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An Acidic Thermostable Recombinant Aspergillus nidulans Endoglucanase Is Active towards Distinct Agriculture Residues.

ABSTRACT: Aspergillus nidulans is poorly exploited as a source of enzymes for lignocellulosic residues degradation for biotechnological purposes. This work describes the A. nidulans Endoglucanase A heterologous expression in Pichia pastoris, the purification and biochemical characterization of the recombinant enzyme. Active recombinant endoglucanase A (rEG A) was efficiently secreted as a 35?kDa protein which was purified through a two-step chromatography procedure. The highest enzyme activity was detected at 50°C/pH 4. rEG A retained 100% of activity when incubated at 45 and 55°C for 72?h. Purified rEG A kinetic parameters towards CMC were determined as K m = 27.5 ± 4.33?mg/mL, V max = 1.185 ± 0.11?mmol/min, and 55.8?IU (international units)/mg specific activity. Recombinant P. pastoris supernatant presented hydrolytic activity towards lignocellulosic residues such as banana stalk, sugarcane bagasse, soybean residues, and corn straw. These data indicate that rEG A is suitable for plant biomass conversion into products of commercial importance, such as second-generation fuel ethanol.

SUBMITTER: Tavares EQ

PROVIDER: S-EPMC3723094 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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An Acidic Thermostable Recombinant Aspergillus nidulans Endoglucanase Is Active towards Distinct Agriculture Residues.

Tavares Eveline Queiroz de Pinho EQ Rubini Marciano Regis MR Mello-de-Sousa Thiago Machado TM Duarte Gilvan Caetano GC de Faria Fabrícia Paula FP Ferreira Filho Edivaldo Ximenes EX Kyaw Cynthia Maria CM Silva-Pereira Ildinete I Poças-Fonseca Marcio Jose MJ

Enzyme research 20130710

Aspergillus nidulans is poorly exploited as a source of enzymes for lignocellulosic residues degradation for biotechnological purposes. This work describes the A. nidulans Endoglucanase A heterologous expression in Pichia pastoris, the purification and biochemical characterization of the recombinant enzyme. Active recombinant endoglucanase A (rEG A) was efficiently secreted as a 35 kDa protein which was purified through a two-step chromatography procedure. The highest enzyme activity was detecte ...[more]

PMID: 23936633

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Project description:BACKGROUND:Filamentous fungi are among the most powerful cellulolytic organisms in terrestrial ecosystems. To perform the degradation of lignocellulosic substrates, these microorganisms employ both hydrolytic and oxidative mechanisms that involve the secretion and synergism of a wide variety of enzymes. Interactions between these enzymes occur on the level of saccharification, i.e., the release of neutral and oxidized products, but sometimes also reflected in the substrate liquefaction. Although the synergism regarding the yield of neutral sugars has been extensively studied, further studies should focus on the oxidized sugars, as well as the effect of enzyme combinations on the viscosity properties of the substrates. RESULTS:In the present study, the heterologous expression of an endoglucanase (EG) and its combined activity together with a lytic polysaccharide monooxygenase (LPMO), both from the thermophilic fungus Myceliophthora thermophila, are described. The EG gene, belonging to the glycoside hydrolase family 5, was functionally expressed in the methylotrophic yeast Pichia pastoris. The produced MtEG5A (75 kDa) featured remarkable thermal stability and showed high specific activity on microcrystalline cellulose compared to CMC, which is indicative of its processivity properties. The enzyme was capable of releasing high amounts of cellobiose from wheat straw, birch, and spruce biomass. Addition of MtLPMO9 together with MtEG5A showed enhanced enzymatic hydrolysis yields against regenerated amorphous cellulose (PASC) by improving the release not only of the neutral but also of the oxidized sugars. Assessment of activity of MtEG5A on the reduction of viscosity of PASC and pretreated wheat straw using dynamic viscosity measurements revealed that the enzyme is able to perform liquefaction of the model substrate and the natural lignocellulosic material, while when added together with MtLPMO9, no further synergistic effect was observed. CONCLUSIONS:The endoglucanase MtEG5A from the thermophilic fungus M. thermophila exhibited excellent properties that render it a suitable candidate for use in biotechnological applications. Its strong synergism with LPMO was reflected in sugars release, but not in substrate viscosity reduction. Based on the level of oxidative sugar formation, this is the first indication of synergy between LPMO and EG reported.

Dataset Information

An Acidic Thermostable Recombinant Aspergillus nidulans Endoglucanase Is Active towards Distinct Agriculture Residues.

Publications

An Acidic Thermostable Recombinant Aspergillus nidulans Endoglucanase Is Active towards Distinct Agriculture Residues.

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