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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.

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. 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.

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 (IC50 423.39 μM for PFA, 329.9 μM for FA). PFA was not cytotoxic at 0.8-100 μM (IC50 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.

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.

Background: Due to its wide range of applications in the food, pharmaceutical and chemical fields, microbial syn- thesis 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 experi- ments 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. Keywords: Basfia succiniciproducens, Arundo donax, Fed-batch, Succinic acid, Pre-pilot scale, Inhibition, Material flow analysis

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. (C) 2017 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.

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.

International audience; 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.

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 K cat and K M 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.

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.

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.

The laccase family of Pleurotus ostreatus has been widely characterized, and studies of the genes coding for laccase isoenzymes in P. ostreatus have so far led to the identification of four different genes and the corresponding cDNAs, poxc, pox1, poxa1b and poxa3. Analyses of P. ostreatus laccase promoters poxc, pox1, poxa1b and poxa3 have allowed identification of several putative response elements, and sequences of metal-responsive elements involved in the formation of complexes with fungal proteins have been identified in poxc and poxa1b promoters. In this work, development of a system for in vivo analysis of P. ostreatus laccase promoter poxc by enhanced green fluorescent protein expression is performed, based on a poly ethylene glycol-mediated procedure for fungal transformation. A quantitative measurement of fluorescence expressed in P. ostreatus transformants is hereby reported for the first time for this fungus.

The genome of the white rot basidiomycete Pleurotus ostreatus includes 12 phenol oxidase (laccase) genes. In this study, we examined their expression profiles in different fungal strains under different culture conditions (submerged and solid cultures) and in the presence of a wheat straw extract, which was used as an inducer of the laccase gene family. We used a reverse transcription-quantitative PCR (RT-qPCR)-based approach and focused on determining the reaction parameters (in particular, the reference gene set for the normalization and reaction efficiency determinations) used to achieve an accurate estimation of the relative gene expression values. The results suggested that (i) laccase gene transcription is upregulated in the induced submerged fermentation (iSmF) cultures but downregulated in the solid fermentation (SSF) cultures, (ii) the Lacc2 and Lacc10 genes are the main sources of laccase activity in the iSmF cultures upon induction with water-soluble wheat straw extracts, and (iii) an additional, as-yet-uncharacterized activity (Unk1) is specifically induced in SSF cultures that complements the activity of Lacc2 and Lacc10. Moreover, both the enzymatic laccase activities and the Lacc gene family transcription profiles greatly differ between closely related strains. These differences can be targeted for biotechnological breeding programs for enzyme production in submerged fermentation reactors.

Conversion of lignocellulosic materials to useful, high value products normally requires a pre-treatment step to transform or deconstruct the recalcitrant and heterogeneous lignin fraction. The development of “green tools” for the transformation of lignocellulosic feedstocks is in high demand for a sustainable exploitation of such resources. This multi-faceted challenge is being addressed by an ever-increasing suite of ligninolytic enzymes isolated from various sources. Among these, fungal laccases are known to play an important role in lignin degradation/modification processes. The white-rot fungus Pleurotus ostreatus expresses multiple laccase genes encoding isoenzymes with different properties. The availability of established recombinant expression systems for P. ostreatus laccase isoenzymes has allowed to further enrich the panel of P. ostreatus laccases by the construction of mutated, “better performing” enzymes through molecular evolution techniques. New oxidative catalysts with improved activity and stability either at high temperature and at acidic and alkaline pH have been isolated and characterized.

Laccases are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. These enzymes are implicated in a variety of biological activities. Most of the laccases studied thus far are of fungal origin. The large range of substrates oxidized by laccases has raised interest in using them within different industrial fields, such as pulp delignification, textile dye bleaching, and bioremediation. Laccases secreted from native sources are usually not suitable for large-scale purposes, mainly due to low production yields and high cost of preparation/purification procedures. Heterologous expression may provide higher enzyme yields and may permit to produce laccases with desired properties (such as different substrate specificities, or improved stabilities) for industrial applications. This review surveys researches on heterologous laccase expression focusing on the pivotal role played by recombinant systems towards the development of robust tools for greening modern industry.

