Cloning, heterologous expression and biochemical characterization of a non-specific endoglucanase family 12 from Aspergillus terreus NIH2624.

The cellulases from Glycoside Hydrolyses family 12 (GH12) play an important role in cellulose degradation and plant cell wall deconstruction being widely used in a number of bioindustrial processes. Aiming to contribute toward better comprehension of these class of the enzymes, here we describe a high-yield secretion of a endoglucanase GH12 from Aspegillus terreus (AtGH12), which was cloned and expressed in Aspergillus nidulans strain A773. The purified protein was used for complete biochemical and functional characterization. The optimal temperature and pH of the enzyme were 55°C and 5.0 respectively, which has high activity against ß-glucan and xyloglucan and also is active toward glucomannan and CMC. The enzyme retained activity up to 60°C. AtGH12 is strongly inhibited by Cu2+, Fe2+, Cd2+, Mn2+, Ca2+, Zn2+ and EDTA, whereas K+, Tween, Cs+, DMSO, Triton X-100 and Mg2+ enhanced the enzyme activity. Furthermore, SAXS data reveal that the enzyme has a globular shape and CD analysis demonstrated a prevalence of a ß-strand structure corroborating with typical ß-sheets fold commonly found for other endoglucanases from GH12 family.

Increased biomass saccharification by supplementation of a commercial enzyme cocktail with endo-arabinanase from Bacillus licheniformis.

OBJECTIVES: The use of endo-arabinanase from Bacillus licheniformis (ABNase) for sugarcane saccharification has been evaluated by enzyme immobilization and commercial cocktail supplement with the immobilized heterologous protein. RESULTS: Biochemical characterization of the purified ABNase showed that the catalytic activity was strongly inhibited by 5 mM Cu(2+), Zn(2+) or Fe(3+). The optimum pH and temperature for activity were 5.5-6.5 and 35-40 °C, respectively. The enzyme stability increased 128-fold when immobilized with glyoxyl agarose, and the hydrolysis of pretreated sugar cane biomass increased by 15 % when a commercial enzyme cocktail was supplemented with immobilized ABNase. CONCLUSION: Pectin hydrolysis by recombinant ABNase plays a role in the effective application of enzymatic cocktails for biomass saccharification.

Development and biotechnological application of a novel endoxylanase family GH10 identified from sugarcane soil metagenome.

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 (ß/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.

Deciphering the synergism of endogenous glycoside hydrolase families 1 and 9 from Coptotermes gestroi.

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-ß-1,4-glucosidase and exo-ß-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.

Characterization of a hexameric exo-acting GH51 α-l-arabinofuranosidase from the mesophilic Bacillus subtilis.

α-l-Arabinofuranosidases (α-l-Abfases, EC 3.2.1.55) display a broad specificity against distinct glycosyl moieties in branched hemicellulose and recent studies have demonstrated their synergistic use with cellulases and xylanases for biotechnological processes involving plant biomass degradation. In this study, we examined the structural organization of the arabinofuranosidase (GH51 family) from the mesophilic Bacillus subtilis (AbfA) and its implications on function and stability. The recombinant AbfA showed to be active over a broad temperature range with the maximum activity between 35 and 50 °C, which is desirable for industrial applications. Functional studies demonstrated that AbfA preferentially cleaves debranched or linear arabinan and is an exo-acting enzyme producing arabinose from arabinoheptaose. The enzyme has a canonical circular dichroism spectrum of α/ß proteins and exhibits a hexameric quaternary structure in solution, as expected for GH51 members. Thermal denaturation experiments indicated a melting temperature of 53.5 °C, which is in agreement with the temperature­activity curves. The mechanisms associated with the unfolding process were investigated through molecular dynamics simulations evidencing an important contribution of the quaternary arrangement in the stabilization of the ß-sandwich accessory domain and other regions involved in the formation of the catalytic interface of hexameric Abfases belonging to GH51 family.

Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus.

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.

Kinetic mechanism and catalysis of Trypanosoma cruzi dihydroorotate dehydrogenase enzyme evaluated by isothermal titration calorimetry.

Trypanosoma cruzi dihydroorotate dehydrogenase (TcDHODH) catalyzes the oxidation of l-dihydroorotate to orotate with concomitant reduction of fumarate to succinate in the de novo pyrimidine biosynthetic pathway. Based on the important need to characterize catalytic mechanism of TcDHODH, we have tailored a protocol to measure TcDHODH kinetic parameters based on isothermal titration calorimetry. Enzymatic assays lead to Michaelis-Menten curves that enable the Michaelis constant (K(M)) and maximum velocity (V(max)) for both of the TcDHODH substrates: dihydroorotate (K(M)=8.6+/-2.6 microM and V(max)=4.1+/-0.7 microMs(-1)) and fumarate (K(M)=120+/-9 microM and V(max)=6.71+/-0.15 microMs(-1)). TcDHODH activity was investigated using dimethyl sulfoxide (10%, v/v) and Triton X-100 (0.5%, v/v), which seem to facilitate the substrate binding process with a small decrease in K(M). Arrhenius plot analysis allowed the determination of thermodynamic parameters of activation for substrates and gave some insights into the enzyme mechanism. Activation entropy was the main contributor to the Gibbs free energy in the formation of the transition state. A factor that might contribute to the unfavorable entropy is the hindered access of substrates to the TcDHODH active site where a loop at its entrance regulates the open-close channel for substrate access.