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Xylan (Beechwood)

Xylan (Beechwood)
Product code: P-XYLNBE-10G

Content:

€125.00

10 g

Prices exclude VAT

Available for shipping

Content: 10 g or 50 g
Shipping Temperature: Ambient
Storage Temperature: Ambient
Physical Form: Powder
Stability: > 10 years under recommended storage conditions
CAS Number: 9014-63-5
Source: Beechwood
Purity: > 95%
Monosaccharides (%): Xylose: Glucuronic Acid: Other sugars = 85.6: 8.7: 5.7
Main Chain Glycosidic Linkage: β-1,4 and α-1,2
Substrate For (Enzyme): endo-1,4-β-Xylanase

Highly purified xylan from beechwood for use in research, biochemical enzyme assays and in vitro diagnostic analysis.

Suitable as a replacement for birchwood xylan as a substrate for β-xylanase in DNSA reducing sugar assay.

Data booklets for each pack size are located in the Documents tab.

Publications
Megazyme publication
Novel substrates for the automated and manual assay of endo-1,4-β-xylanase.

Mangan, D., Cornaggia, C., Liadova, A., McCormack, N., Ivory, R., McKie, V. A., Ormerod, A. & McCleary, D. V. (2017). Carbohydrate Research, 445, 14-22.

endo-1,4-β-Xylanase (EC 3.2.1.8) is employed across a broad range of industries including animal feed, brewing, baking, biofuels, detergents and pulp (paper). Despite its importance, a rapid, reliable, reproducible, automatable assay for this enzyme that is based on the use of a chemically defined substrate has not been described to date. Reported herein is a new enzyme coupled assay procedure, termed the XylX6 assay, that employs a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-45-O-glucosyl-xylopentaoside. The development of the substrate and associated assay is discussed here and the relationship between the activity values obtained with the XylX6 assay versus traditional reducing sugar assays and its specificity and reproducibility were thoroughly investigated.

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Megazyme publication
A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-β-Xylanase.

McCleary, B. V. & McGeough, P. (2015). Appl. Biochem. Biotechnol., 177(5), 1152-1163.

The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.

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Publication

Improving the catalytic activity of thermostable xylanase from Thermotoga maritima via mutagenesis of non-catalytic residues at glycone subsites.

Yang, J., Ma, T., Shang-guan, F. & Han, Z. (2020). Enzyme and Microbial Technology, 139, 109579.

Endo-β-1,4-xylanase from Thermotoga maritima, TmxB, is an industrially attractive enzyme due to its extreme thermostability. To improve its application value, four variants were designed on the basis of multiple sequence and three-dimensional structure alignments. Wild-type TmxB (wt-TmxB) and its mutants were produced via a Pichia pastoris expression system. Among four single-site mutants, the tyrosine substitution of a threonine residue (T74Y) at putative -3/-4 subsite led to a 1.3-fold increase in specific activity at 40°C - 100°C and pH 5 for 5 min, with beechwood xylan as the substrate. T74Y had an improved catalytic efficiency (k>cat/K>m), being 1.6 times that of wt-TmxB. Variants DY (two amino acid insertions) and N68Q displayed a slight increase (1.2 fold) and dramatic decline (1.7 fold) in catalytic efficiency, respectively. Mutant E67Y was totally inactive under all test conditions. Structural modeling and docking simulation elucidated structural insights into the molecular mechanism of activity changes for these TmxB variants. This study helps in further understanding the roles of the non-catalytic amino acids at the glycone subsites of xylanases from glycoside hydrolase family 10.

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Publication

Biochemical Characterization of an Extracellular Xylanase from Aestuariibacter sp. PX-1 Newly Isolated from the Coastal Seawater of Jeju Island in Korea.

Kim, J. H. (2020). Microbiology and Biotechnology Letters, 48(2), 215-222.

