Determination of the modes of action and synergies of xylanases by analysis of xylooligosaccharide profiles over time using fluorescence‐assisted carbohydrate electrophoresis

Abstract

The structure of xylan, which has a 1,4-linked β-xylose backbone with various substituents, is much more heterogeneous and complex than that of cellulose. Because of this, complete degradation of xylan needs a large number of enzymes that includes GH10, GH11, and GH3 family xylanases together with auxiliary enzymes. Fluorescence-assisted carbohydrate electrophoresis (FACE) is able to accurately differentiate unsubstituted and substituted xylooligosaccharides (XOS) in the heterogeneous products generated by different xylanases and allows changes in concentrations of specific XOS to be analyzed quantitatively. Based on a quantitative analysis of XOS profiles over time using FACE, we have demonstrated that GH10 and GH11 family xylanases immediately degrade xylan into sizeable XOS, which are converted into smaller XOS in a much lower speed. The shortest substituted XOS produced by hydrolysis of the substituted xylan backbone by GH10 and GH11 family xylanases were MeGlcA(2) Xyl3 and MeGlcA(2) Xyl4 , respectively. The unsubstituted xylan backbone was degraded into xylose, xylobiose, and xylotriose by both GH10 and GH11 family xylanases; the product profiles are not family-specific but, instead, depend on different subsite binding affinities in the active sites of individual enzymes. Synergystic action between xylanases and β-xylosidase degraded MeGlcA(2) Xyl4 into xylose and MeGlcA(2) Xyl3 but further degradation of MeGlcA(2) Xyl3 required additional enzymes. Synergy between xylanases and β-xylosidase was also found to significantly accelerate the conversion of XOS into xylose.

A Highly Efficient Xylan-Utilization System in Aspergillus niger An76: A Functional-Proteomics Study

“…Endoxylanase isozymes were detected by native PAGE (Figure S2 ), and XynC, XynB, and XynA were in accordance with Xyn1 (GH10), Xyn2 (GH11) and Xyn3 (GH11), respectively, reported in previous studies (Xing et al, 2013 ; Gong et al, 2016b ). In previous study (Xing et al, 2013 ), the genome of A. niger An-76 was not sequenced, the three xylanases secreted by the A. niger An76 were named by Xyn1, Xyn2, Xyn3.…”

“…XynB (GH11) was induced by all carbon sources, except glycerol, indicating that XynB plays a primary role in degrading xylan and identifying numerous enzymes homologous to XynB, which is consistent with previous reports (Krisana et al, 2005 ; Fu et al, 2012 ; Takahashi et al, 2013 ). Although XynA is also in the GH11 family, its expression differed from that of XynB, corresponding to the different product profiles of XynB and XynA reported previously (Gong et al, 2016b ). XynA activity was dramatically enhanced by xylose, XOS, and xylan, but reduced by substrates derived from cellulose, such as glucose, cellobiose, and CMC.…”

“…Supernatant (5 μL) was fluorescently labeled in the dark for 1 h with 7-amino-1,3-naphthalenedisulfonic acid monopotassium salt monohydrate (5 μL, 0.2 M) dissolved in 15% acetic acid, then NaCNBH 3 solution (1 M, 5 μL) in dimethyl sulfoxide was incubated with the mixture at 40°C for 12 h, the labeled products (6 μl/well) were subjected to electrophoresis with a miniaturized vertical gel system utilizing amini-PROTEAN 3 PowerPac Basic Power Supply (Bio-Rad, Hercules, CA, USA), the polyacrylamide gels were made as described in (19). 50% w/v sucrose was used as the loading buffer, and the running buffer was composed of 25 mM Tris-HCl (pH 8.3) and 192 mM glycine, the gels were scanned with a ChemiDoc™ MP imaging system (Bio-Rad) and stored in TIF format (Gong et al, 2016b ).…”

“…Xylose and arabinose were identified as good transcriptional inducers of endoxylanase and arabinan-degrading enzymes, respectively (Battaglia et al, 2011 ); however, the different responses of isozymes having different structures and product profiles have seldom been distinguished (Gong et al, 2016b ). Additionally, the xylan-degradation process is dynamic, leading to rapid changes in extracellular hydrolysate species, especially 4-O-methyl-glucouronic acid substituted xylooligosaccharide (XOS) and unsubstituted XOS (xylobiose, xylotriose, xylotetraose, xylopentaose), which was proved with Fluorescence-assisted carbohydrate electrophoresis (FACE) and electrospray ionization mass spectrometry (Gong et al, 2016b ). A previous study of Neurospora crassa reported that cellodextrins acted as inducers of cellulases (Znameroski et al, 2012 ), whereas few studies have focused on the effect of XOS on xylan utilization (Herold et al, 2013 ).…”

Evaluation of xylooligosaccharide production from residual hemicelluloses of dissolving pulp by acid and enzymatic hydrolysis

“…This may result from the inhibition of the end-products to xylanase activity and the conversion of XOS to xylose. 11,17,35 As shown in Fig. 3, the hydrolysis rate and yield of XOS signicantly increased with the enzyme concentration varied from 50 to 120 IU g À1 substrate.…”