BACKGROUND: Xylan plays an important role in the enzymatic digestion of biomass, but xylan-degrading enzymes are expensive to produce. Current research is focused on improving the activity and efficacy of hydrolytic enzymes by molecular engineering and on understanding the synergistic cooperation of these enzymes.
RESULTS:In this study, xylan-degrading enzymes, including endo--1,4-xylanases (HoXyn11A and AnXyn10C), -xylosidases (AnXln3D) and -L-arabinofuranosidases (AnAxh62A) from Hypocrea orientalis and Aspergillus niger, were expressed at high levels in Pichia pastoris. Substrate specificity and end product analysis verified the mode of actions of the recombinant enzymes, which were specific for xylan degradation. Synergistic cooperation among these recombinant enzymes was clearly demonstrated. AnXyn10C was more efficient than HoXyn11A for producing both xylose and xylo-oligosaccharides from beechwood xylan and wheat arabinoxylan. AnAxh62A displayed better cooperative effects with HoXyn11A than AnXyn10C in the hydrolysis of wheat arabinoxylan and increased the yield of xylobiose and xylotriose 8.6-fold and 7.3-fold, respectively. Although AnXln3D was the key enzyme for producing xylose, the yield of xylose depended strongly on the main-chain and side-group cleaving enzymes.
CONCLUSION: This study will facilitate the development of highly efficient enzyme cocktails for the bioconversion of lignocellulosic biomass into monosaccharides and xylo-oligosaccharides.
Enzyme assayXylanase activity was assayed using 1% BWX as the substrate in sodium citrate buffer (50 mmol L −1 , pH 5.0) at 50 ∘ C for 10 min. 24 wileyonlinelibrary.com/jctb
A gene encoding glycoside hydrolase family 11 xylanase (HoXyn11B) from Hypocrea orientalis EU7-22 was expressed in Pichia pastoris with a high activity (413 IU/ml). HoXyn11B was partly N-glycosylated and appeared two protein bands (19-29 kDa) on SDS-PAGE. The recombinant enzyme exhibited optimal activity at pH 4.5 and 55 °C, and retained more than 90% of the original activity after incubation at 50 °C for 60 min. The determined apparent K and V values using beechwood xylan were 10.43 mg/ml and 3246.75 IU/mg, respectively. The modes of action of recombinant HoXyn11B on xylo-oligosaccharides (XOSs) and beechwood xylan were investigated by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which indicated that the modes of action of HoXyn11B are different from HoXyn11A since it is able to release a significant amount of xylose from various substrates. This study provides an opportunity to better understand the hydrolysis mechanisms of xylan by xylanases from Trichoderma.
Xylan-degrading enzymes from Aspergillus niger and Hypocrea orientalis were characterized by enzyme activity assays and protein profiling with SDS-PAGE and LC-MS/MS. The hydrolysis of Miscanthus xylan by xylan-degrading enzymes from A. niger, H. orientalis, and Trichoderma reesei were comparatively studied by HPLC analysis. It was found that the glycoside hydrolase families 10 xylanase was the main xylanase secreted by H. orientalis and A. niger when using corn cob and wheat bran as inducers. Compared to the enzymes from T. reesei,the enzymes from A. niger showed better efficiency in the hydrolysis of Miscanthus xylan into monosaccharides. Nevertheless, the enzymes from H. orientalis were more preferable for the hydrolysis of Miscanthus xylan into xylo-oligosaccharides (XOS), especially xylobiose and xylotriose. Miscanthus xylan degradation was significantly influenced by the activities of β-xylosidase and α-L-arabinofuranosidase. Xylan-degrading enzymes with high ratios of β-xylosidase and α-L-arabinofuranosidase are necessary for the efficient conversion of Miscanthus xylan into monosaccharides. However, xylan-degrading enzymes with low β-xylosidase activity and high α-L-arabinofuranosidase activity were required for producing XOS.
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