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Colletotrichum lindemuthianum is a phytopathogenic fungus that causes anthracnose in common beans (Phaseolus vulgaris) and presents a great diversity of pathotypes with different levels of virulence against bean varieties worldwide. The purpose of this study was to establish whether pathotypic diversity is associated with differences in the mycelial growth and secretion of plant-cell-wall-degrading enzymes (PCWDEs). We evaluated growth, hemicellulase and cellulase activity, and PCWDE secretion in four pathotypes of C. lindemuthianum in cultures with glucose, bean hypocotyls and green beans of P. vulgaris, and water hyacinth (Eichhornia crassipes). The results showed differences in the mycelial growth, hemicellulolytic activity, and PCWDE secretion among the pathotypes. Glucose was not the preferred carbon source for the best mycelial growth in all pathotypes, each of which showed a unique PCWDE secretion profile, indicating different levels of carbon catabolite regulation (CCR). The pathotypes showed a high differential hemicellulolytic capacity to degrade host and water hyacinth tissues, suggesting CCR by pentoses and that there are differences in the absorption and metabolism of different monosaccharides and/or disaccharides. We propose that different levels of CCR could optimize growth in different host tissues and could allow for consortium behavior in interactions with bean crops. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass holds promise as a renewable feedstock for various applications, but its efficient conversion requires cost-effective degradation strategies. The main objective of this study was to investigate the effect of the growth conditions of Cellulomonas phragmiteti in the production of (hemi)cellulosic supernatants. To meet this aim, different lignocellulosic residues were used as carbon sources for growth using defined mineral or nutritive culture media. Cell-free culture supernatants with xylanolytic activity were produced in all the conditions evaluated, but the highest xylanase activity (15.3 U/mL) was achieved in Luria–Bertani (LB) medium containing 1% waste paper. Under these conditions, almost negligible β-glucosidase, cellobiohydrolase, β-xylosidase, and α-arabinofuranosidase activity was detected. The xylanolytic supernatant showed tolerance to salt and displayed maximal catalytic efficiency at pH 6 and 45 °C, along with good activity in the ranges of 45–55 °C and pH 5–8. As it showed good stability at 45 °C, the supernatant was employed for the hydrolysis of birchwood xylan (50 g/L) under optimal conditions, releasing 10.7 g/L xylose in 72 h. Thus, C. phragmiteti was found to produce a xylanolytic enzymatic supernatant efficiently by utilizing the cheap and abundant lignocellulosic residue of waste paper, and the produced supernatant has promising attributes for industrial applications. [ABSTRACT FROM AUTHOR]

Colletotrichum lindemuthianum is a phytopathogenic fungus that causes anthracnose in common beans (Phaseolus vulgaris) and presents a great diversity of pathotypes with different levels of virulence against bean varieties worldwide. The purpose of this study was to establish whether pathotypic diversity is associated with differences in the mycelial growth and secretion of plant-cell-wall-degrading enzymes (PCWDEs). We evaluated growth, hemicellulase and cellulase activity, and PCWDE secretion in four pathotypes of C. lindemuthianum in cultures with glucose, bean hypocotyls and green beans of P. vulgaris, and water hyacinth (Eichhornia crassipes). The results showed differences in the mycelial growth, hemicellulolytic activity, and PCWDE secretion among the pathotypes. Glucose was not the preferred carbon source for the best mycelial growth in all pathotypes, each of which showed a unique PCWDE secretion profile, indicating different levels of carbon catabolite regulation (CCR). The pathotypes showed a high differential hemicellulolytic capacity to degrade host and water hyacinth tissues, suggesting CCR by pentoses and that there are differences in the absorption and metabolism of different monosaccharides and/or disaccharides. We propose that different levels of CCR could optimize growth in different host tissues and could allow for consortium behavior in interactions with bean crops.

One key factor in the design of enzymatic cocktails for the lignocellulosic biomass breakdown is the performance of bacteria. Bosea sp. FBZP-16, a high-performing bacterium strain producing different types of cellulases and hemicellulases, was evaluated for production of global (hemi)cellulolytic enzymes under different growth conditions. Different (hemi)cellulases were predominantly produced. On Sigmacell 101 and newspaper, the highest specific cellobiohydrolase activity was found with 830.8 U/mg and 1341.2 U/mg, respectively. Beechwood xylan revealed the highest β-xylosidase activity (1625.3 U/mg) consistent with the highest xylose release. Cultivation temperature of 30 and 35 °C produced most (hemi)cellulolytic enzymes between the 3rd and the 9th days of incubation at shaking frequencies from 150 up to 300 rpm, while the highest glucose and xylose yields were observed at 150 rpm. Respiration activity monitoring system analysis confirmed the potential of Bosea sp. FBPZ-16 to grow on xylan. The production of cellobiohydrolase and endoxylanase was further investigated using response surface methodology (RSM) and Box-Behnken matrix. The optimal conditions of the tested parameters were carbon source concentration (10 g/L), nitrogen source concentration (3 g/L), inoculum size (3 mL inoculum/100 mL media), and pH 7.0, where the maximal cellobiohydrolase production was found to be 2797 U/mg, which was close to the predicted activity of 2275.37 U/mg. The maximal endoxylanase production reached 60 U/mg, with a predicted value of 49.75 U/mg. [ABSTRACT FROM AUTHOR]

