Showing papers on "Xylanase published in 2016"

Insights into the mechanism of enzymatic hydrolysis of xylan

Abstract: Hemicelluloses are a vast group of complex, non-cellulosic heteropolysaccharides that are classified according to the principal monosaccharides present in its structure. Xylan is the most abundant hemicellulose found in lignocellulosic biomass. In the current trend of a more effective utilization of lignocellulosic biomass and developments of environmentally friendly industrial processes, increasing research activities have been directed to a practical application of the xylan component of plants and plant residues as biopolymer resources. A variety of enzymes, including main- and side-chain acting enzymes, are responsible for xylan breakdown. Xylanase is a main-chain enzyme that randomly cleaves the β-1,4 linkages between the xylopyranosyl residues in xylan backbone. This enzyme presents varying folds, mechanisms of action, substrate specificities, hydrolytic activities, and physicochemical characteristics. This review pays particular attention to different aspects of the mechanisms of action of xylan-degrading enzymes and their contribution to improve the production of bioproducts from plant biomass. Furthermore, the influence of phenolic compounds on xylanase activity is also discussed.

Production of high-pure xylooligosaccharides from sugarcane bagasse using crude β-xylosidase-free xylanase of Bacillus subtilis KCX006 and their bifidogenic function

Abstract: Xylooligosaccharides (XOS) are used as prebiotics in food industries. XOS with high purity is required for effective prebiotic function. All crude xylanases exhibit β-xylosidase, which forms significant amount of xylose and reduces the yield and purity of XOS. Here, we describe the use of crude xylanase of Bacillus subtilis devoid of β-xylosidase to produce high-pure XOS from ammonia pretreated sugarcane bagasse. MALDI-TOF-MS and HPLC analysis of XOS showed the formation of xylobiose, xylotriose and xylotetraose with xylobiose as the major product. The conversion of bagasse to XOS was >99% with negligible amount of xylose (0.4%). NMR analysis of XOS mixture indicated the presence of arabinosyl and glucuronyl substituted XOS. The relative content of substituted XOS in the mixture was 32%. The XOS mixture supported growth of probiotic bifidobacterial strains under anaerobic conditions. The bifidobacterial strains completely utilized XOS with production of short chain fatty acids indicating their prebiotic function. The concentration of fatty acids formed was in the order of acetate, formate and lactate. Hence, the present method would be useful for further development of low-cost process for production of high-pure prebiotic XOS from lignocellulosic materials.

A GH115 α-glucuronidase from Schizophyllum commune contributes to the synergistic enzymatic deconstruction of softwood glucuronoarabinoxylan

Abstract: Lignocellulosic biomass from softwood represents a valuable resource for the production of biofuels and bio-based materials as alternatives to traditional pulp and paper products. Hemicelluloses constitute an extremely heterogeneous fraction of the plant cell wall, as their molecular structures involve multiple monosaccharide components, glycosidic linkages, and decoration patterns. The complete enzymatic hydrolysis of wood hemicelluloses into monosaccharides is therefore a complex biochemical process that requires the activities of multiple degradative enzymes with complementary activities tailored to the structural features of a particular substrate. Glucuronoarabinoxylan (GAX) is a major hemicellulose component in softwood, and its structural complexity requires more enzyme specificities to achieve complete hydrolysis compared to glucuronoxylans from hardwood and arabinoxylans from grasses. We report the characterisation of a recombinant α-glucuronidase (Agu115) from Schizophyllum commune capable of removing (4-O-methyl)-glucuronic acid ((Me)GlcA) residues from polymeric and oligomeric xylan. The enzyme is required for the complete deconstruction of spruce glucuronoarabinoxylan (GAX) and acts synergistically with other xylan-degrading enzymes, specifically a xylanase (Xyn10C), an α-l-arabinofuranosidase (AbfA), and a β-xylosidase (XynB). Each enzyme in this mixture showed varying degrees of potentiation by the other activities, likely due to increased physical access to their respective target monosaccharides. The exo-acting Agu115 and AbfA were unable to remove all of their respective target side chain decorations from GAX, but their specific activity was significantly boosted by the addition of the endo-Xyn10C xylanase. We demonstrate that the proposed enzymatic cocktail (Agu115 with AbfA, Xyn10C and XynB) achieved almost complete conversion of GAX to arabinofuranose (Araf), xylopyranose (Xylp), and MeGlcA monosaccharides. Addition of Agu115 to the enzymatic cocktail contributes specifically to 25 % of the conversion. However, traces of residual oligosaccharides resistant to this combination of enzymes were still present after deconstruction, due to steric hindrances to enzyme access to the substrate. Our GH115 α-glucuronidase is capable of finely tailoring the molecular structure of softwood GAX, and contributes to the almost complete saccharification of GAX in synergy with other exo- and endo-xylan-acting enzymes. This has great relevance for the cost-efficient production of biofuels from softwood lignocellulose.

