A comprehensive method for the sequential separation of extracellular xylanases and ß-xylosidases/arabinofuranosidases from a new Fusarium species.

Several fungal species produce diverse carbohydrate-active enzymes useful for the xylooligosaccharide biorefinery. These enzymes can be isolated by different purification methods, but fungi usually produce other several compounds which interfere in the purification process. So, the present work has three interconnected aims: (i) compare ß-xylosidase production by Fusarium pernambucanum MUM 18.62 with other crop pathogens; (ii) optimise F. pernambucanum xylanolytic enzymes expression focusing on the pre-inoculum media composition; and (iii) design a downstream strategy to eliminate interfering substances and sequentially isolate ß-xylosidases, arabinofuranosidases and endo-xylanases from the extracellular media. F. pernambucanum showed the highest ß-xylosidase activity among all the evaluated species. It also produced endo-xylanase and arabinofuranosidase. The growth and ß-xylosidase expression were not influenced by the pre-inoculum source, contrary to endo-xylanase activity, which was higher with xylan-enriched agar. Using a sequential strategy involving ammonium sulfate precipitation of the extracellular interferences, and several chromatographic steps of the supernatant (hydrophobic chromatography, size exclusion chromatography, and anion exchange chromatography), we were able to isolate different enzyme pools: four partially purified ß-xylosidase/arabinofuranoside; FpXylEAB trifunctional GH10 endo-xylanase/ß-xylosidase/arabinofuranoside enzyme (39.8 kDa) and FpXynE GH11 endo-xylanase with molecular mass (18.0 kDa). FpXylEAB and FpXynE enzymes were highly active at pH 5-6 and 60-50 °C.

Transformation of corncob into high-value xylooligosaccharides using glycoside hydrolase families 10 and 11 xylanases from Trichoderma asperellum ND-1.

Effective production of xylooligosaccharides (XOS) with lower proportion of xylose entails unique and robust xylanases. In this study, two novel xylanases from Trichoderma asperellum ND-1 belonging to glycoside hydrolase families 10 (XynTR10) and 11 (XynTR11) were over-expressed in Komagataella phaffii X-33 and characterized to be robust enzymes with high halotolerance and ethanol tolerant. Both enzymes displayed strict substrate specificity towards beechwood xylan and wheat arabinoxylan. (Glu153/Glu258) and (Glu161/Glu252) were key catalytic sites for XynTR10 and XynTR11. Notably, XynTR11 could rapidly degrade xylan/XOS into xylobiose without xylose via transglycosylation. Direct degradation of corncob using XynTR10 and XynTR111 displayed that while XynTR10 yielded 77% xylobiose and 25% xylose, XynTR11 yielded much less xylose (11%) and comparable amounts of xylobiose (63%). XynTR10 or XynTR111 has great potential as a catalyst for bioconversion of xylan-containing agricultural waste into high-value products (biofuel or XOS), which is of significant benefit for the economy and environment.

A Novel Multifunctional Arabinofuranosidase/Endoxylanase/ß-Xylosidase GH43 Enzyme from Paenibacillus curdlanolyticus B-6 and Its Synergistic Action To Produce Arabinose and Xylose from Cereal Arabinoxylan.