The primary contributor to global warming has been the careless usage of fossil fuels. Urbanization's threat to the depletion of these resources has made it necessary to find alternatives due to the rising demand. Four different forms of biofuels are now available and constitute a possible replacement for fossil fuels. The first generation of biofuels is generated from the edible portion of biomass, the second generation is made from the non-edible portion of biomass, the third generation is made from algal biomass, and the fourth generation is made using molecular biology to improve the algal strain. Second-generation biofuels are extremely important because they are derived from non-edible biomass, such as agricultural and agro-industrial wastes rich in cellulose, hemicellulose, pectin, and starch impregnated with lignin, and are hydrolyzed after delignification by physio-chemical or biological pretreatments using ligninases. The enzymes involved in the hydrolysis of feedstocks for the production of second-generation bioethanol, a highly acceptable biofuel, are discussed in this article. Furthermore, the article discusses various fermentation technologies as well as significant developments in second-generation biofuel production by combining various microbial enzyme systems. [ABSTRACT FROM AUTHOR]

Abstract Background The microbial production of hemicellulasic cocktails is still a challenge for the biorefineries sector and agro-waste valorization. In this work, the production of hemicellulolytic enzymes by Thermobacillus xylanilyticus has been considered. This microorganism is of interest since it is able to produce an original set of thermostable hemicellulolytic enzymes, notably a xylanase GH11, Tx-xyn11. However, cell-to-cell heterogeneity impairs the production capability of the whole microbial population. Results Sequential cultivations of the strain on xylan as a carbon source has been considered in order to highlight and better understand this cell-to-cell heterogeneity. Successive cultivations pointed out a fast decrease of xylanase activity (loss of ~ 75%) and Tx-xyn11 gene expression after 23.5 generations. During serial cultivations on xylan, flow cytometry analyses pointed out that two subpopulations, differing at their light-scattering properties, were present. An increase of the recurrence of the subpopulation exhibiting low forward scatter (FSC) signal was correlated with a progressive loss of xylanase activity over several generations. Cell sorting and direct observation of the sorted subpopulations revealed that the low-FSC subpopulation was not sporulating, whereas the high-FSC subpopulation contained cells at the onset of the sporulation stage. The subpopulation differences (growth and xylanase activity) were assessed during independent growth. The low-FSC subpopulation exhibited a lag phase of 10 h of cultivation (and xylanase activities from 0.15 ± 0.21 to 3.89 ± 0.14 IU/mL along the cultivation) and the high-FSC subpopulation exhibited a lag phase of 5 h (and xylanase activities from 0.52 ± 0.00 to 4.43 ± 0.61 over subcultivations). Serial cultivations on glucose, followed by a switch to xylan led to a ~ 1.5-fold to ~ 15-fold improvement of xylanase activity, suggesting that alternating cultivation conditions could lead to an efficient population management strategy for the production of xylanase. Conclusions Taken altogether, the data from this study point out that a cheating behavior is responsible for the progressive reduction in xylanase activity during serial cultivations of T. xylanilyticus. Alternating cultivation conditions between glucose and xylan could be used as an efficient strategy for promoting population stability and higher enzymatic productivity from this bacterium.

Abstract Background Hemicellulose acts as one factor contributing to the recalcitrance of lignocellulose that prevents cellulases to degrade the cellulose efficiently even in low quantities. Supplement of hemicellulases can enhance the performance of commercial cellulases in the enzymatic hydrolyses of lignocellulose. Kluyveromyce marxianus is an attractive yeast for cellulosic ethanol fermentation, as well as a promising host for heterologous protein production, since it has remarkable thermotolerance, high growth rate, and broad substrate spectrum etc. In this study, we attempted to coordinately express multiple hemicellulases in K. marxianus through a 2A-mediated ribosome skipping to self-cleave polyproteins, and investigated their capabilities for saccharification and ethanol production from corncobs. Results Two polycistronic genes IMPX and IMPαX were constructed to test the self-cleavage of P2A sequence from the Foot-and-Mouth Disease virus (FMDV) in K. marxianus. The IMPX gene consisted of a β-mannanase gene M330 (without the stop codon), a P2A sequence and a β-xylanase gene Xyn-CDBFV in turn. In the IMPαX gene, there was an additional α-factor signal sequence in frame with the N-terminus of Xyn-CDBFV. The extracellular β-mannanase activities of the IMPX and IMPαX strains were 21.34 and 15.50 U/mL, respectively, but the extracellular β-xylanase activity of IMPαX strain was much higher than that of the IMPX strain, which was 136.17 and 42.07 U/mL, respectively. Subsequently, two recombinant strains, the IXPαR and IMPαXPαR, were constructed to coordinately and secretorily express two xylantic enzymes, Xyn-CDBFV and β-D-xylosidase RuXyn1, or three hemicellulolytic enzymes including M330, Xyn-CDBFV and RuXyn1. In fed-batch fermentation, extracellular activities of β-xylanase and β-xylosidase in the IXPαR strain were 1664.2 and 0.90 U/mL. Similarly, the IMPαXPαR strain secreted the three enzymes, β-mannanase, β-xylanase, and β-xylosidase, with the activities of 159.8, 2210.5, and 1.25 U/mL, respectively. Hemicellulolases of both strains enhanced the yields of glucose and xylose from diluted acid pretreated (DAP) corncobs when acted synergistically with commercial cellulases. In hybrid saccharification and fermentation (HSF) of DAP corncobs, hemicellulases of the IMPαXPαR strain increased the ethanol yield by 8.7% at 144 h compared with the control. However, both ethanol and xylose yields were increased by 12.7 and 18.2%, respectively, at 120 h in HSF of aqueous ammonia pretreated (AAP) corncobs with this strain. Our results indicated that coordinate expression of hemicellulolytic enzymes in K. marxianus promoted the saccharification and ethanol production from corncobs. Conclusions The FMDV P2A sequence showed high efficiency in self-cleavage of polyproteins in K. marxianus and could be used for secretory expression of multiple enzymes in the presence of their signal sequences. The IMPαXPαR strain coexpressed three hemicellulolytic enzymes improved the saccharification and ethanol production from corncobs, and could be used as a promising strain for ethanol production from lignocelluloses.