The marine microorganism PX-1, which can hydrolyze xylan, was isolated from coastal sea water of Jeju Island, Korea. Based on the 16S rRNA gene sequence and chemotaxonomy analysis, PX-1 was identified as a species of the genus Aestuariibacter and named Aestuariibacter sp PX-1. From the culture broth of PX-1, an extracellular xylanase was purified to homogeneity through ammonium sulfate precipitation and subsequent adsorption chromatography using insoluble xylan. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography estimated the molecular weight of the purified putative xylanase (XylA) as approximately 64 kDa. XylA showed xylanase activity toward beechwood xylan, with a maximum enzymatic activity at pH 6.0 and 45°C. Through thin-layer chromatographic analysis of the xylan hydrolysate produced by XylA, it was confirmed that XylA is an endo-type xylanase that decomposes xylan into xylose and xyloligosaccharides of various lengths. The Km and Vmax values of XylA for beechwood xylan were 27.78 mM and 78.13 μM/min, respectively.

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Publication

Identification of a unique 1, 4-β-d-glucan glucohydrolase of glycoside hydrolase family 9 from Cytophaga hutchinsonii.

Jiang, N., Ma, X. D., Fu, L. H., Li, C. X., Feng, J. X. & Duan, C. J. (2020). Applied Microbiology and Biotechnology, 1-16.

Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium that rapidly digests crystalline cellulose. The predicted mechanism by which C. hutchinsonii digests cellulose differs from that of other known cellulolytic bacteria and fungi. The genome of C. hutchinsonii contains 22 glycoside hydrolase (GH) genes, which may be involved in cellulose degradation. One predicted GH with uncertain specificity, CHU_0961, is a modular enzyme with several modules. In this study, phylogenetic tree of the catalytic modules of the GH9 enzymes showed that CHU_0961 and its homologues formed a new group (group C) of GH9 enzymes. The catalytic module of CHU_0961 (CHU_0961B) was identified as a 1,4-β-D-glucan glucohydrolase (EC 3.2.1.74) that has unique properties compared with known GH9 cellulases. CHU_0961B showed highest activity against barley glucan, but low activity against other polysaccharides. Interestingly, CHU_0961B showed similar activity against ρ-nitrophenyl β-D-cellobioside (ρ-NPC) and ρ-nitrophenyl β-D-glucopyranoside. CHU_0961B released glucose from the nonreducing end of cello-oligosaccharides, ρ-NPC, and barley glucan in a nonprocessive exo-type mode. CHU_0961B also showed same hydrolysis mode against deacetyl-chitooligosaccharides as against cello-oligosaccharides. The kcat/Km values for CHU_0961B against cello-oligosaccharides increased as the degree of polymerization increased, and its kcat/Km for cellohexose was 750 times higher than that for cellobiose. Site-directed mutagenesis showed that threonine 321 in CHU_0961 played a role in hydrolyzing cellobiose to glucose. CHU_0961 may act synergistically with other cellulases to convert cellulose to glucose on the bacterial cell surface. The end product, glucose, may initiate cellulose degradation to provide nutrients for bacterial proliferation in the early stage of C. hutchinsonii growth.

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Publication

Effects of Combined α-Amylase and Endo-Xylanase Treatments on the Properties of Fresh and Frozen Doughs and Final Breads.

Kim, H. J. & Yoo, S. H. (2020). Polymers, 12(6), 1349.

Frozen bread doughs usually exhibit less bread volume and poor texture due to dough weakening as well as reduced yeast viability. The objectives of this study were to improve the textural properties of frozen bread dough by applying carbohydrate-active enzymes, α-amylase and endo-xylanase. Each enzyme was applied to dough formulation at 20 (748 and 3.5 units, respectively) and 100 ppm levels of flour, and their combined treatments were also applied. Enzyme-treated doughs were kept frozen at −20°C for 2 weeks, and then baked following the official American Association of Cereal Chemists (AACC) method. A texture profile analysis of oven-baked breads was performed at 25°C after a 5-day storage period. α-Amylase treatment at a 100 ppm level increased the specific bread volume by 24.5% and 21.9% when compared to untreated fresh and frozen bread doughs, respectively, and decreased crumb hardness by 63.4% and 58.3%; endo-xylanase (100 ppm) also decreased crumb hardness by 56.9% and 26.9%. The combined use of α-amylase and endo-xylanase retarded bread hardening synergistically after a 5-day storage period.

