- β-xylosidase from Selenomonas ruminantium: Immobilization, stabilization, and application for xylooligosaccharide hydrolysis
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The tetrameric β-xylosidase from Selenomonas ruminantium is very stable in alkaline pH allowing it to easily immobilize by multipoint covalent attachments on highly activated glyoxyl agarose gels. Initial immobilization resulted only in slight stabilization in relation to the free enzyme, since involvement of all subunits was not achieved. Coating the catalyst with aldehyde-dextran or polyethylenimine, fully stabilized the quaternary structure of the enzyme rendering much more stabilization to the biocatalyst. The catalyst coated with polyethylenimine of molecular weight 1300 is the most stable one exhibiting an interesting half-life of more than 10 days at pH 5.0 and 50 °C, being, therefore, 240-fold more stable than free enzyme. Optimum activity was observed in the pH range 4.0–6.0 and at 55 °C. The catalyst retained its side activity against p-nitrophenyl α-l-arabinofuranoside and it was inhibited by xylose and glucose. Kinetic parameters with p-nitrophenyl β-d-xylopyranoside as substrate were Vmax 0.20 μmol.min?1 mg prot.?1, Km 0.45 mM, Kcat 0.82 s?1, and Kcat/Km 1.82 s?1 mM?1. Xylose release was observed from the hydrolysis of xylooligosaccharides with a decrease in the rate of xylose release by increasing substrate chain-length. Due to the high thermostability and the complete stability after five reuse cycles, the applicability of this biocatalyst in biotechnological processes, such as for the degradation of lignocellulosic biomass, is highly increased.
- Terrasan, César Rafael Fanchini,Aragon, Caio Casale,Masui, Douglas Chodi,Pessela, Benevides Costa,Fernandez-Lorente, Gloria,Carmona, Eleonora Cano,Guisan, Jose Manuel
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p. 161 - 171
(2016/12/16)
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- Two family 11 xylanases from Achaetomium sp. Xz-8 with high catalytic efficiency and application potentials in the brewing industry
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This study identified two family-11 xylanase genes (xynC81 and xynC83) in Achaetomium sp. Xz-8, a thermophilic strain from a desert area with substantial xylanase activity, and successfully expressed them in Pichia pastoris. Their deduced amino acid sequences showed the highest identity of ≤90% to known fungal xylanases and of ≤62% with each other. The purified recombinant xylanases showed optimal activities at pH 5.5 and 60-65 C and exhibited stability over pH 5.0-10.0 and temperatures at 55 C and below. XynC81 had high catalytic efficiency (6082 mL/s/mg), and XynC83 was favorable for xylooligosaccharide production. Under simulated mashing conditions, combination of XynC83 and a commercial β-glucanase improved the filtration rate by 34.76%, which is much better than that of Novozymes Ultraflo (20.71%). XynC81 and XynC83 had a synergistic effect on viscosity reduction (7.08%), which is comparable with that of Ultraflo (8.47%). Thus, XynC81 and XynC83 represent good candidates for application in the brewing industry.
- Zhao, Liang,Meng, Kun,Bai, Yingguo,Shi, Pengjun,Huang, Huoqing,Luo, Huiying,Wang, Yaru,Yang, Peilong,Song, Wei,Yao, Bin
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p. 6880 - 6889
(2013/08/23)
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- Molecular cloning and characterization of a novel thermostable xylanase from Paenibacillus campinasensis BL11
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An open reading frame (XylX) with 1131 nucleotides from Paenibacillus campinasensis BL11 was cloned and expressed in E. coli. It encodes a family 11 endoxylanase, designated as XylX, of 41kDa. The homology of the amino acid sequence deduced from XylX is only 73% identical to the next closest sequence. XylX contains a family 11 catalytic domain of the glycoside hydrolase and a family 6 cellulose-binding module. The recombinant xylanase was fused to a His-tag for affinity purification. The XylX activity was 2392IU/mg, with a Km of 6.78mg/ml and a Vmax of 4953mol/min/mg under optimal conditions (pH 7, 60°C). At pH 11, 60°C, the activity was still as high as 517IU/mg. Xylanase activities at 60°C under pH 5 to pH 9 remained at more than 69.4% of the initial activity level for 8h. The addition of Hg2+ at 5mM almost completely inhibited xylanase activity, whereas the addition of tris-(2-carboxyethyl)-phosphine (TCEP) and 2-mercaptoethanol stimulated xylanase activity. No relative activities for Avicel, CMC and d-(+)-cellobiose were found. Xylotriose constitutes the majority of the hydrolyzed products from oat spelt and birchwood xylan. Broad pH and temperature stability shows its application potentials for biomass conversion, food and pulp/paper industries.
- Ko, Chun-Han,Tsai, Chung-Hung,Tu, Jenn,Lee, Hang-Yi,Ku, Lan-Ting,Kuo, Pei-An,Lai, Yiu-Kay
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supporting information; experimental part
p. 1638 - 1644
(2011/12/03)
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- The impact of dilute sulfuric acid on the selectivity of xylooligomer depolymerization to monomers
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The disappearance of xylose and xylooligosaccharides with degrees of polymerization (DP) ranging from 2 to 5 was followed at 160 °C with sulfuric acid added to adjust the pH from near neutral to 1.45, and the impact on the yields of lower DP xylooligomers and xylose monomer was determined. In addition, the experimental data for the disappearance of these xylooligomers was kinetically modeled assuming first-order reaction kinetics for xylose degradation and xylooligomer hydrolysis to evaluate how the pH affected the selectivity of monomer formation from xylooligomers and direct oligomer degradation to unknown products. The yield of xylose from xylooligomers increased appreciably with increasing acid concentration but decreased with increasing xylooligomer DP at a given acid concentration, resulting in more acid being required to realize the same xylose yields for higher DP species. For example, the maximum xylose yields were 49.6%, 28.0%, 13.2% and 3.2% for DP values of 2, 3, 4, and 5, respectively, at pH 4.75. Kinetic modeling revealed that all the xylooligomers disappeared at a higher rate compared to xylose monomer and the disappearance rate constant increased with DP at all pH. The kinetics for lower DP oligomers of 2 and 3 showed that these species directly degrade to unknown compounds in the absence of acid. On the other hand, higher oligomers of DP 4 and 5 exhibited negligible losses to degradation products at all pH. Therefore, only xylooligomers of DP 2 and 3 were found to directly degrade to undesired products in the absence of acid, but more work is needed to determine how higher DP species behave. This study also revealed that the source of water and the material used for the construction of the reactor impacted xylose degradation kinetics.
- Kumar, Rajeev,Wyman, Charles E.
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p. 290 - 300
(2008/09/19)
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