The xylanolytic potential of endophytic fungi isolated from leaves of Theobroma cacao was explored for the first time. Four fungal strains showed significant amounts of xylanase activity and low cellulase levels when grown on wheat bran as the sole carbon source. Strain Ec220 of Fusarium graminearum had the highest xylanase production (1.79 U/ml), whereas its cellulase activity was minimal (0.24 U/ml). Optimal conditions for xylanase production were: 154 h of incubation time, pH 5.79 and 29.8 °C. Furthermore, two protein spots detected by two-dimensional gel electrophoresis showed molecular weights (26.05 and 27.70 kDa) and isoelectric points (6.18 and 9.20) corresponding to previously reported F. graminearum xylanases, Xyl A and Xyl B, respectively. Therefore, endophytic fungi of T. cacao can be an important source of xylanolytic activities when cultured on wheat bran, and xylanases with low cellulases found in strain Ec220 require further characterization as they show promise for possible industrial applications.

Ultrasound technology is often associated with harmful effects on enzyme reactions, although it is possible to improve the productivity of bioprocesses when suitable conditions are employed. Sugarcane bagasse and straw are the feedstocks widely used in Brazil for second-generation (2G) ethanol production; however, the lignocellulose biomass conversion into fermentable sugars through the enzymatic route is not yet fully optimized. Lignocellulolytic enzymes represent a significant part of the costs related to 2G ethanol production. Nonetheless, they exhibit great potential for cost reduction due to improved enzyme features: mainly increment of its activity and an increase of hydrolysis yield. This enzymatic hydrolysis of feedstock can be enhanced by green technology ultrasound application's combined action on the enzymes and their substrates. The mixed action increases the lignocellulose saccharification; hence, it is considered a promising alternative for fermentable sugar release. The process optimization using green approaches, such as ultrasound and enzymatic treatment, can boost the sugar yield, thus emphasizing the importance of steps integration towards biomass conversion. This review attempts to provide an overview of the effects of ultrasound treatment on lignocellulolytic enzymes used in the 2G ethanol production and those of the process intensification through an unprecedented bibliometric search. [ABSTRACT FROM AUTHOR]

Background: The microbial production of hemicellulasic cocktails is still a challenge for the biorefineries sector and agro-waste valorization. In this work, the production of hemicellulolytic enzymes by Thermobacillus xylanilyticus has been considered. This microorganism is of interest since it is able to produce an original set of thermostable hemicellulolytic enzymes, notably a xylanase GH11, Tx-xyn11. However, cell-to-cell heterogeneity impairs the production capability of the whole microbial population. Results: Sequential cultivations of the strain on xylan as a carbon source has been considered in order to highlight and better understand this cell-to-cell heterogeneity. Successive cultivations pointed out a fast decrease of xylanase activity (loss of ~ 75%) and Tx-xyn11 gene expression after 23.5 generations. During serial cultivations on xylan, flow cytometry analyses pointed out that two subpopulations, differing at their light-scattering properties, were present. An increase of the recurrence of the subpopulation exhibiting low forward scatter (FSC) signal was correlated with a progressive loss of xylanase activity over several generations. Cell sorting and direct observation of the sorted subpopulations revealed that the low-FSC subpopulation was not sporulating, whereas the high-FSC subpopulation contained cells at the onset of the sporulation stage. The subpopulation differences (growth and xylanase activity) were assessed during independent growth. The low-FSC subpopulation exhibited a lag phase of 10 h of cultivation (and xylanase activities from 0.15 ± 0.21 to 3.89 ± 0.14 IU/mL along the cultivation) and the high-FSC subpopulation exhibited a lag phase of 5 h (and xylanase activities from 0.52 ± 0.00 to 4.43 ± 0.61 over subcultivations). Serial cultivations on glucose, followed by a switch to xylan led to a ~ 1.5-fold to ~ 15-fold improvement of xylanase activity, suggesting that alternating cultivation conditions could lead to an efficient population management strategy for the production of xylanase. Conclusions: Taken altogether, the data from this study point out that a cheating behavior is responsible for the progressive reduction in xylanase activity during serial cultivations of T. xylanilyticus. Alternating cultivation conditions between glucose and xylan could be used as an efficient strategy for promoting population stability and higher enzymatic productivity from this bacterium. [ABSTRACT FROM AUTHOR]

Many soil Actinobacteria are potent producers of extracellular enzymes decomposing lignocellulose. Four strains of Actinobacteria with a high potential to hydrolyse cellulose and hemicellulose were identified among environmental isolates. The strains were grown on raw lignocellulosic substrates (olive pomace, oat flakes, sawdust, and wheat straw) under submerged fermentation in a laboratory scale. Modified Melin Norkrans Medium amended with raw lignocellulosic substrates as carbon sources (0.5%) was used to enhance lignocellulosic biomass decomposition. Three strains belonged to the genus Streptomyces and one strain to the genus Mycobacterium. Annotation of genomes showed high proportion of genes encoding for carbohydrate-active enzymes in Streptomyces sp. GESEQ-4 (537, i.e. 6% of 8404 genes), Streptomyces sp. GESEQ-13 (351 (5%) of 6705 genes), Streptomyces sp. GESEQ-35 (608 (6%) of 9788 genes), and Mycolicibacterium fortuitum subsp. fortuitum GESEQ-9 (222 (3%) of 6405 genes). These included plant cell wall-degrading enzymes belonging to the families GH1, GH2, GH3, GH5, GH6, GH9, GH10, GH12, GH16, GH26, GH30, GH39, GH48, GH51, and GH74, of which GH1, GH2, GH3, GH5, GH6, and GH16 were found in all four genomes. Assays for cellulose and hemicellulose degrading extracellular enzymes confirmed the ability of the isolates to decompose cellulose and hemicellulose. The highest endo-cleaving enzyme activities were produced by the strain Steptomyces sp. GESEQ-4 DSM 106287. Our results provide new perspectives into the enzymatic array by which the Actinobacteria break down complex lignocellulosic biomass. It is crucial to assess the genome to determine enzyme function as well as the enzyme families responsible for the degradation process in Actinobacteria. The potential degradation functions for these actinobacterial strains were validated by testing their cellulolytic and hemicellulolytic activities with various lignocellulosic substrates. [ABSTRACT FROM AUTHOR]

