Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol ...production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for β-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl β-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.
•Cellulose nanofibers (CNF) were produced combining hydrolytic and oxidative enzymes.•The set of recombinant enzymes was applied to sugarcane bagasse for the first time.•Enzymatically produced CNF ...are longer and more thermostable than TEMPO-oxidised CNF.•Enzymatic treatment using enzymes holds promise for CNF production.
Cellulose nanofibers (CNF) are renewable and biodegradable nanomaterials with attractive barrier, mechanical and surface properties. In this work, three different recombinant enzymes: an endoglucanase, a xylanase and a lytic polysaccharide monooxygenase, were combined to enhance cellulose fibrillation and to produce CNF from sugarcane bagasse (SCB). Prior to the enzymatic catalysis, SCB was chemically pretreated by sodium chlorite and KOH, while defibrillation was accomplished via sonication. We obtained much longer (μm scale length) and more thermostable (resisting up to 260 °C) CNFs as compared to the CNFs prepared by TEMPO-mediated oxidation. Our results showed that a cooperative action of the set of hydrolytic and oxidative enzymes can be used as a “green” treatment prior to the sonication step to produce nanofibrillated cellulose with advanced properties.
•JdXyn10A is a two-domain GH10 xylanase.•JdXyn10A is halotolerant and ethanol resistant.•A tightening of the +2 subsite prevent the enzyme from releasing xylose.•JdXyn10A can be efficiently used for ...prebiotic production.
Development of a more environmentally sustainable society is based on the maximum use of renewable carbon sources and their valorization of environmentally-friendly green technologies. This includes a thorough use of plant biomass and agricultural residues for the production of value-added bioproducts. Xylan is the second most abundant biopolymer in nature which can be sustainable converted into pentoses and xylooligosaccharides, that have wide applications in the food, feed, pharmaceutical, and cosmetic industry. Within the scope of present study, we biochemically characterized two-domain GH10 xylanase from Jonesia denitrificans (JdXyn10A) and evaluated its applicability for production of xylooligosaccharides (XOS). JdXyn10A has a specific activity of 84 ± 2 U/mg and 65 ± 5 U/mg when acting on beechwood glucuronoxylan and rye arabinoxylan, respectively. The enzyme is stable in a wide pH range and is tolerant to high concentrations of NaCl and ethanol. Interestingly, the profile of products released by the enzyme is predominant in xylobiose and xylotriose, with a very low fraction of xylose which is desirable for XOS production. The efficiencies of enzymatic conversion of beechwood glucuronoxylan and rye arabinoxylan are 47.67 % and 26.01 %, respectively, after 6 h of enzymatic hydrolysis only. Structural comparison between the JdXyn10A homology model and the structure from its homologous that while the glycone region of its active site is well preserved, the aglycone region presents structural differences in the +2 subsite that may explain why JdXyn10A does not release xylose.
•T. composti GH10 xylanase is thermostable, halotolerant and ethanol-resistant.•X-ray structure of TcXyn10A reveals molecular details of xylan recognition.•TcXyn10A improves efficiency of pretreated ...lignocellulosic biomass hydrolysis.•The enzyme liberates xylooligosaccharides from xylan with elevated yields.
Enzymatic transformation of xylans into renewable fuels and value-added products is mediated by xylanases. Here we describe the biochemical and X-ray structural characterization of Thermobacillus composti GH10 xylanase (TcXyn10A) at 2.1 Å resolution aiming to unravel details of its recognition of glucurono- and arabinoxylan at a molecular level. TcXyn10A improves the efficiency of pretreated lignocellulosic biomass hydrolysis by a commercial enzyme cocktail causing a 15.35 % increase in xylose release and 4.38 % glucose release after 24 h of reaction. The enzyme releases predominantly xylobiose and xylotriose, as well as MeGlcA3 × 3 (from beechwood glucuronoxylan) and a range of decorated xylooligosaccharides (XOS) from rye arabinoxylan, with Ara2 × 2 being the major product. The enzyme liberates XOS with the yields of 29.09 % for beechwood glucuronoxylan and 16.98 % for rye arabinoxylan. Finally, TcXyn10A has a high thermal stability, halotolerance, and resistance to ethanol, biochemical properties that can be desirable for a number of industrial applications.