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Publication

Purification and characterization of a native lytic polysaccharide monooxygenase from Thermoascus aurantiacus.

Fritsche, S., Hopson, C., Gorman, J., Gabriel, R. & Singer, S. W. (2020). Biotechnology Letters, 1-9.

Lytic polysaccharide monooxygenases (LPMOs) have emerged as key proteins for depolymerization of cellulose. These copper-containing enzymes oxidize C-1 and/or C-4 bonds in cellulose, promoting increased hydrolysis of the oxidized cellulose chains. The LPMO from Thermoascus aurantiacus, a thermophilic ascomycete fungus, has been extensively studied and has served as a model LPMO. A method was developed to purify the LPMO from culture filtrates of T. aurantiacus along with its native cellobiohydrolase and endoglucanase. The activity of the purified LPMO was measured with a colorimetric assay that established the Topt of the native LPMO at 60°C. Purification of the components of the T. aurantiacus cellulase mixture also enabled quantification of the amounts of cellobiohydrolase, endoglucanase and LPMO present in the T. aurantiacus culture filtrate, establishing that the LPMO was the most abundant protein in the culture supernatants. The importance of the LPMO to activity of the mixture was demonstrated by saccharifications with Avicel and acid-pretreated corn stover.

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Publication

Comparative characterization of extracellular enzymes secreted by Phanerochaete chrysosporium during solid-state and submerged fermentation.

Liu, J., Yang, J., Wang, R., Liu, L., Zhang, Y., Bao, H., Myong Jang, J., M., Wang, E. & Yuan, H. (2020). International Journal of Biological Macromolecules, 152, 288-294.

Influence of water content on the expression of lignocellulolytic enzymes by Phanerochaete chrysosporium remains unclear. This work compares the enzyme production profiles of P. chrysosporium during solid-state and submerged fermentation. There were 110 and 64 extracellular carbohydrate-active enzymes identified in solid-state and submerged fermentation respectively, among which 57 enzymes were common to both of the secretomes. P. chrysosporium secreted more cellulases (especially lytic polysaccharide monooxygenase) and hemicellulases during solid-state fermentation while the proportion of enzyme containing carbohydrate-binding module was higher for submerged fermentation. Although its activities were weaker, the enzyme cocktail from submerged fermentation was surprisingly more effective in hydrolysis at low substrate loading. This advantage of enzymes from submerged fermentation was mainly attributed to carbohydrate-binding module because more xylanases bound with substrate at the beginning of hydrolysis. These results reveal the influence of fermentation conditions on enzyme produced by P. chrysosporium for the first time and show the importance of carbohydrate-binding module in the hydrolysis process of lignocellulose.

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Publication

Characteristics of a XIP‐resistant xylanase from Neocallimastix sp. GMLF 1 and its advantage in barley malt saccharification.

Zhu, D., Liu, X., Xie, X., Yang, S., Lin, H. & Chen, H. (2020). International Journal of Food Science & Technology, 55(5), 2152-2160.

The well‐studied xylanase inhibitor protein (XIP‐I) regularly inhibits fungi‐derived xylanases, yet some fungal xylanases are not inhibited by XIP‐I. Based on the sequence alignment with the known XIP‐I resistant NpXyn11A from Neocallimastix patriciarum, a new annotated xylanase from ruminal fungus Neocallimastix sp. GMLF1 was found, noted as Xyn1B, which shared 77.8% of sequence identity with NpXyn11A and was proved to be resistant to XIP‐I. To evaluate the performance of a XIP resistant xylanase used in barley malt saccharification, a XIP‐I sensitive xylanase ThXyn1 from Trichoderma harzianum was chosen as a control. The barley malt saccharification experiment was carried out on the condition with or without extra XIP‐I added. The results showed that Xyn1B displayed only slight difference in presence and absence of added XIP‐I, with the difference of xylose released (ΔX) and the difference of mash clarity (ΔA) being 0.17 g L-1 (P > 0.05) and 0.007 (P > 0.05), respectively; while those for ThXyn1 group reached 0.96 g L-1 (P < 0.01) and 0.095 (P < 0.05), indicating that XIP‐I did not adversely affect Xyn1B's function, but did affect ThXyn1's function. Our work for the first time suggested that a xylanase with resistance to xylanase inhibitor proteins might have an advantage in barley malt saccharification over an inhibitor sensitive xylanase.

