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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.

Twenty-eight fungal feruloyl esterases (FAEs) were evaluated for their synthetic abilities in a ternary system of n-hexane: t-butanol: 100 mM MOPS-NaOH pH 6.0 forming detergentless microemulsions. Five main derivatives were synthesized, namely prenyl ferulate, prenyl caffeate, butyl ferulate, glyceryl ferulate, and l-arabinose ferulate, offering, in general, higher yields when more hydrophilic alcohol substitutions were used. Acetyl xylan esterase-related FAEs belonging to phylogenetic subfamilies (SF) 5 and 6 showed increased synthetic yields among tested enzymes. In particular, it was shown that FAEs belonging to SF6 generally transesterified aliphatic alcohols more efficiently while SF5 members preferred bulkier l-arabinose. Predicted surface properties and structural characteristics were correlated with the synthetic potential of selected tannase-related, acetyl-xylan-related, and lipase-related FAEs (SF1-2, -6, -7 members) based on homology modeling and small molecular docking simulations.

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

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.

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.

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. Electronic supplementary material The online version of this article (10.1186/s13568-018-0602-7) contains supplementary material, which is available to authorized users.

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.

SixLactobacillusstrains were analyzed to select a bacterium for conversion of brewers’ spent grain (BSG) into lactic acid. Among the investigated strains,L. acidophilusATCC 43121 showed the highest yield of lactic acid production (16.1 g/L after 48 hours) when grown in a synthetic medium. It was then analyzed for its ability to grow on the hydrolysates obtained from BSG after acid-alkaline (AAT) or aqueous ammonia soaking (AAS) pretreatment. The lactic acid production byL. acidophilusATCC 43121 through fermentation of the hydrolysate from AAS treated BSG was 96% higher than that from the AAT treated one, although similar yields of lactic acid per consumed glucose were achieved due to a higher (46%) glucose consumption byL. acidophilusATCC 43121 in the AAS BSG hydrolysate. It is worth noting that adding yeast extract to the BSG hydrolysates increased both the yield of lactic acid per substrate consumed and the volumetric productivity. The best results were obtained by fermentation of AAS BSG hydrolysate supplemented by yeast extract, in which the strain produced 22.16 g/L of lactic acid (yield of 0.61 g/g), 27% higher than the value (17.49 g/L) obtained in the absence of a nitrogen source.

Ethanol production from lignocellulosic biomasses raises a global interest because it represents a good alternative to petroleum-derived energies and reduces the food versus fuel conflict generated by first generation ethanol. In this study, alkaline-acid pretreated brewers' spent grain (BSG) was evaluated for ethanol production after enzymatic hydrolysis with commercial enzymes. The obtained hydrolysate containing a glucose concentration of 75 g/L was adopted, after dilution up to 50 g/L, for fermentation by the strain Saccharomyces cerevisiae NRRL YB 2293 selected as the best producer among five ethanologenic microorganims. When the hydrolysate was supplemented with yeast extract, 12.79 g/L of ethanol, corresponding to 0.28 g of ethanol per grams of glucose consumed (55% efficiency), was obtained within 24 h, while in the non-supplemented hydrolysate, a similar concentration was reached within 48 h. The volumetric productivity increased from 0.25 g/L·h in the un-supplemented hydrolysate to 0.53 g/L h in the yeast extract supplemented hydrolysate. In conclusion, the strain S. cerevisiae NRRL YB 2293 was shown able to produce ethanol from BSG. Although an equal amount of ethanol was reached in both BSG hydrolysate media, the nitrogen source supplementation reduced the ethanol fermentation time and promoted glucose uptake and cell growth. © 2015 by the authors.

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.