Background: Hemicellulose acts as one factor contributing to the recalcitrance of lignocellulose that prevents cellulases to degrade the cellulose efficiently even in low quantities. Supplement of hemicellulases can enhance the performance of commercial cellulases in the enzymatic hydrolyses of lignocellulose. Kluyveromyce marxianus is an attractive yeast for cellulosic ethanol fermentation, as well as a promising host for heterologous protein production, since it has remarkable thermotolerance, high growth rate, and broad substrate spectrum etc. In this study, we attempted to coordinately express multiple hemicellulases in K.marxianus through a 2A-mediated ribosome skipping to self-cleave polyproteins, and investigated their capabilities for saccharification and ethanol production from corncobs. Results: Two polycistronic genes IMPX and IMPαX were constructed to test the self-cleavage of P2A sequence from the Foot-and-Mouth Disease virus (FMDV) in K.marxianus. The IMPX gene consisted of a β-mannanase gene M330 (without the stop codon), a P2A sequence and a β-xylanase gene Xyn-CDBFV in turn. In the IMPαX gene, there was an additional α-factor signal sequence in frame with the N-terminus of Xyn-CDBFV. The extracellular β-mannanase activities of the IMPX and IMPαX strains were 21.34 and 15.50 U/mL, respectively, but the extracellular β-xylanase activity of IMPαX strain was much higher than that of the IMPX strain, which was 136.17 and 42.07 U/mL, respectively. Subsequently, two recombinant strains, the IXPαR and IMPαXPαR, were constructed to coordinately and secretorily express two xylantic enzymes, Xyn-CDBFV and β-D-xylosidase RuXyn1, or three hemicellulolytic enzymes including M330, Xyn-CDBFV and RuXyn1. In fed-batch fermentation, extracellular activities of β-xylanase and β-xylosidase in the IXPαR strain were 1664.2 and 0.90 U/mL. Similarly, the IMPαXPαR strain secreted the three enzymes, β-mannanase, β-xylanase, and β-xylosidase, with the activities of 159.8, 2210.5, and 1.25 U/mL, respectively. Hemicellulolases of both strains enhanced the yields of glucose and xylose from diluted acid pretreated (DAP) corncobs when acted synergistically with commercial cellulases. In hybrid saccharification and fermentation (HSF) of DAP corncobs, hemicellulases of the IMPαXPαR strain increased the ethanol yield by 8.7% at 144 h compared with the control. However, both ethanol and xylose yields were increased by 12.7 and 18.2%, respectively, at 120 h in HSF of aqueous ammonia pretreated (AAP) corncobs with this strain. Our results indicated that coordinate expression of hemicellulolytic enzymes in K. marxianus promoted the saccharification and ethanol production from corncobs. Conclusions: The FMDV P2A sequence showed high efficiency in self-cleavage of polyproteins in K. marxianus and could be used for secretory expression of multiple enzymes in the presence of their signal sequences. The IMPαXPαR strain coexpressed three hemicellulolytic enzymes improved the saccharification and ethanol production from corncobs, and could be used as a promising strain for ethanol production from lignocelluloses. [ABSTRACT FROM AUTHOR]

• Distinct α-arabinofuranosidases boosted MeGlc p A release by α-glucuronidases. • Rates of an α-arabinofuranosidase increased by approximately 300 %. • The crystal structure of α-glucuronidase AxyAgu115A was solved. • A distinct loop in AxyAgu115A might contribute to its coordinated action. The coordinated action of carbohydrate-active enzymes has mainly been evaluated for the purpose of complete saccharification of plant biomass (lignocellulose) to sugars. By contrast, the coordinated action of accessory hemicellulases on xylan debranching and recovery is less well characterized. Here, the activity of two family GH115 α-glucuronidases (SdeAgu115A from Saccharophagus degradans , and AxyAgu115A from Amphibacillus xylanus) on spruce arabinoglucuronoxylan (AGX) was evaluated in combination with an α-arabinofuranosidase from families GH51 (AniAbf51A, aka E-AFASE from Aspergillus niger) and GH62 (SthAbf62A from Streptomyces thermoviolaceus). The α-arabinofuranosidases boosted (methyl)-glucuronic acid release by SdeAgu115A by approximately 50 % and 30 %, respectively. The impact of the α-arabinofuranosidases on AxyAgu115A activity was comparatively low, motivating its structural characterization. The crystal structure of AxyAgu115A revealed increased length and flexibility of the active site loop compared to SdeAgu115A. This structural difference could explain the ability of AxyAgu115A to accommodate more highly substituted arabinoglucuronoxylan, and inform enzyme selections for improved AGX recovery and use. [ABSTRACT FROM AUTHOR]