A process of solid state fermentation (SSF) on tomato pomace was developed with the white-rot fungi Pleurotus ostreatus and Trametes versicolor, using sorghum stalks as support. Operative parameters (humidity, water activity, and size of substrate particles) guaranteeing a good colonization of tomato pomace by both fungi were defined and conditions for production at high titers of the industrially relevant enzymes laccase, xylanase and protease were identified. Significant laccase activity levels (up to 36 U g(-1) dry matter) were achieved without any optimization of culture conditions, neither by nutrient addition nor by O(2) enrichment. Furthermore, protease activity levels up to 34,000 U g(-1) dry matter were achieved, being higher than those reported for the fungi typically considered as the best protease producers such as Aspergillus strains. Moreover, as one of the most significant results of this study, analysis of P. ostreatus tomato SSF samples by zymogram revealed two bands with laccase activity which had not been detected so far.

A comparison of laccase sequences highlighted the presence of a C-terminal extension of sixteen amino acids in POXA1b laccase – that represents the most thermostable isoenzyme among Pleurotus ostreatus laccases and exhibits a notable stability at alkaline pH ( t 1/2 at pH 10 = 30 days) – whereas this tail is missing in the other analysed laccases from basidiomycetes. Site-directed mutagenesis experiments allowed us to demonstrate a role of the C-terminal tail of POXA1b in affecting its catalytic and stability properties. The truncated mutants lose the high stability at pH 10, while they show an increased stability at pH 5. The effect of substituting the residue Asp205 of POXA1b with an arginine was also analysed in the mutant POXA1bD205R. Following the mutation POXA1bD205R, a remarkable worsening of catalytic properties along with a decrease of substrate affinity and of enzyme stability were found. It was demonstrated that introducing Arg205 mutation in a highly conserved region perturbs the structural local environment in POXA1b, leading to a large rearrangement of the enzyme structure. Hence, a single substitution in the binding site introduces a local conformational change that not only leads to very different catalytic properties, but can also significantly destabilize the protein.

BACKGROUND: The ability of the fungi Pleurotus ostreatus and Phanerochaete chrysosporium to decolourize and detoxify 11 (mono-, dis-, poly-azo, and anthraquinonic type) dyes, widely used across the textile and leather industries, was tested. RESULTS: Different substrate specificities were revealed between P. ostreatus and P. chrysosporium in decolourization experiments. The latter fungus provided almost complete decolourization of the tested azo dyes up to 600 ppm and dis-azo dyes up to 1000 ppm, and 80% decolourization of the tris-azo dye DBU1L38 at 1000 ppm, after 6 days. P. ostreatus provided almost total decolourization of the anthraquinone type dye ABU62 (1000 ppm) after just 1 day. P. ostreatus also showed the ability to decolourize the tested dis-azo and tris-azo dyes, giving the best performances against the dis-azo DBU1U1 (600 ppm) dye, which was 100% decolourized after 6 days. Laccases proved to be the main enzymatic activities acting in P. ostreatus decolourization. CONCLUSION: The potential of the fungi P. ostreatus and P. chrysosporium as efficient bio-systems for decolourization and detoxification of several toxic industrial dyes was demonstrated. The role of laccases in the decolourization of dis-azo dyes by P. ostreatus was demonstrated for the first time. (C) 2008 Society of Chemical Industry

The subfamily of POXA3 laccase isoenzymes produced by the fungus Pleurotus ostreatus has been characterized as an example of the complexity and heterogeneity of fungal isoenzyme patterns. Two isoenzymes, POXA3a and POXA3b, were previously purified, exhibiting an unusual heterodimeric structure composed of a large (67 kDa) and a small (18 or 16 kDa) subunit. A unique gene encodes the large subunit of both POXA3a and POXA3b, but alternative splicing produces two variants--differing for an insertion of four amino acids--for each isoenzyme. Two genes encoding POXA3a and POXA3b small subunits have been identified, and the corresponding amino acid sequences show only two amino acid substitutions. The 18- and 16-kDa subunits of both POXA3a and POXA3b differ for N-glycosylation at Asn150 of the 16-kDa subunit. The POXA3 large subunit 3D model allows us to highlight peculiarities of this molecule with respect to the laccases whose 3D structures are known.

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.

