Jinguang Hu

Substrate factors that influence the synergistic interaction of AA9 and cellulases during the enzymatic hydrolysis of biomass

The polysaccharide monooxygenase enzyme AA9 (formerly known as GH61) was shown to interact synergistically with cellulases to enhance the enzymatic hydrolysis of a range of “commercially-relevant” pretreated and “model” cellulosic substrates. Although an exogenous source of reducing power was required when AA9 was added with cellulases to a “pure” cellulosic substrate, it was not required when added to pretreated lignocellulosic substrates. It appears that the non-cellulosic components such as soluble components, lignin, and possibly hemicellulose, can all act as AA9 reducing cofactor. Of the various substrate characteristics that influenced the efficacy of the enzyme mixture, the relative amount of accessible crystalline cellulose, assessed by the specific cellulose binding module (CBM), appeared to be the most critical. Cellulases and AA9 acted synergistically when hydrolysing cellulose I but it did not occur during the hydrolysis of cellulose II and III.

Swollenin aids in the amorphogenesis step during the enzymatic hydrolysis of pretreated biomass

A key limitation in the overall hydrolysis process is the restricted access that the hydrolytic enzymes have due to the macro-and-micro structure of cellulose and its association with hemicellulose and lignin. Previous work has shown that several non-hydrolytic proteins can disrupt cellulose structure and boost the activity of hydrolytic enzymes when purer forms of cellulose are used. In the work reported here, Swollenin primarily disrupted the hemicellulosic fraction of pretreated corn stover, resulting in the solubilisation of monomeric and oligomeric sugars. Although Swollenin showed little synergism when combined with the cellulase monocomponents exoglucanase (CEL7A) and endoglucanase (CEL5A), it showed pronounced synergism with xylanase monocomponents Xylanase GH10 and Xylanase GH11, resulting in the release of significantly more xylose (>300%). It appears that Swollenin plays a role in amorphogenesis and that its primary action is enhancing access to the hemicellulose fraction that limits or masks accessibility to the cellulose component of lignocellulosic substrates.

The impact of glycerol organosolv pretreatment on the chemistry and enzymatic hydrolyzability of wheat straw.

Given that the glycerol organosolv pretreatment (GOP) can effectively improve the hydrolyzability of various lignocellulosic substrates, physicochemical changes of the substrate before and after the pretreatment was characterized to elucidate what is responsible for it. The effect of GOP on the main components and hydrolyzability of wheat straw was revisited. Results demonstrate that the GOP should be a promising candidate for the current pretreatment. Then the composition and structure of substrates was measured at multi-dimensional scales by using various analytic equipment such as TGA, SEM, AFM, CLSM, FT-IR, XRD and solid-state CP/MAS (13)C NMR. This paper reports some new insights on the mechanism behind that, which can be beneficial for further development, optimization, and scale-up of the GOP process.

The influence of lignin on steam pretreatment and mechanical pulping of poplar to achieve high sugar recovery and ease of enzymatic hydrolysis.

With the goal of enhancing overall carbohydrate recovery and reducing enzyme loading refiner mechanical pulping and steam pretreatment (210°C, 5 min) were used to pretreat poplar wood chips. Neutral sulphonation post-treatment indicated that, although the lignin present in the steam pretreated substrate was less reactive, the cellulose-rich, water insoluble component was more accessible to cellulases and Simons stain. This was likely due to lignin relocation as the relative surface lignin measured by X-ray photoelectron spectroscopy increased from 0.4 to 0.8. The integration of sulphite directly into steam pretreatment resulted in the solubilisation of 60% of the lignin while more than 80% of the carbohydrate present in the original substrate was recovered in the water insoluble fraction after Na2CO3 addition. More than 80% of the sugars present in the original cellulose and xylan could be recovered after 48 h using an enzyme loading of 20 mg protein/g cellulose at a 10% substrate concentration.

The Use of Carbohydrate Binding Modules (CBMs) to Monitor Changes in Fragmentation and Cellulose Fiber Surface Morphology during Cellulase- and Swollenin-induced Deconstruction of Lignocellulosic Substrates

Background: Fiber fragmentation is thought to occur at dislocations, which are potential targets for the non-hydrolytic protein, Swollenin. Results: Changes in cellulose morphology within dislocations were assessed using fluorescent CBMs; Swollenin appeared to promote fragmentation at these sites. Conclusion: Swollenin targets and disrupts cellulose at fiber dislocations. Significance: Fragmentation is a key step in cellulose deconstruction and is enhanced by the actions of Swollenin. Although the actions of many of the hydrolytic enzymes involved in cellulose hydrolysis are relatively well understood, the contributions that amorphogenesis-inducing proteins might contribute to cellulose deconstruction are still relatively undefined. Earlier work has shown that disruptive proteins, such as the non-hydrolytic non-oxidative protein Swollenin, can open up and disaggregate the less-ordered regions of lignocellulosic substrates. Within the cellulosic fraction, relatively disordered, amorphous regions known as dislocations are known to occur along the length of the fibers. It was postulated that Swollenin might act synergistically with hydrolytic enzymes to initiate biomass deconstruction within these dislocation regions. Carbohydrate binding modules (CBMs) that preferentially bind to cellulosic substructures were fluorescently labeled. They were imaged, using confocal microscopy, to assess the distribution of crystalline and amorphous cellulose at the fiber surface, as well as to track changes in surface morphology over the course of enzymatic hydrolysis and fiber fragmentation. Swollenin was shown to promote targeted disruption of the cellulosic structure at fiber dislocations.