The transition from an economic model based on resource extraction to a more sustainable and circular economy requires the development of innovative methods to unlock the potential of raw materials such as lignocellulosic biomasses. Corn fiber differs from more traditional lignocellulosic biomasses due to its high starch content, which provides additional carbohydrates for fermentation-based biomanufacturing processes. Due to its unique chemical composition, this study focused on the development of a tailor made enzymatic cocktail for corn fiber saccharification into monosaccharides. Three commercially available hydrolytic enzymes (Cellic® CTec2, Pentopan® Mono BG, and Termamyl® 300 L) were combined to hydrolyze the polysaccharide structure of the three main carbohydrate fractions of corn fiber (cellulose, hemicellulose and starch, respectively). Prior to saccharification, corn fiber was submitted to a mild hydrothermal pretreatment (30 min at 100 °C). Then, two experimental designs were used to render an enzymatic cocktail capable of providing efficient release of monosaccharides. Using 60 FPU/g DM of Cellic® CTec2 and 4.62 U/g DM of Termamyl® 300 L, without addition of Pentopan® Mono BG, resulted in the highest efficiencies for glucose and xylose release (66% and 30%, respectively). While higher enzyme dosages could enhance the saccharification efficiency, adding more enzymes would have a more pronounced effect on the overall process costs rather than in increasing the efficiency for monosaccharides release. The results revealed that the recalcitrance of corn fiber poses a problem for its full enzymatic degradation. This fact combined with the unique chemical composition of this material, justify the need for developing a tailor made enzymatic cocktail for its degradation. However, attention should also be given to the pretreatment step to reduce even more the recalcitrance of corn fiber and improve the performance of the tailored cocktail, as a consequence. • Corn fiber's recalcitrance poses a limit for its enzymatic hydrolysis. • An enzymatic cocktail was tailored for hydrolysis of corn fiber into monosaccharides. • Cellulase was the main enzyme present in the tailored enzymatic cocktail. • An efficient pretreatment is crucial to enhance the tailored cocktail's performance. [ABSTRACT FROM AUTHOR]

Approximately a tenth of all described fungal species can cause diseases in plants. A common feature of this process is the necessity to pass through the plant cell wall, an important barrier against pathogen attack. To this end, fungi possess a diverse array of secreted enzymes to depolymerize the main structural polysaccharide components of the plant cell wall, i.e., cellulose, hemicellulose, and pectin. Recent advances in genomic and systems-level studies have begun to unravel this diversity and have pinpointed cell wall-degrading enzyme (CWDE) families that are specifically present or enhanced in plant-pathogenic fungi. In this review, we discuss differences between the CWDE arsenal of plant-pathogenic and non-plant-pathogenic fungi, highlight the importance of individual enzyme families for pathogenesis, illustrate the secretory pathway that transports CWDEs out of the fungal cell, and report the transcriptional regulation of expression of CWDE genes in both saprophytic and phytopathogenic fungi.

Glucuronoxylans represent a significant fraction of woody biomass, and its decomposition is complicated by the presence of lignin–carbohydrate complexes (LCCs). Herein, LCCs from birchwood were used to investigate the potential coordinated action of a glucuronoyl esterase (TtCE15A) and two α‐glucuronidases (SdeAgu115A and AxyAgu115A). When supplementing α‐glucuronidase with equimolar quantities of TtCE15A, total MeGlcpA released after 72 h by SdeAgu115A and AxyAgu115A increased from 52% to 67%, and 61% to 95%, respectively. Based on the combined TtCE15A and AxyAgu115A activities, ~ 34% of MeGlcpA in the extracted birchwood glucuronoxylan was occupied as LCCs. Notably, insoluble LCC fractions reduced soluble α‐glucuronidase concentrations by up to 70%, whereas reduction in soluble TtCE15A was less than 30%, indicating different tendencies to adsorb onto the LCC substrate. [ABSTRACT FROM AUTHOR]

Biodiesel is an alternative to fossil diesel, non-toxic, and less pollutant. The production of biodiesel occurs with the use of oils, which are extracted from oleaginous seeds such as soybean, rapeseed, and palm fruit. The extraction of oils from oilseeds is one bottleneck of biodiesel production impacting on significant processes' losses and productivity. This review analyzes different technologies to improve the oil extraction focusing on the enzyme-assisted aqueous extraction (EAAE) methods. EAAE is an environmentally friendly technology that takes advantage of the degradation efficiency of the enzymes, in this case hemicellulases preparations, which specifically degrade different structures that are present in vegetable cell walls to improve oil extraction from oilseeds. The enzymes used in this process are industrially produced and are highly efficient, but with high costs. The use of agro-industrial subproducts or the improvement of the enzymes' producing strains could be an interesting solution for viable enzymatic preparations' production to attend the biodiesel industries' demand. EAAE treatment also shows great potential in techno-economic analysis allowing a faster recovery of profits and at a smaller production scale than the hexane extraction methodology. [ABSTRACT FROM AUTHOR]

Synergistic saccharification ability of hemicellulases (endo-xylanase and β-xylosidase) was evaluated in this study for the bioethanol production from plant biomass. Endo-xylanase and β-xylosidase genes from Bacillus licheniformis were cloned and expressed in Escherichia coli BL21 (DE3). Maximum endo-xylanase production was obtained at 200 rpm agitation speed, air supply rate 2.0 vvm, 70% volume of the medium, 20% dissolved oxygen level and with 3% inoculum size. The optimal conditions for maximum production of recombinant β-xylosidase enzyme at pilot scale were 200 rpm agitation speed, 25% dissolved oxygen level, 2.5 vvm aeration rate, 70% volume of the medium with 2% inoculum size. Furthermore, the saccharification potential of these recombinant enzymes was checked for the production of xylose sugar by bioconversion of plant biomass by optimizing individually as well as synergistically by optimizing various parameters. Maximum saccharification (93%) of plant biomass was observed when both enzymes were used at a time with 8% sugarcane bagasse as a substrate and 200 units of each enzyme after incubation of 6 hr at 50 °C and 120 rpm. The results obtained in this study suggested these recombinant hemicellulases as potential candidates for the conversion of complex agricultural residues into simple sugars for ultimate use in the biofuel industry. [ABSTRACT FROM AUTHOR]

