Key amino acid residues for the endo-processive activity of GH74 xyloglucanase

AbstractUnlike endo-dissociative-xyloglucanases, Paenibacillus XEG74 is an endo-processive xyloglucanase that contains four unique tryptophan residues in the negative subsites (W61 and W64) and the positive subsites (W318 and W319), as indicated by three-dimensional homology modelling. Selective replacement of the positive subsite residues with alanine mutations reduced the degree of processive activity and resulted in the more endo-dissociative-activity. The results showed that W318 and W319, which are found in the positive subsites, are essential for processive degradation and are responsible for maintaining binding interactions with xyloglucan polysaccharide through a stacking effect

Cloning and Characterization of Two Xyloglucanases from Paenibacillus sp. Strain KM21

Two xyloglucan-specific endo-β-1,4-glucanases (xyloglucanases [XEGs]), XEG5 and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were isolated from the gram-positive bacterium Paenibacillus sp. strain KM21, which degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively. XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5 lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 type carbohydrate-binding module at its C terminus. The two XEGs were expressed in Escherichia coli, and recombinant forms of the enzymes were purified and characterized. Both XEGs had endoglucanase active only toward xyloglucan and not toward Avicel, carboxymethylcellulose, barley β-1,3/1,4-glucan, or xylan. XEG5 is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while XEG74 has dual endo- and exo-mode activities or processive endo-mode activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses using various oligosaccharide structures revealed that unique structures of xyloglucan oligosaccharides can be prepared with XEG74

Selective fluorescence labeling: time-lapse enzyme visualization during sugarcane hydrolysis

Enzymatic biomass saccharification is an important process for bioethanol production. Hitherto, numerous cellulase cocktails (crude enzyme) have been developed to improve enzymatic activity. For this purpose, the synergy of incorporating hydrolase functionality within a cellulase cocktail is a key function. However, such synergistic action, by potentially numerous different enzyme types, on biomass tissue has not been considered despite the importance toward the realistic case of biomass saccharification. This study aims to visualize the behavior of each of the key cellulase components on biomass tissue during saccharification. Time-lapse fluorescence microscopy observations were conducted during saccharification of a thin transverse sugarcane section to monitor enzymes modified with a fluorescence dye. Statistical image analysis successfully demonstrated a unique adsorption/desorption behavior of each enzyme component. Particularly, the behavior of endoxylanase10 (Xyn10), which was recently discovered from Penicillium sp. as a high-performance xylanase, displayed remarkable adsorption on tissues of sugarcane, which accounts for the superior activity of the cellulase mixture with Xyn10

MOESM1 of The impact of a single-nucleotide mutation of bgl2 on cellulase induction in a Trichoderma reesei mutant

Additional file 1: Figure S1. Secreted protein by transformant of PC-3-7 and QM9414. A: Avicel cultivation; 10 µL of the supernatant from day 5 of PC-3-7 series and day 6 of QM9414 series were subjected to SDS-PAGE. Gels were stained by Coomassie Brilliant Blue. B: Cellobiose cultivation; 20 µL of supernatant from day 2.5 of PC-3-7 series and day 3 of QM9414 series were subjected to SDS-PAGE. Gel of Avicel cultivation was stained by Coomassie Brilliant Blue and that of cellobiose cultivation was silver stained.   Figure S2. Gene expression profile of PC-3-7 transformants on Avicel induction. Genes analyzed were cbh1 (A), egl1 (B), bgl2 (C), and xyr1 (D). Stippled gray bar represents PC-3-7, solid gray bar represents PC-Wbgl2, and the shaded gray bar indicates PC-∆bgl2. Values represent the relative expression of each gene normalized to act1 as an internal control. Values represent the means of triplicate experiments. Error bars indicate standard deviations.   Figure S3. Effect of bgl2 mutation and disruption in T. reesei QM9414. A: specific activity of the intracellular cellobiase from transformants QM9414, QM-Mbgl2 and QM-∆bgl2. Cellobiase activity is derived from the mean of triplicate experiments. Error bars indicate standard deviation. B: HPLC analysis of transglycosylation products using cell-free extracts from transformants. Details are as in Fig. 2. Putative transglycosylation products are indicated by arrows.   Figure S4. Southern analysis of transformants for bgl2 analysis. A: the schematic representation of genomic structure of each transformants. B: results of hybridization by gene specific probe for bgl2 and pyr4. Genomic DNA of each transformants was digested by SacI. Lane M represents molecular marker. Table S1. PCR primers used for plasmid construction. Table S2. primers used for real-time quantitative PCR