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Publication

Utilization of xylan-type polysaccharides in co-culture fermentations of Bifidobacterium and Bacteroides species species..

Zeybek, N., Rastall, R. A. & Büyükkileci, A. O. (2020). Carbohydrate Polymers, 236, 116076.

Although most members of the genus Bifidobacterium are unable to utilize xylan as a carbon source, the growth of these species can be induced by this polysaccharide in the gut environment. This indicates a requirement for an association between Bifidobacterium species and some other members of gut microbiota. In this study, the role of cross-feeding between Bifidobacterium and Bacteroides species in the bifidogenic effect of xylan was investigated using in-vitro pure and co-culture fermentations. The pure culture studies showed that among the Bifidobacterium species tested, only Bifidobacterium animalis subsp. lactis was able to utilize xylooligosaccharides. The co-culture of this strain with Bacteroides species enabled it to grow in the presence of xylan. These results suggest that the ability of Bacteroides species to hydrolyze xylan could allow the proliferation of specific Bifidobacterium species in the gut through substrate cross-feeding.

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Publication

Thermal reactivity of hemicellulose and cellulose in cedar and beech wood cell walls.

Wang, J., Minami, E. & Kawamoto, H. (2020).  Journal of Wood Science, 66(1), 1-10.

Wood cell walls have complex ultra-structures, in which cellulose microfibrils are surrounded by a hemicellulose-lignin matrix. This is the first paper to discuss the effect of cell wall on the pyrolytic reactivity of hemicellulose and cellulose in wood by using Japanese cedar (Cryptomeria japonica, a softwood) and Japanese beech (Fagus crenata, a hardwood), along with isolated xylan and glucomannan. Pyrolysis was conducted by subjecting the samples to temperatures in the range of 220-380°C, at a heating rate of 10°C/min, under a N2 flow with the reactor quenched as soon as the temperature reached the set value. The remaining hemicellulose and cellulose in the heat-treated wood were evaluated by determining the yields of hydrolysable sugar. Isolated xylan containing the uronic acid group, probably acting as a catalyst, was more reactive than isolated glucomannan, whereas the xylan in both woods was remarkably stable and degraded across a similar temperature range to the glucomannan degradation. Thus, the majority of the hemicellulose fractions in cedar and beech unexpectedly exhibited similar reactivity, except for glucomannan in beech that degraded at lower temperatures. Differing thermogravimetric (TG) and derivative TG (DTG) profiles, measured for cedar and beech under similar heating conditions, were explained by the different cellulose reactivity, rather than the hemicellulose reactivity; cellulose decomposed with hemicellulose in cedar, while such decomposition was independent in beech. The observed reactivity is a new finding that is different from the currently understood ideas and may originate from the effects of the cell walls. The research herein provides important information on the kinetics and thermochemical conversion of lignocellulosic biomass.

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Publication

Isolation and Characterization of a Novel Cold-Active, Halotolerant Endoxylanase from Echinicola rosea Sp. Nov. JL3085T.

He, J., Liu, L., Liu, X. & Tang, K. (2020). Marine Drugs, 18(5), 245.

We cloned a xylanase gene (xynT) from marine bacterium Echinicola rosea sp. nov. JL3085T and recombinantly expressed it in Escherichia coli BL21. This gene encoded a polypeptide with 379 amino acid residues and a molecular weight of ~43 kDa. Its amino acid sequence shared 45.3% similarity with an endoxylanase from Cellvibrio mixtus that belongs to glycoside hydrolases family 10 (GH10). The XynT showed maximum activity at 40°C and pH 7.0, and a maximum velocity of 62 μmoL min−1 mg−1. The XynT retained its maximum activity by more than 69%, 51%, and 26% at 10°C, 5°C, and 0°C, respectively. It also exhibited the highest activity of 135% in the presence of 4 M NaCl and retained 76% of its activity after 24 h incubation with 4 M NaCl. This novel xylanase, XynT, is a cold-active and halotolerant enzyme that may have promising applications in drug, food, feed, and bioremediation industries.