PcAxy43B is a modular protein comprising a catalytic domain of glycoside hydrolase family 43 (GH43), a family 6 carbohydrate-binding module (CBM6), and a family 36 carbohydrate-binding module (CBM36) and found to be a novel multifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6. This enzyme exhibited α-l-arabinofuranosidase, endoxylanase, and ß-d-xylosidase activities. The α-l-arabinofuranosidase activity of PcAxy43B revealed a new property of GH43, via the release of both long-chain cereal arabinoxylan and short-chain arabinoxylooligosaccharide (AXOS), as well as release from both the C(O)2 and C(O)3 positions of AXOS, which is different from what has been seen for other arabinofuranosidases. PcAxy43B liberated a series of xylooligosaccharides (XOSs) from birchwood xylan and xylohexaose, indicating that PcAxy43B exhibited endoxylanase activity. PcAxy43B produced xylose from xylobiose and reacted with p-nitrophenyl-ß-d-xylopyranoside as a result of ß-xylosidase activity. PcAxy43B effectively released arabinose together with XOSs and xylose from the highly arabinosyl-substituted rye arabinoxylan. Moreover, PcAxy43B showed significant synergistic action with the trifunctional endoxylanase/ß-xylosidase/α-l-arabinofuranosidase PcAxy43A and the endoxylanase Xyn10C from strain B-6, in which almost all products produced from rye arabinoxylan by these combined enzymes were arabinose and xylose. In addition, the presence of CBM36 was found to be necessary for the endoxylanase property of PcAxy43B. PcAxy43B is capable of hydrolyzing untreated cereal biomass, corn hull, and rice straw into XOSs and xylose. Hence, PcAxy43B, a significant accessory multifunctional xylanolytic enzyme, is a potential candidate for application in the saccharification of cereal biomass. IMPORTANCE Enzymatic saccharification of cereal biomass is a strategy for the production of fermented sugars from low-price raw materials. In the present study, PcAxy43B from P. curdlanolyticus B-6 was found to be a novel multifunctional α-l-arabinofuranosidase/endoxylanase/ß-d-xylosidase enzyme of glycoside hydrolase family 43. It is effective in releasing arabinose, xylose, and XOSs from the highly arabinosyl-substituted rye arabinoxylan, which is usually resistant to hydrolysis by xylanolytic enzymes. Moreover, almost all products produced from rye arabinoxylan by the combination of PcAxy43B with the trifunctional xylanolytic enzyme PcAxy43A and the endoxylanase Xyn10C from strain B-6 were arabinose and xylose, which can be used to produce several value-added products. In addition, PcAxy43B is capable of hydrolyzing untreated cereal biomass into XOSs and xylose. Thus, PcAxy43B is an important multifunctional xylanolytic enzyme with high potential in biotechnology.

Bioconversion of Untreated Corn Hull into L-Malic Acid by Trifunctional Xylanolytic Enzyme from Paenibacillus curdlanolyticus B-6 and Acetobacter tropicalis H-1.

L-Malic acid (L-MA) is widely used in food and non-food products. However, few microorganisms have been able to efficiently produce L-MA from xylose derived from lignocellulosic biomass (LB). The objective of this work is to convert LB into L-MA with the concept of a bioeconomy and environmentally friendly process. The unique trifunctional xylanolytic enzyme, PcAxy43A from Paenibacillus curdlanolyticus B-6, effectively hydrolyzed xylan in untreated LB, especially corn hull to xylose, in one step. Furthermore, the newly isolated, Acetobacter tropicalis strain H1 was able to convert high concentrations of xylose derived from corn hull into L-MA as the main product, which can be easily purified. The strain H1 successfully produced a high L-MA titer of 77.09 g/l, with a yield of 0.77 g/g and a productivity of 0.64 g/l/h from the xylose derived from corn hull. The process presented in this research is an efficient, low-cost and environmentally friendly biological process for the green production of L-MA from LB.

Efficient biological pretreatment and bioconversion of corn cob by the sequential application of a Bacillus firmus K-1 cellulase-free xylanolytic enzyme and commercial cellulases.

We used agricultural residue, corn cob, with biorefinery and bioeconomy concepts. At short-time cultivation in corn cob (12 h), Bacillus firmus K-1 produced cellulase-free xylanolytic enzyme, with xylooligosaccharides (XOSs), X5 and X6, as the main products, which can be used in a variety of applications. The xylanolytic enzyme produced from B. firmus K-1 effectively degraded xylan in corn cob, which was examined by chemical composition, scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FTIR). After cultivation, the xylan contained in the corn cob residue was decreased (as biological pretreatment), causing morphological and structural changes, including creating porosity and increasing the surface area and the exposure of cellulose of pretreated corn cob. These results lead to an improvement of cellulose access by cellulases. Commercially available cellulases, Accellerase® 1500 and Cellic® CTec2, yielded significantly higher glucose concentrations from pretreated corn cob compared to untreated corn cob. After saccharification, the lignin-rich corn cob residue can be used as a raw material for other purposes. Moreover, the B. firmus cells, with a low risk to human health, can be used in some applications. This study presents an efficient method for producing high-value-added products from agricultural residue (corn cob) through biological processes which are environmentally friendly and economically viable. KEY POINTS: • High-value-added products were efficiently produced from corn cob by B. firmus K-1. • After biological pretreatment by B. firmus K-1, cellulase can better reach cellulose. • XOSs and cellulose-derived glucose were the main products from corn cob.