Lignocellulose-Adapted Endo-Cellulase Producing Streptomyces Strains for Bioconversion of Cellulose-Based Materials.

Abstract: Twenty-four Actinobacteria strains, isolated from Arundo donax, Eucalyptus camaldulensis and Populus nigra biomasses 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 Streptomyces (S.) genus and in particular, the most representative species was S. argenteolus including the 50% of strains; while 8 strains were identified as members of species S. flavogriseus (synonym S. flavovirens) and S. fimicarius (synonyms S. acrimycini, S. baarnensis, S. caviscabies and S. flavofuscus), and the other 4 strains belonged to the species S. drozdowiczii, S. rubrogriseus, S. albolongus and S. 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 multi-enzymatic activities (from three to six). The twenty-four 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 of Arundo donax pretreated biomass. The different degrees of cellulose and xylan hydrolysis was evaluated by determining the kinetics of glucose and xylose release during 72 h incubation at 50°C of the pretreated biomass in the presence of cellulose degrading enzymes (cellulase and β-glucosidase) and xylan related activities (xylanase and β-xylosidase). Cellulose and xylan conversion, when conducted using commercial (hemi)cellulolytic, 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 allowed to reach almost the 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. Overall results highlighted the biotechnological potential of S. argenteolus AE58P being an interesting candidate biocatalysts-producing bacterium for the lignocellulose conversion and biochemicals and bioenergy production.

Comparison of three deep eutectic solvents and 1-ethyl-3-methylimidazolium acetate in the pretreatment of lignocellulose: effect on enzyme stability, lignocellulose digestibility and one-pot hydrolysis

Abstract: Certain ionic liquids (ILs) are well-known pretreatment chemicals for lignocellulosic substrates prior to enzymatic total hydrolysis. Deep eutectic solvents (DESs) are closely related to ILs in many properties, but are easier and on occasion cheaper to synthesize and have been claimed to be less inactivating to enzymes used in the hydrolysis, and less toxic for the environment and to micro-organisms used in fermentation. The use of DESs as lignocellulose pretreatment chemicals has not been studied to a similar extent as the use of ILs. In this study, the stability of three Trichoderma reesei cellulases (the endoglucanases Cel5A and Cel7B and the cellobiohydrolase Cel7A) and one T. reesei xylanase (Xyn11) was compared in concentrated solutions (85% w/w) of three DESs (choline chloride : boric acid in molar ratio 5 : 2, choline chloride : glycerol 1 : 1 and betaine : glycerol 1 : 1) and 1-ethyl-3-methylimidazolium acetate ([EMIM]AcO), a powerful lignocellulose-dissolving IL. The pretreatment efficiency of these chemicals was further compared in a mild pretreatment (90% w/w DES or [EMIM]AcO, 80 °C, 24 h, 5% (w/w) lignocellulose consistency) of four different substrates; microcrystalline cellulose, eucalyptus dissolving pulp, shredded wheat straw and spruce saw dust. After pretreatment, the enzymatic digestibility of the pretreated substrates was evaluated in the enzymatic total hydrolysis in three different setups, including hydrolysis of the washed pretreated substrates in buffer, and of the pretreated substrates in solutions containing 30% (w/w) and 80% (w/w) of DES or [EMIM]AcO. The stability analysis identified glycerol-containing DESs to be highly stabilizing for the cellulases, but their pretreatment efficiency was limited. [EMIM]AcO had a high pretreatment efficiency, but was highly inactivating for the used cellulases. The presence of DES or [EMIM]AcO led in all cases to decreased enzymatic hydrolysis yields. Thus, good enzymatic stability in a certain DES does not directly implicate good performance in the hydrolysis of solid lignocellulosic substrates in that DES.

