High pressure homogenization (HPH) is a non-thermal method, which has been employed to change the activity and stability of biotechnologically relevant enzymes. This work investigated how HPH affects ...the structural and functional characteristics of a glucose oxidase (GO) from Aspergillus niger. The enzyme was homogenized at 75 and 150 MPa and the effects were evaluated with respect to the enzyme activity, stability, kinetic parameters and molecular structure. The enzyme showed a pH-dependent response to the HPH treatment, with reduction or maintenance of activity at pH 4.5-6.0 and a remarkable activity increase (30-300%) at pH 6.5 in all tested temperatures (15, 50 and 75°C). The enzyme thermal tolerance was reduced due to HPH treatment and the storage for 24 h at high temperatures (50 and 75°C) also caused a reduction of activity. Interestingly, at lower temperatures (15°C) the activity levels were slightly higher than that observed for native enzyme or at least maintained. These effects of HPH treatment on function and stability of GO were further investigated by spectroscopic methods. Both fluorescence and circular dichroism revealed conformational changes in the molecular structure of the enzyme that might be associated with the distinct functional and stability behavior of GO.
► The Amazon rainforest Trichoderma harzianum is a potential candidate for saccharification of plant biomass. ► Important roles for pectinase and α-l-arabinofuranosidase in the enzymatic synergies. ► ...Sugarcane bagasse hydrolysis is more effective and competitive for second generation ethanol.
Supplementation of cellulase cocktails with accessory enzymes can contribute to a higher hydrolytic capacity in releasing fermentable sugars from plant biomass. This study investigated which enzymes were complementary to the enzyme set of Trichoderma harzianum in the degradation of sugarcane bagasse. Specific activities of T. harzianum extract on different substrates were compared with the extracts of Penicillium echinulatum and Trichoderma reesei, and two commercial cellulase preparations. Complementary analysis of the secretome of T. harzianum was also used to identify which enzymes were produced during growth on pretreated sugarcane bagasse. These analyses enabled the selection of the enzymes pectinase and α-l-arabinofuranosidase (AF) to be further investigated as supplements to the T. harzianum extract. The effect of enzyme supplementation on the efficiency of sugarcane bagasse saccharification was evaluated using response surface methodology. The supplementation of T. harzianum enzymatic extract with pectinase and AF increased the efficiency of hydrolysis by up to 116%.
The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant ...polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from
Aspergillus clavatus
(AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production.
During the past few years, the first industrial-scale cellulosic ethanol plants have been inaugurated. Although the performance of the commercial cellulase enzymes used in this process has greatly ...improved over the past decade, cellulases still represent a very significant operational cost. Depending on the region, transport of cellulases from a central production facility to a biorefinery may significantly add to enzyme cost. The aim of the present study was to develop a simple, cost-efficient cellulase production process that could be employed locally at a Brazilian sugarcane biorefinery.
Our work focused on two main topics: growth medium formulation and strain improvement. We evaluated several Brazilian low-cost industrial residues for their potential in cellulase production. Among the solid residues evaluated, soybean hulls were found to display clearly the most desirable characteristics. We engineered a
strain to secrete cellulase in the presence of repressing sugars, enabling the use of sugarcane molasses as an additional carbon source. In addition, we added a heterologous β-glucosidase to improve the performance of the produced enzymes in hydrolysis. Finally, the addition of an invertase gene from
into our strain allowed it to consume sucrose from sugarcane molasses directly. Preliminary cost analysis showed that the overall process can provide for very low-cost enzyme with good hydrolysis performance on industrially pre-treated sugarcane straw.
In this study, we showed that with relatively few genetic modifications and the right growth medium it is possible to produce considerable amounts of well-performing cellulase at very low cost in Brazil using
. With further enhancements and optimization, such a system could provide a viable alternative to delivered commercial cellulases.
Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the ...isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45°C. The crystal structure was solved at 2.75 Å resolution, revealing the classical (β/α)8-barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the +1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production.
Termites can degrade up to 90% of the lignocellulose they ingest using a repertoire of endogenous and symbiotic degrading enzymes. Termites have been shown to secrete two main glycoside hydrolases, ...which are GH1 (EC 3.2.1.21) and GH9 (EC 3.2.1.4) members. However, the molecular mechanism for lignocellulose degradation by these enzymes remains poorly understood. The present study was conducted to understand the synergistic relationship between GH9 (CgEG1) and GH1 (CgBG1) from Coptotermes gestroi, which is considered the major urban pest of São Paulo State in Brazil. The goal of this work was to decipher the mode of operation of CgEG1 and CgBG1 through a comprehensive biochemical analysis and molecular docking studies. There was outstanding degree of synergy in degrading glucose polymers for the production of glucose as a result of the endo-β-1,4-glucosidase and exo-β-1,4-glucosidase degradation capability of CgEG1 in concert with the high catalytic performance of CgBG1, which rapidly converts the oligomers into glucose. Our data not only provide an increased comprehension regarding the synergistic mechanism of these two enzymes for cellulose saccharification but also give insight about the role of these two enzymes in termite biology, which can provide the foundation for the development of a number of important applied research topics, such as the control of termites as pests as well as the development of technologies for lignocellulose-to-bioproduct applications.
