Dataset Information

Visualizing cellulase adsorption and quantitatively determining cellulose accessibility with an updated fungal cellulose-binding module-based fluorescent probe protein.

ABSTRACT:

Background

Cellulose accessibility to cellulases (CAC) is a direct factor determining the enzymatic digestibility of lignocellulosic cellulose. Improving CAC by pretreatment is a prerequisite step for the efficient release of fermentable sugars from biomass cell wall. However, conventional methods to study the porosimetry of solid materials showed some limitations to be used for investigating CAC. In this work, an updated novel fusion protein comprising a fungal cellulose-binding module (CBM) from Cel7A cellobiohydrolase I (CBH I) of Trichoderma reesei QM6 and a di-green fluorescent protein (GFP₂) was constructed for quantitative determination of CAC.

Results

The obtained probe protein had similar molecular size (e.g., weight) with that of Cel7A and could give detectable signal for quantitative analysis. Several construction strategies were compared with regard to the site of His-tag and order of CBM and GFP₂ modules in the protein sequence, in order to achieve good expression quantity and usability of the probe protein. His6-CBM-GFP₂ has been identified as the best probe protein for investigating the effects of structural features of cellulosic substrates on cellulose accessibility. Substrate samples with different contents of xylan, lignin, and degree of substitution of cellulose -OH by formyl group were obtained, respectively, by mild H₂SO₄ pre-hydrolysis, NaClO₂ selective delignification, and treatment of filter paper cellulose with concentrated formic acid. The determined CAC was in a wide range of 0.6-20.4 m²/g depending on the contents of hemicelluloses, lignin, and formyl group as well as cellulose degree of crystallization.

Conclusions

The obtained fusion probe protein could be used as a versatile tool to quantitatively investigate the impacts of biomass structural features on CAC and hydrolyzability of cellulose substrates, as well as nonproductive adsorption of cellulase enzymes on lignin.

SUBMITTER: Li T

PROVIDER: S-EPMC5890345 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

Visualizing cellulase adsorption and quantitatively determining cellulose accessibility with an updated fungal cellulose-binding module-based fluorescent probe protein.

Li Tian T Liu Nan N Ou Xianjin X Zhao Xuebing X Qi Feng F Huang Jianzhong J Liu Dehua D

Biotechnology for biofuels 20180409

<h4>Background</h4>Cellulose accessibility to cellulases (CAC) is a direct factor determining the enzymatic digestibility of lignocellulosic cellulose. Improving CAC by pretreatment is a prerequisite step for the efficient release of fermentable sugars from biomass cell wall. However, conventional methods to study the porosimetry of solid materials showed some limitations to be used for investigating CAC. In this work, an updated novel fusion protein comprising a fungal cellulose-binding module ...[more]

PMID: 29657580

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Project description:Background:The high cost of enzymes is one of the key technical barriers that must be overcome to realize the economical production of biofuels and biomaterials from biomass. Supplementation of enzyme cocktails with lytic polysaccharide monooxygenase (LPMO) can increase the efficiency of these cellulase mixtures for biomass conversion. The previous studies have revealed that LPMOs cleave polysaccharide chains by oxidization of the C1 and/or C4 carbons of the monomeric units. However, how LPMOs enhance enzymatic degradation of lignocellulose is still poorly understood. Results:In this study, we combined enzymatic assays and real-time imaging using atomic force microscopy (AFM) to study the molecular interactions of an LPMO [TrAA9A, formerly known as TrCel61A) from Trichoderma reesei] and a cellobiohydrolase I (TlCel7A from T. longibrachiatum) with bacterial microcrystalline cellulose (BMCC) as a substrate. Cellulose conversion by TlCel7A alone was enhanced from 46 to 54% by the addition of TrAA9A. Conversion by a mixture of TlCel7A, endoglucanase, and ?-glucosidase was increased from 79 to 87% using pretreated BMCC with TrAA9A for 72 h. AFM imaging demonstrated that individual TrAA9A molecules exhibited intermittent random movement along, across, and penetrating into the ribbon-like microfibril structure of BMCC, which was concomitant with the release of a small amount of oxidized sugars and the splitting of large cellulose ribbons into fibrils with smaller diameters. The dividing effect of the cellulose microfibril occurred more rapidly when TrAA9A and TlCel7A were added together compared to TrAA9A alone; TlCel7A alone caused no separation. Conclusions:TrAA9A increases the accessible surface area of BMCC by separating large cellulose ribbons, and thereby enhances cellulose hydrolysis yield. By providing the first direct observation of LPMO action on a cellulosic substrate, this study sheds new light on the mechanisms by which LPMO enhances biomass conversion.

Project description:Background:The enzymes for efficient hydrolysis of lignocellulosic biomass are a major factor in the development of an economically feasible cellulose bioconversion process. Up to now, low hydrolysis efficiency and high production cost of cellulases remain the significant hurdles in this process. The aim of the present study was to develop a versatile cellulase system with the enhanced hydrolytic efficiency and the ability to synthesize powerful inducers by genetically engineering Trichoderma reesei. Results:In our study, we employed a systematic genetic strategy to construct the carbon catabolite-derepressed strain T. reesei SCB18 to produce the cellulase complex that exhibited a strong cellulolytic capacity for biomass saccharification and an extraordinary high ?-glucosidase (BGL) activity for cellulase-inducing disaccharides synthesis. We first identified the hypercellulolytic and uracil auxotrophic strain T. reesei SP4 as carbon catabolite repressed, and then deleted the carbon catabolite repressor gene cre1 in the genome. We found that the deletion of cre1 with the selectable marker pyrG led to a 72.6% increase in total cellulase activity, but a slight reduction in saccharification efficiency. To facilitate the following genetic modification, the marker pyrG was successfully removed by homologous recombination based on resistance to 5-FOA. Furthermore, the Aspergillus niger BGLA-encoding gene bglA was overexpressed, and the generated strain T. reesei SCB18 exhibited a 29.8% increase in total cellulase activity and a 51.3-fold enhancement in BGL activity (up to 103.9 IU/mL). We observed that the cellulase system of SCB18 showed significantly higher saccharification efficiency toward differently pretreated corncob residues than the control strains SDC11 and SP4. Moreover, the crude enzyme preparation from SCB18 with high BGL activity possessed strong transglycosylation ability to synthesize ?-disaccharides from glucose. The transglycosylation product was finally utilized as the inducer for cellulase production, which provided a 63.0% increase in total cellulase activity compared to the frequently used soluble inducer, lactose. Conclusions:In summary, we constructed a versatile cellulase system in T. reesei for efficient biomass saccharification and powerful cellulase inducer synthesis by combinational genetic manipulation of three distinct types of genes to achieve the customized cellulase production, thus providing a viable strategy for further strain improvement to reduce the cost of biomass-based biofuel production.

Dataset Information

Visualizing cellulase adsorption and quantitatively determining cellulose accessibility with an updated fungal cellulose-binding module-based fluorescent probe protein.

Background

Results

Conclusions

Publications

Visualizing cellulase adsorption and quantitatively determining cellulose accessibility with an updated fungal cellulose-binding module-based fluorescent probe protein.

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