This review summarizes the findings on the use of the Penicillium verruculosum В1-537 (ΔniaD) recipient strain for the development of producers of enzymes—β-glucosidase, polysaccharide monooxygenase, and xylanases—that complement the basic cellulase complex and enhance its effectiveness. A special feature of the P. verruculosum strain is the secretion of a basic cellulase complex consisting of cellobiohydrolases and endoglucanases (EGs) that, in terms of their hydrolytic ability, surpass Hypocrea (Trichoderma) strains that are widely used in the laboratory and in industry. The capabilities of the P. verruculosum В1-537 (ΔniaD) expression system can be used to complement the basic cellulase complex with new enzymes, which are required to enhance its hydrolytic activity, and to develop new enzyme preparations tailored to the conversion of different types of renewable plant biomass (RPB). The use of an expression system based on P. verruculosum fungus makes it possible to enhance the ability of basic cellulase complex to convert different types of RPB. [ABSTRACT FROM AUTHOR]

Recycling biomass is indispensable these days not only because fossil energy sources are gradually depleted, but also because pollution of the environment, caused by the increasing use of energy, must be reduced. This article intends to overview the results of plant biomass processing methods that are currently in use. Our aim was also to review published methods that are not currently in use. It is intended to explore the possibilities of new methods and enzymes to be used in biomass recycling. The results of this overview are perplexing in almost every area. Advances have been made in the pre-treatment of biomass and in the diversity and applications of the enzymes utilized. Based on molecular modeling, very little progress has been made in the modification of existing enzymes for altered function and adaptation for the environmental conditions during the processing of biomass. There are hardly any publications in which molecular modeling techniques are used to improve enzyme function and to adapt enzymes to various environmental conditions. Our view is that using modern computational, biochemical, and biotechnological methods would enable the purposeful design of enzymes that are more efficient and suitable for biomass processing. [ABSTRACT FROM AUTHOR]

To investigate the production of cellulases and hemicellulases from Aspergillus niger MTCC9931, solid state fermentation (SSF) was performed using 10 different lignocellulosic materials derived from agrowastes, i.e., rice straw, rice husk, wheat straw, corn cob, sugar cane bagasse, saw dust, banana stalk, Eichhornia, Parthenium stalk, and residual fruit pulp. Among these agrowastes, the maximum yield of reducing sugars (116.46 ± 2.56 g/mL) was observed with residual fruit pulp. Further, the same substrate showed the highest endoglucanase (389.1 ± 0.44 IU/g), MCC-adsorbable endoglucanase (3.4 ± 0.14 IU/g), cellulase (12.0 ± 0.13 IU/g), and FPase (4.9 ± 0.64 IU/g) activities. Sawdust showed the maximum xylanase activity (7478.0 ± 6.51 IU/g), and corncob showed maximum β-glucosidase activity (79.87 ± 1.15 IU/g). The maximum activities of all enzymes were observed at 72 h of SSF under shaking conditions. A. niger MTCC9931 can be concluded to be an important strain for potential applications in saccharification. The enzymatic hydrolysates of the agrowastes were used as substrates for ethanol production by Saccharomyces cerevisiae MTCC174. The maximum yield (35.34g/L) of ethanol was obtained when residual fruit pulp was used as a substrate. This study has further demonstrated the feasible biotechnological conversion of agrowastes to produce ethanol using both A. niger and S. cerevisiae. [ABSTRACT FROM AUTHOR]

This study evaluated the production of lignocellulose-degrading enzymes by solid-state fermentation (SSF) using a microbial consortium of Aspergillus fumigatus SCBM6 and A. niger SCBM1 (AFN extract). The fungal strains were cultivated in sugarcane bagasse (SCB) and wheat bran (WB) as lignocellulosic substrates for 7 days at 30 °C. After SSF, the highest peaks of enzyme production were 150 and 80 U g−1 for β-xylosidase and β-glucosidase at 48 h, 375 U g−1 for xylanase at 96 h, and 80 U g−1 for endoglucanase and 4 U g−1 for cellulase activity on filter paper (FPase) at 144 h. The efficiency of the produced AFN extract was investigated in the enzymatic hydrolysis of crude biomass sorghum (BS) and after the removal of extractives (ES). After saccharification, the glucose and xylose concentrations were 10× superior in ES than in BS hydrolysate (2.5 g L−1 after 12 h). The presence of inhibitors of alcoholic fermentation, such as formic acid, was also reduced in ES hydrolysates, indicating that the removal of extractives positively contributed to the effectiveness of enzymatic hydrolysis of biomass sorghum using AFN extract. [ABSTRACT FROM AUTHOR]

