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Additional file 1. Schematic representation of the TeTrCBH I expression construct in S. cerevisiae.

Additional file 2. High performance size exclusion chromatography UV and RI chromatograms.

Additional file 1: Figure S1. SDS-PAGE analyses of T. atroviride culture filtrate and purified Trichoderma spp. Cel7A enzymes. Figure S2. Substrate dependence plots and Hanes-Wolff plots from enzyme kinetics experiments with TatCel7A, ThaCel7A and TreCel7A, using pNP-Lac as substrate and cellobiose as inhibitor. Additional information regarding the mathematical model for quasi-steady state kinetics of processive cellulose hydrolysis by GH7 cellobiohydrolases and the derivation of kinetic parameters by non-linear regression fitting to real-time progress curves of the initial stage of cellulose hydrolysis. Figure S3. A) Real-time progress curves. B) Derivative of the progress curves in A). Figure S4. A) Simplified reaction scheme for a processive cellulase. B) Illustration of the molecular steps involved in the reaction scheme. Figure S5. Non-linear regression fit to real-time progress curves. Figure S6. Bar diagram of kinetic parameters derived from initial hydrolysis of BMCC. Additional information regarding correlation of kinetic parameters derived by non-linear regression fit to initial hydrolysis data. Table S1. Parameter correlation matrix for TreCel7A. Figure S7. Kinetic parameter fit to simulated data with 2.5% random noise added, and to experimental data recorded for TreCel7A during initial hydrolysis of BMCC. Table S2. Comparison of kinetic parameters from the fit to simulated data with 2.5% random noise, and to experimental data recorded for TreCel7A during initial hydrolysis of BMCC. Figure S8. Sequence alignment of the GH7 CBH catalytic domains used for RCA analysis. Figure S9. Phylogenetic tree of GH7 catalytic domain protein sequences from Trichoderma spp. and Fusarium spp. Table S3. S scores from RCA analysis for residues of interest for TatCel7A, ThaCel7A and TreCel7A. Additional MD simulation results Figure S10. RMSD as a function of time for each 100-ns, ligand-bound MD simulation of TatCel7A, ThaCel7A and TreCel7A catalytic domains.

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.

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.

Ninety bacteria isolated from raw composting materials were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. The bacteria producing the highest cellulolytic activity levels were identified by 16S rRNA sequencing as Bacillus licheniformis strain 1, Bacillus subtilis subsp. subtilis strain B7B, Bacillus subtilis subsp. spizizenii strain 6, and Bacillus amyloliquefaciens strain B31C. Cellulase activity production by the most productive strain B. amyloliquefaciens B31C was optimized in liquid culture varying the carbon source. Comparison of growth curves of B. amyloliquefaciens B31C at temperatures from 28 to 47 °C indicated its thermotolerant nature. Moreover, analysis of time courses of cellulase activity production in this thermal range showed that increase of temperature from 28 to 37 °C causes an increase of cellulase activity levels. Investigating the enzymes responsible for cellulase activity produced by B. amyloliquefaciens B31C by proteomic analyses, an endoglucanase was identified. It was shown that the purified enzyme catalyzes carboxymethylcellulose's hydrolysis following Michaelis-Menten kinetics with a K(M) of 9.95 mg ml(-1) and a v(max) of 284 μM min(-1) . It shows a retention of 90% of its activity for at least 144 h of incubation at 40 °C and exhibits a range of optimum temperatures from 50 to 70 °C.

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.

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.

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.

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.

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.

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.

Fossil fuel reserves depletion, global warming, costly and problematic waste recycling and population growth greatly induce to fi nd renewable energy sources. Second generation bioethanol produced from lignocellulosic materials exhibits great potential as liquid biofuel to substitute gasoline. Production costs of enzymes involved in cellulose hydrolysis into fermentable sugars represent the main obstacle to achieve competitive production of cellulosic ethanol. Cheaper and more effi cient biocatalysts for the saccharifi cation step are, therefore, required for making the whole process more competitive. The biodiversity of natural niches has been so far exploited for the isolation of new cellulolytic microorganisms whose enzymes are naturally evolved for an effi cient conversion of cellulose into fermentable sugars. This review discusses advances in isolation of bacteria, namely Bacillus spp., from several natural habitats and their ability to produce cellulase activity.