Arundo donax was used to investigate the effect of the enzyme and substrate concentrations on hydrolysis, pre-hydrolysis and simultaneous saccharification and fermentation (PSSF), in comparison to simultaneous saccharification and fermentation (SSF). Hydrolysis was performed at 37 and 50 °C. At the highest biomass (10%) and enzyme (69.6 FPU/g cellulose) loadings, the highest glucose concentration (32.4 g/L) was obtained (at 50 °C). SSF resulted in a cellulose conversion (91.9%) and an ethanol concentration (19.8 g/L) higher than what was obtained using PSSF at 37 °C (86.9% and 18.8 g/L, respectively) and PSSF at 50 °C (81.6% and 17.7 g/L, respectively). A positive correlation between the cellulase concentration, cellulose conversion, and ethanol content was observed. In PSSF, the increase in the solids loadings caused a reduction in the % cellulose conversion, but the ethanol concentration in PSSF and SSF increased. SSF appeared to be the most advantageous process for bioethanol production from A. donax.

Bio-based succinic acid production from lignocellulosic biomass is one of the attractive and prominent alternative technologies to overcome issues associated with the utilization of fossil sources. In this context, it is necessary to find new microorganisms that are able to efficiently ferment this recalcitrant feedstock. The ecological approach developed in this study enabled the isolation of Basfia succiniciproducens BPP7 from a complex rumen ecosystem. This new wild-type strain was able to synthesize up to 6.06 ± 0.05 g/L of succinate (corresponding to 0.84 ± 0.017 g of succinate per gram of consumed glucose + xylose and to 0.14 ± 0.001 g of succinate per gram of glucans + xylans present in the biomass before hydrolysis) from Arundo donax hydrolysate in separate hydrolysis and fermentation (SHF) experiments. Higher titers of succinic acid were obtained through the optimization of growth conditions. The optimal medium composition identified on the smaller scale was then used for 2.5-L batch experiments, which used A. donax hydrolysate and yeast extract as the main C and N sources, respectively. A maximal titer of 9.4 ± 0.4 g/L of succinic acid was obtained after 24 h. The overall results clearly demonstrate the potential of B. succiniciproducens BPP7 for succinate production.

This manuscript describes the analysis of xylanase production by Aspergillus niger NRRL 328 in solid state fermentation (SSF) of soybean husks. A maximum value of extracellular xylanase activity of approximately 950 U g-1 was achieved after 96 h. Proteomic analyses performed on the enzymatic mixture responsible for the maximum value of xylanase activity in SSF revealed the presence of two xylanases. This xylanolytic mixture was partially purified and characterized. It followed Michaelis-Menten kinetics towards xylan, with a KM of 7.92 ±0.97 mg xylan/mL and a Vmax of 262.2 ± 27.8 g L-1 s-1. The optimum pH for the enzyme is 5.3, and the optimal temperature is 50 °C. The enzyme retains 100% of its activity at 40 °C for at least 1 month. It shows very high stability in a broad pH range, with a half-life of 40 days at pH 5.3, pH 6.0, pH 6.5, pH 7.0, and pH 8.0.

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.

Research on regulation of cellulases and hemicellulases gene expression may be very useful for increasing the production of these enzymes in their native producers. Mechanisms of gene regulation of cellulase and hemicellulase expression in filamentous fungi have been studied, mainly in Aspergillus and Trichoderma. The production of these extracellular enzymes is an energy-consuming process, so the enzymes are produced only under conditions in which the fungus needs to use plant polymers as an energy and carbon source. Moreover, production of many of these enzymes is coordinately regulated, and induced in the presence of the substrate polymers. In addition to induction by mono- and oligo-saccharides, genes encoding hydrolytic enzymes involved in plant cell wall deconstruction in filamentous fungi can be repressed during growth in the presence of easily metabolizable carbon sources, such as glucose. Carbon catabolite repression is an important mechanism to repress the production of plant cell wall degrading enzymes during growth on preferred carbon sources. This manuscript reviews the recent advancements in elucidation of molecular mechanisms responsible for regulation of expression of cellulase and hemicellulase genes in fungi.