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Xylanase inhibition by the derivatives of lignocellulosic material.

Hidayatullah, I. M., Setiadi, T., Kresnowati, M. T. A. P. & Boopathy, R. (2020). Bioresource Technology, 300, 122740.

Hydrolysis of lignocellulosic materials into simple sugar plays an important role in biorefinery. Hemicellulosic sugars from the hydrolysis of lignocellulosic materials could be used in xylitol production. However, xylanase activity during hydrolysis process is affected by activators and inhibitors that may present in the reaction system. The pretreatment process was reported to produce compounds that may affect the enzymatic hydrolysis process, such as furans, aliphatic acid, and aromatics. The purpose of this study was to investigate the inhibition effect of these potential inhibitors on xylanase activity. Three groups of potential inhibitors were evaluated including, furan, aliphatic acid, and hydrolysis-fermentation products. The result showed that ethanol, vanillin, and formic acid gave the highest inhibition effect from each group. Ethanol competed with xylanase competitively. Vanillin showed non-competitive inhibition. Formic acid performed mixed-inhibition by reducing maximum hydrolysis rate and giving varied Michaelis constant values at different concentrations.

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Efficient ferulic acid and xylo-oligosaccharides production by a novel multi-modular bifunctional xylanase/feruloyl esterase using agricultural residues as substrates.

Wang, R., Yang, J., Jang, J. M., Liu, J., Zhang, Y., Liu, L. & Yuan, H. (2020). Bioresource Technology, 297, 122487.

Liberating high value-added compounds ferulic acid (FA) and xylo-oligosaccharides (XOSs) from agricultural residues is a promising strategy for the utilization of lignocellulose. In this study, a bifunctional xylanase/feruloyl esterase from bacterial consortium EMSD5 was heterogeneously expressed in Escherichia coli. Depending on the inter-domain synergism of the recombinant enzyme rXyn10A/Fae1A, high yields of FA (2.78, 1.82, 1.15 and 7.31 mg/g substrate, respectively) were obtained from 20 mg in-soluble wheat arabinoxylan, de-starched wheat bran, ultrafine-grinding corn stover and steam-exploded corncob. Meanwhile, 3.210, 1.235, 1.215 and 0.823 mg xylose/XOSs were also released. For cost-saving enzyme production, we firstly constructed a recombinant E. coli, which could secrete the bifunctional xylanase/feruloyl esterase out of cells. When the recombinant E. coli was cultured in medium containing 200 mg de-starched wheat bran, 474 µg FA and 18.2 mg xylose/XOSs were also detected. Hence, rXyn10A/Fae1A and the recombinant strain showed great applied potential for FA and XOSs production.

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Enhanced stability of a rumen-derived xylanase using SpyTag/SpyCatcher cyclization.

Zhou, Y. B., Cao, J. W., Sun, X. B., Wang, H., Gao, D. Y., Li, Y. N., Wu, K. Y., Wang, J. K., Guo-Ying Qian, G. Y. & & Wang, Q. (2020). World Journal of Microbiology and Biotechnology, 36(2), 1-11.

Microbiota from herbivore rumen is of great interest for mining glycoside hydrolases for lignocellulosic biomass biorefinement. We previously isolated a highly active but poorly thermostable xylanase (LXY) from a rumen fluid fosmid library of Hu sheep, a local high-reproductive species in China. In this study, we used a universal enzyme-engineering strategy called SpyTag/SpyCatcher molecular cyclization to improve LXY stability via isopeptide-bond-mediated ligation. Both linear and cyclized LXY (L- and C-LXY, respectively) shared similar patterns of optimal pH and temperature, pH stability, and kinetic constants (km and Vmax). However, the C-LXY showed enhanced thermostability, ion stability, and resilience to aggregation and freeze–thaw treatment than L-LXY, without compromise of its catalytic efficiency. Circular dichroism and intrinsic and 8-anilino-1-naphthalenesulfonic acid-binding fluorescence analysis indicated that the cyclized enzyme was more capable of maintaining its secondary and tertiary structures than the linear enzyme. Taken together, these results promote the cyclized enzyme for potential applications in the feed, food, paper pulp, and bioenergy industries.