The aims of this study were to evaluate the microbial diversity of different lignocellulosic biomasses during degradation under natural conditions and to isolate, select, characterise new well-adapted bacterial strains to detect potentially improved enzyme-producing bacteria. The microbiota of biomass piles of Arundo donax, Eucalyptus camaldulensis and Populus nigra were evaluated by high-throughput sequencing. A highly complex bacterial community was found, composed of ubiquitous bacteria, with the highest representation by the Actinobacteria, Proteobacteria, Bacteroidetes and Firmicutes phyla. The abundances of the major and minor taxa retrieved during the process were determined by the selective pressure produced by the lignocellulosic plant species and degradation conditions. Moreover, cellulolytic bacteria were isolated using differential substrates and screened for cellulase, cellobiase, xylanase, pectinase and ligninase activities. Forty strains that showed multienzymatic activity were selected and identified. The highest endo-cellulase activity was seen in Promicromonospora sukumoe CE86 and Isoptericola variabilis CA84, which were able to degrade cellulose, cellobiose and xylan. Sixty-two percent of bacterial strains tested exhibited high extracellular endo-1,4-beta-glucanase activity in liquid media. These approaches show that the microbiota of lignocellulosic biomasses can be considered an important source of bacterial strains to upgrade the feasibility of lignocellulose conversion for the 'greener' technology of second-generation biofuels.

Dye-decolorizing peroxidases (DyP) are atypical peroxidases showing no homology to other fungal peroxidases and lacking the typical heme binding region conserved among plant peroxidase superfamily. The gene and the corresponding cDNA encoding DyP from Pleurotus ostreatus have been identified on the basis of sequence homology analyses. The deduced amino acid sequence shares 43% identity with DyP from the ascomycete Thanatephorus cucumeris Dec 1. Analyses of the protein sequence by homology searches pointed out some properties of the DyP-type peroxidase family, which includes members from bacteria, ascomycete, and basidiomycete fungi. Some amino acids (C374, H379, and Y501 in the P. ostreatus DyP sequence) are proposed as candidates for the heme ligand, providing a basis for further investigations on the structure of the DyP type peroxidase family members.

In this study, the α- l -arabinofuranosidase from Pleurotus ostreatus was subjected to directed evolution by expressing a library of around 7000 randomly mutated variants by error prone Polymerase Chain Reaction. High-throughput screening of the library for the most active variants was performed by assaying activity towards p-nitrophenyl α- l -arabinofuranoside, and a variant with higher activity than the wild type was selected, purified and characterised. It exhibited a kcat of 7.3 × 103 ± 0.3 min−1, around 3-fold higher than that of the wild type (2.2 × 103 ± 0.2 min−1), and a KM (0.54 ± 0.06 mM) 30% lower than that of the wild type (0.70 ± 0.05 mM) towards this substrate. The mutant also showed improved catalytic properties towards pNP-β- d -glucopyranoside (kcat of 50.85 ± 0.21 min−1 versus 11.0 ± 0.6 min−1) and it was shown able to hydrolyse larch arabinogalactan which is not recognised by the wild type. The mutant was also more active than the wild type towards arabinoxylan and was able to hydrolyse arabinan, which was not transformed by the wild type. The ability of rPoAbf F435Y/Y446F to hydrolyse these insoluble substrates expands its potential for application also to hemicelluloses, which in some types of pretreatment are recovered in solid fractions.

An α-l-arabinofuranosidase produced by Pleurotus ostreatus (PoAbf) during solid state fermentation on tomato pomace was identified and the corresponding gene and cDNA were cloned and sequenced. Molecular analysis showed that the poabf gene carries 26 exons interrupted by 25 introns and has an open reading frame encoding a protein of 646 amino acid residues, including a signal peptide of 20 amino acid residues. The amino acid sequence similar to the other α-l-arabinofuranosidases indicated that the enzyme encoded by poabf can be classified as a family 51 glycoside hydrolase. Heterologous recombinant expression of PoAbf was carried out in the yeasts Pichia pastoris and Kluyveromyces lactis achieving the highest production level of the secreted enzyme (180 mg L−1) in the former host. rPoAbf produced in P. pastoris was purified and characterized. It is a glycosylated monomer with a molecular weight of 81,500 Da in denaturing conditions. Mass spectral analyses led to the localization of a single O-glycosylation site at the level of Ser160. The enzyme is highly specific for α-l-arabinofuranosyl linkages and when assayed with p-nitrophenyl α-l-arabinofuranoside it follows Michaelis–Menten kinetics with a K M of 0.64 mM and a k cat of 3,010 min−1. The optimum pH is 5 and the optimal temperature 40°C. It is worth noting that the enzyme shows a very high stability in a broad range of pH. The more durable activity showed by rPoAbf in comparison to the other α-l-arabinofuranosidases enhances its potential for biotechnological applications and increases interest in elucidating the molecular bases of its peculiar properties.