• Pseudoalteromonas atlantica produced a high diversity of polysaccharide-degrading enzymes. • The marine bacterium Pseudoalteromonas atlantica secreted enzymes active towards hemicelluloses from terrestrial plants. • β-xylosidase, α-arabinofuranosidase and acetylesterase activities were evidenced in the secretome of P seudoalteromonas atlantica. • The GH8 protein present in Pseudoalteromonas atlantica genome is a xylanase with unusual product profile. To address the need for efficient enzymes exhibiting novel activities towards cell wall polysaccharides, the bacterium Pseudoalteromonas atlantica was selected based on the presence of potential hemicellulases in its annotated genome. It was grown in the presence or not of hemicelluloses and the culture filtrates were screened towards 42 polysaccharides. P. atlantica showed appreciable diversity of enzymes active towards hemicelluloses from Monocot and Dicot origin, in agreement with its genome annotation. After growth on beechwood glucuronoxylan and fractionation of the secretome, a β-xylosidase, a α-arabinofuranosidase and an acetylesterase activities were evidenced. A GH8 enzyme obtained in the same growth conditions was further cloned and heterologously overexpressed. It was shown to be a xylanase active on heteroxylans from various sources. The detailed study of its mode of action demonstrated that the oligosaccharides produced carried a long tail of un-substituted xylose residues on the reducing end. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Identification of two novel, thermostable hemicellulolytic enzymes from T. neapolitana. • Design of an enzyme cocktail for the deconstruction of lignocellulose in bioenergy feedstock at high temperatures. • Treatment of complex substrates with the hemicellulolytic cocktail increased the enzymatic degradation of cellulose up to 65%. Abstract The biological conversion of lignocellulose into fermentable sugars is a key process for the sustainable production of biofuels from plant biomass. Polysaccharides in plant feedstock can be valorized using thermostable mixtures of enzymes that degrade the cell walls, thus avoiding harmful and expensive pre-treatments. (Hyper)thermophilic bacteria of the phylum Thermotogae provide a rich source of enzymes for such industrial applications. Here we selected T. neapolitana as a source of hyperthermophilic hemicellulases for the degradation of lignocellulosic biomass. Two genes encoding putative hemicellulases were cloned from T. neapolitana genomic DNA and expressed in Escherichia coli. Further characterization revealed that the genes encoded an endo-1,4-β-galactanase and an α- l -arabinofuranosidase with optimal temperatures of ˜90 °C and high turnover numbers during catalysis (k cat values of ˜177 and ˜133 s−1, respectively, on soluble substrates). These enzymes were combined with three additional T. neapolitana hyperthermophilic hemicellulases – endo-1,4-β-xylanase (XynA), endo-1,4-β-mannanase (ManB/Man5A) and β-glucosidase (GghA) – to form a highly thermostable hemicellulolytic blend. The treatment of barley straw and corn bran with this enzymatic cocktail resulted in the solubilization of multiple hemicelluloses and boosted the yield of fermentable sugars by up to 65% when the complex substrates were further degraded by cellulases. [ABSTRACT FROM AUTHOR]

Soil microorganisms are important mediators of carbon cycling in nature. Although cellulose- and hemicellulose-degrading bacteria have been isolated from Algerian ecosystems, the information on the composition of soil bacterial communities and thus the potential of their members to decompose plant residues is still limited. The objective of the present study was to describe and compare the bacterial community composition in Algerian soils (crop, forest, garden, and desert) and the activity of cellulose- and hemicellulose-degrading enzymes. Bacterial communities were characterized by high-throughput 16S amplicon sequencing followed by the in silico prediction of their functional potential. The highest lignocellulolytic activity was recorded in forest and garden soils whereas activities in the agricultural and desert soils were typically low. The bacterial phyla Proteobacteria (in particular classes α–proteobacteria, δ–proteobacteria, and γ–proteobacteria), Firmicutes, and Actinobacteria dominated in all soils. Forest and garden soils exhibited higher diversity than agricultural and desert soils. Endocellulase activity was elevated in forest and garden soils. In silico analysis predicted higher share of genes assigned to general metabolism in forest and garden soils compared with agricultural and arid soils, particularly in carbohydrate metabolism. The highest potential of lignocellulose decomposition was predicted for forest soils, which is in agreement with the highest activity of corresponding enzymes. [ABSTRACT FROM AUTHOR]

The filamentous fungus Trichoderma reesei is traditionally used as the main industrial producer of cellulases and hemicellulases. Recently, the relevance of carbohydrases hydrolyzing nonstarch polysaccharides of cereals has significantly increased in feed production. In processing of grain raw materials, endodepolymerases, mainly xylanases and endoglucanases, play a key role. Earlier, we carried out gamma mutagenesis of an industrial strain T. reesei BCM18.2/KK to increase the proportion of endodepolymerases in its enzyme complex. Endoglucanase activity of the strain was increased 5-fold, while xylanase activity increased more than 8-fold. It was interesting to determine the carbohydrases composition in enzyme preparations obtained from the original and mutant T. reesei strains. So, the strains were cultured in laboratory fermenters; concentrated preparations were obtained using freeze dryer. It was established that gamma mutagenesis resulted in significant changes in the carbohydrases complex of the strain. Cellobiohydrolase I being the major carbohydrase in the original strain was absent in the enzyme complex of the mutant. The share of xylanases and endoglucanases in the preparation from the mutant strain increased by 6% and 6.5%, respectively, compared with the preparation from the original strain. The obtained data show the ability of gamma irradiation to affect the component composition of T. reesei carbohydrase complex. [ABSTRACT FROM AUTHOR]

In this study, Saccharomyces cerevisiae was engineered to degrade and utilize xylan, one of the major polysaccharide chains present in hemicellulose. Different hemicellulases from Trichoderma reesei , namely: endoxylanase, β-xylosidase, acetylxylan esterase, α- d -glucuronidase and α- l -arabinofuranosidase, were heterologously secreted by S. cerevisiae. A mixture experimental design was adapted to statistically describe the synergistic interactions between the hemicellulases and to determine the optimum formulations for the hydrolysis of xylan substrates. The hydrolytic activities of the hemicellulase mixtures were then improved by displaying the hemicellulases on the yeast surface as whole-cell biocatalysts. The engineered yeast strains displaying hemicellulases were further engineered to express xylose-utilization genes xylose isomerase (XI) and xylulokinase (XK) which enable its utilization of xylose as a sole carbon source. The resulting consortium was then able to grow and produce ethanol from different xylan substrates. [ABSTRACT FROM AUTHOR]