Laccases represent versatile catalysts being able to oxidize a wide range of aromatic substrates and are susceptible of several industrial applications based on both oxidative degradation reactions and synthetic chemistry. The range of laccase based synthetic reactions is extremely wide. Laccases are able to catalyze transformation of antibiotics based on both -lactams functionalization and phtalides functionalization. These enzymes can also catalyze derivatization of amino acids to obtain metabolically stable amino acid analogues, maximizing biological response while minimizing toxicity, thus representing an useful system for drug development. Biomolecules having antioxidative and anticancer activity can also be produced by laccase-mediated reactions of flavonoids oxidative coupling and phenoxazinones synthesis. Application of laccases to production of new derivatives of the hormones resveratrol, 17ß-estradiol, totarol and isoeugenol and oligomerization products of substituted imidazoles was also reported, with applications for pharmacological purposes due to hormonal activity of the products. The enzymatic preparation of aromatic polymeric materials by the action of laccases represents a viable and non-toxic alternative to the usual formaldehyde-based chemical production of these compounds and it has been reported for several substrates such as 2,6-dimethylphenol, 4-hydroxybenzoic acid derivatives, 3,5-dimethoxy-4-hydroxybenzoic acid and 3,5-dimethyl-4-hydroxybenzoic acid, aniline and acrylamide. Moreover, laccase-mediated biografting of phenols or certain other types of low-molecular weight compounds provides a method for tailoring the surface of lignocellulosics or for adhesion enhancement in binderless wood boards under mild conditions and usually without harmful solvents. Laccase-mediated modification of lignocellulosic materials is accomplished through two main routes: coupling of low-molecular weight compounds onto lignocellulosic materials and laccase mediated cross-linking of lignin molecules in situ. Depending on the choice of laccase substrate, properties such as improved strength properties, increased antimicrobial resistance, or hydrophilicity/ hydrophobicity can be imparted to lignocellulosic materials.

Bio-based organic acids are an eco-friendly alternative to petroleum-derived products. In this work, the production of organic chemicals was investigated for the first time in the Cosenzaea myxofaciens species using hydrolysed lignocellulosic biomass from Arundo donax. The strain C. myxofaciens BPM1, isolated from bovine rumen, was able to produce a high amount of lactic acid, followed by acetic and succinic acids in synthetic substrate in microaerophilic and anaerobic conditions. When hydrolysed lignocellulosic biomass from Arundo donax supplemented with several nitrogen sources was used as substrate in separate hydrolysis and fermentation in anaerobic conditions, a significant increase in organic acids was recovered, reaching values up to 12.13 ± 0.17, 1.68 ± 0.1, and 5.23 ± 0.04 g L-1 of lactate, succinate, and acetate, respectively. Moreover, the strain C. myxofaciens BPM1 was capable of synthesizing a small amount of ethanol, with a resulting concentration ranging from 0.67 ± 0.05 to 1.46 ± 0.03 g L-1. This work shows that the strain C. myxofaciens BPM1 is a potential source of interesting bio-based chemicals for a wide range of industrial applications. In addition, the inexpensive fermentation process using A. donax hydrolysate and corn steep liquor as carbon and nitrogen sources could be suitable for economical and efficient production of succinic acid in industrial processes.

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.

Poly-3-hydroxybutyrate (PHB) is a biodegradable polymer produced by many bacteria. Some of the properties are similar to thermoplastics like polypropylene, hence finding application PHB can directly replace non-biodegradable polymers. But the main barrier has been the cost difference. The utilization of mixed microbial cultures facilitates the use of complex substrates and thus can reduce the cost of PHB production. In the present study, mixed culture systems, where metabolite produced by one organism may be assimilated by the other organism, were employed. Bacillus firmus NII 0830, the first organism, was used for the production of PHB since it accumulates a large amount of PHB, while the second organism, Lactobacillus delbrueckii NII 0925, was used to provide acetic acid. Enzyme kinetic studies were performed on the PHB biosynthetic enzymes, such as β-ketothiolase, acetoacetyl CoA reductase and PHB synthase. PHB production by mixed culture was higher when compared to pure cultures. The mathematical model was then fitted to the experimental data, which can describe the dynamics of a mixed culture. The β-ketothiolase and acetoacetyl CoA reductase showed a Vmax value of 0.0093 μM/min and 0.0253 μM/min, respectively. The Km values were 140.8 μM and 183.5 μM, respectively. The enzyme kinetic studies gave an idea about the action of the enzymes.