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Heterologous expression, purification and biochemical characterization of a new endo-1, 4-β-xylanase from Rhodothermaceae bacterium RA.

Liew, K. J., Ngooi, C. Y., Shamsir, M. S., Sani, R. K., Chong, C. S. & Goh, K. M. (2019). Protein Expression and Purification, 164, 105464.

Xylanases (EC 3.2.1.8) are essential enzymes due to their applications in various industries such as textile, animal feed, paper and pulp, and biofuel industries. Halo-thermophilic Rhodothermaceae bacterium RA was previously isolated from a hot spring in Malaysia. Genomic analysis revealed that this bacterium is likely to be a new genus of the family Rhodothermaceae. In this study, a xylanase gene (1140 bp) that encoded 379 amino acids from the bacterium was cloned and expressed in Escherichia coli BL21(DE3). Based on InterProScan, this enzyme XynRA1 contained a GH10 domain and a signal peptide sequence. XynRA1 shared low similarity with the currently known xylanases (the closest is 57.2-65.4% to Gemmatimonadetes spp.). The purified XynRA1 achieved maximum activity at pH 8 and 60°C. The protein molecular weight was 43.1 kDa XynRA1 exhibited an activity half-life (t1/2) of 1 h at 60°C and remained stable at 50°C throughout the experiment. However, it was NaCl intolerant, and various types of salt reduced the activity. This enzyme effectively hydrolyzed xylan (beechwood, oat spelt, and Palmaria palmata) and xylodextrin (xylotriose, xylotetraose, xylopentaose, and xylohexaose) to produce predominantly xylobiose. This xylanase is the first functionally characterized enzyme from the bacterium, and this work broadens the knowledge of GH10 xylanases.

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Consolidated bioprocessing performance of bacterial consortium EMSD5 on hemicellulose for isopropanol production.

Liu, L., Yang, J., Yang, Y., Luo, L., Wang, R., Zhang, Y. & Yuan, H. (2019). Bioresource Technology, 292, 121965.

Consolidated bioprocessing (CBP) of lignocellulose by bacterial consortium for isopropanol production is considered as the most promising strategy. However, low utilization of xylan caused by the complex sidechain structure remains inhibit the conversion of full-biomass. In this study, isopropanol production from different lignocelluloses by the consortium EMSD5 through CBP was performed. A total of 7.00 g/L of isopropanol was obtained from corncob by optimizing fermentation conditions. Isopropanol production by EMSD5 was mainly based on utilizing xylan in corncob and isopropanol titer was increased by 47.71% and reached up to 8.39 g/L using arabinoxylan compared with linear xylan. The analysis of community structures and the isolation of key strain confirmed the enrichment of the isopropanol producer, Clostridium beijierinckii, in the bacterial community when it was incubated with corn glucuronoarabinoxylan and the cooperation between C. beijerinckii and lignocellulose degraders. The unique features of EMSD5 could lead to large-scale isopropanol production using lignocellulose.

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Purification and characterization of an endo-xylanase from Trichoderma sp., with xylobiose as the main product from xylan hydrolysis.

Fu, L. H., Jiang, N., Li, C. X., Luo, X. M., Zhao, S. & Feng, J. X. (2019). World Journal of Microbiology and Biotechnology, 35(11), 171.