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•Structural mechanism of cellulose depolymerization by GH48 cellulase was examined.•BlCel48B has high processivity against crystalline and amorphous cellulose.•Substrate size and ...surface morphology dictate the hydrolytic rate, enzyme binding, and processivity.•Clusters of co-evolved residues drive the recognition and strength of cellulose binding to the active site.•Enzyme low-turnover and high processivity rates correlate with the rigidity of the catalytic tunnel.
Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process. However, the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood. Here, integrated enzymatic assays, protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel48B cellulase from Bacillus licheniformis. Hydrolytic efficiency, substrate binding affinity, cleavage patterns, and the apparent processivity of bacterial BlCel48B are significantly impacted by the cellulose size and its surface morphology. BlCel48B crystallographic structure was solved with ligands spanning -5 to -2 and +1 to +2 subsites. Statistical coupling analysis and molecular dynamics show that co-evolved residues on active site are critical for stabilizing ligands in the catalytic tunnel. Our results provide mechanistic insights into BlCel48B molecular-level determinants of activity, substrate binding, and processivity on insoluble cellulose, thus shedding light on structure-activity correlations of GH48 family members in general.
An interplay between cellulases is fundamental in biomass saccharification. Here, the synergistic action of
Trichoderma harzianum
Cel7A and Cel7B on two cellulosic substrates: bacterial cellulose ...(BC) and a much more heterogeneous filter paper (FP) was investigated by determining their saccharification yields and by analyzing both the released soluble products and the insoluble reducing ends formed in the process. Furthermore, morphological changes of the substrates were evaluated using scanning electron microscopy. Glycoside hydrolase family 7 (GH7) enzymes introduce uniform changes in BC, whereas in FP they preferentially consume thin microfibrils rather than thicker paper fibers. Thus, the size effect, which leads to a smaller surface area per unit of substrate mass for thicker fibers, seems to play a crucial role in higher enzymatic hydrolysis efficiency of BC as compared to FP. These results demonstrate that the morphology-dependent effects could be essential for the industrial breakdown of cellulose-rich plant biomass.
Adsorption of cellulases on the cellulose surface is an integral part of the catalytic mechanism, and a detailed description of the adsorption process is therefore required for a fundamental ...understanding of this industrially important class of enzymes. However, the mode of adsorption has proven intricate, and several key questions remain open. Perhaps most notably it is not clear whether the adsorbed enzyme is in dynamic equilibrium with the free population or irreversibly associated with no or slow dissociation. To address this, we have systematically investigated adsorption reversibility for two cellobiohydrolases (Cel7A and Cel6A) and one endoglucanase (Cel7B) on four types of pure cellulose substrates. Specifically, we monitored dilution-induced release of adsorbed enzyme in samples that had previously been brought to a steady state (constant concentration of free enzyme). In simple dilution experiments (without centrifugation), the results consistently showed full reversibility. In contrast to this, resuspension of enzyme–substrate pellets separated by centrifugation showed extensive irreversibility. We conclude that these enzymes are in a dynamic equilibrium between free and adsorbed states but suggest that changes in the physical properties of cellulose caused by compaction of the pellet hampers subsequent release of adsorbed enzyme. This latter effect may be pertinent to both previous controversies in the literature on adsorption reversibility and the development of enzyme recycling protocols in the biomass industry.
The heteropolysaccharide xylan is a valuable source of sustainable chemicals and materials from renewable biomass sources. A complete hydrolysis of this major hemicellulose component requires a ...diverse set of enzymes including endo‐β‐1,4‐xylanases, β‐xylosidases, acetylxylan esterases, α‐l‐arabinofuranosidases, and α‐glucuronidases. Notably, the most studied xylanases from glycoside hydrolase family 11 (GH11) have exclusively been endo‐β‐1,4‐ and β‐1,3‐xylanases. However, a recent analysis of a metatranscriptome library from a microbial lignocellulose community revealed GH11 enzymes capable of releasing solely xylobiose from xylan. Although initial biochemical studies clearly indicated their xylobiohydrolase mode of action, the structural features that drive this new activity still remained unclear. It was also not clear whether the enzymes acted on the reducing or nonreducing end of the substrate. Here, we solved the crystal structure of MetXyn11 in the apo and xylobiose‐bound forms. The structure of MetXyn11 revealed the molecular features that explain the observed pattern on xylooligosaccharides released by this nonreducing end xylobiohydrolase.