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.

In silico analyses of several laccase promoter sequences have shown the presence of many different responsive elements differentially distributed along the promoter sequences. Analysis of Pleurotus ostreatus laccase promoter poxa1b extending around 1400-bp upstream of the start codon showed the presence of several putative response elements, such as 10 metal-responsive elements. Development of a system for in vivo analysis of P. ostreatus laccase promoter poxa1b by enhanced green fluorescent protein expression was carried out, based on a polyethylene glycol-mediated procedure for fungal transformation. Quantitative measurement of fluorescence expressed in P. ostreatus transformants grown in the presence and in the absence of copper sulfate was performed, demonstrating an increase in expression level induced by the metal.

Laccases are oxidative enzymes linked to biological degradation of lignin. The aim of this work was to evaluate the effect of inducers and different concentrations of nitrogen on production level of total laccase activity and pattern of laccase isoforms, produced in solid state fermentation of sugarcane bagasse by a selected strain of Pleurotus ostreatus. The addition of yeast extract 5g/L, copper sulfate 150μM and ferulic acid 2mM provided highest enzymatic activity (167U/g) and zymograms indicated the presence of six laccase isoforms (POXA1b, POXA3, POXC and three other isoforms). Results of protein identification by mass spectrometry confirmed the presence of POXC and POXA3 as the main isoenzymes, and also identified a glyoxal oxidase and three galactose oxidases. The fact that the isoenzyme POXA1b was not identified in the analyzed samples can be possibly explained by its sensitivity to protease degradation.

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.

Fungal laccases are phenol oxidases widely studied for their use in several industrial applications, including pulp bleaching in paper industry, dye decolourisation, detoxification of environmental pollutants and revalorization of wastes and wastewaters. The main difficulty in using these enzymes at industrial scale ensues from their production costs. Elucidation of the components and the mechanisms involved in regulation of laccase gene expression is crucial for increasing the productivity of native laccases in fungi. Laccase gene transcription is regulated by metal ions, various aromatic compounds related to lignin or lignin derivatives, nitrogen and carbon sources. In this manuscript, most of the published results on fungal laccase induction, as well as analyses of both the sequences and putative functions of laccase gene promoters are reviewed. Analyses of promoter sequences allow defining a correlation between the observed regulatory effects on laccase gene transcription and the presence of specific responsive elements, and postulating, in some cases, a mechanism for their functioning. Only few reports have investigated the molecular mechanisms underlying laccase regulation by different stimuli. The reported analyses suggest the existence of a complex picture of laccase regulation phenomena acting through a variety of cis acting elements. However, the general mechanisms for laccase transcriptional regulation are far from being unravelled yet.

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.

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.

An enzymatic mixture of cellulases and xylanases was produced byPleurotus ostreatususing microcrystalline cellulose as inducer, partially characterized and tested in the statistical analysis ofArundo donaxbioconversion. The Plackett-Burman screening design was applied to identify the most significant parameters for the enzymatic hydrolysis of pretreatedA. donax. As the most significant influence during the enzymatic hydrolysis ofA. donaxwas exercised by the temperature (°C), pH, and time, the combined effect of these factors in the bioconversion byP. ostreatuscellulase and xylanase was analyzed by a 33factorial experimental design. It is worth noting that the best result of 480.10 mg of sugars/gds, obtained at 45°C, pH 3.5, and 96 hours of incubation, was significant also when compared with the results previously reached by process optimization with commercial enzymes.

International audience; 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. 1H NMR analysis was applied for the identification of the products and the assignement of their anomeric configuration.