Fungal endo-β-1,4-xylanases (endo-xylanases) can hydrolyze xylan into xylooligosaccharides (XOS), and have potential biotechnological applications for the exploitation of natural renewable polysaccharides. In the current study, we aimed to screen and characterize an efficient fungal endo-xylanase from 100 natural humus-rich soil samples collected in Guizhou Province, China, using extracted sugarcane bagasse xylan (SBX) as the sole carbon source. Initially, 182 fungal isolates producing xylanases were selected, among which Trichoderma sp. strain TP3-36 was identified as showing the highest xylanase activity of 295 U/mL with xylobiose (X2) as the main product when beechwood xylan was used as substrate. Subsequently, a glycoside hydrolase family 11 endo-xylanase, TXyn11A, was purified from strain TP3-36, and its optimal pH and temperature for activity against beechwood xylan were identified to be 5.0 and 55°C, respectively. TXyn11A was stable across a broad pH range (3.0-10.0), and exhibited strict substrate specificity, including xylan from beechwood, wheat, rye, and sugarcane bagasse, with Km and Vmax values of 5 mg/mL and 1250 µmol/mg min, respectively, toward beechwood xylan. Intriguingly, the main product obtained from hydrolysis of beechwood xylan by TXyn11A was xylobiose, whereas SBX hydrolysis resulted in both X2 and xylotriose. Overall, these characteristics of the endo-xylanase TXyn11A indicate several potential industrial applications.

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Identification and characterization of a hyperthermophilic GH9 cellulase from the Arctic Mid-Ocean Ridge vent field.

Stepnov, A. A., Fredriksen, L., Steen, I. H., Stokke, R. & Eijsink, V. G. (2019). PloS One, 14(9), e0222216.

A novel GH9 cellulase (AMOR_GH9A) was discovered by sequence-based mining of a unique metagenomic dataset collected at the Jan Mayen hydrothermal vent field. AMOR_GH9A comprises a signal peptide, a catalytic domain and a CBM3 cellulose-binding module. AMOR_GH9A is an exceptionally stable enzyme with a temperature optimum around 100°C and an apparent melting temperature of 105°C. The novel cellulase retains 64% of its activity after 4 hours of incubation at 95°C. The closest characterized homolog of AMOR_GH9A is TfCel9A, a processive endocellulase from the model thermophilic bacterium Thermobifida fusca (64.2% sequence identity). Direct comparison of AMOR_GH9A and TfCel9A revealed that AMOR_GH9A possesses higher activity on soluble and amorphous substrates (phosphoric acid swollen cellulose, konjac glucomannan) and has an ability to hydrolyse xylan that is lacking in TfCel9A.

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Coupled chemistry kinetics demonstrate the utility of functionalized Sup35 amyloid nanofibrils in biocatalytic cascades.

Schmuck, B., Gudmundsson, M., Härd, T. & Sandgren, M. (2019). Journal of Biological Chemistry, 294(41), 14966-14977.

Concerns over the environment are a central driver for designing cell-free enzymatic cascade reactions that synthesize non–petrol-based commodity compounds. An often-suggested strategy that would demonstrate the economic competitiveness of this technology is recycling of valuable enzymes through their immobilization. For this purpose, amyloid nanofibrils are an ideal scaffold to realize chemistry-free covalent enzyme immobilization on a material that offers a large surface area. However, in most instances, only single enzyme–functionalized amyloid fibrils have so far been studied. To embark on the next stage, here we displayed xylanase A, β-xylosidase, and an aldose sugar dehydrogenase on Sup35(1–61) nanofibrils to convert beechwood xylan to xylonolactone. We characterized this enzymatic cascade by measuring the time-dependent accumulation of xylose, xylooligomers, and xylonolactone. Furthermore, we studied the effects of relative enzyme concentrations, pH, temperature, and agitation on product formation. Our investigations revealed that a modular cascade with a mixture of xylanase and β-xylosidase, followed by product removal and separate oxidation of xylose with the aldose sugar dehydrogenase, is more productive than an enzyme mix containing all of these enzymes together. Moreover, we found that the nanofibril-coupled enzymes do not lose activity compared with their native state. These findings provide proof of concept of the feasibility of functionalized Sup35(1–61) fibrils as a molecular scaffold for biocatalytic cascades consisting of reusable enzymes that can be used in biotechnology.

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Safety Data Sheet
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