Agricultural by-products such as wheat bran and straw are rich in arabinoxylans. Composed of a main chain of β-(1,4) linked xylose ramified by arabinose and ferulic acid, these polymers are sources of valuable molecules, in particular ferulic acid and monomeric (xylose, arabinose and glucose) and oligomeric carbohydrates. Enzymatic hydrolysis using hemicellulasic cocktails obtained from Thermobacillus xylanilyticus provide an eco-friendly alternative to acid or alkaline deconstruction. In this article, downstream purification of the enzymatic hydrolysate of wheat bran with a weak anion-exchange resin (Amberlyst A21- Dow) in free-base form was studied and optimized to reach concentrated ferulate fractions in environment-friendly conditions. A demineralization step by homopolar electrodialysis prior to anion-exchange allowed most of the competing inorganic anions to be removed from the hydrolysate. The capacity of fixation of ferulic acid on the active sites of the resin was increased, as well as purity and concentration of the basic eluate, consequently. Ferulic acid could therefore be crystallized by adjusting the pH of the eluate. 83% was crystallized and purity (NMR) reached 90–95%. At last, 52% of ferulic acid released from wheat bran by the hemicellulasic cocktail was purified and recovered as a solid. Moreover, more than 50 BV (Bed Volume) of the hydrolysate could be recovered during the resin saturation step as rather pure carbohydrates under monomeric (xylose, arabinose and glucose) and oligomeric (xylobiose, xylotriose and xylotetraose) form. [ABSTRACT FROM AUTHOR]

Bark beetles reproduce and overwinter under the bark of trees, and are associated with bacteria that may influence the fitness of their hosts. As regard the aim of this study was to test the metabolic potential of bacterial strains, isolated from the bark beetle species Cryphalus piceae, Ips typographus and Pityophthorus pityophthorus and collected in the Czech Republic from fir, spruce and pine trees, respectively, to degrade plant cell compounds. The bacterial strains were identified as belonging to the genera Curtobacterium , Erwinia , Pantoea , Pseudomonas, Rahnella, Staphylococcus, and Yersinia . Several activities related to the degradation of lignocellulosic materials, such as cellulose, xylan and starch, were found. Moreover, the genomes of three of these strains were sequenced and analyzed, and the presence of the enzymatic machinery required for biomass hydrolysis was discovered. This finding supports the idea that bacteria aid in the provision of nutrients to the beetle from the hydrolysis of tree compounds, results that are relevant for studying the ecological implication of bacterial strains in the bark beetle life cycle. In addition, the activities found in association with the bacterial strains could be useful in biotechnological processes, such as the production of biofuels from biomass, colorant degradation, in the textile industry and for wastewater treatments. Furthermore, the gene sequences of the lignocellulolytic enzymes found within the genomes serve as a basis for future studies regarding the potential application of these bacteria, and their metabolic machinery, in processes such as biomass hydrolysis and bioremediation. [ABSTRACT FROM AUTHOR]

Abstract: Aims: We investigated the role of carbon and nitrogen sources in the production of cellulase and hemicellulase by Aspergillus strains. Methods and Results: The strains Aspergillus niger SCBM1 and Aspergillus fumigatus SCBM6 were cultivated under solid‐state fermentation (SSF), with biomass sorghum (BS) and wheat bran (WB) as lignocellulosic substrates, in different proportions, along with variable nitrogen sources. The best SSF condition for the induction of such enzymes was observed employing A. niger SCBM1 in BS supplemented with peptone; maximum production levels were achieved as follows: 72 h of fermentation for xylanase and exoglucanase (300·07 and 30·64 U g−1 respectively), 120 h for β‐glucosidase and endoglucanase (54·90 and 41·47 U g−1 respectively) and 144 h for β‐xylosidase (64·88 U g−1). Conclusions: This work demonstrated the viability of the use of BS for the production of hemi‐ and cellulolytic enzymes; the high concentration of celluloses in BS could be associated with the significant production of cellulases, mainly exoglucanase. Significance and Impact of the Study: This is the first study which presents the promising use of biomass sorghum (genetically modified sorghum to increase its biomass content) as an alternative carbon source for the production of enzymes by SSF. [ABSTRACT FROM AUTHOR]

Many species of Penicillium have exhibited great potential for lignocellulose hydrolysis. The filamentous fungus Talaromyces piceus 9-3 ( anamorph: Penicillium piceum), which was isolated from compost wastes in China, was sequenced in this study. Compared with the cellulase producer T. reesei, T. piceus 9-3 processes a lignocellulolytic enzyme system comprising more diverse enzymatic components, especially hemicellulases. This report will facilitate the use of this strain for biomass degradation. [ABSTRACT FROM AUTHOR]

Thermobifidas are thermophilic, aerobic, lignocellulose decomposing actinomycetes. The Thermobifida genus includes four species: T. fusca, T. alba, T. cellulosilytica, and T. halotolerans. T. fusca YX is the far best characterized strain of this taxon and several cellulases and hemicellulases have been cloned from it for industrial purposes targeting paper industry, biofuel, and feed applications. Unfortunately, sequence data of such enzymes are almost exclusively restricted to this single species; however, we demonstrated earlier by zymography that other T. alba and T. cellulosilytica strains encode the same enzyme sets. Recently, the advances in whole genome sequencing by the use of next generation genomics platforms accelerated the selection process of valuable hydrolases from uncharacterized bacterial species for cloning purposes. For this purpose T. cellulosilytica TB100T type strain was chosen for de novo genome sequencing. We have assembled the genome of T. cellulosilytica strain TB100T into 168 contigs and 19 scaffolds, with reference length of 4 327 869 bps, 3 589 putative coding sequences, 53 tRNAs, and 4 rRNAs. The analysis of the annotated genome revealed the existence of 27 putative hydrolases belonging to 14 different glycoside hydrolase (GH) families. The investigation of identified, cloned, and heterologously multiple cellulases, mannanases, xylanases, and amylases may result in industrial applications beside gaining useful basic research related information. [ABSTRACT FROM AUTHOR]