Draft genome sequence data of Paenbacillus curdlanolyticus B-6 possessing a unique xylanolytic-cellulolytic multienzyme system.

Paenibacillus curdlanolyticus B-6 is a facultative anaerobic bacterium that efficiently produces a lignocellulolytic multienzyme complex. The whole genome of P. curdlanolyticus B-6 was sequenced on an Ion GeneStudio S5 system, which yielded 74 contigs with a total size of 4,875,097 bp, 4,473 protein-coding sequences, and a G+C content of 49.7%. The genome data have been deposited in DDBJ/ENA/GenBank under accession numbers BLWM01000001-BLWM01000074. Analyses of average nucleotide identities and phylogenetic relationships of 16S rRNA sequences of Paenibacillus species revealed that strain B-6 is most closely related to Paenibacillus xylaniclasticus TW1. P. curdlanolyticus B-6 should thus be reclassified as a strain of P. xylaniclasticus.

An integrated approach to the sustainable production of xylanolytic enzymes from Aspergillus niger using agro-industrial by-products.

Xylanolytic enzymes were produced by Aspergillus niger NRRL3 grown on agro-industrial by-products obtained from the processing of wheat flour without pretreatment. Significant parameters for xylanase production were screened and optimized. The xylanolytic activity obtained in the optimized extract was 138.3 ± 2.6 U/mL, higher than the activity obtained in an unoptimized medium (14.5 ± 0.3 U/mL) in previous work. The optimized fermentation process was performed in a successful 40-fold scale-up. The optimized enzymatic extract obtained was characterized by LC-MS. Nine enzymes were identified as constituents of the xylanolytic complex. Moreover, the xylanolytic enzymes were stable until 60 °C and over a broad range of pH. Sodium, calcium, cobalt and manganese had no inhibitory effect, meanwhile 1% w/v polyvinylpyrrolidone and 1% w/v dextran increased the xylanolytic activity. The saccharification efficiency was evaluated and the surface morphology of the lignocellulosic substrate was monitored by using scanning electron microscopy (SEM). The synergistic combination of the extracted (o purified) xylanolytic enzymes permitted a higher xylan conversion beneficial for diverse applications, such as bioethanol production. Thus, these agroindustrial by-products can be used within the framework of a circular economy, rendering an added value bioproduct, which is reused in the industry.

An endoxylanase rapidly hydrolyzes xylan into major product xylobiose via transglycosylation of xylose to xylotriose or xylotetraose.

Here, we proposed an effective strategy to enhance a novel endoxylanase (Taxy11) activity and elucidated an efficient catalysis mechanism to produce xylooligosaccharides (XOSs). Codon optimization and recruitment of natural propeptide in Pichia pastoris resulted in achievement of Taxy11 activity to 1405.65 ±â€¯51.24 U/mL. Analysis of action mode reveals that Taxy11 requires at least three xylose (xylotriose) residues for hydrolysis to yield xylobiose. Results of site-directed mutagenesis indicate that residues Glu119, Glu210, and Asp53 of Taxy11 are key catalytic sites, while Asp203 plays an auxiliary role. The novel mechanism whereby Taxy11 catalyzes conversion of xylan or XOSs into major product xylobiose involves transglycosylation of xylose to xylotriose or xylotetraose as substrate, to form xylotetraose or xylopentaose intermediate, respectively. Taxy11 displayed highly hydrolytic activity toward corncob xylan, producing 50.44 % of xylobiose within 0.5 h. This work provides a cost-effective and sustainable way to produce value-added biomolecules XOSs (xylobiose-enriched) from agricultural waste.

ß-Xylosidases: Structural Diversity, Catalytic Mechanism, and Inhibition by Monosaccharides.