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•The description of the endo-b-1,4-glucosidase (CgEG1) and b-glucosidase (CgBG1) from Coptotermes gestroi.•CgEG1 and CgBG1 present high degree of synergy in degrading cellulose polymers into the glucose.•The mode of operation of CgEG1 and CgBG1 was deciphered through extensively biochemical and docking analyses.•CgEG1 features both endo and exo hydrolytic patterns.
► Novel arabinan hydrolytic enzyme. ► High-temperature operating and thermostable endo-1–5-α-arabinanase. ► Determination of mode of operation of the recombinant enzyme along with biophysical ...insights over enzyme hyperthermophilicity. ► Low-resolution structure determination by small angle X-ray scattering of the enzyme.
Arabinan is a plant structural polysaccharide degraded by two enzymes; α-
l-arabinofuranosidase and endo-1,5-α-
l-arabinanase. These enzymes are highly diversified in nature, however, little is known about their biochemical and biophysical properties. We have characterized a novel arabinanase (AbnA) isolated from
Thermotoga petrophila with unique thermostable properties such as the insignificant decrease of residual activity after incubation up to 90
°C. We determined the AbnA mode of operation through capillary zone electrophoresis, which accumulates arabinotriose and arabinobiose as end products after hydrolysis of arabinan-containing polysaccharides. Spectroscopic analyses by Far-UV circular dichroism and intrinsic tryptophan fluorescence emission demonstrated that AbnA is folded and formed mainly by β-sheet structural elements.
In silico molecular modeling showed that the AbnA structure encompasses a five-bladed β-propeller catalytic core juxtaposed by distorted up-and-down β-barrel domain. The low-resolution structure determined by small angle X-ray scattering indicated that AbnA is monomeric in solution and its molecular shape is in full agreement with the model.
Boto, a class II transposable element, was characterized in the Moniliophthora perniciosa genome. The Boto transposase is highly similar to plant PIF-like transposases that belong to the newest class ...II superfamily known as PIF/Harbinger. Although Boto shares characteristics with PIF-like elements, other characteristics, such as the transposase intron position, the position and direction of the second ORF, and the footprint, indicate that Boto belongs to a novel family of the PIF/Harbinger superfamily. Southern blot analyses detected 6-12 copies of Boto in C-biotype isolates and a ubiquitous presence among the C- and S-biotypes, as well as a separation in the C-biotype isolates from Bahia State in Brazil in at least two genotypic groups, and a new insertion in the genome of a C-biotype isolate maintained in the laboratory for 6 years. In addition to PCR amplification from a specific insertion site, changes in the Boto hybridization profile after the M. perniciosa sexual cycle and detection of Boto transcripts gave further evidence of Boto activity. As an active family in the genome of M. perniciosa, Boto elements may contribute to genetic variability in this homothallic fungus. This is the first report of a PIF/Harbinger transposon in the genome of a phytopathogenic fungus.
Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is ...of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCel5A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.
Plant feedstocks are at the leading front of the biofuel industry based on the potential to promote economical, social and environmental development worldwide through sustainable scenarios related to ...energy production. Penicillium echinulatum is a promising strain for the bioethanol industry based on its capacity to produce large amounts of cellulases at low cost. The secretome profile of P. echinulatum after grown on integral sugarcane bagasse, microcrystalline cellulose and three types of pretreated sugarcane bagasse was evaluated using shotgun proteomics. The comprehensive chemical characterization of the biomass used as the source of fungal nutrition, as well as biochemical activity assays using a collection of natural polysaccharides, were also performed. Our study revealed that the enzymatic repertoire of P. echinulatum is geared mainly toward producing enzymes from the cellulose complex (endogluganases, cellobiohydrolases and β-glucosidases). Glycoside hydrolase (GH) family members, important to biomass-to-biofuels conversion strategies, were identified, including endoglucanases GH5, 7, 6, 12, 17 and 61, β-glycosidase GH3, xylanases GH10 and GH11, as well as debranching hemicellulases from GH43, GH62 and CE2 and pectinanes from GH28. Collectively, the approach conducted in this study gave new insights on the better comprehension of the composition and degradation capability of an industrial cellulolytic strain, from which a number of applied technologies, such as biofuel production, can be generated.
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