Comparative genomic, transcriptomic and secretomic profiling of Penicillium oxalicum HP7-1 and its cellulase and xylanase hyper-producing mutant EU2106, and identification of two novel regulatory genes of cellulase and xylanase gene expression

Abstract: The filamentous fungus Penicillium oxalicum is a potential alternative to Trichoderma reesei for industrial production of a complete cellulolytic enzyme system for a bio-refinery. Comparative omics approaches can support rational genetic engineering and/or breeding of filamentous fungi with improved cellulase production capacity. In this study, comparative genomic, transcriptomic and secretomic profiling of P. oxalicum HP7-1 and its cellulase and xylanase hyper-producing mutant EU2106 were employed to screen for novel regulators of cellulase and xylanase gene expression. The 30.62 Mb P. oxalicum HP7-1 genome was sequenced, and 9834 protein-coding genes were annotated. Re-sequencing of the mutant EU2106 genome identified 274 single nucleotide variations and 12 insertion/deletions. Comparative genomic, transcriptomic and secretomic profiling of HP7-1 and EU2106 revealed four candidate regulators of cellulase and xylanase gene expression. Deletion of these candidate genes and measurement of the enzymatic activity of the resultant mutants confirmed the identity of three regulatory genes. POX02484 and POX08522, encoding a putative Zn(II)2Cys6 DNA-binding domain and forkhead protein, respectively, were found to be novel, while PoxClrB is an ortholog of ClrB, a key transcriptional regulator of cellulolytic enzyme gene expression in filamentous fungi. ΔPOX02484 and ΔPOX08522 mutants exhibited significantly reduced β-glucosidase activity, increased carboxymethylcellulose cellulase and xylanase activities, and altered transcription level of cellulase and xylanase genes compared with the parent strain ΔPoxKu70, with Avicel as the sole carbon source. Two novel genes, POX02484 and POX08522, were found and characterized to regulate the expression of cellulase and xylanase genes in P. oxalicum. These findings are important for engineering filamentous fungi to improve cellulase and xylanase production.

Cell surface engineering of Saccharomyces cerevisiae combined with membrane separation technology for xylitol production from rice straw hydrolysate.

Abstract: Xylitol, a value-added polyol deriving from D-xylose, is widely used in both the food and pharmaceutical industries. Despite extensive studies aiming to streamline the production of xylitol, the manufacturing cost of this product remains high while demand is constantly growing worldwide. Biotechnological production of xylitol from lignocellulosic waste may constitute an advantageous and sustainable option to address this issue. However, to date, there have been few reports of biomass conversion to xylitol. In the present study, xylitol was directly produced from rice straw hydrolysate using a recombinant Saccharomyces cerevisiae YPH499 strain expressing cytosolic xylose reductase (XR), along with β-glucosidase (BGL), xylosidase (XYL), and xylanase (XYN) enzymes (co-)displayed on the cell surface; xylitol production by this strain did not require addition of any commercial enzymes. All of these enzymes contributed to the consolidated bioprocessing (CBP) of the lignocellulosic hydrolysate to xylitol to produce 5.8 g/L xylitol with 79.5 % of theoretical yield from xylose contained in the biomass. Furthermore, nanofiltration of the rice straw hydrolysate provided removal of fermentation inhibitors while simultaneously increasing sugar concentrations, facilitating high concentration xylitol production (37.9 g/L) in the CBP. This study is the first report (to our knowledge) of the combination of cell surface engineering approach and membrane separation technology for xylitol production, which could be extended to further industrial applications.

Enhanced cellulase production by Trichoderma harzianum by cultivation on glycerol followed by induction on cellulosic substrates

Abstract: The use of glycerol obtained as an intermediate of the biodiesel manufacturing process as carbon source for microbial growth is a potential alternative strategy for the production of enzymes and other high-value bioproducts. This work evaluates the production of cellulase enzymes using glycerol for high cell density growth of Trichoderma harzianum followed by induction with a cellulosic material. Firstly, the influence of the carbon source used in the pre-culture step was investigated in terms of total protein secretion and fungal morphology. Enzymatic productivity was then determined for cultivation strategies using different types and concentrations of carbon source, as well as different feeding procedures (batch and fed-batch). The best strategy for cellulase production was then further studied on a larger scale using a stirred tank bioreactor. The proposed strategy for cellulase production, using glycerol to achieve high cell density growth followed by induction with pretreated sugarcane bagasse, achieved enzymatic activities up to 2.27 ± 0.37 FPU/mL, 106.40 ± 8.87 IU/mL, and 9.04 ± 0.39 IU/mL of cellulase, xylanase, and β-glucosidase, respectively. These values were 2 times higher when compared to the control experiments using glucose instead of glycerol. This novel strategy proved to be a promising approach for improving cellulolytic enzymes production, and could potentially contribute to adding value to biomass within the biofuels sector.