Structural and biochemical studies were performed to characterize an unusual enzyme from GH11 family which was identified in a compost‐derived consortium. The enzyme has a xylobiohydrolase activity, quite uncommon for members of GH11 family. Structural studies revealed that a lack of ‐3 subsite in the active site of the enzyme restrained a range of its catalytic products, leading to a release of only xylobiose (X2) during xylan hydrolysis.
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•TtLPMO9H oxidizes cellulose at C1 and C4 positions.•Combined activity of TtLPMO9H and GH7 endoglucanase remarkably increased yeild of reducing sugars•Synergy with GH5, GH12 and GH45 ...endoglucanases was significantly smaller.•Light-driven catalysis by TtLPMO9H strongly improved cellulose degradation.•SAXS model reveals molecular organization of two-domain GH9 LPMO.
Lytic polysaccharide monooxygenases (LPMOs), monocopper enzymes that oxidatively cleave recalcitrant polysaccharides, have important biotechnological applications. Thermothelomyces thermophilus is a rich source of biomass-active enzymes, including many members from auxiliary activities family 9 LPMOs. Here, we report biochemical and structural characterization of recombinant TtLPMO9H which oxidizes cellulose at the C1 and C4 positions and shows enhanced activity in light-driven catalysis assays. TtLPMO9H also shows activity against xyloglucan. The addition of TtLPMO9H to endoglucanases from four different glucoside hydrolase families (GH5, GH12, GH45 and GH7) revealed that the product formation was remarkably increased when TtLPMO9H was combined with GH7 endoglucanase. Finally, we determind the first low resolution small-angle X-ray scattering model of the two-domain TtLPMO9H in solution that shows relative positions of its two functional domains and a conformation of the linker peptide, which can be relevant for the catalytic oxidation of cellulose and xyloglucan.
Pretreatment of biomass is one of the most challenging steps in the process of second-generation (2G) ethanol and biochemical production. Dilute acid pretreatment is a widely adapted and convenient ...method to recover pentose (C5) as well as hexose (C6) sugars due to its featured solubilization of hemicellulose and cellulose before and after enzymatic saccharification, respectively. In the present study, dilute sulfuric acid (H
2
SO
4
) pretreatment of sugarcane bagasse (SCB) was statistically optimized using the face-centered composite design (FCCD) of response surface methodology (RSM) in terms of acid concentration (0.1–3% v/v), solid loading (5–20% w/v) and residence time (15–60 min) at constant temperature of 121 °C followed by enzymatic hydrolysis using commercial cellulase (Novozymes Cellic CTec2) for enhanced combined sugar yield (CSY) comprising of C5 and C6 sugars in pretreated as well as saccharified hydrolysates. Optimized process parameters found in the study were 2.18% (v/v) acid; 14.35% (w/v) solid loading; and 29.49 min residence time. CSY under optimized conditions was found to be 521.42 ± 7.2 g/kg raw SCB with 72.06 ± 1.0% sugars recovered out of the maximum theoretical sugars present in the raw biomass. Total reducing sugar yields in pretreated and saccharified hydrolysates were found to be 215.28 ± 2.4 and 306.14 ± 5.3 g/kg raw SCB, respectively. Morphological and structural changes in optimized pretreated and saccharified biomass further validated the efficiency of optimized pretreatment applied in the present study. The maximum ethanol concentration, volumetric productivity and yield from released sugars were calculated as 10.82 ± 2.2 g/L, 0.45 ± 0.9 g/L/h and 0.42 g/g-glucose consumed or 71.45 ± 2.5 g/kg raw SCB, respectively. Ethanol yield obtained from the fermentation of dilute H
2
SO
4
-pretreated SCB was corresponding to 82.4% of the theoretical ethanol yield.