Consolidated BioProcessing (CBP) can provide an important contribution to reducing ethanol production costs and moving from cellulosic feedstock to fuel ethanol tanks. Several efforts have so far been focused mainly on CBP category II engineering an ethanologen yeast or bacterium to be cellulolytic, but the limited ability of the category II CBP system for producing enzymes for lignocellulose degradation remains a challenge. As an alternative, category I CBP, aimed at engineering a cellulase producer to be ethanologenic, could be pursued, but it is still in its infancy. Some cellulolytic thermophilic bacteria have been described as potential candidates for category I CBP. However, only fungi naturally produce the needed titers of cellulases required for the complete saccharification of pretreated lignocellulose. In this review, potential of cellulolytic fungi as candidates for category I CBP is discussed.

This research is aimed at isolating, characterising and selecting cellulolytic bacteria from raw composting materials consisting of wastes from food industry of vegetable. A total of 180 isolates were screened for their cellulosolytic activity and characterized by phenotypic and molecular methods. Moreover, intraspecific DNA polymorphism was evaluated. Time-courses of cellulase production were also analysed by CMC and filter paper activity assays. Only microbial strains exhibiting the highest cellulolytic activity in liquid medium were, then, evaluated for their hydrolytic activity on different soluble and insoluble substrates and cellulase production regulation.

In order to develop improved laccase-based bio-catalysts, semi-rational mutagenesis of the laccase POXA1b from Pleurotus ostreatus was performed through a combination of directed evolution with elements of rational enzyme modification. The R4 laccase was prepared by joining mutations of previously selected POXA1b random variants. An enhancement of stability features was thus obtained, making the novel enzyme R4 more appropriate as scaffold for directed evolution. A library of 1000 randomly mutated variants of R4 was prepared and screened for the ability of oxidising 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). One of the variants selected (V148L) for improved activity was also proved to show higher stability than R4 at pH 5, and to retain its high stability at pH 7 and 10. In comparison with the POXA1b wild-type laccase, the semi-rational approach allowed us to develop a more efficient bio-catalyst, rising specific activity on ABTS up to around 5-fold. The new variant was also proved to be both more versatile and more durable than the wild-type enzyme, exhibiting higher activity in wide temperature and pH ranges and higher stability at acidic (t (1/2) at pH 5 = 35 days), neutral (t (1/2) at pH 7 = 38 days) and alkaline (t (1/2) at pH 10 = 62 days) pH values.

Aims: To select better performing laccase variants among the 2300 randomly mutated variants of Pleurotus ostreatus POXA1b laccase to develop improved laccase-based biocatalysts. Methods and Results: Screening of collections of 2300 randomly mutated variants of POXA1b was performed by assaying activity towards the phenolic substrate 2,6-dimethoxyphenol. Two new variants endowed with higher enzyme activity than the wild-type laccase were characterized, and their ability to decolourize industrial dyes with complex trisazo-, polyazo- and stilbene-type structures, in the absence of mediators, was demonstrated. One of the mutants (2L4A) was also proved to be highly stable at both acidic and alkaline pH values (displaying a half-life of around 1 month at the pH levels of both 5 and 10). Conclusions: In comparison with the wild-type laccase, the new selected 2L4A mutant shows a significant increase in stability at acidic pH, whilst storing its high stability at alkaline pH. This variant also represents a more versatile enzyme with respect to both the variety of xenobiotics degraded and the operative conditions. Significance and Impact of the Study: This work represents the first example of improvement of a basidiomycete laccase for industrial effluents bioremediation by directed evolution.

Among the laccases produced by the white-rot fungus Pleurotus ostreatus, there are two closely related atypical isoenzymes, POXA3a and POXA3b. These isoenzymes are endowed with quaternary structure, consisting of two subunits very different in size. The POXA3 large subunit is clearly homologous to other known laccases, while the small subunit does not show significant homology with any protein in data banks. To investigate on the singular structure of the POXA3 complex, a new system for recombinant expression of heterodimer proteins in the yeast Kluyveromyces lactis has been set up. A unique expression vector has been used and the cDNAs encoding the two subunits have been cloned under the control of the same bi-directionally acting promoter. Expression of the large subunit alone and co-expression of both subunits in the same host have been demonstrated and the properties of the recombinant proteins have been compared. Clones expressing the large subunit alone exhibited always notably lower activity than those expressing both subunits. In addition to the activity increase, the presence of the small subunit led to a significant increase of laccase stability. Therefore, a role of the small subunit in POXA3 stabilisation is suggested.

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.

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.

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.