Xylan, a prominent component of cellulosic biomass, has a high potential for degradation into reducing sugars, and subsequent conversion into bioethanol. This process requires a range of xylanolytic enzymes. Among them, ß-xylosidases are crucial, because they hydrolyze more glycosidic bonds than any of the other xylanolytic enzymes. They also enhance the efficiency of the process by degrading xylooligosaccharides, which are potent inhibitors of other hemicellulose-/xylan-converting enzymes. On the other hand, the ß-xylosidase itself is also inhibited by monosaccharides that may be generated in high concentrations during the saccharification process. Structurally, ß-xylosidases are diverse enzymes with different substrate specificities and enzyme mechanisms. Here, we review the structural diversity and catalytic mechanisms of ß-xylosidases, and discuss their inhibition by monosaccharides.

Pretreatment of mulberry stem by cholinium-based amino acid ionic liquids for succinic acid fermentation and its application in poly(butylene) succinate production.

Cholinium-glycinate ([Ch][Gly]) and cholinium-alanate ([Ch][Ala]) were investigated on the pretreatment of mulberry stem (MS). It resulted in an increase of glucose from 14% to more than 74% compared to the untreated sample. Pretreatment by reused [Ch][Gly] showed good performance for delignification of >60%, and improved structural polysaccharide digestion. Each fractional component has high potential for lignin purification, and succinic acid fermentation. The extracted lignin with [Ch][Gly] showed >90% purity with good qualities of aromatic unit as confirmed by FT-IR and 1H NMR spectra. The carbohydrate rich material was employed for succinic acid fermentation with the highest yield of succinic acid more than 0.89 gsuccinic acid/gglucose. After purification, poly(butylene) succinate (PBS) was synthesized, and was characterized in comparison to commercial PBS.

Agroindustrial biomass for xylanase production by Penicillium chrysogenum: purification, biochemical properties and hydrolysis of hemicelluloses

Background: In this work, the xylanase production by Penicillium chrysogenum F-15 strain was investigated using agroindustrial biomass as substrate. The xylanase was purified, characterized and applied in hemicellulose hydrolysis. Results: The highest xylanase production was obtained when cultivation was carried out with sugar cane bagasse as carbon source, at pH 6.0 and 20°C, under static condition for 8 d. The enzyme was purified by a sequence of ion exchange and size exclusion chromatography, presenting final specific activity of 834.2 U·mg·prot-1. T he molecular mass of the purified enzyme estimated by SDS-PAGE was 22.1 kDa. The optimum activity was at pH 6.5 and 45°C. The enzyme was stable at 40°C with half-life of 35 min, and in the pH range from 4.5 to 10.0. The activity was increased in the presence of Mg+2 and Mn+2 and reducing agents such as DTT and ßmercaptoethanol, but it was reduced by Cu+2 and Pb+2 . The xylanase presented Km of 2.3 mM and Vmax of 731.8 U·mg·prot-1 with birchwood xylan as substrate. This xylanase presented differences in its properties when it was compared to the xylanases from other P. chrysogenum strains. Conclusion: The xylanase from P. chrysogenum F-15 showed lower enzymatic activity on commercial xylan than on hemicellulose from agroindustry biomass and its biochemistry characteristics, such as stability at 40°C and pH from 4.0 to 10.0, shows the potential of this enzyme for application in food, feed, pulp and paper industries and for bioethanol production.

Chemical Pretreatment-Independent Saccharifications of Xylan and Cellulose of Rice Straw by Bacterial Weak Lignin-Binding Xylanolytic and Cellulolytic Enzymes.