Laccases are oxidative enzymes related to the degradation of phenolic compounds, including lignin units, with concomitant reduction of oxygen to water. Delignification is a necessary pretreatment step in the process of converting plant biomass into fermentable sugars. The objective of this work was to optimize the production of laccases and to evaluate the delignification of sugarcane bagasse byPleurotus ostreatusin solid-state fermentation. Among eight variables (pH, water activity, temperature, and concentrations of CuSO4, (NH4)2SO4, KH2PO4, asparagine, and yeast extract), copper sulfate and ammonium sulfate concentrations were demonstrated to significantly influence laccase production. The replacement of ammonium sulfate by yeast extract and the addition of ferulic acid as inducer provided increases of 5.7- and 2.0-fold, respectively, in laccase activity. Optimization of laccase production as a function of yeast extract, copper sulfate, and ferulic acid concentrations was performed by response surface methodology and optimal concentrations were 6.4 g/L, 172.6 μM, and 1.86 mM, respectively. Experimentally, the maximum laccase activity of 151.6 U/g was produced at the 5th day of solid-state fermentation. Lignin content in sugarcane bagasse was reduced from 31.89% to 26.36% after 5 days and to 20.79% after 15 days by the biological treatment of solid-state fermentation.

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.

The fungal arabinofuranosidase from Pleurotus ostreatus PoAbf recombinantly expressed in Pichia pastoris rPoAbf and its evolved variant rPoAbf F435Y/Y446F were tested for their effectiveness to enhance the enzymatic saccharification of three lignocellulosic biomasses, namely Arundo donax, corn cobs and brewer's spent grains (BSG), after chemical or chemical-physical pretreatment. All the raw materials were subjected to an alkaline pretreatment by soaking in aqueous ammonia solution whilst the biomass from A. donax was also pretreated by steam explosion. The capability of the wild-type and mutant rPoAbf to increase the fermentable sugars recovery was assessed by using these enzymes in combination with different (hemi)cellulolytic activities. These enzymatic mixtures were either entirely of commercial origin or contained the cellulase from Streptomyces sp. G12 CelStrep recombinantly expressed in Escherichia coli in substitution to the commercial counterparts. The addition of the arabinofuranosidases from P. ostreatus improved the hydrolytic efficiency of the commercial enzymatic cocktails on all the pretreated biomasses. The best results were obtained using the rPoAbf evolved variant and are represented by increases of the xylose recovery up to 56.4%. These data clearly highlight the important role of the accessory hemicellulolytic activities to optimize the xylan bioconversion yields. (C) 2014 Elsevier Inc. All rights reserved.

This manuscript describes the analysis of the effect of cellulose, carboxymethylcellulose (CMC), xylan, and xylose as inducers of cellulase and xylanase activity production by Ganoderma applanatum MR-56 and the optimization of their production in liquid cultures by statistical methods. The Plackett–Burman screening design was applied to identify the most significant inducers of xylanase and cellulase activities production by G. applanatum MR-56. The most significant effect on xylanase and cellulase activities production was exercised by cellulose, even if xylose and CMC were also effective at some times. The combined effect of cellulose, yeast extract, and pH was analyzed by a 23 factorial experimental design with four central points that showed that the maximum tested cellulose (1 % w/v) and yeast extract (5 g L−1) concentrations gave the maximum production of xylanase (8.24 U mL−1) and cellulase (3.29 U mL−1) activity at pH 6 and 4, respectively. These values achieved for cellulase and xylanase activity represent 12–25 fold and 36 fold higher values than the maximum so far reported for other strains of G. applanatum, respectively.

Pectinolytic enzymes have an important role in the processing of lignocellulosic materials because of their ability to improve the access of cellulases to their substrate by removing pectins. The strain Paenibacillus xylanilyticus 2-6L3 was isolated from mature compost obtained from agro-industrial wastes, and the enzyme pectate lyase from P. xylanilyticus 2-6L3, named Paenxyl Pel, was partially purified and subjected to structural and functional characterisation. The enzyme exhibited an optimum temperature between 60 and 70 °C and optimal pH value of 9.0 for its pectinase activity on pectin from citrus fruit. Paenxyl Pel showed a thermoresistance and pH resistance higher than those of other pectate lyases so far described, with half-lives of 48 and 24 h at 60 and 70 °C, respectively, a retention of around 80% of activity after 96 h at 40 and 50 °C, and a half-life of about 15 days at pH 8.0. Paenxyl Pel followed Michaelis-Menten kinetics toward pectin from citrus fruit, pectin from sugar beet pulp, high-ester pectin extracted from citrus peel (> 50% esterified), and polygalacturonic acid (PLA). The ability to act on both PLA and highly methylated pectins, together with a double peak in the graph of optimum pH at pH 5 and 9, suggest that pectate lyase from P. xylanoliticus shows an unusual activity, combining traits of pectate lyase and pectin lyase. This is the first manuscript on the pectinolytic activity of P. xylanilyticus.