Classical Optimization of Cellulase and Xylanase Production by a Marine Streptomyces Species

Abstract: Cellulase and xylanase are in high demand for application in several industrial processes, consequently necessitating the bioprospecting and manipulation of microbes for novel and greater enzyme productivity. This study reports on the optimal conditions for cellulase and xylanase production by a marine bacterial isolate from Nahoon beach sediment, via the classical process of one variable per time. Furthermore, the inducing effect of mono- and polysaccharides on enzyme production was investigated. The 16S rDNA gene sequence analysis clearly assigned the isolate to the genus Streptomyces, and was deposited at the GenBank under the accession number KU171373. Cellulase and xylanase production was optimal at the following conditions: pH 6 and 8, incubation temperature of 40 and 35 °C, and agitation speed of 100 and 150 rpm, respectively. Under optimum conditions, 0.26 U/mL and 18.54 U/mL activities were attained at 60 and 48 h with specific productivity of 205 and 7417 U/g for cellulase and xylanase, respectively. Xylanase production was induced by the entire mono- and polysaccharides tested, while cellulase production was induced by some. The results from this study signify the resourcefulness of the Streptomyces strain for production of cellulase and xylanase of industrial importance.

High-level expression of thermostable cellulolytic enzymes in tobacco transplastomic plants and their use in hydrolysis of an industrially pretreated Arundo donax L. biomass

Abstract: Biofuels production from plant biomasses is a complex multi-step process with important economic burdens. Several biotechnological approaches have been pursued to reduce biofuels production costs. The aim of the present study was to explore the production in tobacco plastome of three genes encoding (hemi)cellulolytic enzymes from thermophilic and hyperthermophilic bacterium and Archaea, respectively, and test their application in the bioconversion of an important industrially pretreated biomass feedstock (A. donax) for production of second-generation biofuels. The selected enzymes, endoglucanase, endo-β-1,4-xylanase and β-glucosidase, were expressed in tobacco plastome with a protein yield range from 2 % to more than 75 % of total soluble proteins (TSP). The accumulation of endoglucanase (up to 2 % TSP) gave altered plant phenotypes whose severity was directly linked to the enzyme yield. The most severe seedling-lethal phenotype was due to the impairment of plastid development associated to the binding of endoglucanase protein to thylakoids. Endo-β-1,4-xylanase and β-glucosidase, produced at very high level without detrimental effects on plant development, were enriched (fourfold) by heat treatment (105.4 and 255.4 U/mg, respectively). Both plastid-derived biocatalysts retained the main features of the native or recombinantly expressed enzymes with interesting differences. Plastid-derived xylanase and β-glucosidase resulted more thermophilic than the E. coli recombinant and native counterpart, respectively. Bioconversion experiments, carried out at 50 and 60 °C, demonstrated that plastid-derived enzymes were able to hydrolyse an industrially pretreated giant reed biomass. In particular, the replacement of commercial enzyme with plastid-derived xylanase, at 60 °C, produced an increase of both xylose recovery and hydrolysis rate; whereas the replacement of both xylanase and β-glucosidase produced glucose levels similar to those observed with the commercial cocktails, and xylose yields always higher in the whole 24–72 h range. The very high production level of thermophilic and hyperthermophilic enzymes, their stability and bioconversion efficiencies described in this study demonstrate that plastid transformation represents a real cost-effective production platform for cellulolytic enzymes.

Carrier-free co-immobilization of xylanase, cellulase and β-1,3-glucanase as combined cross-linked enzyme aggregates (combi-CLEAs) for one-pot saccharification of sugarcane bagasse

Abstract: Combined cross-linked enzyme aggregates (combi-CLEAs) are an innovative prospect and a lucrative technology. The present study addresses the preparation, characterization and application of combi-CLEAs with xylanase, cellulase and β-1,3-glucanase to achieve one-pot bioconversion of lignocellulosic biomass to fermentable sugars. A three-phase partitioning (TPP) method was used to aggregate the enzymes. Glutaraldehyde (100 mM) was employed as a cross-linker with the cross-linking time of 7.5 h. Scanning electron microscopy of the tri-enzyme biocatalyst has a coarse-grained appearance. Combi-CLEAs were more thermally stable, retaining about 70% of their initial activity at 70 °C compared to 30% for the free enzyme. The storage stability of combi-CLEAs was more than 97% of their activity after incubation for 11 weeks at 4 °C, whereas the free enzymes retained about 65% of initial activity. The residual activity of combi-CLEAs remained constant at 90% until the sixth cycle. Contrary to free enzymes that remain in the hydrolysate, which prevents their recovery, reuse of combi-CLEAs was possible. Free enzymes hydrolyze the ammonia cooked sugarcane bagasse at about 73%, whereas the combi-CLEAs resulted in maximum hydrolysis of about 83.5% in 48 h.