expansion (AFEX) and extractive ammonia (EA) were tested for the pretraetment of NW. Furthermore, based on the previously demonstrated ability of the recombinant enzymes endocellulase rCelStrep, α-L-arabinofuranosidase rPoAbf and its evolved variant rPoAbf F435Y/Y446F to improve the saccharification of different lignocellulosic pretreated biomasses (such as corn stover and Arundo donax), in this study these enzymes were tested for the hydrolysis of pretreated NW, with the aim of valorizing the lignocellulosic fractions of the MSW. In particular, a mixture of purified enzymes containing cellulases, xylanases and accessory hemicellulases, was chosen as reference mix and rCelStrep and rPoAbf or its variant were replaced to EGI and Larb. The results showed that these enzymatic mixes are not suitable for the hydrolysis of NW after AFEX or EA pretreatment. On the other hand, when the enzymes rCelStrep, rPoAbf and rPoAbf F435Y/Y446F were tested for their effect in hydrolysis of pretreated NW by addition to a commercial enzyme mixture, it was shown that the total polysaccharides conversion yield reached 37.32 % for AFEX pretreated NW by adding rPoAbf to the mix whilst the maximum sugars conversion yield for EA pretreated NW was achieved 40.80 % by adding rCelStrep. The maximum glucan conversion yield obtained (45.61 % for EA pretreated NW by adding rCelStrep to the commercial mix) is higher than or comparable to those reported in recent manuscripts adopting hydrolysis conditions similar to those used in this study.

Lignocellulosic biomasses derived from dedicated crops and agro-industrial residual materials are promising renewable resources for the production of fuels and other added value bioproducts. Due to the tolerance to a wide range of environments, the dedicated crops can be cultivated on marginal lands, avoiding conflict with food production and having beneficial effects on the environment. Besides, the agro-industrial residual materials represent an abundant, available, and cheap source of bioproducts that completely cut out the economical and environmental issues related to the cultivation of energy crops. Different processing steps like pretreatment, hydrolysis and microbial fermentation are needed to convert biomass into added value bioproducts. The reactor configuration, the operative conditions, and the operation mode of the conversion processes are crucial parameters for a high yield and productivity of the biomass bioconversion process. This review summarizes the last progresses in the bioreactor field, with main attention on the new configurations and the agitation systems, for conversion of dedicated energy crops (Arundo donax) and residual materials (corn stover, wheat straw, mesquite wood, agave bagasse, fruit and citrus peel wastes, sunflower seed hull, switchgrass, poplar sawdust, cogon grass, sugarcane bagasse, sunflower seed hull, and poplar wood) into sugars and ethanol. The main novelty of this review is its focus on reactor components and properties.

In this study, the recombinant α-L-arabinofuranosidase from the fungus Pleurotus ostreatus (rPoAbf) was subjected to site-directed mutagenesis in order to identify the catalytic nucleophile residue. Based on bioinformatics and homology modelling analyses, E449 was revealed to be the potential nucleophilic residue. Thus, the mutant E449G of PoAbf was recombinantly expressed in Pichia pastoris and its recombinant expression level and reactivity were investigated in comparison to the wild-type. The design of a suitable set of hydrolysis experiments in the presence or absence of alcoholic arabinosyl acceptors and/or formate salts allowed to unambiguously identify the residue E449 as the nucleophile residue involved in the retaining mechanism of this GH51 arabinofuranosidase. (1)H NMR analysis was applied for the identification of the products and the assignement of their anomeric configuration.

In this study, the recombinant α-L-arabinofuranosidase from the fungus Pleurotus ostreatus (rPoAbf) was subjected to site-directed mutagenesis with the aim of elucidating the role of glycosylation on the properties of the enzyme at the level of S160 residue. As a matter of fact, previous mass spectral analyses had led to the localization of a single O-glycosylation at this site. Recombinant expression and characterization of the rPoAbf mutant S160G was therefore performed. It was shown that the catalytic properties are slightly changed by the mutation, with a more evident modification of the Kcat and KM toward the synthetic substrate pN-glucopyranoside. More importantly, the mutation negatively affected the stability of the enzyme at various pHs and temperatures. Circular dichroism (CD) analyses showed a minimum at 210 nm for wild-type (wt) rPoAbf, typical of the beta-sheets structure, whereas this minimum is shifted for rPoAbf S160G, suggesting the presence of an unfolded structure. A similar behavior was revealed when wt rPoAbf was enzymatically deglycosylated. CD structural analyses of both the site-directed mutant and the enzymatically deglycosylated wild-type enzyme indicate a role of the glycosylation at the S160 residue in rPoAbf secondary structure stability., (© 2014 The Authors. Biotechnology and Applied Biochemistry published by Wiley Periodicals, Inc. on behalf of the International Union of Biochemistry and Molecular Biology, Inc.)

Background: Cellulases and xylanases are the key enzymes involved in the conversion of lignocelluloses into fermentable sugars. Western Ghat region (India) has been recognized as an active hot spot for the isolation of new microorganisms. The aim of this work was to isolate new microorganisms producing cellulases and xylanases to be applied in brewer's spent grain saccharification., Results: 93 microorganisms were isolated from Western Ghat and screened for the production of cellulase and xylanase activities. Fourteen cellulolytic and seven xylanolytic microorganisms were further screened in liquid culture. Particular attention was focused on the new isolate Bacillus amyloliquefaciens XR44A, producing xylanase activity up to 10.5 U mL -1 . A novel endo-1,4-beta xylanase was identified combining zymography and proteomics and recognized as the main enzyme responsible for B. amyloliquefaciens XR44A xylanase activity. The new xylanase activity was partially characterized and its application in saccharification of brewer's spent grain, pretreated by aqueous ammonia soaking, was investigated., Conclusion: The culture supernatant of B. amyloliquefaciens XR44A with xylanase activity allowed a recovery of around 43% xylose during brewer's spent grain saccharification, similar to the value obtained with a commercial xylanase from Trichoderma viride , and a maximum arabinose yield of 92%, around 2-fold higher than that achieved with the commercial xylanase. © 2014 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.