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.

This study was aimed at developing a process of solid state fermentation (SSF) with the fungi Pleurotus ostreatus and Trametes versicolor on apple processing residues for wastewater decolorization. Both fungi were able to colonize apple residues without any addition of nutrients, material support or water. P. ostreatus produced the highest levels of laccases (up to 9 U g−1 of dry matter) and xylanases (up to 80 U g−1 of dry matter). A repeated batch decolorization experiment was set up with apple residues colonized by P. ostreatus, achieving 50% decolorization and 100% detoxification after 24 h, and, adding fresh wastewater every 24 h, a constant decolorization of 50% was measured for at least 1 month. A continuous decolorization experiment was set up by a packed-bed reactor based on colonized apple residues achieving a performance of 100 mg dye L−1 day−1 at a retention time of 50 h.

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.

Laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) are blue multicopper oxidases that catalyze the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. In fungi, laccases carry out a variety of physiological roles during their life cycle. These enzymes are being increasingly evaluated for a variety of biotechnological applications due to their broad substrate range. In this review, the most recent studies on laccase structural features and catalytic mechanisms along with analyses of their expression are reported and examined with the aim of contributing to the discussion on their structure-function relationships. Attention has also been paid to the properties of enzymes endowed with unique characteristics and to fungal laccase multigene families and their organization.

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.

The effect of Phanerochaete chrysosporium and Pleurotus ostreatus whole cells and their ligninolytic enzymes on models of colored industrial wastewaters was evaluated. Models of acid, direct and reactive dye wastewaters from textile industry have been defined on the basis of discharged amounts, economic relevance and representativeness of chemical structures of the contained dyes. Phanerochaete chrysosporium provided an effective decolourization of direct dye wastewater model, reaching about 45% decolourization in only 1 day of treatment, and about 90% decolourization within 7 days, whilst P. ostreatus was able to decolorize and detoxify acid dye wastewater model providing 40% decolourization in only 1 day, and 60% in 7 days. P. ostreatus growth conditions that induce laccase production (up to 130,000 U/l) were identified, and extra-cellular enzyme mixtures, with known laccase isoenzyme composition, were produced and used in wastewater models decolourization. The mixtures decolorized and detoxified the acid dye wastewater model, suggesting laccases as the main agents of wastewater decolourization by P. ostreatus. A laccase mixture was immobilized by entrapment in Cu-alginate beads, and the immobilized enzymes were shown to be effective in batch decolourization, even after 15 stepwise additions of dye for a total exposure of about 1 month.

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.

A new laccase isoenzyme (POXA1b, where POX is phenol oxidase), produced by Pleurotus ostreatus in cultures supplemented with copper sulphate, has been purified and fully characterized. The main characteristics of this protein (molecular mass in native and denaturing conditions, pI and catalytic properties) are almost identical to the previously studied laccase POXA1w. However, POXA1b contains four copper atoms per molecule instead of one copper, two zinc and one iron atom per molecule of POXA1w. Furthermore, POXA1b shows an unusually high stability at alkaline pH. The gene and cDNA coding for POXA1b have been cloned and sequenced. The gene coding sequence contains 1599 bp, interrupted by 15 introns. Comparison of the structure of the poxa1b gene with the two previously studied P. ostreatus laccase genes (pox1 and poxc) suggests that these genes belong to two different subfamilies. The amino acid sequence of POXA1b deduced from the cDNA sequence has been almost completely verified by means of matrix-assisted laser desorption ionization MS. It has been demonstrated that three out of six putative glycosylation sites are post-translationally modified and the structure of the bound glycosidic moieties has been determined, whereas two other putative glycosylation sites are unmodified.