Complete utilization of carbohydrate fractions is one of the prerequisites for obtaining economically favorable lignocellulosic biomass conversion. This study shows that xylan in untreated rice straw was saccharified to xylose in one step without chemical pretreatment, yielding 58.2% of the theoretically maximum value by Paenibacillus curdlanolyticus B-6 PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/ß-xylosidase/arabinoxylan arabinofuranohydrolase. Moreover, xylose yield from untreated rice straw was enhanced to 78.9% by adding endoxylanases PcXyn10C and PcXyn11A from the same bacterium, resulting in improvement of cellulose accessibility to cellulolytic enzyme. After autoclaving the xylanolytic enzyme-treated rice straw, it was subjected to subsequent saccharification by a combination of the Clostridium thermocellum endoglucanase CtCel9R and Thermoanaerobacter brockii ß-glucosidase TbCglT, yielding 88.5% of the maximum glucose yield, which was higher than the glucose yield obtained from ammonia-treated rice straw saccharification (59.6%). Moreover, this work presents a new environment-friendly xylanolytic enzyme pretreatment for beneficial hydrolysis of xylan in various agricultural residues, such as rice straw and corn hull. It not only could improve cellulose saccharification but also produced xylose, leading to an improvement of the overall fermentable sugar yields without chemical pretreatment.IMPORTANCE Ongoing research is focused on improving "green" pretreatment technologies in order to reduce energy demands and environmental impact and to develop an economically feasible biorefinery. The present study showed that PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/ß-xylosidase/arabinoxylan arabinofuranohydrolase from P. curdlanolyticus B-6, was capable of conversion of xylan in lignocellulosic biomass such as untreated rice straw to xylose in one step without chemical pretreatment. It demonstrates efficient synergism with endoxylanases PcXyn10C and PcXyn11A to depolymerize xylan in untreated rice straw and enhanced the xylose production and improved cellulose hydrolysis. Therefore, it can be considered an enzymatic pretreatment. Furthermore, the studies here show that glucose yield released from steam- and xylanolytic enzyme-treated rice straw by the combination of CtCel9R and TbCglT was higher than the glucose yield obtained from ammonia-treated rice straw saccharification. This work presents a novel environment-friendly xylanolytic enzyme pretreatment not only as a green pretreatment but also as an economically feasible biorefinery method.

Production of Xylanolytic Enzyme Complex from Aspergillus flavus using Agricultural Wastes.

Five types of agricultural wastes were used for the production of xylanolytic enzyme by Aspergillus flavus K-03. All wastes materials supported high levels of xylanase and ß-xylosidase production. A high level of proteolytic activity was observed in barley and rice bran cultures, while only a weak proteolytic activity was detected in corn cob, barley and rice straw cultures. Maximum production of xylanase was achieved in basal liquid medium containing rice barn as carbon source for 5 days of culture at pH 6.5 and 25℃. The xylanolytic enzyme of A. flavus K-03 showed low thermostability. The times required for 50% reduction of the initial enzyme activity were 90 min at 40℃, 13 min at 50℃, and 3 min at 60℃. Xylanolytic activity showed the highest level at pH 5.5~10.5 and more than 70% of the original activity was retained at pH 6.5 and 7.0. The higher stability of xylanolytic enzymes in the broad range of alkaline pH is useful for utilization of the enzymes in industrial process requiring in alkaline conditions. Moreover, the highest production of xylanolytic enzyme was obtained when 0.5% of rice bran was supplied in basal liquid medium. SDS-PAGE analysis revealed a single xylanase band of approximately 28.5 kDa from the culture filtrates.

Production of Xylanolytic Enzyme Complex from Aspergillus flavus using Agricultural Wastes

Five types of agricultural wastes were used for the production of xylanolytic enzyme by Aspergillus flavus K-03. All wastes materials supported high levels of xylanase and beta-xylosidase production. A high level of proteolytic activity was observed in barley and rice bran cultures, while only a weak proteolytic activity was detected in corn cob, barley and rice straw cultures. Maximum production of xylanase was achieved in basal liquid medium containing rice barn as carbon source for 5 days of culture at pH 6.5 and 25degrees C. The xylanolytic enzyme of A. flavus K-03 showed low thermostability. The times required for 50% reduction of the initial enzyme activity were 90 min at 40degrees C, 13 min at 50degrees C, and 3 min at 60degrees C. Xylanolytic activity showed the highest level at pH 5.5~10.5 and more than 70% of the original activity was retained at pH 6.5 and 7.0. The higher stability of xylanolytic enzymes in the broad range of alkaline pH is useful for utilization of the enzymes in industrial process requiring in alkaline conditions. Moreover, the highest production of xylanolytic enzyme was obtained when 0.5% of rice bran was supplied in basal liquid medium. SDS-PAGE analysis revealed a single xylanase band of approximately 28.5 kDa from the culture filtrates.