Dataset Information

Cellulases dig deep: in situ observation of the mesoscopic structural dynamics of enzymatic cellulose degradation.

ABSTRACT: Enzymatic hydrolysis of cellulose is key for the production of second generation biofuels, which represent a long-standing leading area in the field of sustainable energy. Despite the wealth of knowledge about cellulase structure and function, the elusive mechanism by which these enzymes disintegrate the complex structure of their insoluble substrate, which is the gist of cellulose saccharification, is still unclear. We herein present a time-resolved structural characterization of the action of cellulases on a nano-flat cellulose preparation, which enabled us to overcome previous limitations, using atomic force microscopy (AFM). As a first step in substrate disintegration, elongated fissures emerge which develop into coniform cracks as disintegration continues. Detailed data analysis allowed tracing the surface evolution back to the dynamics of crack morphology. This, in turn, reflects the interplay between surface degradation inside and outside of the crack. We observed how small cracks evolved and initially increased in size. At a certain point, the crack diameter stagnated and then started decreasing again. Stagnation corresponds with a decrease in the total amount of surface which is fissured and thus leads to the conclusion that the surface hydrolysis "around" the cracks is proceeding more rapidly than inside the cracks. The mesoscopic view presented here is in good agreement with various mechanistic proposals from the past and allows a novel insight into the structural dynamics occurring on the cellulosic substrate through cellulase action.

SUBMITTER: Bubner P

PROVIDER: S-EPMC3268433 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Cellulases dig deep: in situ observation of the mesoscopic structural dynamics of enzymatic cellulose degradation.

Bubner Patricia P Dohr Judith J Plank Harald H Mayrhofer Claudia C Nidetzky Bernd B

The Journal of biological chemistry 20111129 4

Enzymatic hydrolysis of cellulose is key for the production of second generation biofuels, which represent a long-standing leading area in the field of sustainable energy. Despite the wealth of knowledge about cellulase structure and function, the elusive mechanism by which these enzymes disintegrate the complex structure of their insoluble substrate, which is the gist of cellulose saccharification, is still unclear. We herein present a time-resolved structural characterization of the action of ...[more]

PMID: 22128148

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Project description:BackgroundAs a green alternative for the production of transportation fuels, the enzymatic hydrolysis of lignocellulose and subsequent fermentation to ethanol are being intensively researched. To be economically feasible, the hydrolysis of lignocellulose must be conducted at a high concentration of solids, which results in high concentrations of hydrolysis end-products, cellobiose and glucose, making the relief of product inhibition of cellulases a major challenge in the process. However, little quantitative information on the product inhibition of individual cellulases acting on cellulose substrates is available because it is experimentally difficult to assess the hydrolysis of the heterogeneous polymeric substrate in the high background of added products.ResultsThe cellobiose and glucose inhibition of thermostable cellulases from Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum acting on uniformly 14C-labeled bacterial cellulose and its derivatives, 14C-bacterial microcrystalline cellulose and 14C-amorphous cellulose, was studied. Cellulases from Trichoderma reesei were used for comparison. The enzymes most sensitive to cellobiose inhibition were glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs), followed by family 6 CBHs and endoglucanases (EGs). The strength of glucose inhibition followed the same order. The product inhibition of all enzymes was relieved at higher temperatures. The inhibition strength measured for GH7 CBHs with low molecular-weight model substrates did not correlate with that measured with 14C-cellulose substrates.ConclusionsGH7 CBHs are the primary targets for product inhibition of the synergistic hydrolysis of cellulose. The inhibition must be studied on cellulose substrates instead of on low molecular-weight model substrates when selecting enzymes for lignocellulose hydrolysis. The advantages of using higher temperatures are an increase in the catalytic efficiency of enzymes and the relief of product inhibition.

Project description:Softening, a common phenomenon in many fruits, is a well coordinated and genetically determined process. However, the process of flesh softening during ripening has rarely been described in hawthorn. In this study, we found that 'Ruanrou Shanlihong 3 Hao' fruits became softer during ripening, whereas 'Qiu JinXing' fruits remained hard. At late developmental stages, the firmness of 'Ruanrou Shanlihong 3 Hao' fruits rapidly declined, and that of 'Qiu JinXing' fruits remained essentially unchanged. According to transmission electron microscopy, the middle lamella of 'Qiu JinXing' and 'Ruanrou Shanlihong 3 Hao' fruit flesh was largely degraded as the fruits matured. Microfilaments in 'Qiu JinXing' flesh were arranged close together and were deep in color, whereas those in 'Ruanrou Shanlihong 3 Hao' fruit flesh were arranged loosely, partially degraded and light in color. RNA-Seq analysis yielded approximately 46.72 Gb of clean data and 72,837 unigenes. Galactose metabolism and pentose and glucuronate interconversions are involved in cell wall metabolism, play an important role in hawthorn texture. We identified 85 unigenes related to the cell wall between hard- and soft-fleshed hawthorn fruits. Based on data analysis and real-time PCR, we suggest that β-GAL and PE4 have important functions in early fruit softening. The genes Ffase, Gns,α-GAL, PE63, XTH, and CWP, which are involved in cell wall degradation, are responsible for the different textures of hawthorn fruits. Thus, we hypothesize that the different textures of 'Qiu JinXing' and 'Ruanrou Shanlihong 3 Hao' fruits at maturity mainly result from cellulose/hemicelluloses degradation rather than from lamella degradation. Overall, we propose that different types of hydrolytic enzymes in cells interact to degrade the cell wall, resulting in ultramicroscopic Structure changes in the cell wall and, consequently, fruit softening. These results provide fundamental insight regarding the mechanisms by which hawthorn fruits acquire different textures and also lay a solid foundation for further research.

Dataset Information

Cellulases dig deep: in situ observation of the mesoscopic structural dynamics of enzymatic cellulose degradation.

Publications

Cellulases dig deep: in situ observation of the mesoscopic structural dynamics of enzymatic cellulose degradation.

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