Pretreating lignocellulosic biomass by the concentrated phosphoric acid plus hydrogen peroxide (PHP) for enzymatic hydrolysis: Evaluating the pretreatment flexibility on feedstocks and particle sizes

In order to seek a high-efficient pretreatment path for converting lignocellulosic feedstocks to fermentable sugars by enzymatic hydrolysis, the concentrated H₃PO₄ plus H₂O₂ (PHP) was attempted to pretreat different lignocellulosic biomass for evaluating the pretreatment flexibility on feedstocks. Meanwhile, the responses of pretreatment to particle sizes were also evaluated. When the PHP-pretreatment was employed (final H₂O₂ and H₃PO₄ concentration of 1.77% and 80.0%), 71-96% lignin and more than 95% hemicellulose in various feedstocks (agricultural residues, hardwood, softwood, bamboo, and their mixture, and garden wastes mixture) can be removed. Consequently, more than 90% glucose conversion was uniformly achieved indicating PHP greatly improved the pretreatment flexibility to different feedstocks. Moreover, when wheat straw and oak chips were PHP-pretreated with different sizes, the average glucose conversion reached 94.9% and 100% with lower coefficient of variation (7.9% and 0.0%), which implied PHP-pretreatment can significantly weaken the negative effects of feedstock sizes on subsequent conversion.

Evaluation of hemicellulose removal by xylanase and delignification on SHF and SSF for bioethanol production with steam-pretreated substrates.

Steam-pretreated sweet sorghum bagasse (SSB) and Douglas-fir (DF) were employed for SHF and SSF to evaluate the effects of xylanase supplementation and delignification on ethanol production. Results indicated final ethanol concentration in SHF could reach 28.4 g/L (SSB) and 20.4 g/L (DF) by xylanase supplementation with the increase of 46% and 61% in comparison with controls. The delignification could significantly enhance final ethanol concentration to 31.2g/L (SSB) and 30.2 g/L (DF) with the increase of 61% and 138%. In SSF, final ethanol concentration in the delignified SSB and DF arrived at 27.6 g/L and 34.3 g/L with the increase of 26% and 157% compared with controls. However, only 2.2 g/L (SSB) and 6.9 g/L (DF) ethanol were obtained with xylanase supplementation. According to these results, it could be concluded that delignification was beneficial to improve ethanol production of SHF and SSF. The xylanase supplementation (0.12 g protein/g glucan) was only positive to SHF while retarded SSF seriously.

Pretreating wheat straw by the concentrated phosphoric acid plus hydrogen peroxide (PHP): Investigations on pretreatment conditions and structure changes.

Wheat straw was pretreated by PHP (the concentrated H3PO4 plus H2O2) to clarify effects of temperature, time and H3PO4 proportion on hemicellulose removal, delignification, cellulose recovery and enzymatic digestibility. Overall, hemicellulose removal was intensified by PHP comparing to the concentrated H3PO4. Moreover, efficient delignification specially happened in PHP pretreatment. Hemicellulose removal and delignification by PHP positively responded to temperature and time. Increasing H3PO4 proportion in PHP can promote hemicellulose removal, however, decrease the delignification. Maximum hemicellulose removal and delignification were achieved at 100% and 83.7% by PHP. Enzymatic digestibility of PHP-pretreated wheat straw was greatly improved by increasing temperature, time and H3PO4 proportion, and complete hydrolysis can be achieved consequently. As temperature of 30-40°C, time of 2.0 h and H3PO4 proportion of 60% were employed, more than 92% cellulose was retained in the pretreated wheat straw, and 29.1-32.6g glucose can be harvested from 100g wheat straw.

Enhanced delignification of steam-pretreated poplar by a bacterial laccase

The recalcitrance of woody biomass, particularly its lignin component, hinders its sustainable transformation to fuels and biomaterials. Although the recent discovery of several bacterial ligninases promises the development of novel biocatalysts, these enzymes have largely been characterized using model substrates: direct evidence for their action on biomass is lacking. Herein, we report the delignification of woody biomass by a small laccase (sLac) from Amycolatopsis sp. 75iv3. Incubation of steam-pretreated poplar (SPP) with sLac enhanced the release of acid-precipitable polymeric lignin (APPL) by ~6-fold, and reduced the amount of acid-soluble lignin by ~15%. NMR spectrometry revealed that the APPL was significantly syringyl-enriched relative to the original material (~16:1 vs. ~3:1), and that sLac preferentially oxidized syringyl units and altered interunit linkage distributions. sLac’s substrate preference among monoaryls was also consistent with this observation. In addition, sLac treatment reduced the molar mass of the APPL by over 50%, as determined by gel-permeation chromatography coupled with multi-angle light scattering. Finally, sLac acted synergistically with a commercial cellulase cocktail to increase glucose production from SPP ~8%. Overall, this study establishes the lignolytic activity of sLac on woody biomass and highlights the biocatalytic potential of bacterial enzymes.

The Accessible Cellulose Surface Influences Cellulase Synergism during the Hydrolysis of Lignocellulosic Substrates

Effective enzymatic hydrolysis of insoluble cellulose requires the synergistic action of a suite of cellulase components. Most previous studies have only assessed cellulase synergism on model cellulosic substrates. When the actions of individual and combinations of cellulases (Cel7A, Cel6A, Cel7B, Cel5A) were assessed on various pretreated lignocellulosic substrates, Cel7A was shown to be the major contributor to overall cellulose hydrolysis, with the other enzymes synergistically enhancing its hydrolytic efficiency, at least partially, by facilitating Cel7A desorption (assessed by a double-sandwich enzyme-linked immunosorbent assay). When the influences of various substrate physicochemical characteristics on the effectiveness of enzyme synergism were assessed, a strong relationship was observed between cellulose accessibility (as determined by the cellulose binding module technique) and the degree of synergism, with greater synergy observed on the more disorganized/accessible cellulose surface.